DiagDiagnosis and Treatment of Infertilityrelated Male Hormonal Dysfunctionnosis and Treatment of Infertilityrelated Male Hormonal Dysfunction

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Diagnosis and treatment of infertilityrelated male hormonal dysfunction

Martin Kathrins1 and Craig Niederberger2
Abstract | Treatment of infertility-related hormonal dysfunction in men requires an understanding

of the hormonal basis of spermatogenesis. The best method for accurately determining male
androgenization status remains elusive. Treatment of hormonal dysfunction can fall into two
categories empirical and targeted. Empirical therapy refers to experience-based treatment
approaches in the absence of an identifiable aetiology. Targeted therapy refers to the correction
of a specific underlying hormonal abnormality. However, the tools available for inferring the
intratesticular hormonal environment are unreliable. Thus, understanding the limitations of
serum hormonal assays is very important for determining male androgen status. Furthermore,
bulk seminal parameters are notoriously variable and consequently unreliable for measuring
responses to hormonal therapy. In the setting of azoospermia owing to spermatogenic
dysfunction, hormonal therapy relying on truly objective parameters including the return
ofsperm to the ejaculate or successful surgical sperm retrieval is a promising treatment.
This approach to the treatment of fertility-related hormonal dysfunction in men contrasts with
the current state of its counterpart in female reproductive endocrinology. Treatment of male
hormonal dysfunction has long emphasized empirical therapy, whereas treatment of the corollary
female dysfunction has been directed at specific deficits.
Division of Urology, Brigham

and Womens Hospital, 45
Francis Street, ASBII, Boston,
Massachusetts 02115, USA.
2
Department of Urology,
University of Illinois at
Chicago, 820S. Wood Street
Clinical Sciences North Suite
505, Chicago, Illinois 60612,
USA.
1
Correspondence to M.K.
mkathrins@bwh.harvard.edu
doi:10.1038/nrurol.2016.62
Published online 19 Apr 2016
Since the first case reports in 1910 of testicular atrophy after canine hypophysectomy, the hormonal basis
of human reproduction has been an area of evolving
investigation1. An array of treatment modalities are
available for hormonal dysfunction in the setting of
male infertility, but the diagnosis of such dysfunction
and its treatment is often empirical, or guided by the
clinicians judgement, and can be open to interpretation.
Our ability to understand the intratesticular hormonal
environment and its effect on spermatogenesis is limited
by current methods of routine clinical investigation.
In contrast to our understanding of hormonal effects
on male infertility, the science of female reproductive
endocrinology has moved toward an algorithmic,
evidence-based approach. Perhaps the best example
of such a rational, evidence-based treatment approach
to female reproductive-associated hormonal dysfunction is provided by controlled ovarian stimulation
before oocyte retrieval to enable invitro fertilization.
Ovulation stimulation follows an algorithmic approach
to achieve both timely ovulation and pregnancy, avoiding ovarian hyperstimulation syndrome, multiple follicular development, and excessive medication costs.
A 2011 Cochrane review, including 45 randomized
controlled trials (RCTs), compared the effectiveness of
gonadotropin-releasing hormone (GnRH) agonists with

