pensar em uso de drogas

Aleksandra

GP1A0 - uma gestação de parto natural e nenhum abortamento

duas gestações*

exames para a gestante

teste de IST’S e tuberculose ética médica - o que fazer se o médico
• hepatite, siflis, hiv não quer fazer o abortamento?
hemograma completo
• deficiência nutricional, dislipidemia
• pedir uma nova ultrassonografia
• alto consumo de carboidratos - diabetes gestacional

formação do cérebro - terceira e quarta semanas

• o que precisa para viver (partes do cérebro)
• notocorda - ectoderme - anencefalia
• como é formado o cérebro?

cartilha de suplementos para gravida casos de abortamento legal

ácido fólico - distúrbios ausência e excesso • risco de vida materno
(revisão ministério da saúde) • estupro
(síndrome de dalton) • anencefalia
1. Explicar e esquematizar a gastrulação e a neurulação
a) Trazer as malformações relacionadas ao tubo neural
2. Justificar a suplementação de ácido fólico para gestação (e sulfato ferroso)
3. Discutir os condicionantes e determinantes de saúde que levam a anencefalia e
a legalização do processo e respaldo legal para a gestante
Sugerir o que o SUS pode oferecer caso a gestante queira manter a gravidez

Menos que dois porcento vem de fatores genéticos

Sífilis Congênita, HIV Congênita
Ultrassom de Anencéfalo
 As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement
with, the contents by NLM or the National Institutes of Health. Learn more about our disclaimer.

Rev Obstet Gynecol. 2011 Summer; 4(2): 52–59. PMCID: PMC3218540

PMID: 22102928

Folic Acid Supplementation and Pregnancy: More Than Just Neural Tube Defect Prevention
James A Greenberg, MD,1 Stacey J Bell, DSc, RD,2 Yong Guan, MD,3 and Yan-hong Yu, MD, PhD4

Abstract

Folate (vitamin B9) is an essential nutrient that is required for DNA replication and as a substrate for a range of enzymatic reactions involved in amino
acid synthesis and vitamin metabolism. Demands for folate increase during pregnancy because it is also required for growth and development of the
fetus. Folate deficiency has been associated with abnormalities in both mothers (anemia, peripheral neuropathy) and fetuses (congenital
abnormalities). This article reviews the metabolism of folic acid, the appropriate use of folic acid supplementation in pregnancy, and the potential
benefits of folic acid, as well as the possible supplementation of L-methylfolate for the prevention of pregnancy-related complications other than neural
tube defects.

Key words: Folic acid, L-methylfolate, Dietary supplements

Folate (vitamin B9) is an essential nutrient that is required for DNA replication and as a substrate for a range of enzymatic reactions involved in amino
acid synthesis and vitamin metabolism. Demands for folate increase during pregnancy because it is also required for growth and development of the
fetus. Folate deficiency has been associated with abnormalities in both mothers (anemia, peripheral neuropathy) and fetuses (congenital
abnormalities). Dietary supplementation with folic acid around the time of conception has long been known to reduce the risk of neural tube defects
(NTDs) in the offspring.1–4 This article reviews the metabolism of folic acid, the appropriate use of folic acid supplementation in pregnancy, and the
potential benefits of folic acid, as well as the possible supplementation of L-methylfolate for the prevention of pregnancy-related complications other
than NTD.

Folate, Folic Acid, and L-Methylfolate

Defining the terminology is important to any discussion of the role of folate in nutrition and reproductive biology. The term folate is typically used as
a generic name for the group of chemically related compounds based on the folic acid structure. Folate, or vitamin B9, is thought of as one of the 13
essential vitamins. It cannot be synthesized de novo by the body, and must be obtained either from diet or supplementation. Dietary folate is a
naturally occurring nutrient found in foods such as leafy green vegetables, legumes, egg yolk, liver, and citrus fruit. Folic acid is a synthetic dietary
supplement that is present in artificially enriched foods and pharmaceutical vitamins. Neither folate nor folic acid is metabolically active. Both must be
reduced to participate in cellular metabolism. L-5-Methyltetrahydrofolate (L-methylfolate) is the predominant micronutrient form of folate that
circulates in plasma and that is involved in biologic processes.5

Folic Acid Metabolism

To become metabolically active, folic acid must first be converted to dihydrofolate (DHF) and then tetrahydrofolate (THF) through enzymatic
reduction, a process catalyzed by the enzyme DHF reductase (DHFR). Thereafter, THF can be converted to the biologically active L-methylfolate by
the enzyme methylenetetrahydrofolate reductase (MTHFR) (Figure 1). This key conversion is necessary to provide L-methylfolate for the one-carbon
transfer reactions (methyl donations) needed for purine/pyrimidine synthesis during DNA and RNA assembly, for DNA methylation, and to regulate
homocysteine metabolism (Figure 2).6,7 MTHFR is the critical enzyme for almost all biologic processes that involve the metabolism of folate and
methionine.
 Figure 1

FORMATION of L-methylfolate from folic acid. CH2, methylene; CH3, methyl group; DHF, dihydrofolate; DHFR, dihydrofolate reductase, F, folic acid; H, hydrogen; SHMT,
serine hydroxy-methyl transferase; THF, tetrahydrofolate. Reproduced with permission from Stahl SM, Stahl’s Essential Psychopharmacology: Neuroscientific Basis and
Practical Applications, 3rd ed. New York: Cambridge University Press; 2008.

Figure 2

Folate metabolic pathway. CBS, cystathionine b synthase; DHF, dihydrofolate; DHFR, dihydrofolate reductase; MS, methionine synthase; MT, methyltransferase; MTHFR,
methylenetetrahydrofolate reductase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SHMT, serine hydroxyl-methyltransferase; THF, tetrahydrofolate. Adapted
with permission from Bodnar LM et al.6

Genetic Polymorphisms and Folic Acid Metabolism

Genetic variations (polymorphisms) are common within the human genome and, in some cases, can result in the production of proteins with altered
biologic activity. Several such polymorphisms have been identified in the genes encoding proteins involved in folate metabolism. As noted, metabolic
processes requiring methyl group donations are regulated by the enzyme MTHFR. In the United States, up to approximately 60% of the population are
intermediate metabolizers of folate or heterozygous for genetic polymorphism of the MTHFR enzyme,8 whereas up to 25% of certain populations are
homozygous for these genetic variations.5 In varying degrees, these polymorphisms impair the conversion of folate to its active form, L-methylfolate.
For example, individuals who are poor metabolizers of folate are homozygous for the common variant MTHFR 677C->T genotype and show
approximately 30% of the enzyme activity found in those with the wild-type (CC) variant, whereas heterozygotes for the same genetic polymorphism
have around 65% of wild-type enzyme activity.9 With another variant, MTHFR 1298A->C, homozygous individuals can display catalytic activity of
the enzyme that is reduced to 68% of the wild-type activity.10

Based on the high prevalence of MTHFR genetic polymorphisms in the general population and concerns about reduced enzymatic activity and,
therefore, less biologically available L-methylfolate, newer research in this area has focused on supplementation with L-methylfolate rather than folic
acid as a means of preventing folate-related pathology.

Folic Acid and the Prevention of NTDs

Dietary supplementation with folic acid around the time of conception has long been known to reduce the risk of NTD in the offspring.1–4 Although
such an intervention is effective, it targets only women planning a pregnancy or recently pregnant. Additional measures to increase the intake of folic
acid in the general population include multivitamin supplementation and fortification of grain-based products such as flour, cereal, and pasta.
Fortification of grain products with folic acid has been mandatory in the United States since January 199811 and in Canada since December 1998.12
Within months, these legislative mandates were associated with a significant increase in the concentrations of erythrocyte folate among women of
childbearing age13,14 and a decrease in the prevalence of infants born with NTDs.15–17 To assure that women have adequate folate stores during
pregnancy, the US National Institutes of Health (NIH) and Institute of Medicine (IOM) have recommended that 600 µg of folic acid be taken daily by
pregnant women, and that this supplementation be continued throughout pregnancy and reduced to 500 µg during lactation.18 Current US
recommendations include (a) for women at high risk of having a child with a NTD (such as those with a personal or family history of NTD, a prior
child with a NTD, or those on anticonvulsant medications), dietary supplementation with 5 mg folic acid daily prior to conception; and (b) for all other
reproductive-aged women, .4 to 1 mg folic acid daily for at least 2 to 3 months prior to conception, throughout pregnancy, and during the postpartum
period. Current recommendations in Canada vary depending on the patient’s demographic characteristics, lifestyle, and a priori risk of having a fetus
with a structural abnormality (summarized in Table 1).19 Furthermore, in a recent double-blind, randomized, placebo-controlled trial of 144 women of
childbearing age, Lamers and colleagues demonstrated that supplementation with L-methylfolate was more effective than folic acid at increasing red
blood cell folate concentrations.20

Table 1

Canadian Recommendations for Folic Acid and Multivitamin Supplementation in Pregnancy

Option Population Folic Acid Dose Duration of Supplementation

A Patients with no personal health risks, planned
pregnancy
B Patients with health risks, family history of neural tube
defect, high-risk ethnic group
Good diet of folate-rich foods; daily
supplementation with folic acid 0.4–1.0 mg
(1) Folate-rich foods, daily supplementation
with 5 mg folic acid
At least 2 to 3 months before conception and throughout
pregnancy and the postpartum period (4–6 weeks and as
long as breastfeeding continues)
(1) Beginning at least 3 months before conception and
continuing until 10 to 12 weeks postconception

C Patients with history of poor compliance with
medications, additional lifestyle issues, variable diet, no
consistent birth control, and possible teratogenic
substance use
(2) Daily supplementation with folic acid 0.4– (2) From 12 weeks postconception and continuing
1.0 mg throughout pregnancy and the postpartum period (4–6
weeks or as long as breastfeeding continues)
Folate-rich foods, daily supplementation with Counsel about folic acid supplementation to prevent
5 mg folic acid birth defects and additional health benefits

Adapted with permission from Wilson RD et al.19

Folic Acid and the Prevention of Anemia

Blood volume expansion resulting from an increase in both plasma and erythrocytes is a normal physiologic change of pregnancy. Although more
plasma than erythrocytes is usually added to the maternal circulation, the increase in erythrocyte volume is considerable, averaging about 450 mL.21
Because of the increase in plasma volume, hemoglobin concentration and hematocrit normally decrease slightly during pregnancy. However, although
hemoglobin concentrations at term average 12.5 g/dL, approximately 5% of women are anemic, with hemoglobin concentrations below 11.0 g/dL (see
Table 2).22

Table 2

Hemoglobin Concentrations in 85 Healthy Women With Proven Iron tores

Hemoglobin (g/dL) Nonpregnant Midpregnancy Late Pregnancy

Mean 13.7 11.5 12.3

Less than 12.0 1% 72% 36%

Less than 11.0 None 29% 6%

Less than 10.0 None 4% 1%

Lowest 11.7 9.7 9.8

Adapted with permission from Cunningham F et al.22

Erythropoiesis is the process by which red blood cells are produced in the hematopoietic tissue of the bone marrow. Among the many requirement for
active erythropoiesis are the need for adequate supplies of three key nutrients: folate, cobalamin (vitamin B12), and iron. Although a complete
detailing of the role of these nutrients in erythropoiesis is beyond the scope of this review, it is important to understand that the reaction in normal
erythropoieses involving both folate and vitamin B12 is the transfer of a methyl group from L-methylfolate to homocysteine via methylcobalamin for
the regeneration of methionine.23 Thus, in settings of low folate and/or vitamin B12, anemia will likely ensue. As an example, in a recent retrospective
analysis of anemia in pregnancy by Bentley and colleagues, pregnant women prescribed prenatal medical food with 1.13 mg of L-methylfolate in
addition to 0.4 mg of folic acid and 500 to 1000 µg vitamin B12 (high folate, high vitamin B12) were compared with pregnant women prescribed
standard prenatal vitamins containing only 0.8 to 1.0 mg of folic acid and 0 to 12 µg of vitamin B12 (low folate, low vitamin B12).24 The women in the
high folate, high-dose vitamin B12 group demonstrated significantly higher hemoglobin levels at delivery (11.8 g/dL vs 10.7 g/dL; P = .001) than the
control standard prenatal vitamin group. How these findings will pan out in prospective, randomized trials and what impact direct L-methylfolate
supplementation will have on other folate-related processes remain to be determined.

Folic Acid and the Prevention of Preterm Birth

Preterm birth (PTB), defined as delivery prior to 37 weeks of gestation, complicates 12.5% (1 in 8) of all deliveries in the United States. It is a major
cause of neonatal mortality and morbidity. Infants born preterm are at risk of short-term respiratory, gastrointestinal, immunologic, and central
nervous system complications, as well as long-term motor, cognitive, and neurobehavioral sequelae. As a result, the societal costs of preterm
deliveries in the United States alone exceed $26 billion a year.25 Preterm labor represents a syndrome rather than a diagnosis because the etiologies are
varied. Approximately 20% of preterm deliveries are iatrogenic and are performed for maternal or fetal indications, including intrauterine growth
restriction, preeclampsia, placenta previa, and nonreassuring fetal testing. Although the causes of the remaining 80% of PTB are not well understood,
four basic pathophysiologic mechanisms are described: (a) premature activation of the fetal hypothalamic-pituitaryadrenal (HPA) axis; (b) intrauterine
infection/inflammation; (c) decidual hemorrhage (placental abruption); and (d) pathological distension of the uterus.26

The treatment of preterm labor has focused primarily on inhibiting uterine contractions, which has been shown neither to reduce the incidence of PTB
nor improve neonatal outcome.27 In the face of such therapeutic nihilism, attention has turned instead to prevention. One of the agents under
investigation to prevent PTB in both low- and high-risk populations is folic acid.

Epidemiologic Evidence

Indirect evidence suggests that folate may indeed be important in the timing of labor. In observational studies, a shorter duration of pregnancy has
been associated with low serum folate levels6,28 and with the absence of folic acid supplementation during pregnancy.29

Initial intervention studies focused on multiple micronutrient supplementation and showed a significant reduction in pregnancy complications with
such supplementation, including low birth weight, small for gestational age (SGA), and maternal anemia30–33; such studies were not sufficiently
powered to show a difference in PTB, preterm premature rupture of membranes (PPROM), or placental abruption. Interestingly, these differences lost
significance when multiple micronutrient supplementation was compared with iron/folic acid supplementation alone,29 suggesting that it may be these
elements that are most important. Subsequent studies suggest that folic acid supplementation alone may protect against PTB, without increasing the
risk of miscarriage, structural anomalies, multiple pregnancy, or stillbirth.29,34–36 The largest such study was a secondary analysis of the prospective
observational FASTER trial carried out in the United States from 1999–2002.36 This secondary analysis included 34,480 women with singleton
pregnancies dated by first trimester ultrasound who delivered between 20-0/7 and 42-0/7 weeks of gestation. Spontaneous PTB was defined as
delivery between 20-0/7 and 36-6/7 weeks in the absence of any medical or obstetric complications or indications; pregnancies that ended in elective
termination or stillbirth and those with fetal chromosomal or structural abnormalities were excluded. All subjects were asked about their diet and
specifically about dose and duration of folic acid supplementation at their initial enrollment in the first trimester. In this cohort, compared with no
supplementation, preconceptional folate supplementation for ≥ 1 year was associated with a significant reduction in spontaneous PTB (hazard ratio
[HR] 0.22; 95% confidence interval [CI], 0.08–0.61; P = .004 for delivery at 20–28 weeks; HR 0.45; 95% CI, 0.24–0.83; P = .010 for delivery at 28–
32 weeks). The authors concluded that preconceptional folate supplementation decreased the risk of spontaneous PTB, and that this association was
strong, specific, dose-dependent, consistent with other studies, biologically plausible, and essentially unchanged after adjustment for potential
confounders.36 Recent data suggest that the duration of folic acid supplementation may be as important as the dose. In the large prospective cohort
study reviewed above,36 the risk of spontaneous PTB was inversely related to the duration of folic acid supplementation, and was lowest in women
who reported using folic acid supplementation for more than 1 year prior to conception.

Biologic Plausibility

If folic acid supplementation is indeed associated with a reduction in PTB, what is the mechanism? This question is complicated by the fact that,
despite years of research, little is known about the molecular mechanisms responsible for the onset of labor in humans, both at term and preterm, let
alone about how to rescue pregnancies complicated by preterm labor. What is becoming clear is that many cases of PTB are associated with an
abnormal inflammatory response, which may be triggered by intrauterine infection or hemorrhage.22 Folate is known to be important for normal
immune function. Folate-deficient individuals, for example, demonstrate dysfunction of both cell-mediated and humoral immunity.37 Moreover, the
phagocytic and bactericidal capacities of polymorphonuclear leukocytes are decreased in folate-deficient humans, thereby increasing their
susceptibility to infections such as asymptomatic bacteriuria.38 In such individuals, dietary supplementation with folic acid has been shown to improve
immune function and decrease circulating biomarkers of inflammation, including α1-acid glycoprotein and C-reactive protein.39

More recently, several genetic variations have been described in key genes involved in folate metabolism that appear to confer an increased risk for
spontaneous PTB. One such variant involves a 19-base pair (bp) deletion in the DHFR gene.40 DHFR is a critical enzyme in the folate metabolic
cascade because, as discussed above, ingested folate must first be fully reduced before further metabolic processing can occur. The DHFR 19-bp
deletion allele thus interferes with folate metabolism and the transport of reduced folate across the placenta.

Another example is a gene sequence variation in the SHMT1 gene, known as the SHMT1(1420)T variant. This variant results in less serine
hydroxymethyltransferase 1 transcriptional activity and is associated with an increased risk of spontaneous PTB; this effect was most pronounced in
patients who had low folic acid intake.41 Findings such as these raise the possibility that even women with “adequate” folate intake may be at risk of
PTB if they carry a particular genetic variant. Whether such women would benefit from higher folic supplementation or supplementation with L-
methylfolate directly is not known at this time.

Additional Benefits of Folic Acid Supplementation

In addition to the prevention of NTD, periconceptional supplementation with folic acid also appears to have other beneficial effects, including the
prevention of congenital heart disease and oral clefts42–45 and possibly preterm birth (discussed above). The mechanism by which folic acid prevents
structural anomalies in the fetus is not known, but may involve the regulation of homocysteine metabolism.46
Several investigators have suggested that folic acid supplementation may have additional benefits on pregnancy outcome. This line of investigation
began because of epidemiologic studies showing that pregnancies exposed to folic acid antagonists have significantly higher rates of placenta-related
pregnancy complications.47–50 Folic acid antagonists encompass a broad spectrum of drugs used for a variety of clinical indications, including the
treatment of seizure disorders, mood disorders, and urinary tract infections. Folic acid antagonists can be divided into two groups: (a) DHFR inhibitors
(eg, sulfamethoxazole-trimethoprim), which block the conversion of folate to its more active metabolites (Figure 1), and (b) other folic acid
antagonists, a group consisting primarily of anticonvulsant medications (phenobarbital, phenytoin, primidone, and carbamazepine) but also including
Spasmophen (an antispasmodic drug that contains low doses of phenobarbital) and cholestyramine. In one study, pregnancies exposed to either DHFR
inhibitors (n = 12,546) or other folic acid antagonists (n = 1565) were noted to be at increased risk of developing preeclampsia (adjusted odds ratio
[OR] 1.52; 95% CI, 1.39–1.66), severe preeclampsia (OR 1.77; 95% CI, 1.38–2.28), placental abruption (OR 1.32; 95% CI, 1.12–1.57), fetal growth
restriction = 10th percentile (OR 1.07; 95% CI, 1.01–1.13), fetal growth restriction < 3rd percentile (OR 1.22; 95% CI, 1.11–1.34), and fetal death
(OR 1.35; 95% CI, 1.07–1.70).49 These adverse events have one thing in common: they all appear to result from abnormalities in implantation and
placentation that occur early in gestation. Because folic acid has been shown to regulate trophoblast invasion,51 it is biologically plausible that folate
deficiency may interfere with the early stages of placental development leading to complications later in gestation.

Risks of High-Dose Folate Supplementation

Although folic acid supplementation to supraphysiologic levels has demonstrated many of the benefits to pregnant women and fetuses noted above, the
potential risk of high-dose folate supplementation must also be considered. First, folate supplementation can mask vitamin B12 deficiency (pernicious
anemia) and care must be taken with susceptible individuals to avoid missing this diagnosis. Also, concerns have been raised about the potentially
untoward effects of unmetabolized synthetic folic acid with regard to cancer, depression, and cognitive impairment. 52 With all these concerns, early
data suggest supplementation with L-methylfolate rather than folic acid may mitigate these risks.53

Conclusions

Periconceptional folic acid supplementation protects against fetal structural anomalies, including NTD and congenital heart defects. Recent data
suggest that it may also protect against preterm birth. The importance of genetic polymorphisms in the genes regulating folate metabolism
(particularly the MTHFR gene) and how it affects the bioavailability of L-methylfolate and thereby strategies for folate supplementation, is not very
well understood. Although additional studies are needed to better define the precise timing, dosing, and formulation, existing data suggest that dietary
folic acid supplementation is a good idea for all reproductiveaged women. Women with known MTHFR mutations may benefit from direct
supplementation with L-methylfolate but, at present, there are insufficient data to conclusively make that determination.

Main Points

Demands for folate increase during pregnancy because it is also required for growth and development of the fetus. Folate deficiency has
been associated with abnormalities in both mothers (anemia, peripheral neuropathy) and fetuses (congenital abnormalities).
The term folate is typically used as a generic name for the group of chemically related compounds based on the folic acid structure. Folate,
or vitamin B9, is thought of as one of the 13 essential vitamins. It cannot be synthesized de novo by the body, and must be obtained either
from diet or supplementation. Folic acid is a synthetic dietary supplement that is present in artificially enriched foods and pharmaceutical
vitamins. Neither folate nor folic acid is metabolically active. Both must be reduced to participate in cellular metabolism. L-5-Methyl-
tetrahydrofolate (L-methylfolate) is the predominant micronutrient form of folate that circulates in plasma and that is involved in biologic
processes.
Based on the high prevalence of methyltetrahydrofolate reductase (MTHFR) genetic polymorphisms in the general population and
concerns about reduced enzymatic activity and, therefore, less biologically available L-methylfolate, newer research in this area has
focused on supplementation with L-methylfolate rather than folic acid as a means of preventing folate-related pathology.
In a recent double-blind, randomized, placebo-controlled trial of 144 women of childbearing age, Lamers and colleagues demonstrated
that supplementation with L-methylfolate was more effective than folic acid at increasing red blood cell folate concentrations.
Periconceptional folic acid supplementation protects against fetal structural anomalies, including neural tube and congenital heart defects.
Recent data suggest that it may also protect against preterm birth. Although additional studies are needed to better define the precise
timing, dosing, and formulation, existing data suggest that dietary folic acid supplementation is a good idea for all reproductive-aged
women.

References

1. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. MRC Vitamin Study Research Group, authors. Lancet. 1991;338:131–137. [PubMed]
[Google Scholar]

2. Rieder MJ. Prevention of neural tube defects with periconceptional folic acid. Clin Perinatol. 1994;21:483–503. [PubMed] [Google Scholar]

3. Pitkin RM. Folate and neural tube defects. Am J Clin Nutr. 2007;85:285S–288S. [PubMed] [Google Scholar]

4. De Wals P, Tairou F, Van Allen MI, et al. Reduction in neural-tube defects after folic acid fortification in Canada. N Engl J Med. 2007;357:135–142. [PubMed] [Google Scholar]

5. Pietrzik K, Bailey L, Shane B. Folic acid and L-5-methyltetrahydrofolate: comparison of clinical pharmacokinetics and pharmacodynamics. Clin Pharmacokinet. 2010;48:535–548.
[PubMed] [Google Scholar]
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Abstract
Vitamin B micronutrients are essential regulators of one carbon metabolism that ensures human health. Vitamin B9, or folate,
lies at the heart of the folate cycle and converges with the methionine cycle to complete the one carbon pathway. Additionally,
vitamin B6 contributes by orchestrating the flux of one carbon cycling. Dysregulation of vitamin B contributes to altered
biochemical signaling that manifests in a spectrum of human diseases. This review presents an analysis of the past, present,
and future work, highlighting the interplay between folate and vitamin B6 in one carbon metabolism. Emerging insights include
advances in metabolomic-based mass spectrometry and the use of live-cell metabolic labeling. Cancer is used as a focal point
to dissect vitamin crosstalk and highlight new insights into the roles of folate and vitamin B6 in metabolic control. This collection
of vitamin-based research detailing the trends of one carbon metabolism in human disease exemplifies how the future of
personalized medicine could unfold using this new base of knowledge and ultimately provide next-generation therapeutics.
Keywords: folate (/search?q=folate); pyridoxine (/search?q=pyridoxine); methionine (/search?q=methionine); one
carbon metabolism (/search?q=one+carbon+metabolism); cancer (/search?q=cancer); methylation (/search?
q=methylation); B6 (/search?q=B6); B9 (/search?q=B9); post-translational modification (/search?q=post-
translational+modification); CEST-MRI (/search?q=CEST-MRI); aptamer (/search?q=aptamer); metabolic probe
(/search?q=metabolic+probe); fluorescent sensors (/search?q=fluorescent+sensors)

1. Introduction
Vitamins dynamically orchestrate cellular metabolic pathways to regulate human physiology. One carbon metabolism is a
vital pathway across living organisms that relies on the levels of several vitamins from the B family. Vitamin B micronutrients are
water-soluble compounds obtained from diet. The era of vitamin discovery occurred in the early 20th century and were aptly
named as ‘vital amines’ or ‘vitamines’ due to the common amine in the chemical structure. While researchers had begun to
understand the importance of this new class of nutrients, Casimir Funk is attributed with the discovery and naming of ‘vitamins’
in 1912; a flurry of other vitamins were discovered shortly after, including the B vitamins [1]. Researchers realized that
deficiencies in this new class of molecules were the cause of several diseases, including scurvy, anemia, and rickets. Over the
past century, the pioneering work of many researchers laid the groundwork of vitamin biology. Many excellent review articles
cover the folate cycle and the one carbon pathway [2,3]. This review focuses on the central roles of vitamin B6 (pyridoxine) and
B9 (folate), essential elements of the one carbon pathway that contribute to functional activity in health and dysregulation in
disease (Figure 1). Folate enters the one carbon pathway through active forms of tetrahydrofolate (THF) that carry methyl-
groups throughout the folate cycle. Pyridoxine is similarly obtained through diet and once converted to the most metabolically
active form, pyridoxal 5′-phosphate (PLP), is a crucial cofactor during one carbon metabolism. This review highlights the recent
advances that have revolutionized our understanding of B6 and B9 vitamins and how these lines of research offer novel insight
to improve treatments in disease.

Figure 1. One carbon metabolism is a collection of cyclical metabolic pathways that orchestrates myriad metabolic
processes. The one carbon pathway is comprised of the folate cycle and the methionine cycle. Folate enters the
pathway through a two-step reaction that generates tetrahydrofolate (THF) with the dihydrofolate reductase enzyme
(DHFR). Within the methionine cycle, dietary methionine is catabolized by methionine adenosyltransferase 2A or 1A
(MAT2A/MAT1A) to produce the universal methyl donor S-adenosylmethionine (SAM). Upon substrate methylation,
SAM is converted to S-adenosylhomocysteine (SAH) and then converted by adenosylhomocysteinase (AHCY) to
homocysteine (Hcy). The methionine cycle is completed by the conversion of Hcy back to methionine by methionine
synthase (MS). Alternatively, Hcy can fed into the transsulfuration pathway for glutathione synthesis and redox
metabolism using vitamin B6-dependent enzymes. The methionine salvage pathway also sparks off the methionine
cycle to produce the by-product methylthioadenosine (MTA) from methionine for polyamine biosynthesis. Figure made
with BioRender.

2. Vitamin B9: Folate

2.1. Dietary and Active Forms
Vitamin B9, or folate, can be obtained directly from diet via leafy greens, seeds, and fruit juice as 5-methyltetrahydrofolate
(5-MTHF) and in fortified grains or supplements as folic acid. Originally discovered in 1931, Dr. Lucy Wills found that this
micronutrient was able to treat anemia during pregnancy [4,5]. Since then, the mechanistic underpinnings of vitamin B9 have
micronutrient was able to treat anemia during pregnancy [4,5]. Since then, the mechanistic underpinnings of vitamin B9 have
been described in embryonic development [6], redox homeostasis [7], immunology [8], and cancer [9]. Dietary B9
recommendation for adults in the United States is set to 400 µg/day (CDC.org). Circulating B9 levels in adults range from 2 to
20 ng/mL [10] where the highest concentrations are the most active B9 vitamer 5-methyltetrahydrofolate (5-MTHF) at 5 µM [11].
Folate can be assessed in patients through various approaches, such as by measuring the levels of circulating homocysteine
(Hcy) or by urinary formiminoglutamate (FIGLU) excretion. Additionally, folate can be directly measured in red blood cells and
through deoxyuridine suppression tests [10].
2.2. Metabolic Pathways and Key Enzymes of the Folate Cycle
The folate family contains a 2-amino-4-hydroxy-pteridine ring and a p-aminobenzoyl moiety linked by a methylene (CH2)
group, which are then linked through an amide bond to the α-amino group of a monoglutamate or poly-γ-glutamate (Figure 2).
Methyl groups occupy the N5 and N10 positions across vitamers [12]. Tetrahydrofolate (THF) is generated from dietary folic
acid and operates as the universal one carbon acceptor [7], where the oxidation states of methyl-units include methanol,
formaldehyde, or formate. Dihydrofolate reductase (DHFR) catalyzes a two-step reaction that converts folic acid to dihydrofolate
(DHF) and then to THF.

