Etiology of recurrent pregnancy loss in women over

the age of 35 years

Kerri Marquard, M.D.,a Lynn M. Westphal, M.D.,b Amin A. Milki, M.D.,b and Ruth B. Lathi, M.D.b
a
Washington University School of Medicine, St. Louis, Missouri; and b Reproductive Endocrinology and Infertility, Stanford
University School of Medicine, Palo Alto, California

Objective: To determine the rate of embryonic chromosomal abnormalities, thrombophilias, and uterine anomalies
in women over the age of 35 years with recurrent pregnancy loss (RPL).
Design: Retrospective cohort study.
Setting: Academic reproductive endocrinology and infertility clinic.
Patient(s): Women R35 years old with R3 first trimester miscarriages.
Intervention(s): None.
Main Outcome Measure(s): Age, number of prior losses, cytogenetic testing of the products of conception (POC),
uterine cavity evaluation, parental karyotype, TSH, and antiphospholipd antibody (APA) and thrombophilia testing.
Aneuploidy in the POC in women with RPL was compared with sporadic miscarriages (%2 losses) in women R35
years.
Result(s): Among 43 RPL patients, there were 50 miscarriages in which cytogenetic analysis was performed. In the
RPL group, the incidence of chromosomal abnormalities in the POC was 78% (39 out of 50) compared with a 70%
incidence (98 out of 140) in the sporadic losses. Thrombophilia results in the RPL patients were normal in 38 pa-
tients, four patients had APA syndrome, and one had protein C deficiency. Forty out of 43 had normal uterine cav-
ities. Both TSH and parental karyotypes were normal in all of the patients tested. When the evaluation of RPL
included karyotype of the POC, only 18% remained without explanation. However, without fetal cytogenetics,
80% of miscarriages would have been unexplained.
Conclusion(s): In older patients with RPL, fetal chromosomal abnormalities are responsible for the majority of
miscarriages. Other causes were present in only 20% of cases. (Fertil Steril 2010;94:1473–7. 2010 by American
Society for Reproductive Medicine.)
Key Words: Recurrent miscarriage, recurrent pregnancy loss, maternal age, karyotype, spontaneous abortion,
chromosome

Recurrent early pregnancy loss (RPL) has traditionally been de-
fined as the loss of three or more pregnancies before 20 weeks’
gestational age; however, newer guidelines from the American So-
ciety of Reproductive Medicine have defined RPL as the loss of
two or more pregnancies (1). The incidence of the disorder will
depend on its definition and population studied, but is generally
accepted to be between 1% and 5% of the reproductive age
population (2, 3). Known causes of RPL include parental genetic
factors, anatomic abnormalities, and antiphospholipid antibodies
(APA), and more controversial causes include endocrine disorders
(4, 5) and thrombophilias (5–17). Unfortunately, standard evalua-
tions for RPL typically leave one-half of patients with this disor-
der unexplained (5, 18, 19).
For patients with unexplained RPL, choosing to pursue additional
pregnancies may be difficult, but studies show that the likelihood of
subsequent live birth is approximately 70%–75% (20, 21). Factors
that influence success for future pregnancy include maternal age,
number of prior losses, and genetic analysis of miscarried products
Received February 2, 2009; revised May 22, 2009; accepted June 23,
2009; published online July 30, 2009.
K.M. has nothing to disclose. L.W. has nothing to disclose. A.M. has
nothing to disclose. R.L. has nothing to disclose.
Presented at Pacific Coast Reproductive Society, Rancho Mirage,
California, April 18–22, 2007.
Reprint Requests: Ruth B. Lathi, M.D., Stanford Fertility and Reproductive
Medicine Center, 900 Welch Road Suite 20, Palo Alto, CA 94306 (FAX:
650-498-4320; E-mail: rlathi@stanford.edu).
of conception (POC) (21). As the number of prior losses increases,
the chance of euploid loss increases and successful future pregnancy
decreases (21–23). In addition, women with a prior live birth or an-
euploid loss have lower subsequent miscarriage rates compared with
women with no history of live birth or a euploid loss (24, 25).
Maternal age is a well known risk factor for sporadic miscarriage
(26) and is a likely a risk factor for RPL as well. Women over the age
of 35 years (‘‘advanced maternal age’’ [AMA]) have an increased
rate of meiotic errors in oocyte development leading to increased
embryonic aneuploidy (25, 27–32). The reported miscarriage rate
among women under 35 years of age is 14% compared with 40%
for women over 40 years old (33). Given this, one would expect
the RPL rate to be more than 20 times higher in women over 40 com-
pared with women under 35 (0.403 vs 0.143). However, the true
incidence of RPL in women over the age of 35 years is unknown.
Embryonic aneuploidy is thought to be the primary cause of age-
related sporadic miscarriage. Controversy exists over whether recur-
rent aneuploidy is a major cause of RPL (34–37), because there are
few age-stratified studies in the literature. In one study by Dr. Ste-
phenson and colleagues (38), the incidence of embryonic abnormal-
ities in miscarriages increased with age in both sporadic and
recurrent pregnancy loss patients. Other studies have reported aneu-
ploidy in 23%–51% of the POC in RPL patients (25, 27, 35, 38–40).
Preimplantation genetic screening has also shown that both age and
RPL seem to be risk factors for embryonic aneuploidy (37, 41, 42).
Because of the association of maternal age with miscarriage and
potential importance of embryonic karyotype on future pregnancy