antagonists for triggering controlled ovarian stimulation.
This review encompassed 7,511 cycles of invitro fertilization and used clinical pregnancy and live-birth rates as
primary outcomes. The authors concluded that similar
live birth rates are achieved using GnRH antagonists or
agonists with a reduced risk of ovarian hyperstimulation
syndrome observed with use of antagonists, based on
high-quality evidence2. Similarly, rigorous investigations
have been undertaken, for example, into the optimum
dose of follicle-stimulating hormone (FSH) for ovarian
stimulation and the use of metformin in the setting of
polycystic ovary syndrome before ovarian stimulation3.
Research into associations between hormonal dysfunction and male infertility have been hampered by
several factors. Firstly, 25% of men presenting with
infertility are ultimately labelled as having idiopathic
infertility, with no identifiable cause4. Treatment of
such patients relies on nonstandardized empirical therapies. Secondly, the several oral medications indicated
for hormonal treatment of infertility are exclusively off-
label (pertaining to FDA-approved indications),which
perhaps contributes to the lack of RCTs for these medications in relation to male infertility3. Furthermore, no
nationwide multi-institutional database that is dedicated
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Key points
Oestradiol is the principal mediator of negative feedback on the hypothalamic
pituitary axis, which illustrates the influence of selective oestrogen receptor
modulators and aromatase inhibitors on male hormonal parameters
Serum hormonal assays are unreliable indicators of intratesticular androgen levels,
and the best approach for determining male androgen status remains elusive
Follicle-stimulating hormone and inhibin B are markers of spermatogenesis and their
relative values in the setting of an intact hypothalamicpituitarygonadal axis provide
important information about testicular function
Targeted hormonal therapy corrects specific hormonal dysfunctions, empirical
hormonal therapy is employed when no underlying cause is identified and the
evidence for empirical therapy is dependent on the type of medication used
A return of sperm to the ejaculate or successful surgical sperm retrieval among men
with azoospermia owing to spermatogenic dysfunction are the most objective
indicators of outcomes of hormonal therapy
to male infertility currently exists in the USA. Indeed,