Figure 2. Vitamin B9 and the Folate Family. Folate or folic acid is obtained from diet and must be converted to
tetrahydrofolate (THF) before it can enter the folate cycle. Dietary 5-methyl-THF can also become incorporated. Folate
is converted first to dihydrofolate (DHF) before becoming THF, the universal methyl-acceptor of the one carbon
pathway. Methylation can occur on the 5 or 10 N groups of THF in 5,10-methylene-THF and 5-methyl-THF.

Upon entering the folate cycle, THF is converted to 5,10-methylene THF (5,10-MTHF) by serine hydroxymethyltransferase
(SHMT1) in the cytosol or by SHMT2 in the mitochondria [7]; this step concomitantly converts serine to glycine. Then, 5,10-
MTHF is reduced further to 5-MTHF by methylenetetrahydrofolate reductase (MTHFR), which uses flavin adenine dinucleotide
(FAD) as a cofactor. In a side pathway, 5,10-MTHF can act as a cofactor for thymidylate synthase (TS), which converts dUMPs
to dTMPs for DNA synthesis and repair [13]. In a second major offshoot of this pathway, methylenetetrahydrofolate
dehydrogenase (MTHFD) is a trifunctional enzyme that interconverts 5,10-MTHF (pyrimidine biosynthesis) and 10-formylTHF
(purine biosynthesis) via a 5,10-methenylTHF intermediate [14].
Together, the folate cycle and the methionine cycle make up the one carbon metabolic pathway. The methionine cycle is
essential for generating the universal methyl-donor of the cell, S-adenosylmethionine (SAM) [15,16]. The folate and methionine
cycles intersect with 5-MTHF and methionine synthase (MS), which uses Hcy to generate THF and methionine. Methionine
adenosyltransferase (MAT) is responsible for the hydrolysis of methionine to SAM. Methionine is an essential amino acid that is
obtained from diet and is among the most variable in circulation [17]. Fluxes in the one carbon metabolism pathway regulate
methionine and directly impact SAM concentrations, which in turn shape cellular signaling and epigenetics.
2.3. Vitamin B9 Regulation
The cell has evolved multiple levels of regulation to ensure the proper concentration of folate is maintained. Enzymes
throughout the one carbon pathway are subjected to multiple forms of regulation through post-translational modifications,
allosteric inhibition, and the abundance of key cofactors (Figure 3 and Table 1). The effects of post-translational modifications
on one carbon metabolic enzymes have been recently investigated.

Figure 3. One-carbon enzymes are modified post-translationally. Enzymes involved in the one carbon metabolism
pathway undergo a variety of post-translational modifications (PTMs), including phosphorylation, acetylation,
methylation, and ubiquitylation. Several modifications (MTHFR phosphorylation and MTHFD1 methylation) affect protein
activity and efficiency, thereby altering one carbon metabolic flux. Since identifying many other PTMs with proteomic
screens, the biological effects of several other PTMs remain undefined. Note that the illustrated modifications only
represent a selection of identified PTMs. Y, tyrosine; K, lysine; S, serine; R, arginine; T, threonine. P, phosphorylation;
Ac, acetylation; Me, methylation; U, ubiquitylation. Figure made with BioRender.

Table 1. One carbon metabolic enzymes are regulated by post-translational modifications and cofactor availability.

Intrinsic feedback systems are essential for tuning the dynamics of one carbon metabolism. For example, MTHFR activity
is sensitive to SAM levels. When present in excess, SAM allosterically inhibits MTHFR to reduce the regeneration of
methionine; SAM levels of ~5 µM decrease MTHFR activity by half [35]. A phosphorylation of MTHFR by dual-specificity
tyrosine phosphorylation-regulated kinase (DYRK1/2) primes a subsequent phosphorylation cascade by glycogen synthase
kinase (GSK3) α/β that further sensitizes MTHFR to allosteric SAM inhibition [36]. Additional allosteric regulation occurs in the
case of SHMT whose activity is inhibited by elevated THF levels above 40 µM [37].
Methylation senses SAM levels and dietary methionine. Since ATP, the cofactor for protein phosphorylation, is highly
abundant in cells (~1–5 mM), kinase active sites are saturated by ATP because kinase Km, ATP is often in micromolar ranges
[38,39,40]. Thus, the dynamics of substrate phosphorylation by kinases and phosphatases is independent of metabolism
[15,41]. In contrast, alternative post-translational modifications that use less abundant metabolites as cofactors are rendered
sensitive to the changes of cellular metabolic fluxes [42,43,44]. Methylation is paradigmatic of this phenomenon as the levels of
methionine, the precursor of SAM, can rapidly change within minutes based on diet [17]. The impact of dietary methionine on
cellular methylation was first appreciated in the regulation of epigenetics and the methylation of histones and DNA [45,46,47].
Similarly, demethylase activity relies on the abundance of a cofactor, α-ketoglutarate, which has been shown to be dynamically
linked to canonical Wnt signaling [48]. More recently, methylation of lipids and proteins has also been shown to link the nutrient
status of a cell with molecular signal transduction [47,49]. In fact, several B9- and other one carbon-metabolizing enzymes are
methylated; in the case of MTHFD1, R173 methylation increases enzymatic activity and subsequently NADPH levels [27].
A finely tuned crosstalk between vitamin B6 and folate plays central roles in several developmental and adult processes
(Figure 4). Beyond the regulation by one carbon metabolites, folate levels are also regulated by other B vitamins such as B6
and B12. Active vitamin B6, PLP, is a necessary cofactor for enzymes linked to B9 metabolism (e.g., SHMT1/2 and
cystathionine-β-synthase) and, therefore, altered B6 levels directly shape the fate of B9 vitamer conversions [50]. This
regulation occurs for both SHMT isoforms, though it is interesting to note that cytosolic SHMT1 is more sensitive to decreased
vitamin B6 than mitochondrial SHMT2 [51]. In the case of a vitamin B6 deficiency, folate cycling is disrupted due to the B6-
dependent conversion of THF to 5,10-MTHF, thereby depriving neural cells of the amino acid precursors of several monoamine
neurotransmitters, including dopamine and serotonin [52].
 Figure 4. Vitamins B6 and B9 are essential in development and in adult tissues. Vitamin B6 and folate are involved in
the biosynthesis of nucleotides for DNA and RNA synthesis, cell growth, neurological development during pregnancy
and infancy, hemoglobin synthesis and red blood cell formation, blood homocysteine balance, immune system
functioning, and nutrient metabolism. Figure made with BioRender.

2.4. Folate Deficiency and Disorders

Folate deficiency (FD) has longstanding links to defective neurological and cardiovascular function [53], which can often be
prevented by dietary supplementation. As 5-MTHF can be readily imported by somatic cells, it is often the recommended
vitamer for B9 supplementation. A hallmark example of this condition is FD-induced spina bifida during embryogenesis, whose
etiology is linked to hypomethylation and disrupted DNA mismatch repair [54]. Folic acid supplementation during the course of
pregnancy can greatly prevent these defects; such recommendations from the WHO and FDA contributed to a 35–50%
reduction in the incidence of FD-induced neural tube defects in the U.S. [55]. Beyond low folate diets, FD can arise through the
loss-of-function mutations in key metabolizing enzymes such as MTHFR, where disrupted conversion of 5,10-MTHF to 5-MTHF
alters DNA and purine synthesis, in addition to global methionine levels, resulting in impaired tissue development [28]. As a
precursor for methionine synthesis, folate deficiencies are often congruent with altered methionine metabolism [56]. Alternative
routes are available, however, to synthesize methionine from betaine and 5-MTHF via betaine-homocysteine methyltransferase
(BHMT), although this route is limited to the liver and kidney [7]. In contrast, an excessive level of folate can in some cases be
detrimental, such as through the masking of symptoms of vitamin B12 deficiencies, delaying diagnosis and therapeutic
intervention [57,58].
2.5. Folate in Cancer
The folate cycle is intertwined with cellular programs that are integral to cancer metabolism and proliferation. Following the
discovery of folic acid, Sidney Farber was inspired to test the hypothesis that folate could restore blood cells in leukemia, given
its role in healing macrocytic anemia [59]. However, clinical trials abruptly stopped as folate was found to promote leukemia in
patients. This unfortunate case led to the revolutionary concept of antifolate therapeutics, with methotrexate (MTX) being the
first of its kind. Indeed, many folate enzymes are overexpressed in cancer. However, the roles of vitamin B9 during cancer cell
initiation, transformation, and metastasis are highly dependent on the cancer type and surrounding tumor microenvironment
(Table 2) [10].

Table 2. Vitamin B9 has been implicated across a spectrum of cancers. Cellular and tissue responses are defined.

Folate deficiencies have been linked to mechanisms of cancer progression [81]. Reducing folate cycling similarly
decreases methionine cycling and SAM production, which may contribute to hypomethylation in cancer [82]. While cellular
methylation dynamics on DNA and histone have been thoroughly studied, recent works highlight the significant impact of SAM
homeostasis on the substrate methylation status of lipids, proteins, and RNA [47,49,83]. Additionally, folate deficiency can
trigger DNA damage by decreasing the flux of dUMP to dTMP, which typically occurs during the TS-mediated side reaction that
generates DHF from 5,10-MTHF. By increasing the dUMP:dTMP ratio, the rate of uracil misincorporation into DNA increases
and could trigger single- or double-stranded breaks, and potentially oncogenic mutations [81,82]. Recent work showed that
folate-supplemented diets protected mice prior to and during xenografting with pancreatic cancer cells [84].
2.5.1. Lung Cancer
Methylenetetrahydrofolate reductase (MTHFR) has been linked to the progression of lung cancer, through the generation of
fresh nucleotides for DNA synthesis and repair in the folate cycle [61]. Meta-analysis of MTHFR polymorphisms revealed that
the most common variant in the MTHFR gene, C677TT, is correlated with an increased risk of lung squamous carcinoma in
East Asian populations [60,85]. Surprisingly, the 677TT mutation reduces MTHFR activity by 70% by disrupting FAD cofactor
binding [86]. A reduction in MTHFR efficiency lowers folate in circulation, decreasing methionine and SAM levels, and causing
an accumulation of Hcy. Notably, high Hcy is a risk factor in cancer and cardiovascular disease by increasing red blood cell
coagulation and pro-inflammatory pathways [87,88,89]. Individuals with this variant have a folate blood concentration 16% lower
than those with the 677CC genotype, indicating that high levels of B9 have also been associated with reduced risk of lung
cancer [90]. Interestingly, a hospital-based case-control study of a non-Hispanic white population found that the 677TT
genotype was associated with a decreased risk of lung cancer in women but not in men [61]. Dietary vitamin B6, vitamin B12,
and methionine in women with C677T genotypes were associated with a decreased risk of cancer. Conversely, the MTHFR
1298CC genotype was associated with an increased risk of lung cancer in women [61].
2.5.2. Colon Cancer
Low folate intake has been associated with an increased risk of colon cancer [91] and long-term folate supplementation
was shown to lower the risk of colon cancer by 75% [92]. Folate has been implicated in colorectal carcinogenesis through
mechanisms of DNA synthesis and methylation [93]. As the folate and methionine cycles are at the center-stage of one carbon
metabolism, low levels of folate lead to decreased methionine in circulation and a reduction in DNA methylation essential for
DNA expression, stability, and repair [94,95]. Folate cycle intermediates acting as cofactors for purine and pyrimidine synthesis
are also essential for DNA synthesis [96,97]. Due to this, low levels of folate have been associated with aberrant DNA regulation
and stability via strand breaks, mutations, and hypomethylation [96,98,99,100]. Human colonocytes cultured in folate-deficient
media were unable to repair DNA strand breaks and showed a five-fold increase in uracil misincorporation. Proteomics revealed
altered activity and expression of proteins involved in proliferation, DNA repair, apoptosis, and malignancy in these cells [101].
Folate deficiency was also shown to induce mitotic aberrations [102]. A meta-analysis investigating the relationship between
folate supplements and colon cancer risk found an inverse correlation between the two [62]. Similarly, folate deficiency has
been shown to aggravate carcinogenesis in colon cancer rat models, while increasing dietary intake reduced neoplasms
[63,64]. Nevertheless, epidemiological studies have failed to find an association between dietary and circulating folate and
colorectal cancer risk [65,66].
2.5.3. Pancreatic Cancer
Similar to colorectal cancer, molecular underpinnings between pancreatic carcinogenesis and folate appear to involve DNA
hypomethylation and impaired DNA synthesis, but specific insights are yet to be uncovered. Conclusions regarding dietary
folate intake yielded inconsistent results [71]. A meta-analysis investigating the link between folate intake and MTHFR
polymorphisms revealed that the 677TT variant was associated with increased risk of gastrointestinal and pancreatic cancer.
This variant lowers circulating folate by preventing 5-MTHF synthesis from 5,10-MTHF, which reduces DNA methylation. In
contrast, meta-analyses based on folate intake instead of genetic mutations revealed a decreased risk of pancreatic cancer
[72,73]. Together, this suggests that MTHFR status may dictate the relationship between folate and pancreatic cancer
prognosis.
2.5.4. Ovarian Cancer
Ovarian cancer is the most lethal cancer of the female reproductive system [103]. Interestingly, 80% of ovarian cancers
have an overexpression of folate receptors (FR), which is largely absent in healthy tissue. Thus, FRα serves as a serum
biomarker [104] in ovarian cancers. Folate receptor α (FRα) binds the active form of folate and transports it inside cells via
receptor-mediated endocytosis. This receptor overexpression leads to a higher intake of folate into the cell, increasing rates of
DNA synthesis that facilitate cancer cell growth. This receptor overexpression on cell surfaces has allowed FRα to emerge as
an attractive target for monoclonal antibody therapies such as farletuzumab. Though epidemiologic studies report no
association or an inverse association between folate intake and ovarian cancer risk [67,70], FRα has been shown to increase
chemotherapy resistance by stabilizing murine double minute 2 (MDM2), an oncogene that can be used as a prognostic factor
in ovarian cancer [68,69].
2.5.5. Esophageal, Liver, and Gastric Cancer
A systematic meta-analysis of esophageal cancer found a decreased cancer risk within a certain folate intake range [105].
Accordingly, vegetarian diets have been shown to be protective against esophageal cancer [106]. Just as FRα is overexpressed
in ovarian cancer, tumor-associated macrophages weaponize folate receptor β (FRβ) to promote liver cancer metastasis [107].
While folate levels are not increased in hepatocellular carcinoma, FRβ can serve as a potential therapeutic target [108] through
FRβ-targeting lipid nanoparticles that deliver anti-neoplastic drugs [109]. Folate deficiency is a risk factor for gastric cancer.
FRβ-targeting lipid nanoparticles that deliver anti-neoplastic drugs [109]. Folate deficiency is a risk factor for gastric cancer.
Interestingly, gastric cancer is frequently related to vitamin B deficiencies such as B12 in pernicious anemia. Whether folate
drives gastric cancer or is merely a byproduct of oncogenic metabolism remains an open area of research [110].
2.5.6. Prostate Cancer
Epidemiologic studies have reported inconclusive correlations between folate and prostate cancer prognosis. Two meta-
analyses found no association between folic acid and prostate cancer risk [74,75], while one meta-analysis found a 24%
increase in risk [76]. Additionally, a case-control study found that low levels of folate and high levels of Hcy were associated
with various cancers including prostate [78]. Another study found a 4% increase risk with every 5 nmol/L increase in serum
folate, although dietary folate intake had little to no effect on cancer risk [77]. During prostate cancer, polyamine levels required
for normal prostate growth are increased [111,112]. High levels of polyamines have been shown to sensitize cells to folate.
Conversely, inhibition of adenosylmethionine decarboxylase 1 (AMD1) blocks polyamine synthesis, increasing SAM and
decreasing folate levels in the cell [113]. This raises the possibility that increased folate sensitivity increases the rate of DNA
synthesis, driving prostate cancer progression.
2.5.7. Breast Cancer
A systematic review and meta-analysis found a U-shaped relationship between folate concentration and breast cancer risk,
where women with dietary folate intake between 153 and 400 µg showed reduced breast cancer risk, unlike those outside of
this range. No correlation was found with circulating folate levels [79]. It is reported that the usual folic acid dosage for breast
cancer patients is less than 400 µg per day [114]. Notably, the chemoprotective effect of folate in this study was more
pronounced in women with high alcohol consumption, as alcohol is an established breast cancer risk factor [115]. Alcohol is a
known antagonist of folate and interferes with folate metabolism by disrupting uptake, storage, and release from hepatocytes
[116]. Folate supplements are recommended to reduce cancer risks associated with alcohol use. Nevertheless, other studies
report no influence of blood or dietary folate levels on breast cancer risk [72,79,80].

3. Vitamin B6: Pyridoxine

3.1. Dietary and Active Forms
Vitamin B6 has been considered to be the forgotten B vitamin as the clinical manifestations are less severe than B9
deficiencies. However, vitamin B6 is an essential cofactor with numerous regulatory functions in glycogen catabolism,
gluconeogenesis, lipid metabolism, amino acid synthesis, heme biosynthesis, neurotransmitter biosynthesis, anaplerosis, and
redox homeostasis [117,118]. It can be directly obtained from diet via fish, beef liver, fortified cereals, dark leafy greens,
chickpeas, and potatoes. PLP and PMP are the primary derivatives found in animal-derived foods, while plant-derived foods are
primarily PN, PNP, and a modified PN, pyridoxine-5′-β-D-glucoside (PNG) [52,119]. The physiological outcomes of these
molecular pathways serve to regulate many tissues. Initially, vitamin B6 was discovered as an anti-dermatitis factor by Paul
Gyürgy in rats on a riboflavin and thiamin diet that developed acrodynia [120]. Recommended vitamin B6 doses have been
clearly defined as 1.3 mg/day, or 1.9 mg/day during pregnancy to support neural development [121]. Supplements can reach
over 5000% of the required daily value. Regardless of this overabsorption, the majority of the supplemented vitamin B6 is
excreted in urine as 4-pyridoxic acid (PA) [122]. Notably, high-performance athletes may benefit from vitamin B6 supplements
as physical exercise increases the excretion of PA [123], with a study reporting lower B6 levels in endurance athletes after
exercise [124]. Plasmatic PLP can increase 10-fold upon supplementation and respond within 1–2 weeks following depletion or
repletion. PLP also decreases within hours of carbohydrate ingestion [125,126]. Interestingly, studies have shown that although
plasmatic PLP in an individual fluctuates with B6 intake, cellular and tissue PLP levels remain relatively steady [127]. Vitamin
B6 levels in patients can be approximated by levels of PLP and Hcy in plasma, levels of PLP in erythrocytes and blood, the
urinary excretion of 4-PA, and tryptophan catabolites [121].
3.2. Metabolic Pathways and Key Enzymes in Pyrixodine Metabolism
Structurally, B6 is a substituted pyridine with a hydroxyl and methyl group at the 5 and 6 positions, respectively (Figure 5).
The 4 position is interconverted between a hydroxymethyl, formyl, or amino group at different stages in the metabolic pathway;
another hydroxymethyl group at the 2 position can be phosphorylated. These variations account for the B6 vitamers: pyridoxine
(PN), pyridoxal (PL), pyridoxamine (PM), and their phosphorylated equivalents, pyridoxine-5′-phosphate (PNP), pyridoxal-5′-
phosphate (PLP) and pyridoxamine 5′-phosphate (PMP). Vitamin B6 is ingested as PN and is converted to PNP via pyridoxal
kinase (PDXK), an ATP-dependent enzyme. Then, PNP is transformed to PLP by pyridoxine 5′-phosphate oxidase (PNPO).
Both PL and PMP can be directly phosphorylated to PLP or PMP, respectively, via PDXK. Finally, PMP is converted to PLP by
PNPO. The active B6 vitamer is PLP, which is the dominant form in circulation and accounts for 60–70% of B6 in humans, while
PL is secondary at 30% [128].
 Figure 5. Vitamin B6 is an umbrella term for various vitamers. Vitamin B6 is ingested as the vitamer pyridoxine (PN,
red) and is phosphorylated by PDXK to become pyridoxine-5′-phosphate (PNP, orange). PNP is finally converted to the
most metabolically active form of vitamin B6, pyridoxal-5′-phosphate (PLP, yellow), via PNPO. PLP can also originate
from pyridoxal (PL, purple) via a one-step phosphorylation reaction by PDXK, or from pyridoxamine (PM, green) via a
two-step enzymatic reaction by PDXK and PNPO, where it is first phosphorylated to become pyridoxamine-5′-phosphate
(PMP, blue) before becoming PLP.

In physiology, PLP is a cofactor for over 160 enzyme reactions [129]. As the dominant vitamer, PLP is an index for general
B6 measurements; however, PL and 4-PA are also measured, usually by employing fluorometric high-performance liquid
chromatography (HPLC) or liquid chromatography-tandem mass spectroscopy (LC-MS/MS) [130]. Some studies have
suggested measuring total vitamin B6 or at least PLP+PL in order to (1) minimize person-to-person variability and (2) account
for different levels of albumin (binds PLP in the bloodstream) and alkaline phosphatase (AP, converts PLP to PL) [131,132].
3.3. Vitamin B6 Regulation
Vitamin B6 is absorbed in the jejunum, metabolized to its active form in the liver, and excreted in urine from the kidney.
Pyridoxal kinase (PDXK) is expressed in many tissues, with its highest expression being in the cerebral cortex and basal levels
in the adrenal gland, lung, breast, gastrointestinal tract, urinary tract, testis, and adipose tissue [133]. As a main regulator of
PLP levels and B6 activity, PDXK regulation occurs at the transcriptional and post-translational levels to shape PLP flux.
Several phosphorylations decorate PDXK (multiple of which are mutated in cancer patients), although the effects of these post-
translational modifications (PTMs) have yet to be defined [20,31,134] (Figure 3). To restrict PDXK enzymatic output, PDXK can
be tagged for proteasomal degradation at eight identified ubiquitination sites [135]. It can also be pharmacologically inhibited
with drugs such as 4′-O-methylpyridoxine, which competitively inhibits the PDXK active site [136]. Beyond vitamer
interconversion, B6 activity can also be suppressed via PLP sequestration by a PLP-binding protein [137]. Natural PN
antagonists such as 1-amino-D-proline exist in foods such as flaxseed, which reduce PLP levels and the output of several PLP-
dependent enzymes [138].
Cellular uptake requires PLP dephosphorylation by a membrane-bound AP, which is expressed across several organs
(bone, kidney, and liver) [81]. PLP and PMP are the primary derivatives found in animal-derived foods, while plant-derived foods
are primarily PN, PNP, and a modified PN, pyridoxine-5′-β-D-glucoside (PNG) [52,119]. Interestingly, a study reported a 13%
loss of vitamin B6 in food after cooking, though the remaining vitamin B6 was still sufficient to meet daily requirements [139].
Vitamin B6 is also available as supplements as either a complex with other B vitamins or as PN (specifically as pyridoxine
hydrochloride).
3.4. Pyridoxine Deficiencies and Disorders
Vitamin B6 deficiency is caused by reduced intake, poor absorption, or increased utilization. Low intake of vitamin B6 has
been associated with malnutrition, whereas low absorption can be caused by alcoholism where PLP fails to be released from
the liver [140]. In areas with adequate access to food, diet-based B6 deficiencies are less common and are instead exacerbated
by environmental factors [141], smoking, and adverse drug interactions [142,143,144]. Increased demand for vitamin B6 is
common in pregnancy, where it promotes central and peripheral nervous system development [145]. Beyond its role as a
cofactor for over 160 enzymes, B6 deficiency alters neurological functioning via depressed N-methyl-D-aspartate (NMDA)
receptor function [145]. Medications such as isoniazid can also interfere with vitamin B6 metabolism. Isoniazid is used to treat
tuberculosis but can lead to peripheral neuropathy by interfering with the metabolism of PN, an essential component of the
nervous system. Isoniazid reacts with PL to form hydrozone derivatives that act as a PDXK inhibitor, therefore reducing PLP
levels required for central nervous system function [146,147].
Vitamin B6 levels directly impact a wide array of physiological processes and deficiencies can present as pruritic rash,
glossitis (tongue swelling), cheilitis (cracks on lips and skin), and neurological disorders [148]. Despite divergent manifestations,
many of these disorders converge with mechanisms of poor antioxidant defense, lower purine synthesis, and disrupted
glutathione synthesis. Severe deficiency can result in dermatitis, where B6-dependent enzymes involved in creating collagen
amino acid precursors lack their necessary cofactor [149], and anemia, as low levels of PLP hinder heme synthesis. Other
consequences of low vitamin B6 are nausea, confusion, depression, and even dream loss [150].
 Similar to folate, vitamin B6 is critical for neurological function and red blood cell formation. The neuroprotective role of B6
stems from its capacity as a cofactor for glutathione, thereby lowering the aging brain’s susceptibility to oxidative stress-based
damage [151]. Thus, B6 deficiencies directly skew oxidative stress balance, thereby contributing to neurodegeneration as cells
accrue damage from reactive oxidative species [152]. In the bone marrow, PLP is used as a coenzyme in heme synthesis and
subsequently hemoglobin formation [153]. Interestingly, Parkinson’s patients treated with L-DOPA often report vitamin B6
deficiency, which can induce anemia [154]. The classic vitamin B6 antioxidant role has also been shown to support diabetes
prognosis. Diabetic rats treated with vitamin B6 showed marked reductions in oxidative stress markers in tissues [155,156].
Notably, the role of vitamin B6 as an antioxidant also allows for the quenching of reactive oxygen species resulting from UV light
[157].
In addition to diet, low PLP can also result from genetic mutations in PDXK, PDXP, or PNPO enzymes [119]. Loss-of-
function mutations in PDXK are found in Charcot-Marie-Tooth disease, which could be remedied by PLP supplementation, and
PNPO enzyme mutations have been reported in early onset epileptic encephalopathy [158,159,160].
Conversely, high levels of vitamin B6 also contribute to pathologies across different tissues. Most notably, excess PN, both
from PNPO mutations as well as supplement abuse, has been linked to sensory neuropathy characterized by numbness, nerve
damage, and muscle pain [161]. Interestingly, a cohort study of post-menopausal women revealed that those treated with high
doses of vitamin B6, a common treatment of menopausal symptoms, had an increased risk of hip fractures [162]. Similarly,
vitamin B6 has recently emerged as a novel biomarker for ankle fractures and osteoporosis, where high levels of vitamin B6 are
thought to overstimulate bone remodeling and erode bone tissue [163]. Additionally, pellagra, a B3 deficiency characterized by
scaly skin, has been reported to be aggravated by high levels of vitamin B6 [164]. Several other mechanisms of B6 involvement
in disease include immune suppression by downregulating pro-inflammatory cytokines such as IL-1β and IL-18, thereby
shutting down NF-κβ and JNK activation, and suppressing the NLRP3 inflammasome [165].
3.5. Pyridoxine and Cancer
The physiological roles of vitamin B6 in coordinating cellular metabolism and amino acid synthesis are misregulated in
numerous cancers. Vitamin B6 was first linked to cancer in the late 1960s when increased urinary excretion of tryptophan in
Hodgkin’s lymphoma patients was related to plasma PLP deficiency, as PN deficiencies are known to impact the metabolism of
amino acids including L-tryptophan [51]. Since this discovery, vitamin B6 has been implicated across a spectrum of cancers in
tissues derived from the colon, lung, breast, and blood. Intensive efforts have focused on distinguishing which cancers are
exacerbated or shunted by vitamin B6 supplementation. The multifaceted roles of vitamin B6 in cancer and recent mechanistic
insights are detailed in Table 3.

Table 3. Vitamin B6 has been implicated across a range of cancers. Cellular and tissue responses are defined.