0015-0282/$36.00 Fertility and Sterility Vol. 94, No. 4, September 2010 1473
doi:10.1016/j.fertnstert.2009.06.041 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
prognosis, we feel that embryonic karyotype is an important part of
the evaluation of RPL. The objective of the present study was to
evaluate the causes of RPL in women over the age of 35 years by
examining the results of a thorough evaluation for recurrent loss.
MATERIALS AND METHODS
A retrospective analysis of miscarriages occurring between 1999 and 2006 at
an academic reproductive endocrinology and infertility clinic was per-
formed. Miscarriages were identified from a database of miscarriages with
cytogenetic analysis maintained by the authors. Women who had passed their
35th birthday at the time of the miscarriage were included in this study.
Recurrent miscarriage was defined as three or more clinical pregnancies end-
ing in a first-trimester loss. Patients with only one or two miscarriages were
classified as sporadic losses. The evaluation included a uterine cavity evalu-
ation, parental karyotypes, TSH, APA, and thrombophilia testing, and karyo-
type of the POC. Patients were classified as having unexplained RPL if their
evaluation was negative. Women with RPL who did not have an evaluation
for underlying causes were excluded. Patients undergoing PGD and oocyte
donation were also excluded.
Missed abortion was diagnosed by transvaginal ultrasound between 6 and
10 weeks’ gestational age. In our practice, patients with miscarriages are rou-
tinely offered dilatation and curettage (D&C) with karyotype analysis. The
D&C was typically performed in the office setting with a paracervical block
(1% lidocaine) and IV conscious sedation. After the procedure, chorionic
villi were separated from maternal tissue, thoroughly washed, and sent for
cytogenetic analysis as previously described (43).
Uterine cavity evaluation was performed by either hysterosalpingogram or
hysteroscopy. Thrombophilia workup included anticardiolipin antibodies,
lupus anticoagulant, proteins C and S, prothrombin gene, antithrombin III, fac-
tor V Leiden, and methylenetetrahydrofolate reductase (MTHFR). Any patients
with MTHFR mutations had further evaluation of fasting homocysteine levels.
Only patients with abnormal homocysteine levels were considered to have an
increased risk of miscarriage due to a thrombophilia.
Incidence of aneuploidy in POC of women with recurrent miscarriage was
compared with women with sporadic miscarriages. All twin pregnancies were
considered to be a single miscarriage. If two karyotype results were obtained
in a twin miscarriage and there was one normal and one abnormal result, the
abnormal result was recorded as the likely cause of the miscarriage.
Data were compared using chi-squared and Fisher exact test, where P<.05
determined statistical significance. Institutional Review Board approval was
obtained for this study.
RESULTS
Chromosomal analysis on POC was performed on 270 miscarriages
from 1999 to 2006 in 256 women who underwent D&C for first-
trimester pregnancy loss. One hundred ninety specimens were
examined in 180 AMA women. Forty-three of these patients had
R3 pregnancy losses. The incidence of aneuploidy in the sporadic
group was 70% (98 out of 140), which was not significantly different
than the 78% aneuploidy rate (39 out of 50) seen in the RPL group
(P¼.28). In the sporadic group, 42 were euploid, with a ratio of
46XX to 46XY of 25:17. This compared with 11 euploid specimens
in the RPL group, with a ratio of 46XX to 46XY of 9:2. With the
higher rate of 46XX results, maternal contamination appears to be
more common in the women with recurrent loss. However, confir-
matory studies were not routinely done. If we performed the analysis
using only 46XY results in the normal category, the abnormality
rates are similar in the RPL group compared to the control subjects
(95% vs. 85%; P¼.096).
We identified six patients in the RPL group and three patients in
the sporadic loss group over age 35 with more than one miscarriage