the Society for Assisted Reproductive Technology database (which includes thousands of annual USA-based
invitro fertilization cycle results) does not differentiate between testicular sperm obtained from men with
azoospermia owing to obstruction (including congenital bilateral absence of the vas deferens, nondisjunction,
infectious aetiology, or ejaculatory duct obstruction) or
severe spermatogenic dysfunction two diametrically
opposed underlying pathologies5.
Despite the presence of various factors that hinder
our understanding of hormonal therapy for male infertility, variations in semen analysis remain the greatest
barrier to a rational clinical approach. Bulk seminal
parameters historically the prime pathway for determining male fertility potential are fraught with intrasubject variability and ever-evolving normal reference
parameters. For example, in a multi-institutional study of
765 infertile couples and 696 fertile couples, the investigators observed such considerable overlapping values of
sperm concentration, motility, and morphology that no
single parameter had acceptable discriminatory power
between groups6. Furthermore, considerable intrasubject
variability over time even among fertile men hampers the utility of semen analysis as an outcome measure
for medical or surgical interventions intended to correct
male infertility7,8.
The diagnosis and treatment of female reproductive
hormonal dysfunction is well elucidated and understood
by physicians. That physicians treating parallel hormonal problems in infertile men are as well informed of
the pathophysiology of these conditions is imperative.
Furthermore, by relying on rational, objective outcomes the diagnosis and treatment of male infertility
can move beyond the use of empirical treatments and
towards a more targeted approach. Currently, the development of sperm in the ejaculate or successful surgical sperm retrieval (SSR) in men with azoospermia
owing to spermatogenic dysfunction (ASD) are the
most objective clinical outcomes available to us. In this
Review, we describe and discuss the pathophysiology,
diagnosis, and treatment of fertility-associated male
hormonaldysfunction.
Pathophysiology of hypoandrogenism
Hypothalamicpituitarygonadal axis
Secretion of the anterior pituitary gonadotropin hormones
luteinizing hormone (LH) and FSH are stimulated by the
pulsatile release of GnRH and inhibited by the release of
gonadotropin-inhibiting hormone from the hypothalamus9 (FIG.1). LH and FSH share a common subunit and a
distinct subunit10. LH stimulates and promotes testicular
androgen production by the Leydig cells and FSH stimu
lates spermatogenesis supported by Sertoli cells. FSH
and testosterone are both necessary for spermatogenesis
to occur. Indeed, FSH gene mutations in men invari
ably lead to azoospermia and diminished testicular size11.
However, the small number of case reports regarding FSH
receptor mutations in men suggest heterogeneous effects
on male fertility. Case reports have revealed findings ran
ging from severe oligozoospermia to normozoospermia,
including instances of natural conception12. These reports
correlate well with experimental data from FSH-receptor
knockout mouse models13. LHB-knockout mouse models
exhibit diminished serum and intratesticular testosterone
and late-maturation-arrest pathology10,14. Animal models
in this setting are useful for elucidating the underlying
function of gonadotropins in male reproductive physio
logy, but treatment decisions should not be based on such
data alone. In 2001, the peptide kisspeptin was discovered and results from subsequent functional studies have
been published, revealing an agonistic effect on GnRH
release and pulsatile LH release. Kisspeptin has been used
experimentally in men to induce gonadotropin secretion. Increases in LH secretion are more dramatic than
increases in FSH in response to experimental kisspeptin
supplementation; however kisspeptin is not currently
used in clinical practice15.
Other serum indicators of male fertility exist, beyond
FSH and LH. Inhibin B, a glycoprotein heterodimer also
composed of and subunits, is secreted by the testicular
germinal epithelium (primarily Sertoli cells) in response
to FSH (FIG.1). Inhibin B subsequently acts on the anterior
pituitary in a negative-feedback loop, thus inhibiting FSH
production16. Inhibin B is an important indicator of the
degree of spermatogenesis. For example, in instances of
hypospermatogenesis pathology, inhibinB secretion is
decreased and FSH secretion is increased17. In one prospective study, inhibin B and FSH were both found to be
significantly correlated with bulk seminal parameters;
however, the correlation coefficient values were modestly
higher for inhibin B than for FSH (inhibinB:0.48;
P<0.0001, FSH:0.41; P=0.0007). Thus, inhibin B could
be a more sensitive indicator of semen parameters than
FSH18. Conversely, another prospective study of fertile and
infertile men found inhibin B and FSH to be equally predictive of bulk seminal parameters19. Nevertheless, inhibin
B has yet to be widely adopted as part of the routine evalu
ation of male infertility, perhaps owing to increased assay
costs20. Finally, activin is a heterodimer or homodimer
of subunits and is also produced in the testes. Activin
has an agonistic effect on the pituitary secretion of FSH
and its release is inhibited by inhibin B16. However, activin
does not presently have a role in the clinical evaluation of
infertile men.
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Hypothalamus
GnRH
GnIH
Anterior pituitary
FSH
Inhibin B
Activin
Testes
Sertoli cells
In a remarkable demonstration of evolutionary conservation, it is the female hormone oestradiol that exerts
the principle inhibitory feedback on gonadotropin secreSelective oestrogen
tion in men. Oestradiol is generated from testosterone
receptor modulators
by aromatase, a member of the cytochrome p450 super
family (FIG.1). The integral feedback role of oestradiol was
demonstrated in a study involving four men with idiopathic hypogonadotropic hypoandrogenism who were
treated with pulsatile GnRH to normalize pituitary gonadotropin secretion. The men were subsequently treated
Oestradiol
with infusions of testosterone, oestradiol and dihydro
testosterone, a nonaromatizable androgen. Only the testosterone and oestradiol infusions resulted in diminished
serum LH and FSH levels. Dihydrotestostone, which has
Aromatase
inhibitors no potential to be converted to oestradiol had no inhibi
tory effect on gonadotropin secretion24. In other studies,
investigators have localized the negative-feedback effects
Aromatase
of oestradiol to the hypothalamus, where it acts to reduce
LH pulse frequency, and the anterior pituitary, where it
reduces sensitivity to GnRH25,26.
Oestradiol-receptor-knockout mouse models are
Testosterone
(serum ~500 ng/dl)
infertile and have and abnormal spermatogenesis.
Further investigations of such models have revealed
LH
that infertility stems from aberrant proximal epididymal lumen fluid reabsorption and subsequent increases
in intraluminal pressure27,28. Conversely, at the testicular
level, basal oestradiol is necessary for normal spermato
genesis, as proven by case studies of infertile men with
Leydig cells
congenital aromatase deficiency. In fact, the ARO1
(also known as CYP19A1) and oestradiol receptors
are also expressed in Sertoli, Leydig, and germ cells of
Testosterone
(~60,000 ng/dl)
fertile men26,29,30.
Figure 1 | The hypothalamicpituitarygonadal axis. Secretion of the anterior pituitary