3.5.1. Colorectal Cancer

High levels of vitamin B6 have been correlated to a reduced risk of colon cancer [167] in population-based studies as
marked by plasma PLP concentrations. A population-based study revealed that a diet high in vitamin B6 reduced the risk of
alcohol-associated colon cancer in women that consumed moderate and high amounts of alcohol, as alcohol leads to
decreased vitamin B6 by interfering with methionine synthase and vitamin B6 synthesis and absorption [168].
In a xenograft model of colorectal carcinoma, vitamin B6 metabolism was among the most upregulated in an exercise-
induced model [170]. Additionally, mechanistic insight into the role of vitamin B6 in colorectal cancer cell lines found that PL
significantly stimulated p21 mRNA expression, which is well known as a negative regulator of the cell cycle [185]. Similarly, PL
raised phospho-p53 levels, which promote p21 expression, though no changes in p53 protein levels were observed. These
results were recapitulated in mice, where mice fed diets low in vitamin B6 displayed low expression of p21 mRNA. As p21 and
p53 are known to suppress cell proliferation, vitamin B6 levels may play a role in the p21 and p53 pathways leading to
tumorigenesis [169].
3.5.2. Pancreatic Cancer
A meta-analysis examining the association between vitamin B6, vitamin B12, methionine levels, and risk of pancreatic
cancer revealed that increasing concentrations of vitamin B6 inversely correlated with risk of pancreatic cancer. Similarly,
increasing blood PLP levels also showed an inverse association with pancreatic cancer risk, with risk decreasing by 9% for
every 10 nmol/L increment [178]. Consistent with this, a Singaporean–Chinese health study revealed that at a 16-year follow-
up, 271 pancreatic cancer cases were identified out of over 63,000 men and women enrolled, with a higher intake of vitamin B6
statistically correlating with a decrease in pancreatic cancer risk [186].
3.5.3. Lung Cancer
Lung cancer is the most common cause of cancer death in all sexes [187]. Similar to colorectal cancer, high levels of
vitamin B6 have also been reported to reduce lung cancer risk. In non-small cell lung cancer (NSCLC) patients under cisplatin
regimens, vitamin B6 sensitizes cancer cells to cisplatin-mediated DNA damage and cell apoptosis [171]. Low PDXK levels also
correlated with poor disease prognosis. Consistent with this, a case-control study revealed that among diverse participants,
high vitamin B6 serum levels correlated with a decreased risk of lung cancer [172]. It should be noted, however, that
contradictory studies showed correlations between high vitamin B6 levels and high incidence of lung cancer, and vice versa
[173,174]. Nevertheless, there have also been reports finding no association between sera and dietary B6 levels with increased
cancer risk in lung tumor sites [175].
3.5.4. Breast Cancer
Breast cancer is the most common cancer in women (cancer.gov). Unlike the established role of vitamin B6 in cancers
such as that of the colon and the lung, there is a less convincing association between vitamin B6 and breast cancer prognosis.
Population-based studies have reported different results. A 2001 study reported that breast cancer patients presented with
higher serum vitamin B6 levels than the control group [176], while a 2004 study reported no correlation between breast cancer
risk and vitamin B6 intake or serum PLP levels [177]. A study on the link between folate and breast cancer risk revealed that
vitamin B6 increased the protective effect of folate on breast cancer, lowering risk [176]. This effect suggests that different
players of the one carbon metabolism may also collaborate in preventing or reducing cancer incidence. It would be interesting
to evaluate how folate-based dietary interventions combine with methionine-related chemotherapeutics, such as
methyltransferase inhibitors like MS023, which reduces triple negative breast cancer development [188].
3.5.5. Prostate Cancer
Prostate cancer is the second most common cancer among men (cancer.gov). A meta-analysis of observational and
interventional studies assessing the association between vitamin B6 intake, PLP levels, and cancer risk revealed that there was
no statistically significant association [175]. Nevertheless, a previous study observed that an increasing consumption of vitamin
B6 reduced risk [180], and suggested it could be due to the role of vitamin B6 in organ sensitivity to hormone uptake and action,
which is increased with low levels of the vitamin [189].
3.5.6. Skin Cancer
Though vitamin B6 has been shown to have protective properties on the skin, such as its anti-oxidant and anti-inflammatory
roles, there is little to no evidence linking it to cancers of the skin, warranting more clinical and scientific attention. A 1985 in
vitro study revealed that B16 melanoma cells grown with 5.0 mM PN or 0.5 mM PL had an 80% reduction in cell proliferation
[181]. Furthermore, B16 melanoma xenografted mice pretreated with PL showed a 62% reduction in tumor growth compared to
those without the pretreatment. Similarly, a 2018 study revealed that PL at 500 µM suppressed B16F10 cell proliferation, while
PN had a weak inhibitory effect [182], with both studies supporting that PL has a stronger inhibitory effect on melanoma cell
growth at lower concentrations compared to other B6 vitamers. Though there appears to be potential, whether vitamin B6 may
serve as an antineoplastic drug for skin cancers requires further investigation.
3.5.7. Kidney Cancer
Vitamin B6 is part of the vital renal vitamins, and is commonly supplemented to Chronic Kidney Disease (CKD) patients at
5 mg/day on non-dialysis and 10 mg/day on dialysis regimens [190]. The role of vitamin B6 in the context of renal cancer
remains to be closely investigated. A World Cancer Research Fund-funded study revealed that patients with higher plasma
concentrations of vitamin B6 had reduced risks of renal cell carcinoma and overall better prognosis [183]. In support of this, a
case-cohort study revealed that higher vitamin B6 concentrations were associated with lower risk of death in renal cell
carcinoma patients [184]. Similarly, PLP depletion in diabetic patients has been linked to an increased risk of developing several
malignancies, including that of the kidney [191], though a meta-analysis study revealed no evidence of association between
high vitamin B6 intake and kidney tumors [175].
3.5.8. Acute Myeloid Leukemia
Vitamin B6 was originally thought to play a role in blood cancers, such as leukemia. This was attributed to the role of
vitamin B6 in red blood cell production [192]. Following this discovery, low levels of vitamin B6 have been continuously reported
in blood cancers, where low levels are correlated with worse outcomes. In the context of acute myeloid leukemia (AML), high
vitamin B6 levels are associated with increased risk of cancer. Vitamin B6 is low in circulation of AML patients, which may
indicate an addiction of AML cells to vitamin B6 [136,166]. In normal tissues, PDXK phosphorylates inactive into active vitamin
B6 when cellular levels are low. In AML cancerous states, PDXK constitutively phosphorylates PL and PN into PLP, thereby
promoting cancer cell proliferation, while a dominant negative PDXK mutant was unable to restore PLP levels or the
proliferative effects of AML cells [136]. These surprising findings were uncovered using a CRISPR-Cas9 screen to determine
proliferative effects of AML cells [136]. These surprising findings were uncovered using a CRISPR-Cas9 screen to determine
which metabolic enzymes required PLP for AML proliferation, revealing GOT2 and ODC [136], which may also support
dysregulated cell growth by increasing one carbon metabolic flux towards biomacromolecule precursor synthesis. This deeply
mechanistic experimental approach provided new insights into a central mystery in the field of vitamers and oncology.
3.5.9. Brain Cancer
It is interesting to note the absence of vitamin B6 involvement in brain-related cancers given the high levels of PDXK
expression across neuronal tissues, with the exception of some neuroblastoma studies [193]. One could speculate that high
PDXK may be chemoprotective by promoting PLP production.

4. Key Advances and Implementation of Innovative Tools and Instrumentation

Vitamin B6 and B9 levels have been historically measured Ex vivo from blood or sera samples using microbiological or
enzymatic assays [194,195]. However, approximating one carbon metabolite levels within cells was limited until recent
advances of metabolomics-based mass spectrometry [196]. An additional challenge surrounds the measurement of metabolic
fluxes, particularly in real time, where the metabolism within a cell can dramatically change within seconds [197]. Modeling
metabolic flux in vitro with cell lines also suffers from current culture methods, where excess nutrient levels can skew the
metabolism of a cell [198]. Exciting advances using in vivo imaging, physiologic cell culture media, metabolite labeling, and
organelle proteomics have resulted in new discoveries surrounding one carbon metabolism in physiology and pathological
states. Indeed, metabolomics enables the precise quantification of intracellular metabolites with resolution to distinguish
between different vitamers [199,200,201,202,203]. When combined with metabolite labeling in vivo, these advances have
enabled metabolic flux measurements and paradigm-shifting insight for the fields studying folate and PN metabolism [204].
Metabolomics has also offered novel biomarkers and new areas for therapeutic intervention in researching the etiologies of
B6- and B9-related disorders. In the case of PN-dependent epilepsy, untargeted metabolomics uncovered several candidate
biomarkers in blood (α-aminoadipic semialdehyde, piperideine-6-carboxylate, and pipecolic acid) that had been previously
disregarded given their instability in urine-based sampling [205]. Given the higher sensitivities of modern analytical tools, clinical
sampling is more practical as in the case of cerebrospinal fluid detection of several B6 vitamers [206,207]. In vivo metabolite
measurements overcome major obstacles in the field by correlating metabolic activity with gene expression and patient
histology. A particularly exciting innovative technology overcomes these limitations and even enabled quantitative metabolic flux
monitoring of human tumors. The De Bernardis lab applied multiparametric, preoperative imaging with intraoperative infusions
of isotope-labeled nutrients (e.g., 13C-glucose) to resolve the dynamics of metabolism in lung and brain tumors and even
across organ systems [208]. Metastasis rates in a profile of 17 patient-derived xenograft melanoma mouse models were directly
linked to variable usages of methionine metabolism, which was explained by H3K9 and H3K27 methylation patterns [27].
Modulating the levels of methionine in the diets of cancer patients has become of increasing clinical interest, where methionine
usage directly explains features such as metastatic potential [27,209]. Since B6 and B9 both control one carbon metabolic flux,
combining B6 and/or B9 restrictions with methionine restriction therapies could offer synergistic benefits for patient health. Use
of new techniques that can decipher the differences between the tumor metabolic environment from those in adjacent and
benign tissues could provide insight into new vulnerabilities for targeting cancer tissues specifically.
Additional transformative techniques include those to visualize metabolite levels in vivo or in vitro. Folate uptake by cells
has historically been imaged using radiolabeled or fluorophore-conjugated folate analogs [210]. Such techniques have
progressed to the point of observing fluorescence in operating rooms, as is the case with Cytalux. Cytalux is a fluorescent folate
analog that was approved by the FDA in 2021 to illuminate cancer lesions for diagnosis, as well as during surgery for immediate
identification of malignant versus benign tissue [211]. Cytalux binds to folate receptors, which are overexpressed in ovarian and
lung cancers, and can be excited via near-infrared light [210].
Imaging B6 has proved elusive until a first-of-its-kind probe was developed by Jun et al. in 2019. Utilizing the reactivity of
acyl-hydrazides with aldehydes, they designed a rhodamine-based probe that selectively fluoresces upon binding PLP [212].
The probe demonstrated excellent selectivity among other biological aldehydes—including other B6 vitamers. The probe was
employed in vitro to monitor the conversion of PL to PLP upon addition of PDXK, with fluorescence building over an 18-h time
course. The probe was further tested in control and PDXK-knockdown cells. Interestingly, PLP levels in PDXK-deficient cells
remained similar to that of control cells, hypothesized to be a result of PNPO conversion of PMP and PNP to PLP. However, the
addition of exogenous PL specifically enhanced fluorescence. Another B6 probe was recently developed by Brun et al. in the
form of a hydrazine-derived contrast agent (2-hydrazinonicotinic acid, or 2-HYNIC) that reacts with an aldehyde to form a
hydrazone in 2021 [213]. 2-HYNIC adopts a unique planar conformation when bound to PLP, allowing for enhanced contrast
and hence imaging using chemical exchange saturation transfer MRI (CEST-MRI). With this technology, B6 metabolism was
tracked in vitro in lung cancer cell lines, as well as in vivo with animals bearing tumor xenografts. Differences in CEST-MRI
contrast between the cell lines reflected metabolic differences of tumor cells [171].
Nucleic acid-based imaging modalities likewise offer a unique approach that could promote the rapid development of
selective metabolite sensors for vitamins B6, B9, and their constituent vitamers. Aptamers are short nucleic acid sequences that
bind a ligand, and thus can be harnessed for use as sensors. Upon binding to a target of interest, aptamer sensors fold into a
structure that resembles the fluorophore of fluorescent proteins [214,215]. Aptamer sensors have already been used and
optimized to detect metabolites in one carbon metabolism, including SAM [216,217]. While fluorescent aptamers are now well-
characterized, more difficulty lies in designing a high-affinity ligand-binding sequence. Conveniently, bacteria contain
endogenous small-molecule-binding RNA regulatory elements among their mRNA known as riboswitches. A riboswitch has
been discovered for THF [214,218,219]. In principle, the appropriate riboswitch could be coupled to a fluorescent aptamer,
granting dynamic imaging of folate metabolism. It is predicted that thousands of other riboswitches exist for metabolites, which
may enable complex imaging of the metabolic interplay between B6, B9 and other metabolites [220]. Other imaging approaches
have exploited inducible CRISPR-dCas9 transcription activation of a fluorescent protein reporter combined with a split
riboswitch system that associates upon SAM binding [221], or riboswitches for ZTP, a molecule that indicates a lack of folate
biosynthesis [222].
As long-awaited goals of sensing small molecules in living contexts have been realized and continue to advance, it will be
easier to illuminate these metabolic mechanisms to advance biomedical sciences. The complementation of these sensors can
further expand the chemical toolbox to improve understanding of one carbon metabolism and pathogenicity.

5. Exploiting One Carbon Metabolism for New Therapeutics

Metabolic enzymes across the folate and methionine cycles have been shown to be overexpressed in cancer. Recent
advances have already led to exciting new therapies that target key enzymes in the one carbon pathway. Advances in
technology have allowed deeper mechanistic insight and offer a vulnerability for designing novel therapies.
The DHFR enzyme is the target of MTX, the first antimetabolite therapeutic that was initially developed for the treatment of
acute lymphoblastic leukemia (ALL) [58]. The success of MTX-based interventions gave rise to a series of antifolate cancer
therapies such as 5-fluorouracil (5-FU) [223], which targets TS in the folate cycle. MTX has been prominently used to treat
psoriasis, rheumatoid arthritis, and several cancers [72,224,225]. MTX functions through several mechanisms related to
inflammation and proliferation; within the context of cancer, its inhibitory effects on DHFR block conversion of DHF to THF,
thereby slowing metabolic flux towards nucleotide synthesis and halting proliferation. Challenges surrounding toxicity limit the
extended use of MTX treatments, depleting THF pools from healthy cells [226]. A CRISPR-Cas9 screen of MTX-treated
leukemia cells revealed a possible mechanism to attenuate MTX-induced cellular toxicity of healthy cells.
Formimidoyltransferase cyclodeaminase (FTCD) is an enzyme that uses THF as a cofactor to perform histidine catabolism. In
the presence of histidine supplementation, available pools of THF were redirected to the histidine catabolic pathway, increasing
the sensitivity of cells to MTX and reducing its toxicity [227,228]. This landmark discovery highlights the fundamental
importance of environment folate sources to antifolate therapies and provides mechanistic insight into improving therapeutic
conditions. Indeed, several exciting reports have followed this and similarly demonstrate how cancer cells overexpressing folate
pathway enzymes can be targeted with higher specificity. Either MS or MTR convert 5-MTHF, the predominant form of folate in
circulation, to THF and are overexpressed in many cancer cell types. Exposing cancer cells to dietary 5-MTHF was sufficient to
increase resistance to MTX-based treatments [229,230].
Small-molecule SHMT inhibitors have recently been shown to block the growth of many human cancer cells [231]. Cancers
with defective amino acid transport systems such as B-cell lymphoma are particularly vulnerable to SHMT inhibitors because
glycine is the byproduct of SHMT reactions. MTHFD2 is overexpressed in cancer and contributes to genomic instability during
the early stages of cancer initiation that are associated with an activated DNA-damage response (DDR) [50]. MTHFD2
inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress.
Tumor cell metabolism is rewired to enable aberrant proliferation and expansion [232]. However, cancer cells do still rely on
metabolites provided by the patient’s diet. For this reason, dietary restriction of specific amino acids has been deeply
investigated as a potential therapy across different cancer types. Methionine is a prime example of this new avenue of research
[233]. In KRAS-driven lung and pancreatic cancers, reduction of asparagine showed promising results [234]. Within the one
carbon pathway, limiting dietary methionine reduced tumorigenesis in chemotherapy-resistant KRAS-driven colorectal cancer
and radiation-resistant KRAS-driven soft tissue sarcoma [235,236]. Similarly, lowering the levels of non-essential amino acids
serine and glycine was sufficient to reduce tumor progression in APC-inactivated models of intestinal cancers [237]. It is
interesting to note that cancer cells may use 50% of glucose-derived carbon for serine biosynthesis and catabolism [215]. The
amplification of breast and melanoma cancers has an upregulation of 3-phosphoglycerate dehydrogenase (PHGDH), a rate-
limiting serine biosynthesis enzyme that was found to be required for growth in vitro and in vivo models [238,239].
The combination of metabolomics and metabolite tracing analyses led to the discovery that tumor-initiating cells have high
activity of the methionine cycle via MAT2A overexpression. Importantly, the high consumption of methionine outcompetes
regeneration and renders these cancer cells auxotrophic to methionine and vulnerable to pharmacological inhibition [240]
(Figure 6). Similarly, genetic mutations in one carbon metabolic enzyme methyl-5′-thioadenosine phosphorylase (MTAP) offer a
point to help identify tumor cell populations that could be vulnerable to reducing dietary methionine [241,242,243,244]. The
mechanism for this underlies the methionine salvage pathway and the generation of metabolite MTA, which acts as an inhibitor
mechanism for this underlies the methionine salvage pathway and the generation of metabolite MTA, which acts as an inhibitor
of methylation. Reports from multiple labs have now shown that MTA accumulation with either methionine deprivation or
methylation inhibitors led to dramatic decreases in proliferation of many cancers. This exemplifies how genetics can help to
inform clinicians in the design of dietary regimens during chemotherapeutic interventions [17]. As metabolic disease states
cannot be determined using classic genomic analysis, these lines of research elucidating the contributions of metabolic
regulation and enzyme mutations in disease will provide key insight for clinicians and basic researchers.

Figure 6. Dietary vitamin B6 and folate are essential for human health. Diets high in vitamin B6- and B9-rich foods (left)
correlate with decreased risks of several cancers, but may mask the symptoms of a vitamin B12 deficiency, increase
risk of ovarian and AML cancers, aggravate pellagra symptoms, and lead to numbness, nerve damage, and muscle
pain. Diets low in vitamin B6- and B9-rich foods (right) can lead to dermatitis, glossitis, cheilitis, nausea, confusion,
dream loss, as well as an increased risk of several cancers and a decreased risk of AML. Figure made with BioRender.

6. Future Perspective and Ending Notes

The progress of folate research has unfolded at an accelerated rate and new therapeutics are on the horizon [3]. The rapid
development of new tools to monitor the dynamic control of metabolism through multidisciplinary approaches that incorporate
synthetic biology and systems biology provide clear readouts at the transcriptional, translational, and post-translational levels.
For example, one carbon metabolism was recently implicated in viral replication and infection [245]. Methionine metabolism
determined viral latency by controlling the B cell EBV epigenome [246].
Going forward, advances that tackle challenges associated with the complexity of vitamer chemical structures and the vast
number of low quantity metabolites could aid in current limitations. Between advanced metabolomics, new models of disease,
isotopic labeling approaches, and sensors, new technologies will have revolutionary impacts on medicine and forge a future for
dietary regimens that enhance therapeutic interventions. Over the next decade, user-friendly and affordable metabolomics-
based instrumentation will play a major role in the clinical diagnosis across a spectrum of diseases and cancer to enable
precision medicine.

Author Contributions
Conceptualization, writing and editing was performed by C.N.F., L.J.S., J.T.L., S.T.N. and L.V.A. Project supervision was
overseen by L.V.A. All authors have read and agreed to the published version of the manuscript.

Funding
This review article received no external funding.

Acknowledgments
We would like to thank all the members of the one carbon metabolism community for inspiring the field through the work.

Conflicts of Interest
The authors declare no conflict of interest.
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Birth Defects Res. Author manuscript; available in PMC 2020 Nov 15. PMCID: PMC6511489
Published in final edited form as: NIHMSID: NIHMS986149
Birth Defects Res. 2019 Nov 15; 111(19): 1455–1467. PMID: 30421543
Published online 2018 Nov 12. doi: 10.1002/bdr2.1380

Overview on Neural tube defects: from development to physical characteristics

Laura Avagliano,#1 Valentina Massa,#1 Timothy M. George,2 Sarah Qureshy,3 Gaetano Bulfamante,1 and Richard H. Finnell3,4

Abstract

Neural tube defects (NTDs) are the second most common congenital malformations in humans affecting the development of the central nervous
system. Although NTD pathogenesis has not yet been fully elucidated, many risk factors, both genetic and environmental, have been extensively
reported. Classically divided in two main sub-groups (open and closed defects) NTDs present extremely variable prognosis mainly depending on the
site of the lesion. Herein we review the literature on the histological and pathological features, epidemiology, prenatal diagnosis and prognosis, based
on the type of defect, with the aim of providing important information based on NTDs classification for clinicians and scientists.

Keywords: Neural Tube Defects, Spina bifida, Anencephaly

Development of Neural Tube Defects

Neural Tube Defects (NTDs) arise secondary to abnormal embryonic development of the future central nervous system. The two most common types
of NTDs are spina bifida and anencephaly, affecting different levels of the brain and spine, normally reflecting alterations of the embryonic processes
that form these structures. Birth defects such as NTDs are relatively uncommon, with a global prevalence among live births in the US of 1 in 1200,
and a worldwide prevalence ranging from 1 in 1,000 (in Europe and the Middle East) to 3–5 in 1,000 (in northern China as of 2014 with folate
supplementation campaigns, bringing the prevalence down from 10 per 1,000 for years 2000–2004) (Blencowe, Kancherla, Moorthie, Darlison, &
Modell, 2018; Khoshnood et al., 2015; Liu et al., 2016; Salih, Murshid, & Seidahmed, 2014). Despite their public health significance, surprisingly
little is known about the etiology of NTDs in humans.

The central nervous system (brain and spinal cord) is formed in vertebrates during a process known as neurulation. This process occurs in human
embryos between days 17 and 28 post-fertilisation. In the previous developmental phase (gastrulation), the ectoderm is formed, which will thicken in
response to specific molecular signals released by the underlying notochord, giving rise to the neural plate. This plate of ectodermal cells will form the
neural tube by elevating, juxtaposing and fusing along the midline (primary neurulation) of the body axis. In the caudal region, neurulation
(secondary) involves cellular condensation and mesenchymal-to-epithelial transition to close the neural tube (Saitsu, Yamada, Uwabe, Ishibashi, &
Shiota, 2004). In mammals, primary neurulation is a multi-site process and recent evidence suggest that in humans two closure sites are recognisable
(one at the prospective cervical region and one over the mesencephalon-rombencephalic boundary) (Copp, Stanier, & Greene, 2013; Nakatsu, Uwabe,
& Shiota, 2000). Mammalian neurulation is tightly regulated and energetically highly demanding, involving the formation of an anterior (ANP) and
posterior neuropore (PNP). These neuropores or openings will progressively reduce in size until final fusion completes the process of neural tube
closure (NTC) (Figure 1).

Figure 1.

A: Transverse section of normal fetal vertebra with typical spinal cord section. B. Craniorachischisis. Note the absence of the cranial vault, the defect extends to vertebral arch
into upper dorsal area, resulting in anencephaly and spina bifida
Different types of NTDs reflect the site of the interrupted neurulation. For example, craniorachischisis, which affects the brain and spinal cord, results
from a failure of the initial closure site resulting in an open brain and spine, while anencephaly arises from abnormalities in the cranial neurulation
process, and spina bifida results from incomplete caudal neurulation

Epidemiology and risk factors

Chromosomal anomalies such as trisomy 13, trisomy 18 and triploidy represent less than 10% of all NTDs cases (Kennedy, Chitayat, Winsor, Silver,
& Toi, 1998), while non-syndromic isolated cases represent the vast majority of NTDs, exhibiting a sporadic pattern of occurrence (Copp et al., 2015).
The prevalence of the different types of NTDs is not always reported in publications, given the difficulties in data ascertainment following legal
pregnancy terminations and spontaneous abortions. The prevalence of NTDs in miscarriage or stillbirths appears higher than in term pregnancies
(Padmanabhan, 2006), and the estimated prevalence reflects temporal, regional and ethnic variations (Blom, Shaw, den Heijer, & Finnell, 2006;
Wallingford1, Niswander, Shaw, & Finnell, 2013). For example, the NTD prevalence in Mexico (~3.2 in 2000 births) is higher than in the United
States, (CDC 2000), while the prevalence in some regions of China are 20 times higher than in the United States (Li et al., 2006). Interestingly, the
different prevalence of NTD cases between ethnic groups persists after migration of the racial groups to other geographical areas, indicating a genetic
contribution to NTD susceptibility (Leck, 1974; Shaw, Velie, & Wasserman, 1997).

In terms of genetic predisposition, women who have had an affected fetus have an empirical recurrence risk of 3% in any subsequent pregnancy, yet
this risk only rises to approximately 10% after conceiving a second NTD embryo (Copp et al., 2015). This underscores the fact that while genetic
factors are important, one cannot ignore the impact of the environment on the development of the NTD phenotype. In twins, the NTDs’ concordance
rates among monozygotics is reported to be 7.7%, significantly higher than the rate for dizygotic twins (4.4%).

Finally, a gender predisposition has been reported for some types of NTDs: a female excess has been reported for neural tube defects, possibly due to a
sex-related genetic or epigenetic effect. Despite the decades long attempts to elucidate genetic factors causative of NTDs in humans, no conclusive
evidence has been identified. In studies based on vertebrate animal models, a great number of genes have been implicated in the causation of NTDs. In
the mouse, for example, more than 400 genes have been found responsible for failed NTC (Harris & Juriloff, 2010; Wallingford1 et al., 2013). Some of
these genes are highly evolutionarily conserved, and their role in neurulation has been shown in multiple vertebrate animal models (Wallingford,
2005). In humans cohort studies of NTDs, genetic variants have been associated with increased risk (N. D. E. Greene, Stanier, & Copp, 2009).
However, some variants are present in different NTDs subtypes and some have also been reported in healthy patients, highlighting the difficulties of
finding a clear genotype/phenotype association in humans.

Many factors, both genetic and non-genetic, are involved in the abnormal closure of the neural tube, suggesting that multi-factorial causes lead to the
development of NTDs (Copp et al., 2015). It is likely that each factor individually is insufficient to disrupt normal NTC; however, together these
genetic and non-genetic factors produce synergistic effects leading to failed NTC and the abnormal embryonic phenotype. This working hypothesis
represents the so-called “multifactorial threshold model” (Harris & Juriloff, 2007).

Non-genetic risk factors include exposure to a broad range of environmental exposures such as air pollution (Brender, Felkner, Suarez, Canfield, &
Henry, 2010; Carmichael et al., 2014; Cordier et al., 1997; Hutchins et al., 1996; Zhiwen Li et al., 2011; Lupo et al., 2011; Padula et al., 2013; Ren et
al., 2011; Righi et al., 2012) and maternally toxic factors including disease, nutrition, exposure to occupational chemicals or physical agents and abuse
of substance (Table 1).
 Table 1:

Modifiable risk factors for NTDs

Risk Factor Action Risk References

Maternal diabetes Teratogenic effect due to embryonic exposure to 2-10-fold increase (Ray, 2001; Shaw et al., 2003; Yazdy, Mitchell, Liu,
high glucose concentrations leading to increased cell & Werler, 2011)
death in the neuroepithelium

Maternal obesity Teratogenic effect due to embryonic exposure to 1.5-3.5-fold increase.  (Anderson et al., 2005; Carmichael, Rasmussen,
hyperinsulinemia, metabolic syndrome, and The risk increases with Lammer, Ma, & Shaw, 2010; Dietl, 2005;
oxidative stress related to adiposity increased maternal body mass Hendricks, Nuno, Suarez, & Larsen, 2001; Shaw,
index Velie, & Schaffer, 1996; Werler, Louik, Shapiro, &
Mitchell, 1996)

Maternal Hyperthermia Teratogenic effect due to embryonic exposure to 2-fold increase (Moretti, Bar-Oz, Fried, & Koren, 2005; Suarez,
(sauna, hot water tube, heat stress Felkner, & Hendricks, 2004; Waller et al., 2017)
fever)

Drugs (particularly Teratogenic effect due to embryonic exposure to 10-fold increase (Kanai, Sawa, Chen, Leeds, & Chuang, 2004;
valproate) valproate action as inhibitor of histone deacetylases, Lammer, Sever, & Oakley, 1987; Meador et al.,
disturbing the balance of protein acetylation and 2006; Pai et al., 2015; Yildirim et al., 2003)
deacetylation, leading to neurulation failure

Inadequate maternal Teratogenic effect due to embryonic exposure to low Undetermined
nutritional status folate intake, low methionine intake, low zinc
intake, low serum vitamin B12 level, low vitamin C
level, caffeine abuse, alcohol use, smoking, all
conditions disturbing the folate-related metabolism
(Grewal, Carmichael, Ma, Lammer, & Shaw, 2008;
Kirke et al., 1993; Ray & Blom, 2003; Schmidt et
al., 2009; Suarez, Hendricks, Felkner, & Gunter,
2003; Velie et al., 1999)

Physical characteristics of neural tube defects

NTDs have been classically divided into open defects such as craniorachischisis, exencephaly-anencephaly and myelomeningoceles, and closed
defects, including encephalocele, meningocele and spina bifida occulta (Copp & Greene, 2013; McComb, 2015). In general, open defects are
characterized by the external protrusion and/or exposure of neural tissue. Closed defects have an epithelial covering (either full or partial skin
thickness) without exposure of neural tissue (McComb, 2015). Biochemically, during pregnancy open defects are detectable due to the high levels of
amniotic fluid α-fetoprotein and amniotic fluid acetylcholinesterase, whereas closed defects do not deviate from normal levels of amniotic fluid α-
fetoprotein or acetylcholinesterase. Clinically, open defects trend towards having worse functional neurological outcomes in children, compared to
closed defects.

Open neural tube defects

Craniorachischisis

Definition. Craniorachischisis is a defect of NTC that involves both the cranial and spinal portions of the neural tube (Golden & Harding, 2004). It is
the most severe expression of an open NTD.

Epidemiology. This is a very rare congenital malformation of the central nervous system whose actual prevalence is not known. Reported prevalence
is about 0.1 per 10,000 live births for cases of 20 weeks gestation or greater in Atlanta, a prevalence that had been adjusted upward 30% to account for
prenatal terminations (Moore et al., 1997); 0.51 per 10,000 births in a Texas-Mexico border population whose prevalence ascertainment included
prenatal terminations (Johnson, Suarez, Felkner, & Hendricks, 2004); 10.7 per 10,000 births in Northern China and 0.9 per 10,000 births in Southern
China (Moore et al., 1997).

Gross Dysmorphology. The neural tube is open from the midbrain to the spine. The defect of the skull and vertebral arch results both in anencephaly
and an open spina bifida (Figure 1). Fetuses show absence of the cranial vault, with the defect extending with various degree to the vertebrae. The
neck may be short or absent, facial dysmorphisms may be present and include a broad nose, exopthalamos, low-set and folded ears. Typically,
posterior telencephalon, hindbrain and spinal cord tissue is externally exposed.

Histology. In these cases, both the brain and spinal cord are exposed to the intra-amniotic environment resulting in destruction of the nervous tissues
due to the inherent toxicity of the amniotic fluid. The same histological characteristics of anencephaly and myelomeningocele are detectable in these
specimens (see below).