studied. In the recurrent miscarriage group, four patients had two an-
euploid losses, one patient miscarried one aneuploid and two 46XX
embryos, and one patient lost one abnormal and one 46XX embryo.
1474 Marquard et al. Etiology of RPL in women over 35
Regarding the sporadic group, two patients had both one aneuploid
and one euploid POC, and one patient had two normal results.
There was no significant difference in age between the recurrent
and sporadic loss groups. In the RPL patients, the mean age was
39.3 years, compared with 39.2 years in the sporadic loss patients
(Table 1). On average, patients in the RPL group had three prior los-
ses, making the pregnancy examined the fourth loss. Among the spo-
radic group, there were 41 prior miscarriages in 137 patients, an
average of 0.3 prior losses per patient. Although control subjects
had higher utilization of fertility treatments, both the RPL and the
control groups had a significant percentage of patients undergoing
fertility treatments (62% vs. 83%; P¼.002).
The most common abnormality in both RPL and sporadic miscar-
riage was autosomal trisomies (Table 2). Trisomy 16 was most com-
mon in both groups. The distribution of other trisomies is shown in
Figure 1. Trisomies 2, 8, 12, and 14 were more frequent in the RPL
group, and, of note, trisomies 2 and 12 would not have been detected
on standard fluorescence in situ hybridization preimplantation ge-
netic screening panels.
The incidence of underlying etiologies of pregnancy loss in these
AMA women is shown in Table 3. Of the 43 RPL patients studied, 38
had normal thrombophilia and APA testing. There was one case of
protein C deficiency, which was found in a patient with a history
of a submucosal fibroid. Despite having two presumptive causes
for her loss, this patient had a trisomic embryo. Four patients in
this group had been previously diagnosed with APA syndrome and
were taking heparin. Three of them had autosomal trisomies in their
POC.
Three women had either congenital or acquired uterine cavity ab-
normalities. These included one T-shaped uterus, one partial bicorn-
uate versus septum, and the previously described patient with
a submucosal uterine fibroid that was resected. Interestingly, all
three RPL patients with uterine cavity abnormalities miscarried an-
euploid embryos and had a prior term delivery. Five patients with
Asherman syndrome (11.6%) had been treated with lysis of intra-
uterine adhesions and had anatomically normal hysteroscopy before
the loss that was evaluated cytogenetically. Three of these five pa-
tients had aneuploid losses.
Complete parental karyotypes were available in 30 of the 43 RPL
patients. In all eight patients with deletions or chromosomal rear-
rangements in the POC, both parental karyotypes were available
and normal. Parental karyotypes were missing for one or both
partners in 13 cases. In 9 of these 13 miscarried aneuploid embryos,
no deletions or translocations were seen in the embryos of these
patients.
Normal TSH values were documented within 1 year of the mis-
carriage in 40 out of 43 patients. The three patients whose results
were unavailable had no symptoms or history of thyroid disease,
and all of them miscarried aneuploid embryos.
DISCUSSION
Recurrent pregnancy loss is a challenging problem for both physi-
cians and patients, because extensive evaluations often leave the dis-
order unexplained. We routinely order parental karyotypes, APA and
thrombophilia testing, thyroid function tests, and a uterine cavity
evaluation for patients with R3 miscarriages. Although RPL is
commonly thought to be due to maternal causes, we found a low in-
cidence of underlying maternal causes in the AMA population. In-
stead, the present study showed a high rate of chromosomal
abnormalities, 78%, in the POC of recurrent miscarriage patients.
This rate was not significantly different than the abnormality rate
Vol. 94, No. 4, September 2010
 TABLE 1 FIGURE 1
Demographics of study population (women R35 years
Trisomic distribution.
old).
30
RPL Sporadic
(n [ 43) (n [ 137) 25