Nature Reviews | Urology
gonadotropin hormones luteinizing hormone (LH) and follicle-stimulating
hormone (FSH) are stimulated by the pulsatile release of gonandotropinreleasing hormone (GnRH) and inhibited by the release of gonadotropin-inhibiting
hormone (GnIH) from the hypothalamus. Selective oestrogen receptor modulators
(SERMs) competitively inhibit hypothalamic oestrogen receptors, which leads to
increased anterior pituitary gonadotropin release and subsequent endogenous
testosterone production. Inhibin B is secreted by the testicular germinal epithelium
(primarily Sertoli cells) in response to FSH and subsequently acts on the anterior pituitary
in a negative-feedback loop, inhibiting FSH production. Activin has an agonistic effect
on the pituitary secretion of FSH and its release is inhibited by inhibin B. Aromatase
inhibitors are used to correct a diminished testosterone:oestrogen ratio.
Hormonal biomarkers to determine fertility potential

are an important and validated part of a routine female
infertility evaluation, but reliable hormonal markers
are lacking in the realm of male fertility. For instance,
anti-Mllerian hormone is used in routine clinical
practice and is as an excellent marker, according to
Level 1a evidence, of both ovarian reserve and invitro
fertilization outcomes21. FSH is a useful indicator of the
status of the male germinal epithelium, with published
reference values ranging widely from 1.4 to 18.1IU/l,
although it is far from perfect. However, a large retrospective analysis of infertile men found that FSH values
>4.5IU/l indicate an increased risk of abnormal bulk
seminal parameters22. Unfortunately, unstimulated FSH
is a poor predictor of successful surgical sperm retrieval
in men with ASD23.
Associated clinical conditions

Hypoandrogenism can be subcategorized as hypo
gonadotropic or hypergonadotropic. The latter present
ation is commonly referred to as primary testicular
failure. Hypogonadotropic hypoandrogenism is charac
terized the presence of low or normal serum gonadotropin levels in the setting of low serum testosterone.
Congenital hypogonadotropic hypogonadism (CHH)
is a condition that is associated with severely impaired
GnRH secretion and is related to delayed puberty, and
is often diagnosed early in life31 (BOX1). Approximately
50% of instances of CHH are associated with anosmia
referred to as Kallman Syndrome resulting from
the common embryological origin of the olfactory
nerve bulb and the hypothalamus. Kallman syndrome
is a rare condition (occurring in one in 10,000 births)
with a polygenic aetiology, and is associated with several
known genetic mutations including KAL1 (also known
as ANOS1), PROKR2, and FGFR1 (REF.32). Whether
syndromic or not, CHH can also present with micropenis, cryptorchidism, and/or bone fractures resulting
from severe hypoandrogenism and patients with this
condition can benefit from genetic counselling31.
Adult-onset idiopathic hypoandrogenism with
complete or near-complete absence of gonadotropins,
similar to Kallman Syndrome, is thought to be a rare
infertility-associated presentation The diagnosis refers to
men with normal pubertal development and secondary

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Box 1 | Types of hypogonadotropic hypoandrogenism
Congenital hypogonadotropic hypoandrogenism
Kallman syndrome (when associated with anosmia)
Adult-onset hypogonadotropic hypoandrogenism
Underlying medical condition:
-- Hyperprolactinaemia
-- Pituitary adenoma
-- Hypopituitarism
-- Hypothyroidism
-- Sickle cell disease
-- Haemochromatosis
-- Opiate-induced
-- Exogenous testosterone or anabolic steroid use
onset of hypogonadotropic hypoandrogenism 33,34