Prenatal diagnosis. Absence of the cranial vault and spinal dysraphism is detectable by ultrasound. A disorganized mass of cerebral tissue is visible
and cervical hyperextension may also be observed. Differential diagnosis includes: nuchal tumors (such as teratomas and lymphangiomas), Klippel–
Feil syndrome (a disease caused by a failure of segmentation of the cervical vertebrae during early fetal development), and Jarcho–Levin syndrome,
also known as spondylocostal dysostosis, an autosomal recessive disorder characterized by a shortened trunk, opisthotonus position of the head, short
neck, barrel-shaped thorax, multiple wedge-shaped and block vertebrae, spina bifida, and rib anomalies (Chen, 2007).
Prognosis. Craniorachischisis is a lethal condition: there is no cure or surgical intervention and the death of the newborn is unavoidable.

Anencephaly

Definition. When the primary defect involves failure to close just the cranial portion of the neural tube, the defect is refered to as exencephaly
(anencephaly). The degeneration of the cerebral-neural tissues due to the destructive exposure of the brain to the intra-amniotic environment converts
the exencephaly defect to anencephaly (Golden & Harding, 2004; Timor-Tritsch, Greenebaum, & Monteagudo, 1996; Wilkins-Haug & Freedman,
1991)

Epidemiology. The Center for Disease Control and preventin (CDC) estimates that each year, about 3 pregnancies per every 10,000 births in the
United States will be affected by anencephaly.

Gross Dysmorphology. Anencephaly is a defect in which there is absence of the structures derived from the forebrain and the skull. The calvarium is
generally absent, the parietal, frontal and squama of the temporal and occipital bones appears as rudimentary fragments; the base of the skull is nearly
normal (Goldstein & Filly, 1988) but is thick and flattened; the sphenoidal bone is abnormally shaped (Golden & Harding, 2004) resembing a “bat
with folded wings” (Marin-Padilla, 1965). The facial bones appear normal and the face is generally normally structured (Figure 2), although the eyes
often appear to protrude because of the shallowness of the orbits and the forehead is absent or shortened. Moreover, other associated facial
abnormalities have been reported such as flattened nasal bridge and low-set ears (Stumpf et al, 1990).

Figure 2.

Macroscopic and histologic features of anencephaly. The cranial vault is absent, the brain is not covered with bones, the skin is in continuity with the nervous tissue and the
nervous tissue, called area cerebrovasculosa, is a mass of degenerated tissue. Histologically the typical aspect of area cerebrovasculosa are shown with the enlarged venous
vessels with various dimension immersed in nervous tissues.

The brain remnants is called the area cerebrovasculosa (Figure 2). Its macroscopic aspect appears as a dark brown undifferentiated mass (Anand, Javia,
& Lakhani, 2015); the residual amount of brain tissues varies, generally there is an absence of the structures of the forebrain (both diencephalon and
telencephalon structures including thalamus and cerebrum) and midbrain, whereas the brainstem may appear to have been spared, or less severely
involved. The pituitary is present, but it is hypoplastic and without the intermediate and posterior lobes.

Histology. The residual cerebral tissue appears as an irregular mass containing vascular tissue, glia, and some neuroblasts or neurons surrounded by
meninges (Golden & Harding, 2004) although some authors failed to find neurons in the area cerebrovasculosa (Ashwal et al., 1990). The overview
shows a poorly structured mass consisting of blood vessels (Figure 2) scattered with connective tissue and islets of nervous tissue including
interspersed astroglial cells, nerve cells, and cavities surrounded by the epithelium (Anand et al., 2015). The exposed cerebral area is covered by non-
keratinising squamous epithelium that laterally is continuous with epidermis (Figure 2) (Golden & Harding, 2004). Interestingly, even though there is
a severe rostral neural tube abnormality, the spinal cord in the anencephalic foetuses appears structurally normal (Anand et al., 2015).

Prenatal diagnosis. The diagnosis is primarily based on the absence of a normally formed calvarium and brain above the orbital line (Goldstein &
Filly, 1988). The area cerebrovasculosa may be detected as fluctuant echogenic tissue. Often polyhydramnios are present. The differential diagnosis
between anencephaly and other causes of an absence of the cranial vault may include cases of head destruction related to amniotic bands (Stumpf et
al., 1990). This distinction is very important for counseling families, because the destruction related to amniotic bands represents a sporadic event
which would have a very low recurrence risk, whereas the presence of anencephaly increases the risk of occurrence of another NTD in any subsequent
pregnancy. The differential diagnosis between these two entity with an of absence of the cranial vault relies on the presence or absence of othe
malformations as anencephalic infants are usually isolated with no other associated malformations. Moreover, the cranial defect in cases of amniotic
bands syndrome is asymmetric, whereas in the case of anencephaly it is symmetric (Goldstein & Filly, 1988; Keeling & Kjaer, 1994).

Prognosis. Anencephaly is a lethal condition. Detection of anencephaly during early gestation is generally followed by legal interruption of the
pregnancy. In cases of pregnancies that continue to term, most anencephalic newborns die within the first day or two post-parturition (Stumpf et al.,
1990).

Myelomeningocele

Definition. In myelomeningocele, the developmental defect involves the failure to close the posterior spinal portion of the neural tube, more
frequently the lumbar portion is the region which fails to fuse. In this defect, the meningeal sac herniates through a bony defect of the vertebral arch (
Figures 3–4). In some cases, a clear open defect that involves the spinal cord, but without a protruding meningeal sac, is defined as a myelocele.
Therefore, a myelocele is an open defect without the cystic component (Figure 3–4).
 Figure 3.

Macroscopic and histologic aspect of myelocele (upper panel) and myelomeningocele (lower panel).

In myelocele the foetus presented a clear open defect in the lumbar-sacral region. The spinal cord is displayable in the depth of the cleft. The defect is open and no meningeal
sac is protruding. The histological sections show the bone defect of the vertebral arches and the exposed spinal cords. In this case the spinal cord is not closed in its typical
shape but is divided in two halves appearing as an “open book”. The remnant of the ependymal layer is visible at the surface, covering the center of the two halves.

In myelomeningocele the foetus presented a clear meningeal cystic sac that contains cerebro-spinal fluid and nervous tissue. Note the translucent appearance of the nervous
tissue. The histological sections show the protrusion of the medulla through the bone defect of the vertebral arches. Meninges are also herniated forming the cystic mass.

Figure 4.

Schematic representations of central nervous system appearance in normal foetus, meningocele, myelocele and myelomeningocele.

In meningocele meninges are displaced through a bone defect of the vertebral arches; spinal cord is not involved in the protrusion and the brain is normal.

In cases with myelocele and melomeningocele the neural tube defect is associated with Chiari malformation type II, characterised by the herniation of cerebellar vermis
through the foramen magnum

Epidemiology. It is the most common form of spina bifida aperta in humans, with an estimated incidence according to the CDC of 1.8 per 10,000 live
births in the United States.

Gross Dysmorphology. In myelomeningocele, a cystic mass protruding though a bony defect in the vertebral arches is detectable. The size and shape
of the lesion can vary significantly and may include cerebrospinal fluid drainage. The neural tissues appears translucent through the protruded
meningeal sac and the neural placode, a segment of flat, non-neurulated embryonic neural tissue, is externally shown and protrudes above skin surface
(Figure 4). In case of myelocele the cystic mass is absent and the neural tissue is clearly detectable though the vertebral cleft and the placode is flush
with the surface of the skin (Figure 4). The spinal cord above the defect may be distorted in position and shape, but it is not overtly malformed (John
L. Emery & Lendon, 1973; Naik & Emery, 1968).

Myelomeningecele and myelocele are usually associated with Chiari malformation type II (Figures 4–5), because of the traction of the brain stem from
below due to tethering of the open spinal cord through the vertebral defect. Indeed, the brain stem is elongated, there is caudal elongation of the
medulla and the fourth ventricle, cerebellar vermis is displaced into the foramen magnum (Figures 4–5). As a result, the normal flow of cerebrospinal
fluid through the ventricles is compromised, resulting secondarily in hydrocephalus. There is also an abnormal orientation of the cervical nerve roots
that lack their usual downward oblique orientation. Moreover, in these cases, the cerebellum appears smaller than the normal dimension and presents
an altered contour (Del Bigio, 2010).

Figure 5.

Prenatal signs of open neural tube defects

Differences between the normal fetus and fetus affected by open neural tube defects are shown. Note the appearance of the transverse section of cerebellum at the ultrasound
scan in normal fetus (typical butterfly shape) and the appearance of the herniated cerebellum in Chiari malformation type II (characteristic banana shape, asterisk). Note also
the appearance of the transverse section of the skull comparing the shape of the normal fetus with the lemon shaped skull of the NTD fetus. Lemon sign represents the
scalloping of the frontal bones (arrows).

Histology. The meningeal cystic sac contains cerebrospinal fluid, nerve roots and spinal cord. Both spinal cord and meninges are damaged and
displaced through the bony opening (Figure 3). Histologically, the medulla is generally hypervascularised and abnormal: in some cases the spinal cord
could be closed with dilated central canal, while in other cases the spinal cord appears open as a flat mass (Golden & Harding, 2004). In Chiari
malformation type II, the cerebellar structure appears modified due to its flattened elongation that extend downward below the level of the foramen
magum: the structure may be atrophied with decreased neurons and chronic astroglial changes (Del Bigio, 2010). Historical studies based on cell
count demonstrated that in these cases, the central lobes of the cerebellum develop normally but subsequently acquire an irregular degeneration and
arrest of growth (J L Emery & Gadsdon, 1975). This cerebellar local atrophy might depend on local ischemia (Poretti, Prayer, & Boltshauser, 2009).

Prenatal diagnosis. The detection rate of this type of open spina bifida by ultrasound is very high (virtually 100%) thanks to the indirect cranial signs
including the “lemon” and the “banana” sign (Figure 5) (Nicolaides, Gabbe, Campbell, & Guidetti, 1986). The lemon sign describes the shape of the
skull and represents the scalloping of the frontal bones: loss of the convex outward shape of the frontal bones with mild flattening. It is present in
virtually all fetuses with myelomeningocele between 16 and 24 weeks of gestation. The banana sign describes the shape of the cerebellum and is due
to the downward traction of the cerebellum, most likely related to the leakage of spinal fluid from the open spinal defect. Foetal repair of
myelomeningocele has been shown to reverse the cerebellar and brainstem displacement (Adzick et al., 2011).

Direct findings of open spina bifida may be also detected by ultrasound or fetal MRI: sections of the spine demonstrates vertebral dysraphism with an
associated fluid-filled posterior bulging of meninges forming the cystic sac often containing internal septations. Sometimes spinal dysraphism without
posterior cyst may be observed; this is the case of myelocele. During pregnancy a progressive deterioration of leg movements may be observed due to
the damage of the spinal cord and nerves. Clubfoot, which is a deformity characterized by abnormal foot position in which the foot is internally
rotated, is virtually always detectable.

Prognosis. In the absence of other additional serious congenital malformations, newborns with myelomeningocele or myelocele survive with various
degree of neurological impairment. Although controversial, cesarean section may be indicated to prevent additional neurological defects related to the
mode of delivery (Thompson, 2009). Clinical characteristics depend on the level of the lesion: motor and sensorial deficit occur at levels below the
spinal lesion and may be associated with bladder and rectal incontinence (Golden & Harding, 2004) and sexual dysfunction (Thompson, 2009).
Intellectual disability is relatively infrequent (20–25% of cases) and generally related to hydrocephalus (Copp et al., 2015). According to the results of
the Management of Myelomeningocele Study trial (MOMS), to improve the prognosis of the affected foetuses, pre-delivery surgical solutions may be
offered in selected cases, performing an in utero surgical intervention. In fact, the results of the MOMS study show that surgery performed prior to 26
weeks of gestation may preserve neurologic function, reverse the hindbrain herniation of Chiari II malformation, and decrease the need to place
postnatally a ventriculo-peritoneal shunt (Adzick et al., 2011). However, the prenatal surgery increases the risk of premature rupture of the membranes
and preterm delivery (Adzick et al., 2011).

Closed neural tube defects

Encephalocele

Definition. Encephalocele is a herniation such as a sac-like protrusion of the brain and/or the meninges through an opening in the skull. According to
the type of tissue involved in the herniation, cephaloceles are classified as meningocele (herniation of meninges), encephalomeningocele (herniation
of meninges and brain), and encephalomeningocystocele (herniation of meninges, brain and ventricle) (Figure 6) (Pilu, Buyukkurt, Youssef, & Tonni,
2014).

Figure 6.

Type and site of cephalocele

Upper panel shows a schematic representation of possible cephalocele localization.

Middle panel shows a schematic representation of different type of cephalocele classified according to the content of the herniated mass.

Lower panel shows macroscopic aspect of human fetuses affect by cephalocele.

Epidemiology. CDC estimates that each year about 1 out of every 10,000 babies are born with an encephalocele in the United States.

Gross Dysmorphology. Encephaloceles can occur in any part of the cranial vault; cystic mass passes through a cleft of the calvarium squamae and
approximately 90% of cases involve the midline. According to the site of the herniation, encephaloceles are classified as: anterior, with lesion located
between the bregma and the anterior aspect of the ethmoid bone; parietal, with lesion located between the bregma and the lambdoid suture; occipital,
with lesion located between the lambdoid suture and foramen magnum (Figure 6). This location is the most common, involving almost 75% of all
encephaloceles.

The anterior encephalocele is further sub-classified into frontal, sincipital and basal varieties, according to the localisation of the defect. The frontal
encephaloceles are external lesions that cause craniofacial abnormalities and are typically found near the glabella, the root of the nose. They are
subdivided into naso-frontal, naso-ethmoid and naso-orbital types. Basal encephaloceles are internal lesions which occur within the nose, the pharynx,
or the orbit. They are normally classified into three types: spheno-orbital, spheno-maxillary and spheno-pharyngeal (Pilu et al., 2014).

The occipital encephalocele may occur in association with a Chiari III malformation. The Chiari type III malformation is characterised by occipital
and/or high cervical encephalocele associated with caudal displacement of the lower brain stem into the spinal canal, and herniation of the cerebellar
tissues into the herniated sac.

The macroscopic aspect of the encephalocele is a round, soft, compressible nodule with sub-scalp localisation (Figure 6) (Gao, Massimi, Rogerio,
Raybaud, & Di Rocco, 2014). The mass may appear solid or cystic according to the content of the herniation, and it is generally covered by alopecic
skin that could be surrounded by a ring of hair determining the so called “hair collar signs” (Gao et al., 2014), although sometimes hypertrichosis may
be present over the lesion (Sewell, Chiu, & Drolet, 2015). Sometimes the surface of encephalocele appears bluish, translucent or glistening, and
capillary malformations are detectable (Sewell et al., 2015)

Due to its patent intracranial communication, encephaloceles modify its dimension (enlargement/reduction) with the modification of intracranial
pressure: it enlarges during increase of pressure such as during Valsalva maneuver (Sewell et al., 2015) including crying and suction.
Histology. Solid variants results from the proliferation of neuroglial and fibrous tissues and hyperplasia of the meningeal cells. The herniated mass
may contain various types of tissues including cerebral and cerebellar portion, ventricles with choroid plexus, gray matter heterotopias. Brain tissues
involved in the herniation are generally nonfunctional and appears abnormal with gliosis, necrosis, reactive astrocytosis. Meningeal inflammation has
been detectable (Castillo, Quencer, & Dominguez, 1992; Isik, Elmaci, Silav, Celik, & Kalelioglu, 2009; Ivashchuk, Loukas, Blount, Tubbs, & Oakes,
2015). Aberrant deep veins and ectopic venous sinuses are also often observed (Ivashchuk et al., 2015). Cystic variants contain cerebro-spinal fluid
(Gao et al., 2014).

Prenatal diagnosis. A sac protruding through a bony defect may be detected by ultrasound. Associated brain abnormalities may include cerebral
ventriculomegaly and microcephaly (Cameron & Moran, 2009). In case of a Chiari III malformation, a protrusion through a defect of the occipital
squama and or the arch of the first cervical vertebra may be observed, associated with a small posterior cranial fossa with low tentorial attachment,
scalloping of the clivus and herniation of the cerebellum into the defect and hydrocephalus (Caldarelli, Rea, Cincu, & Di Rocco, 2002; Ivashchuk et
al., 2015). In the absence of identifying a skull defect, the differential diagnosis includes a wide range of craniofacial mass, according to the site of the
lesion, such as a teratoma, a lynphangiona, a haemangioma, nasolacrimal duct cyst, neuroglia heterotopia, lipoma (Cameron & Moran, 2009; Connor,
2010).

Prognosis. Clinical characteristic depends on the localisation and content of the herniated mass. The more rostral the site, the better the prognosis
(Thompson, 2009). Moreover, the underlying brain involvement and hydrocephalus affects the prognosis. In cases with mechanical effects of distortion
and traction of the brain stem thought the herniation, various degree of developmental delay, epilepsy and motor and sensorial nerve dysfunction may
occur (Caldarelli et al., 2002; Gao et al., 2014). The treatment of encephaloceles requires a surgical correction.

Meningocele.

Definition. Although macroscopically similar to a myeolomeningocele (Figure 3), meningocele is a closed spina bifida, comparable to encephalocele,
whereby the defect consists in herniation of meninges through the vertebral column. Although the herniation of the meninges through the vertebral
arch defect, the spinal cord resides within the spinal canal (McComb, 2015). Differences between meningocele, myeolomeningocele and myelocele are
summarized in Table 2.

Table 2:

Differential diagnosis between meningocele, myelomeningocele and myelocele

Meningocele Myelomeningocele Myelocele

Type of defect Closed Open Open

Ultrasound aspects
Posterior anechogenic cystic mass (sac-like protrusion) from the spine + + −
Presence of septa in the sac − + //

Abnormality of vertebral bones (absence of the arches) + + +

Abnormal shape of skull (lemon sign) − + +
Abnormal shape of cerebellum (banana sign) − + +

Association with Chiari type II malformation − + +

Association with hydrocephalus − + +

Association with clubfoot − + +

Macroscopic aspects of the lesion

Absence of vertebral arches + + +
Meningeal herniation though the bones defect + + −
Presence of neural tissues in the meningeal sac (medulla and/or nerves) − + //
External exposition of placode − + +

Covered by skin + − −

Although macroscopically similar, meningocele and myelomeningocele represent two opposite types of spina bifida, a closed and open defect respectively with different
prognosis. Because of the similar macroscopic aspect of the herniated sac, prenatal ultrasound differentiation of the cystic lesion may be difficult.
In contrast, myelomeningocele and myelocele are macroscopically different but they represent the same type of spina bifida, an open defect, with the same clinical
implications. The macroscopic distinction between myelomeningocele and myelocele is based on the location of the neural placode. When the placode is pushed out of the
confines of the canal and through the vertebra, and a cutaneous defect is created by expansion of the subarachnoid space, a myelomeningocele is present. Otherwise, in case of
myelocele, the subarachnoid space is not expanded, the placode remains in plane with or deep to the cutaneous surface

Epidemiology. The actual incidence of meningoceles is unknown.

Gross Dysmorphology. In meningocele, the dura and the arachnoid herniate through the vertebral arch defect whereas the spinal cord remains in the
normal position into the spinal canal. The herniated mass is covered by skin that is characterised by atrophic epidermis without skin appendages
(Golden & Harding, 2004). Sometimes the skin may display different degree of dysplasia (McComb, 2015). The lesion is peduncolated to varying
degrees and is generally easily compressible and well transilluminable.
Histology. A protrusion in continuity with epidermal tissue is detectable. The protruded mass is formed by extremely thick meninges and generally
contains vascularized stromal tissue. The surface of the herniated mass generally does not presented skin appendages.

Prenatal diagnosis. By ultrasound may be seen spinal dysraphism with an associated cystic mass. The characteristics of the cystic mass are
comparable with that of the myelomeningocele, although they lack septa in the herniated mass, and the cranial anatomy is unremarkable (Ghi et al.,
2006).

Prognosis. Cases with meningocele generally have normal neurologic examination without deformity of the lower extremities or sphincter
dysfunction (McComb, 2015). The treatment of meningocele is a surgical correction.

Spina bifida occulta.

Definition. Spina bifida occulta represents a spectrum of a spinal cord abnormalities related to abnormal development of the embryonic tail bud. The
defect involves the low lumbar and sacral regions, and results in closed defects with incomplete vertebral arches. Spina bifida occulta is often
associated with other skeletal defects including sacral agenesis.

Epidemiology. The actual incidence of spina bifida occulta is unknown (Hertzler, DePowell, Stevenson, & Mangano, 2010).

Gross Dysmorphology. The presence of cutaneous stigmata of the low back may be the only signs of occult spina bifida. Cutaneous markers include
nevi, lumps, depigmented region, subcutaneous lipomas, capillary hemangiomas, hair tufts (localized hypetricosis) and dermal sinus tract (Hertzler et
al., 2010; Sewell et al., 2015). Structural abnormalities may also been detected such as an asymmetrical gluteal cleft, scoliosis and leg length
discrepancy.

Prenatal diagnosis. No secondary cranial findings are detectable thus the prenatal diagnosis is hard and in such cases is a challenge (Coleman, Langer,
& Horii, 2014). The commonest form of closed spinal dysraphism detectable in utero is the type associated with intradural lipoma. Its aspect may
affect the sensitivity of differential diagnosis and the ability of the correct identification during the prenatal diagnosis: during intra-uterine life,
meningoceles and lipomas have a very similar appearance and may be very difficult or impossible to distinguish (Pierre-Kahn & Sonigo, 2003). In
fact, by ultrasound, lipomas typically appear as echogenic masses (Ghi et al., 2006) much like a meningocele. Post-natally, high resolution ultrasound
may be employed until the 6th month of life, before the ossification of the vertebral body, but the sensitivity is low, especially in cases of a
subcutaneous mass (Sewell et al., 2015). The more appropriate diagnostic technique is magnetic resonance, but this procedure is limited by its cost,
availability, and the need for sedation for many children.

Prognosis. This group of defects represents a less severe form of malformation: more often, this problem may be detected only later in life, because
nerves and the spinal cord are not affected and therefore it does not usually result in disabilities. In other cases spinal cord anomalies may occur and
include hydromyelia (overdistension of the central canal), diplomyelia (longitudinal duplication of the spinal cord), diastematomyelia (longitudinal
split of the spinal cord) and tethering of the lower end of the cord (Golden & Harding, 2004). Neurological symptoms start when the damage and/or
traction of the cord occurs. During the intrauterine life, the spinal cord grows slower than the spinal column. The caudal spinal cord (also called conus
medullaris) reaches its normal level only after birth, and the symptoms may start through the extrauterine life when the cord is insulted (Sewell et al.,
2015). Sometimes this occurs in childhood, other times this occurs later in life, in case of thickening of filum terminale (increased fibrosis leading to
progressive loss of elasticity), increased physical activity, development of spinal stenosis or occurrence of lumbar trauma (Hertzler et al., 2010).
Neurosurgical intervention is the therapy of choice in cases of symptomatic patients with neurological deterioration.

Prevention of Neural Tube Defects

NTDs are known as one of the few birth defects in which primary preventive strategies are available and effective. Pioneering studies by Smithells and
colleagues in the UK (Smithells et al., 1980; Smithells, Sheppard, & Schorah, 1976) showed the importance of vitamin intake during pregnancy with
respect to NTD outcome. Subsequently, a randomised double-blind, placebo-controlled trial was initiated in 1991 by the UK Medical Council (MRC
Vitamin Study Research Group, 1991). This study demonstrated that supplementation with 4 mg folic acid per day resulted in a threefold reduction in
NTD recurrence risk. Together with several non-randomised trials, this data indicate that folic acid supplementation during pregnancy in a dose range
of ~0.4–5 mg per day prevents NTD births. The biological and molecular mechanisms of this prevention are yet to be elucidated, despite the large
number of studies conducted using animal studies to test various hypotheses mechanistically (Blom et al., 2006), as folate plays a pivotal role in
numerous cellular reactions, including purines and thymidylate production and SAM synthesis (S-adenosyl-methionine), which is the cellular methyl
donor used in methylation reactions for DNA, proteins including histones and lipids (Crider, Yang, Berry, & Bailey, 2012).

Maternal folic acid periconceptional supplementation strategies have been implemented in a number of countries (Blom et al., 2006) however
epidemiological studies have shown that not all NTDs are prevented and a sub-group of “folic acid-resistant” NTDs still occur (Mosley et al., 2009;
MRC Vitamin Study Research Group, 1991). Among other possible supplements fundamental for correct neural tube closure, inositol may represent a
tool for those non folate responsive NTDs. Indeed, in non-randomszed studies supported by experimental evidence on animal models (Copp et al.,
2013; N. Greene & Copp, 1997), inositol supplementation appears to reduce NTDs recurrence. In a recent randomised, placebo-controlled trial no
recurrence was observed in women supplemented both with folic acid and inositol (N. D. E. Greene et al., 2016). Larger and statistically powerful
studies are expected to investigate this possible new preventive strategy.

ACKNOWLEDGMENTS

The authors appreciate the financial support of the various funding agencies that contributed to this effort. RHF was supported by NIH grants
HD081216, HD067244, HD083809, as well as support from the March of Dimes (16-FY16–169). VM was supported by Università degli studi di
Milano grant (Linea 2–2017).
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Trends Neurosci. Author manuscript; available in PMC 2021 Jul 1. PMCID: PMC7321880
Published in final edited form as: NIHMSID: NIHMS1588125
Trends Neurosci. 2020 Jul; 43(7): 519–532. PMID: 32423763
Published online 2020 May 15. doi: 10.1016/j.tins.2020.04.009

Closing In on Mechanisms of Open Neural Tube Defects

Sangmoon Lee1,2 and Joseph G. Gleeson1,2

Abstract

Neural tube defects (NTDs) represent a failure of the neural plate to complete the developmental transition to a neural tube. NTDs are the most
common birth anomaly of central nervous system. Following mandatory folic acid fortification of dietary grains, a dramatic reduction in the incidence
of NTDs was observed in areas where the policy was implemented, yet the genetic drivers of NTDs in humans, and the mechanisms by which folic
acid prevents disease remain disputed. Here we discuss current understanding of human NTD genetics, recent advances regarding potential
mechanisms by which folic acid might modify risk through effects on the epigenome and transcriptome, and new approaches to study refined
phenotypes for a greater appreciation of the developmental and genetic causes of NTDs.

Keywords: Anencephaly, folate, folic acid, myelomeningocele, neural tube defect, spina bifida

Neural tube defect is a genetically complex disease

Neural tube defects (NTDs; see Glossary) are the most common birth anomaly of the central nervous system (CNS), encompassing a range of defects
of varying severity. NTDs can occur anywhere along the neural axis and present with a range of clinical severity, and their subtypes are named based
upon the anatomic site and severity of the defect. The most severe forms of NTD are anencephaly or craniorachischisis, in which either the forebrain
or the entire CNS, respectively, fails to transition from a neural plate to a neural tube. The least severe forms show near complete closure of the neural
tube, in conditions such as spinal lipoma or spina bifida occulta, occurring in 4–6% of the general population, identified incidentally during spine
imaging in many cases [1]. NTDs account for approximately 88,000 deaths/year worldwide (disease burden and mortality estimates of World Health
Organization: https://www.who.int/healthinfo/global_burden_disease/estimates/en/index1.html) [2]. In the US, NTDs are one of the conditions tracked
by the Centers for Disease Control and Prevention (CDC) longitudinally across the country, to define disease incidence, and identify populations at
risk. Most patients with symptomatic disease remain wheelchair bound throughout life, with urinary or fecal incontinence and learning difficulties [3].
Moreover, it is likely many more fetuses with NTD are spontaneously or electively terminated. Despite its frequency, the genetic basis of NTDs in
humans remains mostly unknown, and therefore molecular characterization and effective quantification of risk remains unachievable.

Up to 70% of the NTD risk is predicted to be attributable to genetic factors, although these estimates were made over 40 years ago, prior to dietary
folic acid (FA) supplementation, so these bear repeating [4]. There are multiple lines of evidence supporting NTD as a genetic disease, but the lack of
mendelian inheritance in most cases suggests it is a genetically complex disease (Fig. 1). Most evidence is from twin studies, where higher
concordance was observed in same-sex twins (which includes monozygotic and dizygotic twins) than in opposite-sex twins (1.93 vs 1.00 per 1,000)
[5, 6], which was further validated in a more recent study [7]. However, the concordance rate varies with NTD subtypes, implying that there might be
multiple genetic mechanisms or environmental factors influencing the genetic architecture. In familial cases, NTDs phenotypes tend to ‘breed true’
within families; in other words, if the proband has spina bifida then the recurrence tends to have spina bifida rather than a different NTD [8]. This
observation suggests that not all NTDs are equivalent and that different genetic factors may determine the location and severity of the NTD.
 Figure 1.

Neural tube defects (NTDs) are likely to be impacted by many types of genetic variants.

These variants include coding and noncoding ones, structural ones, and chromosomal aneuploidy. Sources of variants include de novo, inherited, modifiers, somatic and
combinations of these. Genetic variants are influenced by epigenetic factors and the environment to establish the NTD risk. Newborn image adopted from the CDC Public
Library of Images.

Can an understanding of genetic architecture of NTDs be achieved using similar approaches to those used for conditions like congenital heart disease
and autism spectrum disorder (ASD)? These conditions share features with NTDs in that: 1) they show complex inheritance; 2) most cases are
sporadic; 3) in the severe case of the disease, most affecteds are unlikely to bear children, and thus mutations are likely under strong purifying
selection [9, 10]. What was most insightful for these conditions was the realization that a large collection of phenotypically diverse patients could
capture both rare and common variants contributing to risk and that simplex cases could take the place of large pedigrees that were used for traditional
linkage associations. Current trends are to apply similar approaches to what was successful for congenital heart disease and ASD, by recruiting large
and phenotypically-diverse cohorts of NTD simplex cases, stratified based upon FA exposure. The goal of this review is to discuss the current
understanding of human NTD genetic architecture, NTD risk factors, and potential NTD candidate genes. We also address potential ways in which FA
works to reduce risk of NTDs through transcriptional and epigenetic mechanisms, which has potential implications on the genetic architecture of
disease.