20

Percentage
Average age 39.3  2.61 39.2  2.71
No. of miscarriages before 129 41 15
current loss
Average losses before 2.98 0.29 10

current loss 5

Note: RPL ¼ recurrent pregnancy loss. 0

2 3 4 7 8 9 11 12 13 14 15 16 17 18 20 21 22
Marquard. Etiology of RPL in women over 35. Fertil Steril 2010.
Trisomies Sporadic RPL ≥ 35

Marquard. Etiology of RPL in women over 35. Fertil Steril 2010.

of 70% seen in the POC of similarly aged women with their first or
second miscarriages. Although the presence of fetal aneuploidy did
not exclude the possibility of an additional underlying disorder, the from the same couple, and PGD data reveal some interesting find-
rate of maternal causes of RPL was low in this study, with a 9% in- ings. When only seven chromosomes are analyzed, aneuploidy
cidence of APA syndrome, a 7% incidence of congenital uterine can be found in 70% of embryos undergoing PGD from patients
anomalies, and a 2% incidence of inherited thrombophilias. Rates with RPL compared with 45% from age-matched control subjects
of APA syndrome in RPL patients range from 5% to 15% (10, 11, (37). In addition, 22% of cycles in the present study revealed all em-
17, 44, 45), which is consistent with the present finding of a 9% bryos as abnormal. Moving toward extended panels and compara-
RPL APA syndrome rate. The 7% uterine anomaly rate in the present tive genomic hybridization may show an even higher aneuploidy
study is slightly lower than earlier literature reports of 10%–15% rate. Retrospective data indicates that in AMA women with RPL,
uterine abnormality rates in RPL patients (5, 46). Overall, 80% of PGD decreases pregnancy loss (41, 42), and increases viable preg-
miscarriages would have been unexplained without chromosome nancies (41). However, these same studies show little or no benefit
testing of the products of conception. Based on this finding and in younger patients (41).
others in the literature with similar findings (27, 38), we conclude Despite high aneuploidy rates in AMA women with RPL, no large
that the vast majority of cases of miscarriages traditionally thought prospective trials have been performed on preimplantation aneu-
to be unexplained may have fetal chromosomal errors as the cause of ploidy (genetic) screening (PGS) in this population. In the AMA in-
the loss. fertility population, randomized prospective trials of PGS/IVF fail
The high rate of aneuploidy seen in POC is not surprising given to show an advantage over conventional IVF regarding live birth
the current literature showing that couples with RPL have higher or miscarriage rates (48–53). Although these trials did not directly
than expected aneuploidy rates in their embryos. Second-trimester address PGS in RPL patients, a subgroup analysis of RPL patients
prenatal testing data has shown that chromosomal abnormalities in- from the Twisk et al. (54) data set showed no benefit of PGS in pa-
crease with the number of previous miscarriages (47). Preimplanta- tients with RPL. The fact that no significant reduction in miscarriage
tion genetic diagnosis (PGD) allows us to look at multiple embryos was seen in these studies may reflect the technical challenges asso-
ciated with PGS (55–57) or the overall low oocyte quality in these
patients, and further study is needed in this area.
TABLE 2 There are several limitations to a study of this design. We had
only one available POC karyotype result on most RPL patients,
Details of abnormal embryonic karyotype (women R35 and therefore it is impossible to know if all losses were due to aneu-
years old). ploidy or just the most recent one. In addition, karyotype analysis
only detects gross chromosomal rearrangements. There are many
RPL Sporadic
potential genetic and embryonic defects leading to a loss that would
n % n % be missed on this type of testing. Therefore, it is possible that even
more of the miscarriages would be explained by embryonic, rather
Trisomy 28 71.8 67 68.4
than maternal, factors. Another limitation was the small sample
Monosomy 1 2.6 5 5.1
size in the RPL group: only 50 cases using the strict definition of
Triploidy 2 5.1 4 4.1
Tetraploidy 2 5.1 1 1 R3 clinical losses. We could have increased the numbers by consid-
Complex other 3 7.7 16 16.3 ering RPL as R2 losses. With this definition, the number of RPL
Separate rearrangementsa 3 7.7 5 5.1 losses would increase to 91, resulting in 76% aneuploidy in the re-
Total 39 100 98 100 current loss group, which is still not statistically significantly differ-
ent than 69% aneuploidy in the sporadic group (P¼.27).
Note: RPL ¼ recurrent pregnancy loss.
a
RPL: 46XYadd(12)p13; 46XYdel(4)q32; 46XYadd(4)q32. Sporadic Despite relatively small numbers, the present data supports ear-
miscarriage: 46XYdel(6)p22; 46XXdel(8)p12; 46XXdel(5)p14; lier larger studies looking at chromosomal status of miscarriages
46XYadd(12)p13; 46XYdel(8)t. in the AMA RPL population and complements those findings with
Marquard. Etiology of RPL in women over 35. Fertil Steril 2010. additional information regarding rates of other causes of RPL in
this age group (27, 38). One of those studies reported 60%