(BOX 1) . Its presentation includes severely impaired
spermatogenesis, and higher serum testosterone and
inhibin B levels than those typically observed in men
with CHH. Gonadotropin levels are similar to those
observed in men with CHH (mean FSH1.51.2IU/l
and LH2.91.7IU/l). Interestingly, Sussman etal.20, in
a single-institution review of the coincidence of hypo
androgenism and abnormal sperm parameters, pointed
out that the majority of these men did experience compensatory increases in LH levels above 12.0IU/l. Thus,
the authors surmised that the incidence of hypogonado
tropic hypoandrogenism, with gonadotropins in the
normal range and an inadequate pituitary response,
could be much higher than previously suspected20.
Medical conditions associated with hypogonadotropic hypoandrogenism must be excluded, including
haemochromatosis using serum ferritin determination,
genetic testing for sickle-cell anaemia, hypothyroidism
by serum assay of thyroid stimulating hormone, and a
pituitary adenoma by serum prolactin assay or cranial
MRI (BOX1). Treatment of such underlying conditions
might correct the hormonal dysfunction. In one series,
investigators found partially empty sella turcica in 40%
of patients35. Opiate-induced androgen deficiency is
an under-appreciated but important cause of hypogonadotropic hypoandrogenism36,37. These men should
be strongly encouraged to wean themselves off of their
opiate medications to optimize their fertility potential.
Prolactinomas are the most common secretory pituitary tumour, but are a much rarer cause of infertility in
men than in women. The clinical presentation ranges
from dramatic visual-field defects and galactorrhoea to
less conspicuous complaints including erectile dysfunction and diminished libido. In addition to the mass effect
on the hypothalamicpituitary axis by a pituitary macroadenoma (defined as >1cm in diameter), prolactin
secretion itself can suppress gonadotropin secretion38.
Routine determination of serum prolactin in the absence
of the above symptoms has a low clinical yield, with the
prevalence of elevated prolactin levels among infertile
men ranging widely from as low as 0.5% to as high as
33%3941. The cut-off serum prolactin value at which
use of cranial imaging is warranted is poorly defined.
Macroprolactinomas are more closely associated with
serum prolactin levels >250ng/ml. However, MRI

should be considered if underlying medical conditions
or the use of causative medications, including many
psychotropic medications, which might block dopamine
receptors, are excluded42. Therapy with dopamine receptor agonists can considerably improve sperm count and
motility, with parameters continually improving with
increasing treatment time43.
Hypergonadotropic hypoandrogenism is a sign of
testicular end-organ failure. Klinefelter syndrome is
the most common cause of hypergonadotropic hypo
androgenism and the most common genetic cause of
male infertility. Klinefelter syndrome is characterized
by a supranumery Xchromosome, diminished testicular volume, gynaecomastia, and severely diminished
spermatogenesis. Chromosomal mosaicism might miti
gate the presenting phenotype of men with Klinefelter
syndrome44,45. The pathophysiology includes both Leydig
cell dysfunction and a decreased t estosterone:oestradiol
ratio secondary to increased aromatase (CYP19A1)
expression46,47. Beyond Klinefelter syndrome, hypergonadotropic hypoandrogenism secondary to Leydig cell
dysfunction could be a consequence of prior chemotherapy or the testicular dysgenesis syndrome, postulated
by Skakkebaek. The testicular dysgenesis syndrome is
notable for associations with cryptorchidism and testicular malignancy48. Finally, men with complete or partial
androgen insensitivity, the latter of which might present
with a normal male phenotype and impaired spermatogenesis, have elevated serum gonadotropins and normal
or elevated serum testosterone levels49.
Determination of androgen status