Mouse models of NTDs

To understand mechanism of NTDs, mouse has proven to be a tractable model, but of relatively limited impact on our understanding of human NTDs,
mostly attributable to the polygenic nature of human NTDs. In mouse models, most results rely on recessive or dominant models, which are not
common in humans. Over 300 NTD mouse models are reported (see Table S1 in the supplemental information online) [11], some of which show
reduced penetrance or severity with FA supplementation (e.g. Pax3, Cart1), whereas others are FA resistant (e.g. Ct, Axd) [12]. One of the best known
models is the FA-responsive crooked tail (Cd), a fully penetrant recessive NTD model linked to a damaging variant in LRP6 encoding Low-density
lipoprotein receptor-related protein 6, a component of the Wnt-Fzd-LRP5-LRP6 complex that triggers beta-catenin signaling [13]. Several NTD
patients with damaging variants in LRP6 were described [14], suggesting an evolutionarily conserved role, but few such examples exist. Multiple
reasons could possibly account for the fundamental differences between humans and mice NTDs. Some possibilities are that (1) NTDs have different
developmental mechanism in rodents; (2) body size differences impart differential risk; and (3) the human orthologs of mouse NTD genes predispose
to risk in humans in ways that are not yet fully understood.

Genetic studies of human NTDs

Early linkage analysis within multiplex NTD families suggested candidate loci on chromosomes 2, 7, 10, and X [15–17]. However, logarithm of odds
(LOD) scores were below genome-wide significance, and fine mapping was not successful. Moreover, there are no published large-scale genome-wide
association studies (GWASs) of NTDs to date. Instead, most published NTD genetics studies have focused on candidate genes derived from the folate
metabolic pathway (e.g. MTHFR), or mouse NTD models (see Table S2 in the supplemental information online). With the advent of next-generation
sequencing (NGS), and the availability of approaches like whole exome (WES) and whole genome sequencing (WGS), the numbers of genes tested as
candidates has increased. A number of consortia have started to emerge to aggregate cohorts and sequencing data, such as the Spina Bifida Sequencing
Consortium (https://sbseqconsortium.org) which we took part in initiating, given the need for multi-site and multi-ethnic aggregation for
comprehensively studying disease architecture.

De novo mutations
De novo mutations (DNMs) can increase risk of both rare and common disease, and are the most likely to be under purifying selection [9, 10]. Given
the success of DNM discovery in ASD and congenital heart disease [9, 10], investigating DNMs in NTDs would seem like an obvious strategy, further
evidenced by positive results from small scale studies [18]. In a cohort of 43 NTD trios (parents and affected child) of mixed clinical presentation,
about 10% carried loss-of- function (LoF) DNMs [18], some in genes associated with mouse NTD models. For instance, SHROOM3, mutated in the
mouse NTD model Shrm [19], showed LoF DNMs in two offspring, one with myelomeningocele and one with anencephaly. SHROOM3 is a strong
NTD candidate, but since even healthy offspring show LoF DNMs at a basal rate, genes identified with this approach require validation, and a
statistical framework. Such a framework would not be difficult to develop. Assuming 100 different genes that can lead to NTDs with a LoF DNM, and
with a 50% excess burden of such haploinsufficient LoF DNM in cases vs. controls, and a false discovery rate of <10%, we calculate about 1000 trios
would need to be sequenced in order to identify 10 recurrently mutated genes (Fig. 2).

Figure 2.

Estimation of cohort size for causative variants discovery.

Power calculation curve indicating estimated number of trios necessary to discover a given numbers of genes as recurrently mutated, assuming that there are 100
‘discoverable’ genes for haploinsufficient neural tube defects (NTDs) in humans. The variable ‘v’ is defined as the ratio of the number of de novo loss-of-function (LoF)
mutations in affected individuals divided by the number of de novo LoF mutations in unaffected individuals across the population. For these calculations, we estimate v as 1.5
or 2.5, meaning that subjects with NTD will have 1.5 or 2.5 as many de novo LoF mutations as controls. Assuming v=2.5, then a cohort of 1000 trios would discover 40
recurrently mutated genes with a false discovery rate of 10% (blue line). If on the other hand v = 1.5 then 1000 trios would only discover 10 recurrently mutated genes. These
calculations are based upon the basal de novo mutation rate, and assumes a total of 20,000 human genes that can be mutated. Abbreviation: FDR, false discovery rate.

Beyond the success with coding DNM, some NTD risk alleles are likely to reside in the noncoding genome. The noncoding genome contains (among
other elements) regulatory elements that determine gene expression specificity, and thus extending discovery of rare genetic variants of high effect to
the noncoding genome is essential to gain high-resolution insight into the spatiotemporal and cell type specific dynamics of the biology of NTD. WGS
analysis holds the potential to detect the most noncoding genome variation, but a major challenge is a lack of a priori evidence for association testing,
relying instead on an ever-increasing number of annotations describing noncoding genome functionality [20]. Such high noncoding genome
dimensionality will require consideration of multiple comparisons problem, requiring again a statistical framework to test associations. Recently,
category-wide association study (CWAS) has been developed to test multiple hypotheses for noncoding variants from WGS data [21]. From the WGS
of 1,902 ASD families, CWAS has successfully identified noncoding DNMs that contribute to ASD risk, with the strongest contributors in conserved
promoter regions [22]. These combined approaches have the potential to paint a complete picture of risk associated with noncoding variation, but will
require enormous cohort sizes.

Rare inherited coding variants

The plethora of individual genetic factors contributing to NTDs in mice, and the genes involved in folate metabolism, have provided ample fodder for
candidate gene testing in NTD singletons. Efforts to detect rare coding variants using genome-wide approaches have been pursued with WES mainly
on singleton patients [23, 24]. These early efforts generally relied on publicly available cohorts like the Genome Aggregation Database (gnomAD) to
establish baseline minor allele frequencies [25], and as such may suffer errors from population stratification. One effort applied WGS analysis from
ethnically diverse NTD cases, revealing enrichment of rare LoF variants in cases [23], and suggesting that NTDs may be caused by the sum of effects
of multiple genes, similar to the oligogenic model. A study of a cohort of 51 NTD families showed cases had a greater burden of damaging variants in
MYO1E, encoding an unconventional nonmuscle class I myosin [24]. These approaches, while promising, will require confirmation and genotype-
phenotype correlations in larger cohorts.

Functional validation of human NTD genetic studies

Functional validation is a critical step that should follow the discovery of candidate genes or variants. A hurdle is overcoming the potential complexity
of human NTDs, including zygosity, modifiers, and environmental factors. The lack of simple genetically faithful and highly penetrant model of
human NTD is a potential hurdle, yet still important information can be gained by studying the impact of FA on existing or new animal models.
Higher throughput than achievable in mouse can be realized in frog or zebrafish, but a caveat is that neural tube closure mechanisms differ across
species [26]. Although in vivo vertebrate animal models are the most robust validation approach currently, other tools such as stem cell models, neural
tube organoids, or 3-dimensional bioprinting have advantages, for instance in incorporating human-specific variants, and may prove fruitful in
modeling human NTD [27].

Genes and pathways implicated in NTD

Folate metabolism pathway

Since folate was implicated in NTD risk, folate metabolic pathway genes have been studied as candidates in NTD. MTHFR encodes
methylenetetrahydrofolate reductase, required for the conversion of folate into its active form, L-methylfolate. The MTHFR c.677C>T SNP
(rs1801133) p.V222A reduces enzyme activity in cells by 35% when heterozygous and 70% when homozygous [28], and is the most reproduced single
marker of NTD risk, conferring an odds ratio between 1.34–1.44 when present in the mother or the child, but not the father [29]. However, there are
still some discrepancies in its effect on NTD risk, as the effect does not replicate in some populations [29, 30]. Similarly, the MTHFR promoter SNP
(rs3737965) regulates MTHFR expression and contributes to spina bifida risk [31, 32], hinting at the kinds of non-coding variants likely to function in
NTDs. There are other MTHFR SNPs studied but since they may be in linkage disequilibrium with other SNPs, it is difficult to separate their
individual effects. SNPs in other folate-related genes such as MTHFD1, MTRR, MTR, TYMS, SLC19A1 or LRP2 have been associated with only a
moderate effect on NTD risk [24, 33–36], and similarly the mitochondrial folate metabolism glycine cleavage system genes AMT, GCSH, and GLDC
are attracting attention as NTD risk factors [37, 38].

Planar cell polarity (PCP) and canonical Wnt pathways

PCP has been appreciated as a critical pathway for neural tube closure, ever since rare coding variants in VANGL1 were found enriched in human
NTDs [39], and a missense variant in Vangl2 was identified as the cause for the partially-penetrant Loop-tail (Lp) NTD mouse [40, 41]. The PCP
pathway determines cellular orientation necessary for shape transitions from a neural plate to a tube. Moreover, PCP is a key driver of ‘convergent
extension’, the process in which cellular movements converge (narrow) along one axis and extend (elongate) along the perpendicular axis. A PCP
defect should thus result in an abnormally wide neural plate, hampering proper neural tube closure [41]. Since convergent extension of the embryo
precedes neural tube closure, the idea is that early defects could lead to mis-patterning of the embryo that could increase NTD risk indirectly [41], and
that defects in early developmental processes preceding neural tube closure could potentially impact NTD risk. PCP pathway gene mutations have
been identified in 1–20% of NTD cases, depending upon the study, including genes SCRIB, DACT1, CELSR1, FUZ, FZD6, PRICKLE1, VANGL1 and
VANGL2 [42], but the relative importance of these genes remains unclear. The canonical Wnt pathway has been also implicated in NTD risk. For
example, LRP6, a Wnt co-receptor mutated in a mouse NTD model, was assessed for polymorphisms in 190 individuals with NTD, identifying four
individuals with predicted deleterious variants, some of which impacted Wnt signaling (a key pathway regulating PCP) [14].

Sonic Hedgehog (Shh) pathway

While Shh loss-of-function mouse mutants often show gross patterning defects, it is hyperactivation of Shh signaling that leads to NTDs [43], and
thus disruption of negative regulators of Shh pathway often leads to NTDs [44]. For instance, mutations in the Shh negative regulator GPR161 were
enriched in 384 human spina bifida patients [45]. The Shh pathway repressor GLI3 was also linked to NTD risk in a WES study in a French cohort of
23 individuals [35]. Other Shh pathway genes, such as DISP1 and PTCH1, have also been implicated in NTD pathogenesis [46, 47]. PRKACB is
important for the formation of the GLI3 repressor and has been implicated in human NTD risk [48]. NTD phenotypes resulting from Shh pathway
mutations may share important components with ciliary pathway genes and PCP pathway mutants, since these pathways are known to interact [49].

Cytoskeleton and cilia genes

Genes controlling the cytoskeleton are strong candidates for NTD risk. However, it remains to be clarified in which cell populations these genes
function, since there are many cell populations interacting during neural tube closure. These cell populations include neural cells, paraxial mesoderm,
overlying fibroblasts, bone progenitors, etc. To date, only a handful of cytoskeletal genes are associated with human NTDs. The aforementioned
SHROOM3 is a PDZ-domain containing actin regulator (sometimes included in the PCP pathway) and was implicated as an NTD risk gene in two
separate studies with de novo and gene burden analyses [18, 24]. ITGB1, a signal transduction protein functioning between the cytoskeleton and
extracellular matrix, and DLC1, a GTPase-activating protein important for cytoskeletal remodeling and cell migration/proliferation [18, 24], were also
implicated. A number of ciliary genes lead to NTD when mutated, such as Tulp3 [50], because the graded response to Shh depends upon cilia
architecture [51]. Sequencing of 281 candidate NTD genes in 373 NTD patients and 222 healthy controls revealed eight rare mutations in the cilia-
related gene DNAAF1, which encodes a protein for preassembly of the dynein-arm complex within the cilium [52].

Inositol metabolism genes

NTDs were identified in mice lacking Itpk1, a regulator of inositol hexakisphosphate [53], and subsequently polymorphisms in ITPK1 were identified
in human NTD patients [54]. Inositol is required for neural tube closure, and inositol supplementation can dramatically reduce curly tail NTD
penetrance from ~70% to ~3–7% [55]. This led to the model that deficiency in inositol may impart risk of NTD, particularly in mothers found to be FA
resistant. Currently there is a clinical trial of inositol as an add-on to FA supplementation in NTD prevention [56].

Chromosomal abnormality and structural variation

Given the complex genetic architecture of NTDs, it is not surprising that chromosomal abnormalities and structural variations (SVs) contribute to
NTD risk. Large chromosomal abnormalities such as trisomy 13 or 18, among others, have been reported as risk factors for various NTDs [57, 58],
accompanied by multiple congenital anomalies.

Structural variations

SVs such as chromosomal microdeletions or microduplications have not been studied much because most of the genetic assays used were unable to
detect SVs, and therefore, effects of SV on NTD risk is not well established. For instance, NTD cases with a 2q35–36.2 deletion containing PAX3 have
been reported in patients showing NTD with or without Waardenburg syndrome, which is an autosomal dominant PAX3 haploinsufficiency
characterized by pigment abnormalities and sensorineural deafness [59–61]. Deletions in a few genes associated with proteoglycans or cilia also have
been implicated in the NTD risk [62, 63].

DiGeorge/Velocardiofacial 22q11.2 deletion syndrome (22q11.2DS) is relatively common (1 per 3,000–6,000 live births), since the locus harbors four
low copy number repeat sequence blocks (LCRs) which predispose to unequal crossing-over, leading to de novo deletions (or duplications) [64].
Although a few NTD cases with 22q11.2DS have been reported, it is still unclear which, if any, gene(s) among the ~90 genes in the interval confer
NTD risk [64–66]. Likewise, SOX3 duplication (Xq27.1) has been reported in three female myelomeningocele patients [67], and duplication of
15q24.2-q26.2 containing ~100 genes was also reported in a single case of anencephaly and NTD [68]. Presumably, these SVs may contain either
gene(s) of which decreased dosage increases risk NTDs, or noncoding regulatory elements including the ones impacting long-range chromatin effects,
although it is not possible to distinguish them without refinement of the minimal interval or animal modeling.

Monogenic syndromes potentially increasing risk of NTDs

Several human syndromes where mutant gene function is well established display an increased risk of NTDs, which can provide insight into the
pathogenesis of NTDs. The risk of NTD appears to be increased in individuals with Waardenburg syndrome, caused by haploinsufficiency of PAX3.
PAX3 is a member of the paired box transcription factor family expressed by multipotent neural crest and somitic mesoderm precursors, implicating
these cell types in NTD. In the literature there are reports of six of 617 Waardenburg syndrome patients with NTDs [61], i.e. risk of roughly 1:100,
which is many times higher than the baseline population risk. This is supported by the splotch mouse Pax3 recessive mutation, that results in NTDs in
8–56% of offspring, depending upon the background and folate status [69]. TBXT aka brachyury, encoding the T-box transcription factor T, was
identified in a human monogenic NTD with sacral agenesis, abnormal ossification of the vertebral bodies and persistent notochordal canal, as well as
in isolated chordoma [46, 70]. Focal dermal hypoplasia, or Goltz syndrome, is an X-linked dominant disease caused by mutation of PORCN [71],
encoding a homologue of porcupine, an O-acyltransferase involved in Wnt protein processing. Although only a few cases are reported, this condition
has been associated with NTDs such as spina bifida occulta or myelomeningocele and Chiari 2 malformation [72]. A case study reported recessive
variants of APAF1 and CASP9, which are key apoptotic genes, as potential causative genes for FA resistant NTD such as spina bifida,
holoanencephaly, or spinal rachischisis in patients with multiple anomalies [73].

Gene-environment and gene-gene interactions

The effect of folate on NTD risk offers a striking example of a gene-environment interaction. Evidence for gene-environment interaction by FA
include: 1) FA alters the sex ratio in human NTDs (a male/female ratio: from 0.48–0.77 to about 1), 2) FA may reduce NTD severity in human [74,
75], and 3) FA reduces the incidence and severity of mouse NTD models, such as splotch (Pax3 mutant) or Lrp2null [76, 77]. However, despite the
impact of folate in reducing NTD incidence, genes and pathways regulated by folate remain mostly unknown [78]. Other environmental factors
influencing NTD risk are maternal hyperglycemia, obesity, elevated body temperature, disadvantaged socio-economic status, and drugs such as
valproic acid, opioids or potentially the antiretroviral dolutegravir [79–85].

There are now over 20 genes implicated in digenic pathogenesis of mouse NTD [86–88]. Gene-gene interaction examples in mouse include digenic
interaction between PCP genes (e.g. Vangl1 and Vangl2) [87]. Digenic inheritance was proposed in human NTD, especially for SNPs near folate
metabolism genes [89, 90], and between PCP genes. As an example, 1% of more than 500 patients were double heterozygous for PCP mutations,
meaning they carried damaging variants in two different genes [91], but establishing the specific combinations of these gene mutations in determining
risk requires studies in additional patients.

While risk for NTDs is generally assumed to depend upon the genetic constitution of the offspring, it is conceivable that some risk might trace back to
the parents’ genetic makeup or risk factors, which could interact with the fetal genetic constitution. One obvious place to look would be the mother’s
genotype for folate metabolism genes. Multiple meta-analyses have shown the association of MTHFR rs1801133 with NTD maternal risk, although
they are limited to candidate SNP genotyping [92]. Perhaps the greatest maternal factors will turn out to be genetic risk of diabetes, which in turn
imparts risk of fetal NTDs. Supporting this model, interaction between maternal hyperglycemia and insulin resistance genes with fetal glucose
homeostasis genes increases risk of NTDs from a cohort of 737 NTD trios [89]. Paternal risk is generally not considered, but an important
contribution to the number of DNMs in an offspring is paternal age at the time of conception. Indeed, paternal age correlates with the risk of NTDs,
with an odds ratio of 1.35 for 40-year old vs. 20-year old father, about the same risk attributed to the MTHFR rs1801133 [93].

Epigenetic regulation in NTD

Given that FA is a methyl donor, researchers have long hypothesized that FA can impact genes expression, through FA-dependent DNA and histone
methylation, which are major components of ‘epigenome’ (Fig. 3) [36, 94, 95]. Since most or all DNA methylation marks are removed post-
fertilization, and then undergo re-marking [96], there is probably a substantial demand for methyl groups, which folate provides. In one example,
genetic risk of birth defects (including NTDs) in mice was attributed to the grandparents genotype, specifically the enzyme methionine synthase
reductase (Mtrr), necessary for utilization of methyl groups from the folate cycle [97]. Moreover, findings from Fbp1 (folate binding protein 1,
partially responsible for folate uptake) knockout mice suggest a requirement in gene transcription for G-proteins, transcription factors, growth factors,
methyltransferases, and cell cycle mRNAs [98], but assessment in the neural tube remains to be explored. Mouse studies also support the potential for
other epigenetic regulation pathways (in addition to folate-related ones) in NTDs. For instance, exencephaly is observed in several murine chromatin
regulator mutations, including DNA methylases, histone methylases, acetylases and deacetylases [94]. Moreover, maternal exposure to valproate,
which is an histone deacetylase inhibitor, is a risk factor for NTDs in humans [83].
 Figure 3.

Schematic of folate as a methyl group donor.

Either dietary folates or synthetic folic acid are incorporated into folate one-carbon metabolism pathway and serve as methyl group donors for various substrates including
protein, DNA, RNA and lipids. Epigenetic regulation by DNA and histone methylation plays crucial roles in transcriptional regulation and embryonic development. DNMT,
DNA methyltransferase; DHFR, dihydrofolate reductase; HMT, histone methyltransferase; K, lysine; mC, methylcytosine; MTHFR, methylenetetrahydrofolate reductase;
MTR, methionine synthase; PRMT: protein arginine methyltransferase; R, arginine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SHMT, serine
hydroxymethyltransferase; THF, tetrahydrofolate.

How does folic acid reduce NTD incidence?

What can we learn from the decades of FA supplementation? Incidence of NTDs has dropped by ~15–70% in every region where FA supplementation
policy has been implemented, with a strong female sex bias. NTDs previously showed a female preponderance, but this bias has disappeared with FA
supplementation [6, 74, 99]. In addition to the sex-specific bias, differences are also evident between NTD subtypes. Specifically, FA supplementation
had a greater impact on reducing anencephaly than other types of NTD [6, 74, 99]. Although further studies are needed, this drop seems to have
coincided with a decrease in severity of NTDs. There are two possible explanations for this observation (although a combination of the two is feasible
as well): 1) FA has prevented NTDs only in those that would have had the most severe phenotypes. 2) FA has reduced the severity of NTD across the
board, essentially rescuing those at the mild end of the spectrum, and shifting those from the severe end to the mild end (Fig. 4). The latter explanation
seems more plausible, and is supported by observations in mice [13, 76, 77].

Figure 4.

A. Model of impact of folic acid (FA) supplementation on neural tube defect (NTD) incidence and severity.

In the absence of FA, a hypothetical gray curve represents the relationship between incidence and severity. FA supplementation may decrease NTD incidence (arrow 1, curve
moves to left) or severity (arrow 2, curves moves down), or may impact both (arrow 3, curve moves both left and down). B. Cumulative NTD risk along y-axis and individual
example risk profiles numbered at bottom. Yellow: genetic risk; green: environment risk attributed to FA deficiency or other factors. Individuals 1 and 2 have risk below the
threshold. Individual 3 has cumulative risk above threshold for mild disease, but if exposed to folate, would fall below threshold for mild disease. Individuals 4 and 5 have
cumulative risk above threshold for severe disease, but if exposed to folate, 4 would fall into mild disease and 5 would fall below mild disease threshold. Individual 6 has
genetic risk above threshold for severe disease that is not modified by folate.

This phenomenon is somewhat reminiscent of ‘female protective effect’ in ASD. The female protective effect was proposed to explain the observation
of a ~5:1 sex bias of ASD toward males, together with a 2-fold enrichment of de novo protein-truncating variants in female ASD individuals [100]. In
other words, females require a greater genetic insult to manifest ASD compared to males. Many hypotheses have been put forward to explain the
female protective effect, but there is still lack of clarity regarding the mechanisms involved.

The ‘female protective effect’ in ASD, where a greater mutation burden seems to be required for disease in females, is reminiscent of the effect of FA
supplementation in the protection from NTDs. Consider the potential outcomes of a hypothetical experiment comparing DNMs in NTD cases
conceived in folate-deficient areas vs. folate-replete areas (or in cases born before vs. after FA implementation). If the effect of FA was to reduce the
burden of DNMs as a means of protecting from NTDs, then we would expect to observe a drop in DNM burden after FA fortification. As a specific
example, since the entire US population was subjected to FA supplementation since 1998, we would expect to observe fewer DNMs in NTD cases
conceived after fortification compared with those conceived before fortification, and since the entire population was subject to FA fortification, we
would expect this drop in DNM to extend to the generation population. While it is possible that FA supplementation coincided with reduced DNM
rate in humans, no such observation has been reported in the literature. In fact, the DNM rate in offspring seems to be increasing in recent years,
probably correlating with advanced parental age [101]. If the effect of FA was independent of DNMs, then we would expect the DNM rate to be
constant before and after fortification. However, if the effect of FA was to increase the threshold of mutations required for NTD, much like females
with ASD, then we would expect to observe an increased burden of DNMs after FA supplementation in cases of the same NTD severity. In other
words, the same clinical severity now requires a greater burden of DNMs, and thus FA preferentially rescued cases that had milder DNM burden (
Fig. 4). To our knowledge, such a test has not yet been performed.

It remains unclear whether FA reduces all NTD subtypes globally or certain subtypes more specifically; it also remains unclear whether FA reduces
NTD incidence across the board or decreases disease severity in terms of the spinal level of the defect and the extent of open neural tissue [74, 75,
102, 103]. Large scale studies so far indicate that the answer to these questions may depends on sex, ethnicity, geographic region, and background
nutritional status [36, 74, 75, 103]. For example, for females in NTD prevalent areas such as high-elevation Argentina and Northern China, FA
supplementation tends to have more impact on anencephaly prevention, while many other populations show comparable rates on both anencephaly and
spina bifida [74, 75]. Here again, genetic factors may play a role of modifying the severity of NTD. Studies with detailed phenotyping, including
defect level and extent would be necessary to answer these questions.
Unaccounted-for variables in persistent cases post-FA supplementation include genetic and/or environmental factors regulating maternal folate levels.
For instance, there may be some pregnant women with very low levels of circulating folate despite their being exposed to supplementation. So far, a
few small-scaled SNP genotyping genetic studies in humans have uncovered variants in folate metabolic genes in mothers contributing to risk [33],
although further studies are needed. It is interesting that among rare essential B vitamins like B1, B2, B9 and B12, only folate (B9) shows no evidence
of genetic regulation, meaning that genotypes do not seem to determine folate levels [104]. What then determines folate levels? Obviously dietary
intake is one driver, and population level studies demonstrate that this significantly increases serum folate levels [105]. But a largely unexplored area is
the role of the gut microbiome in this context. Specifically, it has been noted that the gut microbiome can produce folate, which is subsequently
absorbed by the host [106]. It remains to be tested whether alterations in this process are associated with NTD risk.

Concluding Remarks and Future Perspectives

Understanding the genetic architecture of human NTDs and the role of folate is crucial, not only to achieve a mechanistic understanding of this most
common CNS structural defect, but also for effective clinical screening or further prevention and treatment. However, the heterogeneous nature of
NTDs in terms of phenotypes and genetic architecture, together with the recognized contribution of environmental factors, have hindered progress.
The other consequence of this complexity is a lack of large-scale genetic studies stratified by NTD subtypes, which is partially responsible for low
yield of the expected insight by prior genetic studies applying approaches like GWAS and candidate gene testing. Yet, noticeable advances have been
made, including the discovery of an association between NTDs and folate metabolism, PCP, and environmental risk factors. As sequencing costs
decrease, new NGS studies are likely to reveal proposed and novel candidate genes, enabling the detection of rare alleles including SNVs, indels, short
tandem repeats and SVs using exome- or genome-wide sequencing applied to trios or quads (trio + unaffected sibling), as have been performed for
ASD and congenital heart disease.

Large scale sequencing of cohorts from diverse ethnic groups is needed to disentangle the population-level genetic architecture of NTDs (see
Outstanding Questions). To solve this problem, researchers from all over the world are organizing consortia to aggregate NTD samples, including
preexisting cases that were conceived prior to FA supplementation, or from parts of the world yet to include FA fortification. With the gradual
replacement of “phenotype-driven” syndrome delineation by “genotype-driven” by virtue of high-throughput sequencing and extensive collaborative
efforts, not only the size of a cohort but also its quality (in terms of refined phenotyping and detailed description of environmental factors) will be
crucial for future research [107].

Outstanding Questions

- Has folic acid (FA) supplementation changed the landscape of de novo mutations in terms of their rate and severity?

- Is the genetic contribution to neural tube defect (NTD) risk different before and after FA supplementation? In other words, are the mutations
found in NTD patients severer or milder after FA was introduced into the diet?

- Are there mutated genes or affected pathways in that are specific to affected individuals that were conceived after FA supplementation that
are ‘folate-nonresponsive’?

- Could residual cases of NTD in the US or other parts of the world already supplementing be prevented if the dose of FA was higher?

- Are there other dietary supplements that could added to further reduce NTD rates, such as inositol supplementation?

- Why has the rate of NTDs dropped more significantly following FA supplementation in geographies with higher baseline NTD rates?

- How much do noncoding variants contribute to NTD risk and what is their pathogenic mechanisms?

- Are there subtypes- (e.g. anencephaly or myelomeningocele) specific or level- (e.g. thoracic, lumbar, or sacral) specific genetic factors?

- Are there population-specific genetic risk factors?

- Can the risk of NTDs be predictable by polygenic risk score?

Highlights

- Neural tube defects (NTDs) are the most common birth anomaly of the central nervous system (CNS). They result from a failure of the
neural plate to complete the developmental transition to a neural tube.

- Mandatory folic acid (FA) supplementation of dietary grains led to a significant decrease in the incidence of NTDs, but the mechanisms
involved remain disputed.
 - NTD is a relatively common phenotype in mouse knockouts, showing both simple and complex inheritance, as well as responses to folate,
yet few of the genes identified in mouse models have been established as causes in human NTDs.

- Candidate gene approaches in human studies support a role for Wnt/planar cell polarity, cilia, Sonic Hedgehog (Shh), bone morphogenetic
protein (BMP) signaling factors in NTD risk.

- Whole genome sequencing, as opposed to SNP genotyping or candidate gene resequencing, allows for detection of most coding, noncoding,
and structural variants, including rare, common, de novo, and somatic variants.

- Recently, there have been collaborative efforts to assemble larger cohorts of phenotypically diverse patients from diverse ethnic groups, both
prior to and after FA fortification, for comprehensive risk assessment.

Supplementary Material

Click here to view.(171K, docx)

Acknowledgements

The authors wish to acknowledge the UCSD CTRI UL1TR001442 of CTSA funding, support from the Rady Children’s Institute for Genomic
Medicine, and Gabriella Miller Kids First research program. The content is solely the responsibility of the authors and does not necessarily represent
the official views of the NIH. The authors wish to thank Kathleen Fisch and Sara Brin Rosenthal of the Center for Computational Biology &
Bioinformatics of University of California, San Diego for power calculation (Fig. 2), Aakash Patel for literature review, and Allison Elizabeth Ashley-
Koch, José F. Cordero, Osvaldo Mutchinick, Andrew J. Copp, Lee Niswander, Kit Sing Au, Joon-Yong An, members of the Spina Bifida Sequencing
Consortium, and members of the Gleeson lab for useful feedback.