Fertility and Sterility 1475

 our results show that patients with a presumptive diagnosis for
TABLE 3 a cause of RPL had aneuploidy in the POC in 80% of the cases.
Etiology of RPL in women R35 years old. This is higher than the 20%–60% rate reported in other studies
(29, 35, 58) and is likely due to the advanced maternal age specifi-
Abnormal results %
cally examined in this study. Aneuploidy may be a common cause of
TSH 0 of 40 0 treatment failure and is important to diagnose in a failed pregnancy
Parental karyotype 0 of 30 0 even when another presumptive cause is present.
APAS 4 of 43 9.30 Although not all miscarriages require chromosomal analysis, we
Uterine anomalies 3 of 43 7.00 recommend testing in cases where it may affect clinical manage-
Asherman syndrome (treated) 5 of 43 11.60 ment. Patients with RPL show high rates of anxiety and depression,
Inherited thrombophilias 1 of 43 2.30
which is likely to be worsened by lack of an explanation. In our ex-
Aneuploidy in POC 39 of 50 78
perience, performing karyotype analysis not only provides an expla-
Note: APAS ¼ antiphospholipid antibody syndrome; RPL ¼ recurrent nation for the loss but in many cases it gives a patient emotional
pregnancy loss; POC ¼ products of conception; TSH ¼ thyroid-stim- closure. If a loss or losses are unexplained, patients may be more
ulating hormone.
likely to seek unnecessary or unproven testing and treatments. The
Marquard. Etiology of RPL in women over 35. Fertil Steril 2010. data in this study can be used to counsel our patients without karyo-
type analysis of the POC regarding the likelihood of aneuploidy in
their miscarriages.
aneuploidy in 186 specimens from women >35 years old with RPL In conclusion, aneuploidy is the most common cause of miscar-
(38), rates that were not significantly different than the those for the riage in patients with recurrent (R3) miscarriage over the age of
sporadic loss group. Although Sullivan et al. (27) found decreased 35 years. Without karyotype of the POC on these losses, 80% (40
overall aneuploidy rates in RPL versus sporadic loss, the mean ma- out of 50 had negative evaluation) of cases remain unexplained,
ternal age was lower and exact number of patients >35 years old was and of these, 78% (31 out of 40) had aneuploid POC. Only 18%
unclear. However, they did still see an increase in aneuploidy with of losses remain unexplained if chromosomal analysis of POC is
AMA, and when they stratified by maternal age, aneuploidy rates performed. Aneuploidy accounted for more losses than all other
were similar in RPL versus control subjects (27). causes combined. Patients with no obvious underlying cause of
The standard work-up for RPL continues to be important in eval- RPL should be encouraged to continue attempts at pregnancy, be-
uating potential factors responsible for pregnancy loss. In fact, we cause prospective studies show that these women, even with ad-
identified a potential etiology in 20% of cases with these tests. Chro- vanced maternal age, have a high rate of live births with their
mosomal analysis remains crucial even in these patients, because subsequent pregnancies (21).

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