Glossary
 Anencephaly a condition of absent skull and major portion of brain due to developmental abnormality

Craniorachischisis a condition whereby the neural tube is not closed along both of the brain and spinal cord

Epigenome sum of any modifications on DNA or histones without alterations in the DNA sequence itself

a generic term referring to any kinds of natural food folate (in tetrahydrofolate form). Folate in fortified
Folate foods and most of dietary supplements is in the form of folic acid, which is artificially synthesized as a
pure chemical compound

many countries mandate manufacturers to add FA to various dietary grains based on the previous clinical
Folic acid (FA) fortification trials showing FA’s effect on neural tube defect prevention. Concentration of fortified FA varies from
country to country, but is typically in the range 140–220 μg/100 g of flour

understanding of the genetic factors responsible for heritable phenotypic variability, which also includes
Genetic architecture their interactions with each other (genetic interaction) and with the environment (gene-environment
interaction)

Genome-wide association
an approach to associate genetic variations with particular diseases
study (GWAS)

Haploinsufficient a single copy of a certain gene that does not produce sufficient protein product for normal phenotypes

Linkage association (linkage

an approach to find co-segregated genetic segments within family
analysis)

Loss-of-function (LoF) mutations (usually coding ones) that result in the gene product having no function, including stopgain,
mutation frameshift, or splicing mutations

Mendelian inheritance inheritance patterns of single gene (monogenic) diseases

relatively small chromosomal deletion/duplication that usually cannot be detected by conventional

Microdeletion/microduplication
cytogenetic method under light microscope

statistical problem whereby an increase in simultaneous tests increases the likelihood of an erroneous
Multiple comparisons problem
result

a condition of protrusion of meninges and spinal cord through vertebra and skin defects. Also known as
Myelomeningocele
‘spina bifida’

Neural tube defect a group of developmental disorders that is caused by failed neural tube closure during development

Oligogenic model an inheritance model involving several genes

a systematic difference in allele frequencies of genetic variants among subpopulations due to different
Population stratification
ancestry

Purifying selection an evolutionary tendency to eliminate protein-altering or -damaging gene variants

Regulatory elements genetic regions that control the transcription of nearby genes

Spina bifida see myelomeningocele

Footnotes

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Conflict statement: Neither author has any conflicts to report.

ORIGINAL
ARTICLES
Neural Tube Defects and Associated Anomalies before and after Folic Acid
Fortification
Jane H. Dean, RN, Rini Pauly, MS, and Roger E. Stevenson, MD

Objective To examine the prevalence and types of neural tube defects and the types of anomalies co-occurring
with neural tube defects in 6 years before fortification of cereal grain flour with folic acid (1992-1998) and 20 years
after fortification (1999-2018) in South Carolina, a state with a historically high prevalence of these birth defects.
Study design The prevalence of neural tube defects was determined by active and passive surveillance methods
in South Carolina since 1992. The types of neural tube defects and co-occurring malformations were determined by
prenatal ultrasound and post-delivery examination.
Results In the 6 prefortification years, 363 neural tube defects were identified among 279 163 live births and fetal
deaths (1/769), 305 (84%) of which were isolated defects of the calvaria or spine. In the 20 fortification years, there
were significant reductions in the prevalence and percentage of isolated defects: 938 neural tube defects were
identified among 1 165 134 live births and fetal deaths (1/1242), 696 (74.2%) of which were isolated. The current
prevalence of neural tube defects in South Carolina (0.56/1000 live births and fetal deaths) is comparable with
that nationwide.
Conclusions The continued occurrence of neural tube defects, the majority of which are isolated, after folic acid
fortification of cereal grain flours suggests that additional prevention measures are necessary to reduce further the
prevalence of these serious defects of the brain and spine. (J Pediatr 2020;226:186-94).

B
irth defects occur as isolated anomalies affecting a single anatomical structure and as more complex disorders in the
company of anomalies in other portions of the anatomy.1,2 Isolated birth defects far outnumber nonisolated birth de-
fects affecting multiple anatomical systems. Among birth defects with other anomalies, there are those that have spe-
cific genetic or environmental causes, a number of clinically recognizable phenotypes for which specific causation is not known,
and those that do not comprise clinically recognizable syndromes nor have a plausible genetic or environmental cause.
Most neural tube defects are isolated malformations of the central nervous system and its protective encasements. In surveys,
15%-25% of neural tube defects are accompanied by anomalies of other systems.3-5 Enhancement of folic acid intake in the
periconceptional period is a well-established method of lowering the risk of neural tube defects. This survey examines the neural
tube defect prevalence and types of anomalies associated with neural tube defects in 6 prefortification years (1992-1998) and 20
fortification years (1999-2018) in South Carolina. Folic acid supplementation in the periconceptional period was the major
prevention strategy during the prefortification years, whereas both supplements and fortification of cereal grain flours were
used during the fortification years.6

Methods
The South Carolina Neural Tube Defects Prevention Program began on October 1, 1992, and has operated continuously. In the
first year, surveillance and prevention activities covered 14 of the state’s 46 counties and thereafter included all 46 counties.
From 1992 to 2006, surveillance and prevention activities were conducted by the Greenwood Genetic Center. Neural tube de-
fects were identified through the state’s 3 genetics referral services and through all the state’s prenatal diagnostic centers, obste-
trician offices, maternal alpha-fetoprotein laboratories, and delivery hospital medical records. In 2006, surveillance was
transferred to the South Carolina Department of Health and Environmental Control and uses hospital medical records as
the major source of case identification. Prevention activities continued to be conducted by the Greenwood Genetic Center.
The South Carolina Neural Tube Defects Prevention Program followed the recommendation made in 1992 by the US Centers
for Disease Control and Prevention for increased folic acid use (400 mg/d) for all women of childbearing years and the 1991
recommendation for women with a previous affected pregnancy to use 4000 mg/d in the periconceptional period to prevent
recurrences.7,8 In addition, enriched cereal grain products in the state were for-
tified beginning in 1998 as mandated by the US Food and Drug Administration.9
Details collected on the neural tube defect–affected pregnancy, fetus, or infant
included sex, race, nature of the neural tube defect (location, and isolated or From the Greenwood Genetic Center, Greenwood, SC
Supported by the South Carolina Department of Dis-
abilities and Special Needs (2020-43 [to R.S.]). The au-
thors declare no conflicts of interest.

OEIS Omphalocele, Exstrophy, Imperforate anus, Sacral anomalies 0022-3476/$ - see front matter. ª 2020 Elsevier Inc. All rights reserved.
https://doi.org/10.1016/j.jpeds.2020.07.002

186

nonisolated), pregnancy complications or exposures, gesta-
tional age at delivery, laboratory test results, surgical proced-
ures, and current status of liveborn infants. The number of
live births, fetal deaths, and terminations each year were ob-
tained from South Carolina Vital Records.
Women with neural tube defect–affected pregnancies were
offered enrollment in a neural tube defect recurrence preven-
tion program. Those enrolled were consented using the form
reviewed and approved by the Self Regional Healthcare insti-
tutional review board. Enrollees were contacted every
3 months to reinforce prevention information and every
month when a pregnancy occurred.
Terms Used
Anencephaly, spina bifida, and encephalocele are the 3 cate-
gories of neural tube defects used by the South Carolina Neu-
ral Tube Defects Prevention Program. If 2 defects are present,
the case is included in the more severe category. Iniencephaly
(n = 8) is included in the anencephaly category because of de-
fects in the calvaria and spine. Cutis aplasia of the scalp is
included in the encephalocele category. The single case of
multiple vertebral anomalies is included as spina bifida.
Isolated neural tube defects have 1 of the 3 types of neural
tube defects and no other major malformation except those
that are considered secondary to the neural tube defects (hy-
drocephalus, tethered cord, omphalocele, or club foot). Non-
isolated neural tube defects include 3 categories: Category I:
Nonisolated neural tube defects with specific genetic or envi-
ronmental causes known include neural tube defects with
other birth defects and known causes. The genetic causes
(Category Ia) include chromosomal aberrations, microdupli-
cations, microdeletions, and single-gene disorders. Environ-
mental influences (Category Ib) include maternal diseases,
obesity, and pregnancy exposures; Category II: Nonisolated
neural tube defects with clinically recognizable phenotypes
include neural tube defects with other birth defects that
constituted a recognizable pattern of malformation but
with cause unknown; Category III: Nonisolated neural tube
defects with nonrecognizable phenotype include neural
tube defects with other birth defects that do not constitute
a recognizable pattern of malformation and which do not
have a specific known cause.
Rates of neural tube defects are given as rates per 1000 live-
births and fetal deaths. Pregnancy terminations are not
included in these numbers.
Folate-resistant neural tube defects are those neural tube
defects that occurred even though the mother was taking folic
acid supplements in the periconceptional period (prefortifi-
cation years) or consumed fortified food products with or
without supplements in the periconceptional period (fortifi-
cation years). Periconceptional use of folic acid supplements
indicates intake for at least 1 month before conception and 1
or more months following conception.
Statistical Analyses
The Fisher exact test was used to test the effect of fortification
on isolated and complex cases separately for all neural tube
Volume 226 ! November 2020
defects, spina bifida, anencephaly, and encephalocele cases.
OR, CIs, and P values were calculated using the Baptista–
Pike method using GraphPad Prism 8.3.1 (GraphPad Soft-
ware, San Diego, California).
Results
All Surveillance Years
Since initiation of surveillance for neural tube defects and use
of folic acid prevention/treatment strategies in 1992, the
overall prevalence of neural tube defects in South Carolina
has decreased from 1.87 cases to 0.56 cases per 1000 live
births and fetal deaths, which includes spontaneous fetal
deaths, but does not include pregnancy terminations. During
this 26-year period, 1301 neural tube defect cases were iden-
tified. During the prefortification years (1992-1998), live-
births accounted for 151 (41.6%) of cases, terminations for
180 (49.6%), and fetal deaths for 32 (8.8%). During the forti-
fication years (1999-2018), live births accounted for 475
(50.6%) cases, terminations for 299 (31.9%), and fetal deaths
for 161 (17.2%). The source of ascertainment was unknown
for 3 cases in the fortification years (Table I; available at
www.jpeds.com). The 1301 neural tube defects identified in
the prefortification and fortification years included 466
cases of isolated spina bifida and 128 cases of nonisolated
spina bifida, 416 cases of isolated anencephaly and 89 cases
of nonisolated anencephaly, and 119 cases of isolated
encephalocele and 83 cases of nonisolated encephalocele
(Figure 1 and Table II [Table II available at www.jpeds.
com]). The type of neural tube defect and the presence of
other anomalies was determined by prenatal
ultrasonography only (202, 15.5%), postnatal examination
(931, 71.6%), and autopsy (168, 12.9%). The year-by-year
prevalence rates are shown in graphic form in Figure 1.
Recurrences accounted for only 18 (1.4%) of the cases.
Genetic laboratory testing was performed on a minority of
the 1301 neural tube defects. Chromosomal analysis was
completed on 405 cases (121 prefortification), microarray
analysis for microduplications and microdeletions on 36
cases (6 prefortification), whole-exome sequencing on 39
cases (13 prefortification), and whole-genome sequencing
on 58 cases (23 prefortification).
In South Carolina, similar to other states, Hispanic
subjects had the greatest prevalence of neural tube defects,
white subjects an intermediate prevalence, and black sub-
jects the lowest prevalence. The prevalence of isolated
neural tube defects among Hispanic subjects was 0.87 in
1000 live births and fetal deaths, among white subjects
0.78 in 1000, and among black subjects 0.49 in 1000.
The prevalence for nonisolated neural tube defects was
0.29 in 1000 live births and fetal deaths for Hispanic sub-
jects, 0.20 in 1000 for white subjects, and 0.20 in 1000 for
black subjects. The prevalence in the prefortification and
fortification years by race is provided in Table III
(available at www.jpeds.com). The male/female ratio for
all neural tube defects was 0.9 for the 1144 cases in
187
THE JOURNAL OF PEDIATRICS ! www.jpeds.com Volume 226

A Prefor fica on Years For fica on Years

2.0
(1992-1998) (1999-2018)
2.0

1.8 1.8
Rate of NTDs per 1000 livebirths and fetal deaths

1.6 1.6

1.4 1.4

1.2 1.2

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
Project Year 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
All NTDs 1.88 1.51 1.28 1.24 1.33 0.96 0.87 0.96 0.94 0.92 0.75 0.81 0.76 0.81 0.85 0.99 0.81 0.72 0.77 0.66 0.73 0.85 0.97 0.79 0.54 0.61
Isolated NTDs 1.45 1.29 1.08 1.09 1.20 0.71 0.71 0.84 0.67 0.56 0.48 0.62 0.64 0.68 0.65 0.80 0.60 0.49 0.53 0.59 0.58 0.64 0.58 0.52 0.33 0.35
With Anomalies 0.42 0.23 0.19 0.16 0.13 0.26 0.16 0.12 0.27 0.36 0.27 0.19 0.12 0.13 0.21 0.19 0.21 0.24 0.24 0.07 0.16 0.21 0.39 0.28 0.21 0.26
Livebirths and
Fetal Deaths 16.5 52.9 51.7 51.6 52.5 53.9 55.0 56.2 56.4 55.3 55.7 56.8 57.8 61.6 63.3 63.6 61.8 59.5 58.1 57.7 57.3 57.9 58.5 58.0 57.5 57.1

Figure 1. A, Year-by-year prevalence for all isolated and nonisolated neural tube defects. B, Year-by-year prevalence for iso-
lated and nonisolated spina bifida, anencephaly, and encephalocele cases. Annual deliveries (live births and fetal deaths) are
provided at the bottom of A. Note the scale change in the encephalocele rates. (Continues)

which the sex was known. The male/female ratio was 1.05 prenatal methimazole exposure was included in Category Ib
among spina bifida cases, 0.7 among anencephaly cases, (in fortification years).
and 0.94 among encephalocele cases. Neural tube defects with clinically recognizable phenotypes
Of the 1301 neural tube defects identified in the 26-year of unknown cause (Category II) included amniotic bands syn-
period (1992-2018) in South Carolina, 300 (23%) had additional drome (43/300, 14.3%), OEIS complex, ie, Omphalocele, Exs-
anomalies (Figure 1 and Table II). A specific cause or a clinically trophy, Imperforate anus, Sacral anomalies (12/300, 4%),
recognizable pattern of malformations (Category Ia, Ib: hemifacial microsomia (3/300, 1%), sirenomelia (3/300,
nonisolated neural tube defects with specific cause known and 1%), renal and M€ ullerian dysgenesis (3/300, 1%), and short
Category II: non-isolated neural tube defects with clinically cord syndrome (2/300, 0.7%). One case with sirenomelia
recognizable phenotype of unknown cause) could be identified also had trisomy 18. The distribution of these recognizable
in slightly more than one-half (161/300; 53.7%) of instances in phenotypes by type of neural tube defect is shown as Table IV.
which neural tube defects were associated with other Overall, 139 of 300 (46.3%) neural tube defects were
anomalies (Figure 2 and Table IV [Table IV available at www. accompanied by other anomalies that did not comprise clin-
jpeds.com]). Chromosomal aberrations (n = 77), single-gene ically recognizable phenotypes (Category III, Table VI).
disorders (n = 19), and prenatal environmental influences Among these, the most common anomalies associated with
(n = 1) were the specific causes found among this group. spina bifida were cardiac and genitourinary defects, with
Among the chromosomal aberrations, trisomy 18 was found anencephaly were cleft lip/palate and cardiac defects, and
in 40 cases (32 spina bifida, 4 anencephaly, and 4 with encephalocele were cardiac defects and cleft lip/palate.
encephalocele), trisomy 13 in 8 cases (5 spina bifida and 3 In this 26-year study, the presence or absence of maternal
encephalocele), and triploidy in 8 cases (all spina bifida). diabetes was known for 1095 of the 1301 pregnancies
Other trisomies (4), mosaic trisomies (3), and chromosomal (Table VII; available at www.jpeds.com). Pregestational
translocations, duplications, deletions, or markers (14) diabetes existed in 42 pregnancies that resulted in isolated
accounted for the remaining chromosomally abnormal cases neural tube defects (12 spina bifida, 21 anencephaly, 9
(Table V). Single-gene disorders included Meckel syndrome encephalocele) and in 8 pregnancies that resulted in neural
(18 cases, 7 in prefortification years, 11 in fortification years), tube defects with other anomalies (4 spina bifida, 1
and 1 CASP9 mutation (in fortification years). A single case of anencephaly, 3 encephalocele). Gestational diabetes

188 Dean, Pauly, and Stevenson

November 2020 ORIGINAL ARTICLES

B Prefor fica on Years For fica on Years

(1992-1998) (1999-2018)
1.2 1.2

Spina Bifida
1.0 Isolated
Spina Bifida Rates

1.0

With Anomalies
0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0

Project Year 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Spina Bifida 0.97 0.78 0.62 0.47 0.61 0.37 0.31 0.44 0.43 0.38 0.38 0.33 0.40 0.34 0.43 0.30 0.40 0.32 0.41 0.35 0.31 0.36 0.56 0.38 0.21 0.32
Isolated 0.85 0.70 0.58 0.43 0.53 0.33 0.29 0.36 0.28 0.27 0.23 0.25 0.31 0.31 0.30 0.28 0.28 0.22 0.31 0.28 0.24 0.29 0.31 0.26 0.09 0.18
With Anomalies 0.12 0.08 0.04 0.04 0.08 0.04 0.02 0.09 0.14 0.11 0.14 0.09 0.09 0.03 0.13 0.02 0.13 0.10 0.10 0.07 0.07 0.07 0.26 0.12 0.12 0.14

1.0 1.0

Ancencephaly
0.8
Anencephaly Rates

Isolated 0.8

With Anomalies
0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0

Project Year 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Ancencephaly 0.67 0.57 0.41 0.56 0.59 0.39 0.40 0.43 0.39 0.40 0.25 0.39 0.24 0.34 0.27 0.53 0.26 0.34 0.19 0.28 0.33 0.31 0.26 0.22 0.24 0.19
Isolated 0.42 0.51 0.33 0.48 0.57 0.24 0.27 0.41 0.30 0.24 0.20 0.32 0.24 0.29 0.24 0.42 0.24 0.22 0.14 0.28 0.30 0.26 0.21 0.16 0.19 0.18
With Anomalies 0.24 0.06 0.08 0.08 0.02 0.15 0.13 0.02 0.09 0.16 0.05 0.07 0.00 0.05 0.03 0.11 0.02 0.12 0.05 0.00 0.03 0.05 0.05 0.07 0.05 0.02

0.4 0.4
Encephalocele Rates

Encephalocele
0.3 Isolated 0.3

With Anomalies
0.2 0.2

0.1 0.1

0.0 0.0

Project Year 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Encephalocele 0.24 0.17 0.25 0.21 0.13 0.20 0.16 0.09 0.12 0.14 0.13 0.09 0.12 0.13 0.16 0.16 0.15 0.07 0.17 0.03 0.09 0.17 0.15 0.19 0.09 0.11
Isolated 0.18 0.06 0.17 0.14 0.10 0.13 0.15 0.07 0.09 0.05 0.05 0.05 0.09 0.08 0.11 0.09 0.06 0.05 0.09 0.03 0.03 0.09 0.07 0.10 0.05 0.00
With Anomalies 0.06 0.11 0.08 0.08 0.04 0.07 0.02 0.02 0.04 0.09 0.07 0.04 0.03 0.05 0.05 0.06 0.08 0.02 0.09 0.00 0.05 0.09 0.09 0.09 0.03 0.11

Figure 1. Continued.

occurred in 41 pregnancies that resulted in isolated neural anus, and 1 with cleft lip/palate and ambiguous genitalia). In
tube defects (19 spina bifida, 13 anencephaly, 9 49 of the sets of twins, only 1 of the twins was affected
encephalocele) and in 16 pregnancies that resulted in (25 with spina bifida, 18 with anencephaly, and 6 with ence-
neural tube defects with other anomalies (6 spina bifida, phalocele). In 3 instances both twins had an neural tube
4 anencephaly, 6 encephalocele). Valproate was taken in 7 defect. One set had anencephaly in one twin and encephalo-
pregnancies that resulted in isolated spina bifida and in 4 cele in the other. In 2 sets, both twins had spina bifida. Three
pregnancies that resulted in neural tube defects with other sets of triplets occurred and one triplet in each set had spina
anomalies. Obesity as an independent risk factor was bifida. One infant in a quadruplet pregnancy had anenceph-
identified in 243 (25.6%) of 916 mothers on whom height aly. Twelve of the affected twins or multiple births occurred
and weight were known (Table VII). in the prefortification years and 50 in the fortification years
The 62 affected twins and multiple births included 3 (Table VIII; available at www.jpeds.com).
conjoined twins, all 3 affected with spina bifida and other In a subset of 707 neural tube defects (695 occurrent, 12
anomalies (1 with diaphragmatic hernia, 1 with imperforate recurrent) who enrolled in the South Carolina Neural Tube
Neural Tube Defects and Associated Anomalies before and after Folic Acid Fortification 189
THE JOURNAL OF PEDIATRICS ! www.jpeds.com Volume 226

Figure 2. Findings in the 3 most common chromosome aberrations associated with A-C, neural tube defects D, E, in Meckel
syndrome, F, G, in amniotic band syndrome and H, I, in OEIS syndrome. A, Trisomy 18 with microcephaly, overlapping fingers,
and rocker bottom feet. B, Trisomy 13 with microcephaly, cleft lip/palate, and polydactyly. C, Triploidy with craniosomatic
disproportion and syndactyly. D and E, Meckel syndrome with small occipital encephalocele, enlarged abdomen due to cystic
kidneys, and polydactyly. F, Amniotic band syndrome with cranio-placental fusion and digit amputations. G, Amniotic band
syndrome with facial clefting, syndactyly, digit amputations, and cranial disruption. H and I, OEIS syndrome.

Defects Prevention Program, more complete family history,
maternal health information, pregnancy history, folic acid
use, dietary recall, and demographic data were collected.
Folic acid supplements in doses of 400-4000 mg/d were
used in the periconceptional period of 192 occurrent preg-
nancies (35 in the prefortification years, 157 in the fortifica-
tion years), 142 (74%) of which resulted in isolated neural
tube defects and 50 (26%) of which resulted in nonisolated
neural tube defects (Table IX; available at www.jpeds.com).
In total, 400 mg was used in 88 of these pregnancies, 800 mg
in 64 pregnancies, 1000 mg in 28 pregnancies, 1200-2200 mg
in 11 pregnancies, and 4000 mg in 1 pregnancy.
In addition, 311 women with previous neural tube defect
pregnancies took 4000 mg/d folic acid in a subsequent preg-
nancy. Only one isolated neural tube defect and three
non-isolated neural tube defects occurred among these preg-
nancies.
Prefortification Years
During the 6 years of the prefortification era (1992-1998),
363 neural tube defects were identified in South Carolina;
84% (305/363) were isolated and 16% (58/363) had addi-
tional major malformations (Table II). This represents a
significantly greater percentage of isolated cases (OR 1.828,
95% CI 1.350-2.525, P < .001) when compared with the
percentage in the fortification years. Encephalocele had the
lowest percentage of isolated cases (35/55, 63.6%) and
spina bifida had the greatest percentage of isolated cases
(149/165, 90.3%). Anencephaly was accompanied by other
anomalies in 15.4% of occurrences (22/143).
The 58 nonisolated neural tube defects included 6 (10%)
with a chromosomal etiology, 7 (12%) with Meckel syn-
drome, 14 (24%) with amniotic bands or limb–body wall
defect, 1 with short cord syndrome, and 30 (55%) with de-
fects that did not comprise recognizable phenotypes
(Tables IV, V, and VI).
Fortification Years
During the 20 years of the fortification era (1999-2018), 938
neural tube defects were identified in South Carolina: 696
(74.2%) were isolated and 242 (25.8%) had other major mal-
formations. Among the nonisolated neural tube defects, a
chromosomal aberration, a single-gene disorder, a terato-
genic exposure, or a clinically recognizable pattern of malfor-
mations (Categories Ia, Ib, and II) were identified in 161 of
1001 (16%). A chromosomal etiology was most common
(71/242, 29.3%). Single-gene etiologies included Meckel syn-
drome (n = 11) and a single CASP9 mutation. The recogniz-
able phenotypes of uncertain etiology (Category II) included
amniotic bands (n = 31), limb–body wall defect (n = 3), OEIS
complex (n = 12), short cord syndrome (n = 2), hemifacial
microsomia (n = 3), sirenomelia (n = 3), and renal and
M€ullerian dysgenesis (n = 3).
Among the nonisolated neural tube defects that did not
comprise a recognizable phenotype (Category III), cardiac
malformation was the most common associated defect

190 Dean, Pauly, and Stevenson

November 2020 ORIGINAL ARTICLES

Table V. Chromosomal aberrations including microdeletions and microduplications (n = 77) among 300 nonisolated
neural tube defects (prefortification years, fortification years)
Aberrations SB* A† E‡ Total
Trisomy 18 32 (1, 31) 4 (0, 4) 4 (1, 3) 40 (2, 38)
Triploidy 8 (0, 8) 0 0 8 (0, 8)
Trisomy 13 5 (1, 4) 0 3 (0, 3) 8 (1, 7)
Trisomy 21 1 (0, 1) 0 0 1 (0, 1)
Mosaic trisomy 22 0 0 1 (1, 0) 1 (1, 0)
XXX 1 (0, 1) 0 0 1 (0, 1)
XXY 0 1 (0, 1) 0 1 (0, 1)
Mosaic trisomy 9 1 (0, 1) 0 1 (0, 1) 2 (0, 2)
Mosaic trisomy 8 1 (0, 1) 0 0 1 (0, 1)
Deletion 1p 0 0 1 (1, 0) 1 (1, 0)
Deletion 13q 0 1 (0, 1) 2 (0, 2) 3 (0, 3)
Ring 13 0 1 (1, 0) 0 1 (1, 0)
Deletion 15q 1 (0, 1) 0 1 (0, 1) 2 (0, 2)
Deletion 16p 1 (0, 1) 0 0 1 (0, 1)
Deletion 18p 0 1 (0, 1) 0 1 (0, 1)
Deletion 21q 1 (0, 1) 0 0 1 (0, 1)
Translocation 15/20 0 0 1 (0, 1) 1 (0, 1)
Marker 1 (0, 1) 0 0 1 (0, 1)
Tetrasomy 1q 0 1 (0, 1) 0 1 (0, 1)
Duplication 2p 0 1 (0, 1) 0 1 (0, 1)
53 (2, 51) 10 (1, 9) 14 (3, 11) 77 (6, 71)
*Includes 1 case with multiple vertebral anomalies but no external spina bifida.
†Includes 4 cases of craniorachischisis and 8 cases of iniencephaly.
‡Includes 2 cases of cutis aplasia but no external encephalocele.

(43/139, 31%) and facial clefting was second most common
(30/139, 22%) (Table VI).
Discussion
From the landmark studies of Smithells et al, the United
Kingdom Medical Research Council, and Czeizel et al in
the 1980s and early 1990s, it became clear that enhanced folic
acid intake in the periconceptional period would lower the
occurrence and recurrence rates of neural tube defects.10-12
Although it might be assumed that the folate-resistant neural
tube defects would be predominantly those caused by chro-
mosomal aberrations, single-gene disorders, and environ-
mental influences unrelated to folate metabolism, this
appears to not be the case. Although there was a significantly
greater percentage of syndromal neural tube defects in the
fortification years, the majority of folate-resistant neural
tube defects continued to be isolated malformations.
Neural tube defects are the most common of serious
malformations affecting the brain and spine. Neural
tube defect prevalence varies widely among different pop-
ulations, with a very high prevalence recorded in some lo-
cations (eg, >10 per 1000 live births and fetal deaths in
Ukraine, China, and Ethiopia) and very low prevalence
in others (eg, <1 per 1000 live births and fetal deaths
in the US and Canada).6,13-17 The latest population-
based prevalence of neural tube defects in the US is for
the years 2010-2014.17 The combined prevalence for the
3 types of neural tube defects was 0.735 in 1000 live
births and fetal deaths, which is nearly equivalent to the
prevalence in South Carolina (0.747/1000 live births and
fetal deaths) for those years and greater than the preva-
lence in South Carolina for year 26 (0.56/1000 livebirths
and fetal deaths) of the survey presented here. The na-
tional figures reported by Mai et al do not separate iso-
lated and nonisolated neural tube defects.17

Table VI. Nonisolated neural tube defects (n = 139) that did not comprise recognizable phenotypes–Category III
(prefortification years, fortification years)
Holopro- Ocular Diaphragmatic
Type of neural tube defects sencephaly anomaly CL/CP Cardiac hernia GI GU Limb Other
Spina bifida 4 (3, 1) 2 (0, 2) 5 (0, 5) 18 (1, 17) 4 (3, 1) 15 (2, 13) 17 (4, 13) 8 (0, 8) 4* (0, 4)
Anencephaly 2† (0, 2) 2 (0, 2) 20 (7, 13) 14 (4, 10) 5 (3, 2) 5 (0, 5) 4 (2, 2) 10 (2, 8) 2‡ (0, 2)
Encephalocele 3 (0, 3) 5 (1, 4) 13 (1, 12) 18 (2, 16) 1 (1, 0) 4 (0, 4) 7 (0, 7) 0 1§ (0, 1)
All neural tube defects 9 (3, 6) 9 (1, 8) 38 (8, 30) 50 (7, 43) 10 (7, 3) 24 (2, 22) 28 (6, 22) 18 (2, 16) 7 (0, 7)

CL/CP, cleft lip/cleft palate; GI, gastrointestinal abnormalities; GU, genitourinary abnormalities.
Note that some cases had more than 1 other anomaly.
*Includes 1 laryngeal atresia, 1 cystic lungs, 1 laryngeal paralysis, and 1 multiple anomalies not specified.
†Diagnosed from facial manifestations.
‡Includes 1 microtia and 1 multiple anomalies not specified.
§Includes 1 craniosynostosis.

Neural Tube Defects and Associated Anomalies before and after Folic Acid Fortification 191
THE JOURNAL OF PEDIATRICS ! www.jpeds.com
Most neural tube defects are sporadic, and specific genetic
or environmental causes have been identified in <20% of
cases. As in the survey reported here, numerical chromo-
somal aberrations are found in "5% of cases, genomic mi-
crodeletions or microduplications in <1%, and single-gene
disorders in <2%.15-23 Environmental causes include preges-
tational maternal diabetes ("5%), maternal obesity ("10%),
maternal hyperthermia (<1%), prenatal exposure to anticon-
vulsants (<1%), and prenatal exposure to other medications,
pollutants, and toxins (unknown).24-26 In South Carolina,
the percentage of women with periconceptional obesity
(body mass index >29 kg/m2) during the period 1992-2018
was 25.6%, more than twice the percentage reported by
Shaw et al in women from California.25 The recurrence risk
for neural tube defects of 3%-5% is consistent with multifac-
torial causation with contributions from a large number of
genetic and environmental influences. Although more than
250 different genes are involved in neural tube closure in
mouse, only a few of them, notably genes involved in planar
cell polarity and folate metabolism, have been implicated in
human neural tube defects.27-29
Chromosomal aneuploidies, segmental deletions, and
segmental duplications figure prominently in the causation
of neural tube defects.5 In this survey, chromosomal aberra-
tions, including microduplications and microdeletions, ac-
counted for 5.9% of all neural tube defects and 25.7% of
300 neural tube defects with other major malformations
(Table V). In the prefortification years 6 of 58 (10.3%) of
neural tube defects with other anomalies had chromosomal
aberrations. In the fortification years 71 of 242 (29.3%) of
neural tube defects with other anomalies had chromosomal
aberrations. Spina bifida had the greatest rate of
chromosomal errors (53/594, 8.9%), encephalocele the
second greatest (14/202, 6.9%), and anencephaly the lowest
(10/505, 2.0%).
Meckel syndrome, an autosomal-recessive disorder, is
recognized as the most common single-gene condition
among neural tube defects (Figure 2 and Table IV). In
addition to an encephalocele or other central nervous
system anomaly, Meckel syndrome typically has a triad of
other findings: polydactyly, cystic kidneys, and hepatic
fibrosis. In this survey, 18 cases of Meckel syndrome were
identified, comprising 6% of neural tube defects with
associated anomalies and 1.4% of all neural tube defects.
The only other single-gene disorders were biallelic
mutations in APAF1 (1 case) and CASP9 (1 case).30
Two recognizable phenotypes of unknown cause (Cate-
gory II)—OEIS complex and amniotic bands/limb–body
wall defect—number prominently in this survey and
others.4,5 Both occur as sporadic conditions with amniotic
bands/limb–body wall defect being the more common and
more variable in craniofacial, central nervous system, ventral
body wall, and limb manifestations (Figure 2). The neural
tube defect most commonly affects the cranium but may be
difficult to classify. When the cranium is completely or
partially absent, the defect may be considered anencephaly
192
Volume 226
or meroanencephaly. When central nervous system tissues
extend beyond the cranial vault but remain skin covered,
the defect may be considered to be single or multiple
encephaloceles. Atypical facial clefts that do not follow the
typical lines of clefts often accompany the cranial
malformation. Limb defects are also variable and include
single-limb amelia, distal limb or digit amputations,
syndactyly, and ring constrictions. Strands of amnion may
be found attached to any of the craniofacial, truncal, or
limb defects and/or floating free in the amniotic fluid
cavity. There is no consensus regarding the origin of
amniotic bands or whether the associated neural tube
defects are primary or secondary.31-33
Because of the overlapping findings, amniotic bands syn-
drome and limb–body wall defect are classified together in
this survey. The 43 cases comprised 3.3% of all neural tube
defects and 14.3% of neural tube defects with other anoma-
lies. Amniotic bands/limb–body wall defect accounted for
2.5% of all neural tube defects in the prefortification years
and 3.6% of all neural tube defects in the fortification years.
OEIS complex was diagnosed in 12 cases comprising 0.9% of
all neural tube defects and 4% of neural tube defects with
other anomalies (Table IV).
Maternal diabetes, obesity, and valproic acid exposure are
the best known of a limited number of environmental influ-
ences which increase the risk of neural tube defects with or
without associated anomalies.25,34-37 Warfarin exposure
and maternal hyperthermia have been associated with neural
tube defects and other anomalies.38-40 Anencephaly and
spina bifida have been noted in infants with prenatal expo-
sure to the folic acid antagonist, aminopterin, and prenatal
exposure to methimazole, an antithyroid medication, has
been associated with cranial aplasia cutis, choanal atresia
and other anomalies.41,42
The continued occurrence of neural tube defects, the ma-
jority of which are isolated, after folic acid fortification of
cereal grain flours suggests that additional prevention mea-
sures are necessary to further reduce the prevalence of these
serious defects of the brain and spine. Measures that poten-
tially could contribute to further reduction include identifi-
cation of cryptic environmental or genetic/genomic
influences that could be mitigated preconceptionally, daily
intake of a greater dose of folic acid for all women or for
women in the childbearing years, or specifically for women
with obesity, diabetes, or epilepsy treated with valproic acid
or carbamazepine, and the addition of other micronutrients,
such as vitamin B12 or other one-carbon sources, to the pre-
vention strategy.16,43,44 Failure to consider additional mea-
sures is equivalent to stating that the continued occurrence
of 35 isolated neural tube defects per year in South Carolina
and more than 2500 isolated neural tube defects per year in
the US is acceptable.
Lowry et al report that more than 20% of women of repro-
ductive age did not achieve the optimal red blood cell folate
concentration for neural tube defect prevention (906 nmol/
L) after folic acid fortification of cereal grain flours in
Dean, Pauly, and Stevenson
November 2020
Canada.16 Fayyaz et al45 found similar suboptimal red blood
cell folate levels during the first 2 trimesters of pregnancy, but
noted that deficiencies of vitamins B12 and B6 were rare
(<1%).
Although 4000 mg/d folic acid was used in the Medical
Research Council’s neural tube defect recurrence study
without reported adverse effects and is currently the Center
of Disease Control and Prevention’s recommendation for
recurrence prevention, increasing the daily dose of folic
acid for all women of childbearing age must be weighed
against potential side effects. Patel and Sobczy" nska–Malefora
have enumerated the potential effects that might occur in
certain populations, including diarrhea, rashes, sleep distur-
bances, masking of vitamin B12 deficiency–related neuro-
logic damage, insulin resistance, increase in cancer risk or
promotion of the growth of pre-existing cancers, and
lowering the effectiveness of anticonvulsant medications.46
Other concerns relate to potential effects on the fetus, the
most plausible being alteration of the methylation profile.
In contrast, Wald et al have suggested that the risks are over-
stated and that the concept of a tolerable upper intake level of
folic acid should be abandoned.47
A strength of the study is the complete or nearly complete
ascertainment and classification of neural tube defects that
occurred before and after fortification over 26 years in a state
with a historically high neural tube defect occurrence rate. No
claim for complete ascertainment of all neural tube defects
can be made because early pregnancy spontaneous and
induced terminations and out-of-state terminations could
have eluded the surveillance system. Other limitations
include the inability to examine and to conduct comprehen-
sive laboratory testing on all neural tube defect fetuses/in-
fants, the inability to obtain serum and erythrocyte folate
levels on the mothers, and incomplete information on peri-
conceptional folic acid us especially during the prefortifica-
tion years. Hispanic families typically use unfortified corn
flour, so the assumption that they received adequate folic
acid from their diet during the fortification era is incorrect.
Eight percent of neural tube defects in the 26-year study
period were born into Hispanic families. Although the
impact of early pregnancy terminations on the results could
not be determined, it is plausible that nonisolated neural tube
defects might lead to spontaneous or medical terminations
more frequently than isolated neural tube defects. Further,
it would have been optimal to have observations during a
greater number of prefortification years.
Reduction in the occurrence and recurrence of neural tube
defects with folic acid may be properly considered the birth
defects prevention success story of the past several decades.
This reduction, achieved by folic acid supplementation in
the periconceptional period and by folic acid fortification
of cereal grain flours, has been most notable in locations
with the greatest rates of neural tube defects. Some author-
ities consider that an irreducible rate of neural tube defects
is approaching or has been reached in areas with folate-
fortified cereal grain products. Yet, neural tube defects
continue to occur and the majority of these are isolated
Neural Tube Defects and Associated Anomalies before and after Folic Acid Fortification
ORIGINAL ARTICLES
defects, presumably of the type most amenable to folate
prevention. n
We thank the many families and clinicians who have contributed infor-
mation about neural tube defects that have occurred in South Carolina
and to the Birth Defects Program of the SC Department of Health and
Environmental Control through which surveillance for neural tube de-
fects is conducted.
Submitted for publication Dec 13, 2019; last revision received Jun 15, 2020;
accepted Jul 1, 2020.
Reprint requests: Roger E. Stevenson, MD, J. C. Self Research Institute of
Human Genetics, Greenwood Genetic Center, 113 Gregor Mendel Circle,
Greenwood, SC 29646. E-mail: res@ggc.org
Data Statement
Data sharing statement available at www.jpeds.com.
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Dean, Pauly, and Stevenson
Neural Tube Defects and Associated Anomalies before and after Folic Acid Fortification

November 2020
Table I. Outcome of pregnancies with neural tube defects in 6 prefortification and 20 fortification years
Year
Prefortification 1 2 3 4 5 6 Total
Live births 12 34 27 23 30 25 151
Terminations 12 40 36 38 34 20 180
Fetal demise 4 1 1 2 1 0 9
Stillbirths 2 3 1 0 4 6 16
Spontaneous abortion 1 2 1 1 1 1 7
Unknown 0 0 0 0 0 0 0
Total neural tube defects 31 80 66 64 70 52 363
Pregnancies 21 512 65 430 63 063 62 736 63 564 63 965 340 270
Livebirths 16 382 52 359 51 162 51 057 51 930 53 426 276 316
Fetal deaths 181 568 513 515 547 523 2847
Terminations 4949 12 503 11 388 11 164 11 087 10 016 61 107
Year
Fortification 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Total
Live births 18 26 17 20 19 20 23 24 37 25 25 19 25 18 23 25 37 32 18 24 475
Terminations 20 18 26 24 16 18 16 23 11 32 18 18 15 15 12 6 7 0 2 2 299
Fetal demise 0 0 1 2 0 0 0 1 0 0 0 0 0 1 1 16 13 9 10 6 60
Stillbirths 10 9 7 3 5 7 5 2 5 5 6 6 5 4 6 2 0 5 1 0 93
Spontaneous 0 1 2 2 1 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 8
abortion
Unknown 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 3
Total neural 48 54 53 51 42 46 44 50 54 63 50 43 45 38 42 49 57 46 31 32 938
tube defects
Pregnancies 65 251 66 278 66 445 66 050 67 125 67 560 69 379 74 437 76 244 74 989 73 315 70 960 68 553 67 510 66 499 66 925 69 455 68 939 68 184 67 830 1381 921
Live births 54 488 55 650 55 802 54 777 55 209 56 273 57 289 61 028 62 748 63 041 61 281 58 914 57 585 57 160 56 832 57 409 58 009 57 537 57 107 56 759 1154 894
Fetal deaths 523 561 563 532 534 505 517 594 589 551 563 551 520 515 460 484 455 454 411 360 10 240
Terminations 10 240 10 067 10 080 10 741 11 382 10 782 11 573 12 815 12 907 11 397 11 471 11 495 10 448 9835 9207 9032 10 991 10 948 10 666 10 711 216 787

ORIGINAL ARTICLES
194.e1
THE JOURNAL OF PEDIATRICS ! www.jpeds.com Volume 226

Table II. Isolated and nonisolated neural tube defects in all surveillance years, South Carolina
Prefortification
All 26 years (October 1,
surveillance 1992-September Fortification years (October 1,
Type of neural tube defects years 30, 1998) 1998-September 30, 2018) Statistical significance
All neural tube 1301 363 938
defects
Isolated 1001 (77%) 305 (84%) 696 (74%) OR 1.828, 95% CI 1.330-2.525, P £ .0001*
Nonisolated 300 (23%) 58 (16%) 242 (26%) OR 0.5469, 95% CI 0.3960-0.7521, P £ .0001*
Spina bifida 594 165 429
Isolated 466 (78%) 149 (90%) 317 (74%) OR 3.290, 95% CI 1.886-5.676, P £ .0001*
Nonisolated 128 (22%) 16 (10%) 112 (26%) OR 0.3039, 95% CI 0.1762-0.5303, P £ .0001*
Anencephaly 505 143 362
Isolated 416 (82%) 121 (85%) 295 (81.5%) OR 1.249, 95% CI 0.7354-2.105, ns†
Nonisolated 89 (18%) 22 (15%) 67 (18.5%) OR 0.8005, 95% CI 0.4751-1.360, ns†
Encephalocele 202 55 147
Isolated 119 (59%) 35 (64%) 84 (57%) OR 1.313, 95% CI 0.7077-2.435, ns†
Nonisolated 83 (41%) 20 (36%) 63 (43%) OR 0.7619, 95% CI 0.4106-1.413, ns†

ns, not significant.

*Significantly greater percentage of isolated defects and significantly smaller percentage of nonisolated defects in prefortification years compared with fortification years (P < .0001).
†Greater percentage of isolated defects and smaller percentage of nonisolated defects in prefortification years ns.

Table III. Prevalence of neural tube defects in the prefortification and fortification years by race
Prevalence (neural tube defects/deliveries)
Race Prefortification years (1992-1998) Fortification years (1999-2018)
White 265/175 284 (1/661) [A: 105, SB: 124, E: 36] 562/684 464 (1/1218) [A: 228, SB: 249, E: 85]
Black 86/97 835 (1/1138) [A: 32, SB: 35, E: 19] 250/380 749 (1/1523) [A: 85, SB: 125, E: E#40]
Hispanic 7/4833 (1/690) [A: 3, SB: 4, E: E#0] 100/90 193 (1/902) [A: 41, SB: 41, E: 18]
Other/unknown 5/3847 (1/769) [A: 2, SB: 3, E: 0] 26/27 032 (1/1040) [A: 8, SB: 14, E: 4]
All races 363/281 799 (1/776) [A: 142, SB: 166, E: 55] 938/1 182 438 (1/1261) [A: 362, SB: 429, E: 147]

A, anencephaly; E, encephalocele; SB, spina bifida.

194.e2 Dean, Pauly, and Stevenson

November 2020 ORIGINAL ARTICLES

Table IV. Type of neural tube defects associated with

chromosomal aberrations, Meckel syndrome, and
selected other malformations
Associated defects SB A E
Chromosome (n = 77*) 53 (69%) 10 (13%) 14 (18%)
Amniotic bands/L-BWD 5 (12%) 21 (49%) 17 (40%)
(n = 43)
Diaphragmatic 8 (50%) 5 (31%) 3 (18%)
hernia (n = 16†)
Meckel (n = 18) 0 1 (6%) 17 (94%)
Holoprosencephaly 6 (40%) 2 (13%) 7 (47%)
(n = 15‡)
OEIS (n = 12) 12 (100%) 0 0

L-BWD, limb–body wall defect.

*Among the chromosome aberrations were 2 copy number variations <500 kb (1 spina bifida
with 435 kb loss of 16p and 1 anencephaly with 334 kb loss of 18p).
†Six neural tube defects with diaphragmatic hernia had chromosome abnormalities (2 trisomy
18, 2 trisomy 13, 1 mosaic trisomy 9, 1 XXY).
‡Six neural tube defects with holoprosencephaly had a chromosome or single gene abnormality
(3 trisomy 13, 2 13q deletion, 1 Meckel).

Table VII. Maternal diabetes mellitus and obesity as risk factors

Prefortification Fortification years
Risk factors All neural tube defects Isolated Nonisolated All neural tube defects Isolated Nonisolated
Gestational diabetes 19 15 4 38 26 12
Spina bifida 9 9 0 16 10 6
Anencephaly 6 4 2 11 9 2
Encephalocele 4 2 2 11 7 4
Pregestational diabetes 10 9 1 40 33 7
Spina bifida 3 3 0 13 9 4
Anencephaly 5 4 1 17 17 0
Encephalocele 2 2 0 10 7 3
Without diabetes 270 226 44 718 520 198
Unknown 64 56 8 142 117 25
Total neural tube defects 363 306 57 938 696 242
All mothers with BMIs available 241 204 37 675 493 182
BMIs >29 kg/m2 as an independent risk 59 56 3 184 147 37
factor

BMI, body mass index.

Neural Tube Defects and Associated Anomalies before and after Folic Acid Fortification 194.e3
THE JOURNAL OF PEDIATRICS ! www.jpeds.com Volume 226

Table VIII. Twins and other multiple births with

neural tube defects (n = 62)
Prefortification Fortification
Types of multiple birth years (6) years (20) All years (26)
Twins—1 affected 8 41 49
Spina bifida 5 20 25
Anencephaly 3 15 18
Encephalocele 0 6 6
Twins—both affected 1 2 3
Spina bifida 2 2 4
Anencephaly 0 1 1
Encephalocele 0 1 1
Conjoined twins 1 2 3
Spina bifida 1 2 3
Anencephaly 0 0 0
Encephalocele 0 0 0
Quads, 1 w/anencephaly 1 0 1
Triplets, 1 w/spina bifida 0 3 3

Table IX. Use of folic acid supplements in a subset of 707 neural tube defect pregnancies following which the mothers
enrolled in the recurrence prevention component of the South Carolina Neural Tube Defects Prevention Program
Folic acid dose/d Prefortification years Fortification years All years
Occurrent neural tube defects 252 (198 isolated) 443 (328 isolated) 695 (526—76% isolated)
Folic acid used 35 (30 isolated) 157 (112 isolated) 192 (142—74% isolated)
400-1000 mg 33 (28 isolated) 147 (106 isolated) 180 (134—74% isolated)
1200-2200 mg 2 (2 isolated) 9 (6 isolated) 11 (8—73% isolated)
4000 mg 0 1 (0 isolated) 1 (0 isolated)
Recurrent neural tube defects 3 (2 isolated) 9 (4 isolated) 12 (6—50% isolated)
Folic acid used 0 5 (1 isolated) 5 (1—20% isolated)
400-1000 mg 0 2 (0 isolated) 2 (0 isolated)
1200-2200 mg 0 0
4000 mg 0 3 (1 isolated) 3 (1—33% isolated)

194.e4 Dean, Pauly, and Stevenson


Iulia Bicu Fernandes (https://orcid.org/0000-0002-2857-2938) 1
Rozania Bicego Xavier (https://orcid.org/0000-0003-3435-8038) 2
Paulo Alexandre de Souza São Bento (https://orcid.org/0000-0002-1598-3340) 3
Andreza Rodrigues (https://orcid.org/0000-0002-1873-5828) 4
1
Hospital Municipal de
Petrópolis Alcides Carneiro.
R. Vigário Correas 1345,
Correas. 25720-320
Petrópolis RJ Brasil.
iulia.bicu.86@gmail.com
2
Departamento de
Ginecologia e Obstetrícia.
IFF, Fiocruz. Rio de Janeiro
RJ Brasil.
3
Núcleo Interno de
Regulação, IFF, Fiocruz. Rio
de Janeiro RJ Brasil.
4
Escola de Enfermagem
Anna Nery, Universidade
Federal do Rio de Janeiro.
Rio de Janeiro RJ Brasil.
Nas vias de interromper ou não a gestação: vivências de gestantes
de fetos com anencefalia
On the way to interrupting the gestation or not: experiences of
pregnant women with anencephalic fetuses
Abstract Anencephaly is a malformation cha-
racterized by the total or partial absence of the
brain, and Brazil records the fourth largest num-
ber of births of anencephalic fetuses in the world.
Fetal anencephaly is associated with a more sig-
nificant number of maternal complications. As of
2012, women with anencephalic gestation were
empowered with the right to carry the pregnan-
cy to term or terminate it, if they so desired, wi-
thout any judicial authorization. Objectives: to
understand the experiences of women with fetal
anencephaly and to identify the determinant fac-
tors for interrupting the gestation or not. This is
a qualitative study using the Life Narratives me-
thod with 12 women over 18 years old diagnosed
with an anencephalic fetus, who interrupted ges-
tation or delivery in a public maternity hospital in
Rio de Janeiro. Data were collected between June
and November 2016, and the process was finalized
when the narrative patterns reached progressive
saturation from the recurrences. The statements
that emerged following floating and in-depth re-
ading were articulated in Narrative Nuclei, and
data comparative and comprehensive analysis
was performed. The reports brought to light the
intense experiences of these women, as well as the
weaknesses existing concerning care and the preg-
nancy termination issue.
Key words Anencephaly, Legal abortion, Abor-
tion candidates, Women’s health, Public health
DOI: 10.1590/1413-81232020252.14812018 429
ARTIGO ARTICLE
Resumo A anencefalia é uma malformação ca-
racterizada pela ausência total ou parcial do en-
céfalo e o Brasil é o quarto colocado em número
de nascimentos de fetos anencéfalos no mundo.
Existe associação entre anencefalia fetal e maior
número de complicações maternas. A partir de
2012 a mulher com gestação de anencéfalo pode-
rá manter ou interromper a gestação, se assim o
desejar, sem necessidade de autorização judicial.
Objetivos: compreender as vivências das mulhe-
res de fetos com anencefalia e identificar os fatores
determinantes para a escolha de interromper ou
não interromper a gestação. Estudo qualitativo e
método das narrativas de vida, com 12 mulhe-
res, maiores de 18 anos e com diagnóstico de feto
anencéfalo, que realizaram a interrupção da ges-
tação ou o parto em uma maternidade pública do
Rio de Janeiro. A coleta dos dados foi entre junho e
novembro de 2016 e encerrada quando os padrões
narrativos alcançaram a saturação progressiva, a
partir das recorrências. Os enunciados emergidos
após leitura flutuante e aprofundada foram arti-
culados em Núcleos Narrativos e realizada análise
comparativa e compreensiva dos dados. Os relatos
trouxeram à tona as vivências intensas dessas mu-
lheres, como também as fragilidades existentes em
relação ao cuidado e a problemática da interrup-
ção da gestação.
Palavras-chave Anencefalia, Aborto legal, Aspi-
rantes a aborto, Saúde da mulher, Saúde pública
 430
Fernandes IB et al.
Introdução
A anencefalia é uma malformação caracterizada
pela ausência total ou parcial do encéfalo, sendo
esta última a forma mais comum; ela é incom-
patível com a vida extrauterina, sobrevivendo o
feto somente por horas ou dias após o parto1-3.
De acordo com dados da Organização Mundial
da Saúde, Brasil é o quarto colocado no número
de nascimentos de fetos anencéfalos (1/1600 nas-
cidos vivos), ficando atrás somente do México,
Chile e Paraguai2,4. Das gestações que são levadas
a termo, aproximadamente 75% dos anencéfa-
los são natimortos e o restante morre no perío-
do neonatal4,5. Deparar-se com o diagnóstico de
um filho incompatível com a vida extrauterina
leva os pais a enfrentarem grande sofrimento e
intensas vivências emocionais6, além de compli-
cações de saúde na gestante, como hipertensão
arterial, gravidez prolongada, polidramnia, entre
outros4,5.
Apesar do risco de complicações físicas e
emocionais, somente em abril de 2012 o Supre-
mo Tribunal Federal decidiu pela descriminali-
zação da interrupção da gestação de fetos anen-
céfalos. Estabeleceu-se o direito pela interrupção
terapêutica da gestação sem precisar de autori-
zação judicial prévia ou pela manutenção desta,
se a mulher assim o desejar5,7. A prática da inter-
rupção da gestação em casos de anencefalia não
pode ser configurada como abortamento, pois se
trata de interrupção terapêutica da gestação ou
antecipação terapêutica do parto, pelos riscos au-
mentados à saúde das gestantes e fetos sem possi-
bilidade de vida após o nascimento3,5.
Estudos sobre anencefalia e aborto legal refe-
rente à realidade brasileira nos últimos 15 anos
apontou somente para três estudos4,6,8 que dis-
cutiram especificamente os aspectos emocionais
da gestação de feto anencéfalo. Assim, são obje-
tivos deste artigo: compreender as vivências das
mulheres de fetos com anencefalia e identificar
os fatores determinantes para a escolha de inter-
romper ou não interromper a gestação.
Métodos
O método de estudo foi das Narrativas de Vida
nas proposições de Bertaux9, com participação de
12 mulheres maiores de 18 anos e com diagnósti-
co de feto anencéfalo, que realizaram a interrup-
ção da gestação ou o parto em uma maternidade
pública na cidade do Rio de Janeiro. A coleta de
dados se deu após a apreciação e aprovação pelo
Comité de Ética em Pesquisa da maternidade,
respeitando as normas da Resolução 466/1210. A
entrevista ocorreu em local e horário de disponi-
bilidade das participantes, após a assinatura do
Termo de Consentimento Livre e Esclarecido, no
período entre junho e novembro de 2016. Uma
única pergunta disparadora foi realizada: “Você
pode me contar como foram as suas gestações?”.
Demais inferencias foram feitas respeitando o
que era dito pela participante. As entrevistas fo-
ram realizadas com um intervalo de, no mínimo,
um ano após o parto referente ao bebê anencé-
falo, permitindo à entrevistada refletir acerca da
sua vivência, buscando através desta estratégia,
minimizar desconfortos pelo processo de ressig-
nificação da história vivida. Os padrões narrati-
vos alcançaram a saturação progressiva na quinta
entrevista, a partir das recorrências e a coleta dos
dados foi encerrada na décima segunda entre-
vista, permitindo a pesquisa de caso negativo9,11.
Após as trascrições e leituras das entrevistas, os
enunciados emergidos foram articulados em
dois Núcleos Narrativos, de modo a possibilitar
uma análise compreensiva e comparativa à luz
de Bertaux9. Foi respeitado o anonimato e cada
entrevista foi identificada utilizando a letra E (de
entrevista) seguida por sistema alfanumérico de
1 a 12.
Resultados e discussão
As doze mulheres entrevistadas encontravam-se
em idade reprodutiva, na faixa etária entre 23 e
42 anos. A maioria declarou ter um filho vivo,
com variação de zero a três filhos por participan-
te. Nove identificaram-se como casadas ou em
união estável e as outras se declararam solteiras.
Dez possuíam ensino médio e superior e nove re-
lataram estar formalmente empregadas. A renda
familiar variou entre zero e 11 salários mínimos.
Em relação à gestação do bebê anencéfalo, em
metade dos casos se tratou da primeira gestação
e 75% das mulheres declararam ter se tratado de
gestação planejada. Em relação à entrada no Ju-
diciário a fim de obter a autorização para a inter-
rupção terapêutica da gestação, quatro das mu-
lheres recorreram, apesar de em oito casos, existir
jurisdição para o procedimento.
Algumas mulheres tiveram que sepultar seus
filhos, pois se tratava de fetos com idade gestacio-
nal superior a seis meses. Se a interrupção ocor-
rer com idade gestacional inferior a 20 semanas
de gestação, o feto pesar menos de 500 gramas
e estatura menor que 25 centímetros, a declara-

ção de óbito é facultativa, conforme Legislação
vigente, sendo emitida quando há o desejo fami-
liar de sepultamento. Caso contrário, o feto ficará
no hospital, sendo posteriormente incinerado ou
entregue à adequada coleta hospitalar12.
Do ventre pleno ao colo vazio: um trânsito
entre a expectativa e realidade
Um primeiro eixo a ser considerado nesta
análise é que a concretização da maternidade
biológica permanece, para algumas mulheres,
como um forte desejo e que se confunde com os
papéis de gênero socialmente construídos. É sa-
bido que na contemporaneidade – considerando
os senões que esta classificação evoca – as mulhe-
res têm conquistado diversos espaços do âmbito
público, buscando realização profissional, inser-
ção no mercado de trabalho, vocalização política
e autonomia econômica.
As mulheres, mesmo sendo maioria numéri-
ca no Brasil (51% da população), ainda recebem
30% menos do que homens na realização dos
mesmos trabalhos, possuem menos cargos de li-
derança e, não somente contribuem com a renda
de suas famílias, mas são arrimo de muitas13. Para
uma parte destas mulheres um dos seus projetos
pessoais é o desejo da maternidade. Mais do que
isso, a sociedade ainda demanda, fortemente, esta
questão enquanto um papel social da mulher4.
As noções de vocação e obrigatoriedade em
gestar e parir um filho biológico possuem uma
historicidade que confere aspectos identitários
atrelados à mulher, que reúne representações
capazes de estabelecer nexos entre ‘ser mulher’ e
‘ser mãe’. Cumprir este papel social, que possui
raízes socioculturais patriarcalistas e utilitaristas,
é como atender a uma ampla tradição cultural
que é, diretamente ou indiretamente, imposta às
mulheres4.
Neste caminho, uma pergunta é estruturan-
te para este núcleo narrativo, isto é: quais as ex-
pectativas sobre a gestação compartilhadas pelas
mulheres? A enfática afirmativa de E2 quando
soube do diagnóstico da gravidez – “a gente fi-
cou super feliz” – representa de maneira índex as
expectativas das participantes deste estudo e seus
parceiros, pois grande parte das gestações era de-
sejada pelos casais e, nestes casos, a gravidez foi
vivida com alegria e realização pessoal. Apenas
no depoimento de E3, mãe de três filhos, encon-
trou-se traços de insegurança devido a uma situ-
ação financeira difícil, ao mesmo tempo em que
se tratava de uma gestação não planejada. Mes-
mo assim, a participante compartilhou do mes-
431
Ciência & Saúde Coletiva, 25(2):429-438, 2020
mo sentimento de alegria quando do diagnóstico
de sua gestação.
A valoração da maternidade desde os idos do
que se considera antiguidade leva muitas mulhe-
res a depositarem um significado pessoal e muito
importante em gerar um filho. A gravidez pode
representar a confirmação da sua feminilidade
e posição de aparente prestígio social14. Há tam-
bém um conjunto de sonhos, esperanças e ex-
pectativas que as mulheres depositam na futura
gestação, idealizando e imaginando o ‘ser’ que
está em seu útero, sendo a mulher e muitas vezes
o cônjuge envolvidos por sentimentos de realiza-
ção pessoal e a idealização de uma criança perfei-
ta15,16. Em situações de vulnerabilidade, como no
caso supracitado de E3, a literatura aponta que
as mães solteiras possuem problemas com baixa
renda em decorrência de oferta de emprego defi-
ciente, com a falta de apoio do pai, dos escassos
benefícios públicos, além de terem maior neces-
sidade de redes de apoio17.
Assim, a realidade idílica da maternidade en-
quanto um atributo das mulheres, juntamente
com as elevadas expectativas das participantes
deste estudo com suas gravidezes, impuseram
outras reflexões. Afinal, o que a gravidez de um
feto com anencefalia apresenta de realidade con-
creta a estas mulheres? Se por um lado o diag-
nóstico da gravidez trouxe alegria, o diagnóstico
da anencefalia trouxe uma gama de sentimentos
negativos. Expectativas foram colocadas à pro-
va. Tais sentimentos negativos foram comparti-
lhados pela mulher, cônjuge e família trazendo
uma ruptura relacionada à idealização do bebê
perfeito15. A gestação se torna uma realidade de
intenso sofrimento e angústia emocional devido
à frustração de expectativas16. É o difícil trânsi-
to entre as expectativas relacionadas a felicidade
para a árdua realidade em deparar-se com um
feto malformado – um duplo que foi vivido, de
forma agudizada no diagnóstico, passando por
decisões, pelo parto, pelo destino do bebê morto
e no retorno ao lar.
A gravidez de um feto anencéfalo gerou nas
mulheres e seus cônjuges reações que envolveram
palavras, tais como relatadas nos depoimentos:
choque; angústia; tristeza; sofrimento; desespero;
pânico e desorientação. Uma palavra do verná-
culo popular surgiu em quatro depoimentos e
carrega a noção do sentimento destas mulheres,
ou seja, elas verbalizaram que foi um baque des-
cobrir que o bebê era malformado:
perdi o meu chão, eu tava sozinha […] aí eu
desabei, chorei, […] foi dias assim terríveis [...]
levei quase uma semana naquela situação – E12.
 432
Fernandes IB et al.
Defronte às expectativas frustradas e sen-
timentos angustiantes, as mulheres narraram
sobre outro sentimento que foi a culpa. A cul-
pabilização diante do ocorrido se deu por uma
autocobrança, assim como, na evidente relação
com o homem, uma vez que ela “carrega o bebê”
elevando o seu status de maior propensão em se
considerar responsável pelo desfecho da gesta-
ção16. Nestes meandros, elas questionaram a situ-
ação inesperada em busca de explicações e justifi-
cativas, nem sempre obtidas com êxito18:
“porque comigo? que que eu fiz de errado?” você
se pergunta – E6.
Outra realidade foi desconhecer o que era
anencefalia. Das mulheres entrevistadas, nove
delas nunca tinham ouvido falar neste problema
e o desconhecimento foi um fator que atrelou ao
processo sentimentos de ansiedade e estresse. É
preciso considerar que, diante deste tema delica-
do e pouco falado, os profissionais responsáveis
em noticiar este diagnóstico a uma mulher de-
vem exercitar aquilo que se chama de comunica-
ção de notícias difíceis.
O exame radiológico é um procedimento téc-
nico, lugar onde o diagnóstico da anencefalia é
obtido. É preciso pontuar que o procedimento
é realizado em um indivíduo que possui subje-
tividades que não podem ser desconsideradas
e, neste sentido, é iminente que os profissionais
tenham consciência, pois também possuem suas
subjetividades, de que ao realizar o procedimen-
to, estão diante de alguém que se apresenta com
uma forte carga emocional. Afinal, em última
instância, é precisamente este profissional que
apresenta, pela primeira vez, a imagem do filho
à sua mãe e está em jogo nesta cena clínica a in-
formação relacionada a saúde, ou não, do bebê19.
Das participantes deste estudo, sete deram a
luz fetos com idade gestacional superior a seis
meses e, por conseguinte, os bebês tiveram que
ser enterrados. Eis uns dos acmes da ruptura en-
tre o imaginário e a realidade posta a estas mulhe-
res – o sepultamento. O diagnóstico de um bebê
com anencefalia impôs, de pronto, a vivência de
um luto, no plano das ideias, paulatinamente ex-
perienciado até o momento do parto. Diante do
bebê nascido e morto, o luto passa para o plano
concreto – visível e palpável. Este movimento
implicou em um processo de reajuste emocional
por parte das mulheres, seus parceiros e família,
de acordo com os recursos subjetivos disponí-
veis18. As outras cinco participantes da pesquisa
não precisaram viver a situação de sepultar o
filho, mas passaram por outra, a de autorizar o
estudo genético dos seus bebês.
Considerando que a sensação de frustração e
perda não se encerra com o nascimento e destino
dos bebês mortos, o retorno ao lar se caracteri-
zou como o segmento da experiência vivida de
uma realidade não esperada. As participantes do
estudo também utilizaram palavras carregadas
de intensidade sobre o assunto: tristeza; perda;
dor; inaceitação; revolta; culpa e vazio. O trânsito
entre o idealizado e a dura realidade da ausên-
cia do bebê foi presente independente do tempo
de gestação em que a mesma foi interrompida18.
Para muitos pais, o nascimento de um filho é um
pórtico de esperança para o futuro. Estar dian-
te de um filho com defeitos é interromper esta
idealização, é deparar-se com um futuro imper-
feito onde o luto é uma resposta em relação, não
somente à perda de uma criança idealizada, mas
também às expectativas malogradas20,21:
a parte pra mim mais difícil foi a chegada em
casa […] você de cesárea […] doía muito olhar pra
marca e […] não ter o neném – E5.
Uma das entrevistadas (E7) relatou ter sofri-
do de estresse pós-traumático após a interrupção
por cesariana no término da gestação. Apesar da
decisão de não antecipar o parto ter sido unâni-
me entre o casal, a mulher teve o diagnóstico de
estresse pós-traumático na volta para o trabalho
e apontou que com o apoio incondicional do es-
poso e família, conseguiu superar a situação. Os
sintomas de estresse pós-traumático podem ser
verificados em mulheres que realizam a interrup-
ção da gestação, principalmente, em idade gesta-
cional mais avançada6.
Para E2, a perda de um bebê anencéfalo sig-
nificou romper com o desejo de uma nova gesta-
ção – “não senti a vontade de ter outro filho, fiquei
muito traumatizada”. Para outras participantes,
o desejo de engravidar falou mais alto, como foi
o caso de três delas. Para duas entrevistadas, a ex-
periência da perda anterior as levou a uma gesta-
ção vivida com medo e apreensão relacionados
a gerar um bebê malformado. O trauma gerado
pela perda anterior potencializa uma memória
nada silenciosa, aquela que não quer tornar a vi-
venciar uma gestação frustrada, tampouco, o luto
posterior18:
Dessa minha gestação agora […] toda ultra
que eu vou bater eu vou com aquele desespero – E5.
Diante desta realidade, a presença do com-
panheiro e demais familiares, que demonstrem
preocupação e que providenciem apoio, tem um
papel fundamental para contribuir com a recupe-
ração do equilíbrio emocional necessário à mu-
lher que sofreu a perda. A família pode cooperar
no processo de luto e o suporte fornecido, sem

dúvida, auxiliará o casal a elaborar a perda a par-
tir do que se estabelece enquanto rede de apoio18.
A maioria das participantes deste estudo teve
apoio do companheiro e/ou da mãe, um aspec-
to central para a vivência do problema, desde o
diagnóstico até o retorno ao lar. Mas nem sempre
esta realidade se apresentou para todas as mulhe-
res como foram os casos de E3 que não teve ne-
nhuma rede de suporte, E4 sem apoio do marido
e E8 que buscou ajuda na figura do marido e da
mãe, não obtendo em nenhum dos dois. Vale en-
fatizar que na presença de companheiros, mães
e demais familiares, o profissional pode, além
de acolher e orientar a mulher, fazer o mesmo
com sua rede familiar explicando que o apoio é
fundamental para a vivência deste momento de
vida. Existem recursos pessoais e socioecológicos
de enfrentamento no processo de gestar um bebê
com malformação e aqueles que perpassam pelas
crenças ideológicas e pelo relacionamento conju-
gal, assim como, as redes sociais22:
a família muito do meu lado, minha mãe [...]
meu esposo sempre perto – E10.
A importância da rede de apoio pode ser
identificada também por sua ausência, através
dos relatos das mulheres que declararam ter vi-
vido um percurso solitário. Narrativas que apon-
tam para ausência de suporte do companheiro,
família e pessoas próximas, assumidos enquanto
entraves para atravessar a situação:
eu sempre fui sozinha, meu marido nunca quis
me acompanhar […] Para mim assim, eu me senti
sozinha, sozinha, sozinha – E8.
Não suficiente a ausência de suporte, a par-
ticipante E6 revelou que sofreu perseguição por
parte de um grupo de pessoas religiosas. Uma vez
tendo manifestado o desejo de interromper a ges-
tação e por ter recorrido ao judiciário foi acusada
de ser uma assassina. Não ter uma rede de apoio
é, para a mulher, viver uma experiência solitá-
ria e angustiante diante de uma gravidez de feto
anencéfalo. Ser acusada de assassina por decidir
interromper a gestação, além de ferir seu direito
adquirido, é fazê-la passar por uma situação de
violência, aquela perpetrada por pessoas que, a
princípio, deveriam dar apoio e acolhimento, é
ser julgada moralmente/espiritualmente por pes-
soas e instituições:
tava negócio de igreja me perturbando, chegou
a ir na minha casa, […] iam na minha porta, falar
que eu era uma assassina, porque eu tava querendo
tirar – E6.
Os julgamentos em questão possuem raízes
na polemização da questão do aborto no Brasil,
que a literatura levanta com frequência, mes-
433
Ciência & Saúde Coletiva, 25(2):429-438, 2020
mo em casos de fetos gravemente acometidos. A
oposição à interrupção da gravidez em casos de
anencefalia é de fundo religioso23 e como a grande
maioria da população brasileira se autodeclara re-
ligiosa, é possível presenciar tais comportamentos
voltados à mulher que decide pela interrupção da
gestação, uma negação de direitos.
Ante as expectativas compartilhadas pelas
mulheres desta pesquisa versus a concretude com-
pulsória da realidade vivida de um feto anencéfa-
lo, este núcleo narrativo assenta que a ausência de
rede de apoio e julgamentos diversos constituem
uma violência contra as mulheres. Em contra-
partida, a gestante precisa encontrar um ambien-
te seguro e acolhedor, um facilitador para que a
mesma verbalize e reflita seus sentimentos, pre-
ocupações, significações, crenças e necessidades
pessoais, sem medo de represálias. Ela deve ser
respeitada, antes de tudo, evitando-se juízos de
valor, independente de sua decisão, diminuindo
assim possíveis sentimentos de culpa8.
Neste difícil trânsito das expectativas à rea-
lidade, com todas as vicissitudes presentes, um
aspecto merece relevo, o da resiliência das mulhe-
res. A maioria das participantes deste estudo ma-
nifestou ter se tratado de uma vivência que lhes
trouxe um grande aprendizado. Elas consegui-
ram significar o acontecido, superar limites dan-
tes inimagináveis, verbalizando um amadureci-
mento gerador de fortes transformações de sua
vida, de seus companheiros e de suas famílias20:
é uma experiência de vida… a gente começa a
amadurecer sem querer – E11.
O percurso empreendido pelas mulheres:
decisões e cuidados
Para a compreensão do percurso empreendi-
do pelas mulheres é preciso, de imediato, situar
onde e como se deu o diagnóstico do problema
e a assistência prestada pelos profissionais, para
depois compreender escolhas e decisões. Foi no
espaço das consultas de pré-natal ou nos centros
de ultrassonografia que as mulheres receberam a
difícil notícia de que estavam vivendo uma gra-
videz de um bebê anencéfalo. As mulheres deste
estudo relataram que os profissionais foram cui-
dadosos em noticiar o diagnóstico e esclarecer
os pormenores que envolviam informações rela-
cionadas ao total desconhecimento deste tipo de
problema, das especificações técnicas sobre a do-
ença e das possibilidades de escolha frente a esta
situação, que envolvia o pensar, escolher e decidir
por interromper ou não a gestação, uma vez que
o desfecho já era sabido.
 434
Fernandes IB et al.
Neste sentido, foi neste espaço relacional – a
consulta pré-natal ou centro de ultrassonografia
– que, por um lado, profissionais cuidaram destas
mulheres seguindo os protocolos assistenciais,
fornecendo esclarecimentos que incluíam os re-
lacionados aos seus direitos e legislação vigente6
e noutra face, as mulheres e seus companheiros
frente as suas subjetividades que influenciariam
suas escolhas. Embora pareça, a princípio, uma
decisão focal – a opção pela interrupção da gesta-
ção ou de levá-la a termo – as escolhas se mostra-
ram complexas dada a pluralidade de situações
que atravessam este processo. Cumpre pontuar
também que, em se tratando de um espaço rela-
cional, a relação profissional/mulheres e compa-
nheiros foi construída a partir das intersubjetivi-
dades envolvidas.
A equipe médica tem a responsabilidade de
informar a gestante sobre a malformação e prog-
nóstico letal, já que nenhuma abordagem tera-
pêutica poderá trazer melhora. O médico deverá
ainda expor a possibilidade de interromper a ges-
tação, seja pelos meios legais, através do alvará de
autorização, seja através de uma ultrassonogra-
fia com a assinatura de dois médicos. Não cabe
a ele impor ou manifestar sua opinião e deve-se
buscar imparcialidade nas informações forneci-
das, tal como esclarecimentos completos sobre os
procedimentos diagnósticos e terapêuticos6,8, fato
revelado nas entrevistas:
“a decisão é de vocês”, explicou todos os direitos
legais que a gente teria [...] ela foi muito delicada,
atenciosa e falou isso tudo segurando na mão, foi
bem, bem, bem humana – E7.
Segundo literatura, a disponibilidade de in-
formação e uma atenção especializada da rede de
saúde são elementos essenciais para a gestante e
sua família se reorganizarem diante desse diag-
nóstico e ajudarem no enfrentamento e manejo
da situação16.
O cuidado de qualidade oferecido pelos pro-
fissionais é um resultado esperado uma vez que
esta pesquisa foi desenvolvida em uma institui-
ção que possui um histórico de atendimento às
gravidezes de fetos anencéfalos antes mesmo da
legislação vigente. Isto é, além de habituados, os
profissionais são qualificados para este cuidado,
cuidado que deve estar presente nos demais espa-
ços de cuidado à saúde das mulheres, pois diante
da condição de gestar um bebê com malforma-
ção, a mulher necessita de uma atenção especial
por parte dos profissionais de saúde, sendo aco-
lhida e respeitada16. Este papel deve ser assumi-
do pelos profissionais de saúde em todos os mo-
mentos da assistência, a começar pelo pré-natal15.
A interrupção da gestação em si pode ser
classificada como um dilema nestes casos, onde
comparecem questões variadas desde a escolha
decidida, a não escolha decidida e a do âmbito
das dúvidas. Esta última revelou desfechos diver-
sos, desde complicações com sua saúde materna,
questões judiciais e religiosas.
Nos casos das participantes E1, E3 e E8 esta-
va-se diante de mulheres decididas a interromper
a gestação, com escolhas bem situadas. Dentre seus
motivos aponta-se: o desejo de findar com aque-
la gestação, de uma vez por todas, abreviando o
tempo para poder tentar engravidar novamente
(E1); e o fato de uma delas, uma vez sabendo que
sua gestação atual não lhe daria um filho saudá-
vel, não quis “ficar presa” (E3) e priorizou voltar
logo para casa e para o cuidado com seus três fi-
lhos.
Das 12 mulheres, apenas duas não quiseram
interromper e conseguiram manter suas escolhas
decididas até o fim (E5 e E7), seus bebês nasce-
ram através de uma cesariana a termo. Para E5 a
decisão de não interromper teve íntima relação
com aquilo que relatou como falta de coragem
relacionada aos sentidos atribuídos ao amor ma-
terno. No caso de E7, seu desejo era de dar um
nome ao bebê e após nascer, que este pudesse ser
enterrado junto da sua família.
Muitas mulheres conseguem encontrar sen-
tido no fato de prosseguirem com a gestação,
mesmo diante da inviabilidade do feto e devem
ser respeitadas na sua vontade de continuarem
grávidas8. Para algumas há a necessidade de dar
um nome e de enterrar24.
Quatro mulheres interromperam suas ges-
tações por motivos relacionados com sua saúde
(E2, E4, E10 e E12). No caso da E2 e E10, estas
não demoraram na decisão de interromper, devi-
do às complicações sentidas. Em contrapartida, a
E12 decidiu pela interrupção somente no terceiro
trimestre, quando as complicações começaram a
comprometer a qualidade de vida do casal:
eu estava com arritmia direto e outras compli-
cações – E10.
Apesar da presença de complicações na saú-
de, a decisão de interromper no caso da E4 se
mostrou complexa, já que ela não queria, de fato,
interromper a gestação, pela crença de que a mãe
não deve interferir no tempo de vida fetal24:
eu não conseguia aceitar o fato que eu tinha
que interromper, eu queria deixar correr natural
– E4.
Gestações de fetos anencéfalos estão relacio-
nadas a complicações obstétricas diversas, eis um
dos pontos levantados pela literatura a favor da

interrupção da gestação. Dentre algumas com-
plicações estão: doenças hipertensivas; compli-
cações no momento do parto; insuficiência renal
e cardíaca; descolamento prematuro da placen-
ta; ruptura prematura das membranas; e infec-
ção são identificadas nesses casos. Mais do que
somente as intercorrências clínico-obstétricas, é
preciso valorizar as consequências psicológicas
de uma gestação de feto anencéfalo8,24. Tanto as
complicações físicas quanto as de ordem emocio-
nal foram fatores que influenciaram nas decisões
das mulheres deste estudo.
É interessante observar as narrativas de E6,
E9 e E11, mulheres que, a princípio, desejaram
interromper, mas que tiveram trajetórias que en-
volveram dificuldades na justiça, desistência da
interrupção e interrupção em idade gestacional
avançada. Assim, cumpre aqui situar pontos ne-
vrálgicos que atravessaram suas decisões iniciais.
Como abordado anteriormente, quatro mulheres
tiveram que judicializar sua interrupção, dentre
elas estavam E6 e E9. De início, as duas queriam
interromper, tanto que recorreram a justiça, mas
com o passar do tempo mudaram de ideia por
diferentes motivos.
O caso da depoente E6 chamou a atenção de-
vido à peregrinação sofrida, em busca da auto-
rização para interromper a gestação, o que mais
tarde resultou na divulgação deste caso na mídia.
Inclusive, esse foi um dos fatores que auxiliaram
na promulgação da súmula da interrupção de
gestações de fetos anencéfalos. Esta mulher en-
frentou a constrangedora negação do seu pedido
por três vezes, fato frequente no Brasil, antes da
promulgação da súmula8:
foi muito difícil, todo dia eu ia […] só levava
não e não […] ele (o desembargador) achou um
absurdo […] foi e jogou pra mídia […] com esse
depoimento do caso da minha neném [...] passou
a liberar – E6.
Após tanto tempo de negativas, ela já havia de-
sistido da interrupção. Por fim, aos sete meses de
gestação chegou a decisão favorável e a depoente
resolveu interromper a gestação por cesariana.
No caso de E9, ela entrou em trabalho de par-
to aos seis meses e, naturalmente, a gestação foi
interrompida. Ocorre que ela, neste percurso, foi
intimada judicialmente para interromper, mas foi
algo que como ela mesma disse “deixou para lá”:
eu entrei pra tentar fazer o aborto, mas no fun-
do eu não queria tirar – E9.
Para E11 a mudança de ideia se deu por mo-
tivos religiosos, em seu caso o bebê acabou vindo
a óbito aos seis meses e o parto foi induzido. A
literatura mostra que gestar um feto anencéfalo
435
Ciência & Saúde Coletiva, 25(2):429-438, 2020
está relacionado com trabalho de parto prema-
turo e morte intraútero1,4,24 e assim foi no caso
destas mulheres.
A depoente E2 desejou interromper, acionou
a justiça e, de fato, concretizou a interrupção tan-
to pelo feto ser anencéfalo quanto por compli-
cações em sua saúde. Entretanto, é um caso que
revela exemplarmente a angústia vivida por algu-
mas mulheres diante da situação de interromper
ou não a gestação, onde escolhas são postas em
contraditório. Para E2, os motivos também esta-
vam relacionados aos seus valores religiosos. Ela
afirmou de maneira veemente que era “contra o
aborto”, mesmo tendo optado pela interrupção
da gestação. No contradito entre interromper ou
não a gestação, optou pela primeira opção. Sua
assertiva enfática foi uma maneira de assumir
um mea-culpa:
quando eu fui pegar o alvará, eu me senti mui-
to mal no tribunal, eu num lugar criminal, uma
bandida igual um bandido – E2.
Neste caso, a ambiguidade de sentimentos
foi notável e revelada em um conflito interior
entre interromper ou não a gestação, levando-a
a repensar conceitos condenatórios daquilo que
ela intitulava como prática abortiva. Notam-se
questões de foro íntimo geradoras de inconfor-
midades, entre elas, a culpabilização de si mesma,
a sensação de estar cometendo um crime e a de-
cisão de ‘esconder o cometido’ perante os amigos.
Com sentimentos de culpa, as mulheres podem
vivenciar a interrupção como uma marca de ca-
ráter e no sentido de não serem estigmatizadas
pelos amigos e pela sociedade, há uma necessida-
de de silenciar essa prática25. A religião é um dos
fatores referidos pelas gestantes para não dar fim
a gestação8.
Se o espaço relacional do pré-natal foi evi-
denciado enquanto um sítio de cuidado de qua-
lidade, os processos que envolveram a internação
hospitalar foram diferentes. O cuidado humanís-
tico dos profissionais manteve-se presente, mas
as dificuldades encontradas situavam-se nos as-
pectos subjetivos das mulheres relacionados à
aproximação do momento do parto e de aspectos
objetivos relacionados à infraestrutura da mater-
nidade.
Internar-se para parir um feto inviável é estar
diante de uma vivência dolorosa. Sentimentos
diversos envolveram as mulheres por ocasião das
suas admissões hospitalares: nervosismo, deses-
pero, tristeza, dor e sofrimento intenso. E2 foi um
caso extremo, pois teve ideal suicida devido à ex-
periência de passar pelo trabalho de parto e não
conseguir levar o bebê para casa:
 436
Fernandes IB et al.
fiquei muito depressiva, fiquei muito doente
[…] coisas que eu nunca pensei na minha cabeça,
de me jogar em uma janela, eu tive – E2.
Na aproximação do desfecho não desejado,
as mulheres referiram que o momento do parto
com a visualização do bebê foi um dos piores mo-
mentos vividos. Nem todas as mulheres quiseram
ver seus filhos, quatro delas, por acreditarem que
o fato de entrar em contato e ver as deformidades
iria dificultar o processo do luto6:
não quis ver, não quis saber o sexo, não quis
saber nada – E2.
O restante das entrevistadas fez questão de
ver o bebê logo após o parto, tanto pela curio-
sidade de visualizar as deformidades, como para
se despedir. Deve-se considerar que elas já esta-
vam lidando com um luto ainda não palpável, de
um bebê intraútero, a vontade de despedida é o
início da concretização do luto a partir do real
visualizado:
eu quis […] dei tchau pra ele, eu, sabe, eu quis
me despedir dele – E4.
O parto e o nascimento representam momen-
tos de confronto entre a, ainda, esperança de ter
ocorrido um erro no diagnóstico de malforma-
ção e as reais condições do recém-nascido, além
de ser o primeiro encontro direto com o bebê.
Esse momento é angustiante e marcado pela ex-
pressão do luto refletido na ausência de um bebê
saudável15. Algumas mães necessitam ver o feto
com todos os problemas, para que consigam crer
plenamente, e a partir de uma realidade visível,
no diagnóstico da malformação, para estabelecer
um sentido psíquico para essa vivência6. Cabe a
equipe que presta os cuidados acatar a escolha da
mulher, em respeito ao momento vivido e pro-
porcionar o que for necessário para que esta mu-
lher possa experienciar este encontro com o bebê
da maneira menos traumática possível.
Uma vez dados por encerrados o parto e a
(não) despedida do bebê morto, essas mulheres
passaram para o período em que foram encami-
nhadas, na instituição, para os setores de inter-
nação pós-parto. Existe um marcador objetivo
que pode facilitar ou dificultar a vivência deste
momento – o local onde esta mulher passou por
seu período puerperal na instituição.
Isso ocorre, pois geralmente elas são encami-
nhadas a uma enfermaria chamada Alojamento
Conjunto. O problema não é o local propriamen-
te dito, mas por ser um espaço compartilhado
com mulheres que estão acompanhadas de bebês.
Para uma mulher que perdeu seu filho estar alo-
cada no mesmo espaço com mães de bebês, em
geral, saudáveis, é um ato violento. Isso não ocor-
re por insensibilidade dos profissionais, mas, em
geral, por uma ausência de um espaço restrito a
estas mulheres e/ou, até mesmo, por hiperlotação
das maternidades. Ainda que sejam bem tratadas
e cuidadas, o local será sempre um deflagrador
de memórias negativas, para grande maioria das
mulheres.
O próprio conceito de ‘maternidade’ enquan-
to ambiente hospitalar encerra a ideia de que ali
é um lugar onde se encontram as mães e seus
bebês. E, de fato, é assim que ocorre. Rotinas e
situações das mais diversas preenchem este es-
paço de sentidos: banhos, cuidados com o coto
umbilical, amamentação, troca de fraldas, choro
dos bebês etc. Ou seja, não são espaços para mu-
lheres que perderam seu filho, não propiciando a
vivência do luto. Ao contrário, potencializa ques-
tões do âmbito emocional trazendo fragilidades
incomensuráveis. Os relatos das participantes
apontaram para o desejo de experimentar colo-
car os bebês no colo e, desta forma, retornaram
ao imaginário de poder sair com ele nos braços
na alta hospitalar.
Dividir o espaço com outras mulheres e seus
filhos coloca as que vivenciaram a perda em con-
tato direto com tudo o que o seu filho represen-
tava deixando evidente a falta, a sensação do não
ganhar18. A internação proporciona contato com
gestações que terminaram em sucesso, o que pro-
voca sentimentos mais intensos de fracasso, mais
pela incapacidade sentida no gestar e pela ausên-
cia do filho imaginado e perdido, do que propria-
mente pela interrupção6,8.
O depoimento de E6 é capaz de mostrar a
importância de não estar internada no mesmo
espaço, coabitando com mães e bebês, naquele
período de tempo. Ela relatou que a equipe teve
o cuidado de deixá-la em uma enfermaria de ges-
tantes após o parto, na tentativa de minimizar a
dor da perda:
quando eu ganhei […] me botaram junto com
as outras que iam ganhar, pra não ficar junto das
que já tiveram neném – E6.
É preciso valorizar o preparo e a sensibiliza-
ção dos profissionais de saúde que atuam dire-
tamente com as mulheres enlutadas como, por
exemplo, na escolha da enfermaria que a mulher
será alocada após o parto18. Devem-se conside-
rar, evidentemente, as condições, infraestrutura
e lotação das enfermarias. Mas, se possível, não
se deve colocar mulheres que sofrem perda em
enfermarias compartilhadas com mães e bebês.
Diante disso, o cuidado humanizado, direito
da mulher garantido pelas diretrizes do Sistema
Único de Saúde (SUS), é fundamental na elabo-

ração do luto vivenciado pelos pais desde o mo-
mento do diagnóstico de malformação, passando
pelo momento da internação e após a alta16. Des-
ta forma, cabe aos profissionais de saúde ofertar
uma assistência qualificada e o suporte emocio-
nal adequado para promover o enfrentamento
dessa situação18.
Considerações finais
Conhecer as narrativas de mulheres que viven-
ciaram gestação de fetos anencéfalos nos impul-
siona a pensar sobre o despreparo dos serviços
– enquanto instituições e enquanto pessoas que o
concretizam – em lidar com o processo de decisão
vivenciado por mulheres, seus parceiros e suas fa-
437
Ciência & Saúde Coletiva, 25(2):429-438, 2020
mílias. Em cada narrativa de vida, somos defron-
tados pela escassez de literatura que dialogue com
as (novas) questões analisadas nos depoimentos.
Os sentimentos vivenciados pelas mulheres
durante toda a trajetória (tornados notórios du-
rante as entrevistas), o problema da interrupção
da gestação de fetos anencéfalos e as dificuldades
enfrentadas durante todo o percurso são pontos
cruciais que o estudo identificou. No entanto,
as fragilidades que precisam ser geridas para a
construção de um SUS pleno, visando melhorias
à atual (des)organização do cuidado a estas mu-
lheres, e a garantia de seus direitos reprodutivos,
muitas vezes desrespeitados, são reflexões neces-
sárias para avançarmos no campo da assistência
às mulheres que enfrentam gravidezes de fetos
inviáveis.
Colaboradores
Todos os quatro autores participaram ativamente
da concepção, da discussão dos resultados, da re-
visão e aprovação da versão final do estudo.