The most important first step in the hormonal evaluation of an infertile man is to determine if a problem
truly exists. The Endocrine Society defines the total testosterone lower-limit reference range for young men as
280300ng/dl. This reference cut-off value is based on
morning serum assays, owing to the existence of a normal hormonal circadian rhythm. A finding of abnormal
testosterone levels should be confirmed with a second
assay50. However, the accepted total testosterone cutoff value of ~300ng/dl might not be broadly applicable
for the diagnosis of symptomatic hypoandrogenism, an
important end-point in determining true androgen status. For instance, a single-centre retrospective review of
352 men <40 years of age, presenting with symptoms
of hypoandrogenism based on the validated Androgen
Deficiency in Aging Male questionnaire, found that
a total testosterone cut-off value of 400ng/dl was an
equally effective predictor of symptoms compared with
a cut-off value of 300ng/dl51.
Among infertile men, however, serum total testosterone level might not be the most accurate indicator
of androgen status. The vast majority of testosterone
in serum is protein bound either tightly bound to
sex-hormone binding globulin (SHBG) or loosely
bound to albumin. To account for this, subcategories
of total testosterone are employed including free testosterone (completely unprotein-bound testosterone) and
bioavailable testosterone (the combination of free and
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loosely albumin-bound testosterone)52. Several conditions exist that might predispose men to derangements
in serum SHBG concentrations and affect the serum
concentration of free testosterone and bioavailable
testosterone, including liver dysfunction, thyroid dysfunction, and obesity. Advanced age is one of the most
important of these conditions, and is associated with
increases in serum SHBG concentrations independent
of body-mass index53,54.
The Endocrine Society clinical practice guidelines
also recommend measurement of free testosterone or
bioavailable testosterone in the event of equivocal findings from total testosterone assays50. Indeed, serum total
testosterone concentrations ranging from 280350ng/dl
lack sufficient sensitivity to rule out diminished free testosterone. However, a total testosterone threshold of
400ng/dl has a sensitivity of 98.2% for ruling out low free
testosterone levels55. In a retrospective analysis of 73 men
presenting for fertility evaluation that used bioavailable
testosterone as the most accurate indicator of androgen
status, investigators found that low total testosterone
yielded a positive-predictive value (PPV) of 50% for
abnormal bioavailable testosterone. Specifically, among
men >40years of age, the PPV of total testosterone was
59% and among men <40years of age the PPV of total
testosterone was only 20%, which is unsurprising given
the naturally increased SHBG serum concentrations in
older men. Thus, overall, total testosterone is a poor predictor of androgen status when bioavailable testosterone
is employed as the gold standard56. In our clinical practice,
we routinely rely on bioavailable testosterone as the gauge
of androgen status for men p
resenting withinfertility.
The specific determination of serum androgen levels
is also fraught with uncertainty. A survey conducted
by the College of American Pathologists used a single
sample from a hypoandrogenic man that was processed
independently by 1,100 pathology laboratories across
the USA. The results of these analyses showed a 32.2%
coefficient of variability for total testosterone, indicating highly inconsistent laboratory results. Specifically,
free testosterone can be directly measured by ultrafiltration, equilibrium dialysis, or immunoassay, the latter
of which is particularly unreliable, whereas equilibrium dialysis is the most reliable method. Bioavailable
testosterone levels can be measured using ammonium
sulfate precipitate, which is unreliable52,57. Moreover,
these methods are time-intensive, costly, not suited to
automation, and subject to difficult to control laboratory variables including temperature, which can affect
the final results52. To address these limitations, several
algorithms for calculating bioavailable testosterone or
free testosterone levels have been developed from assays
of total testosterone, SHBG, and albumin. These algorithms, if adopted, must be validated on a casebycase
basis by individual clinical laboratories58. Perhaps the
most widely adopted algorithm for calculating free testosterone and bioavailable testosterone was published by
Vermeulen and colleagues59, who proposed lower-limit
reference values for young, healthy men of 0.23nmol/l
(6ng/dl) for free testosterone and 5.3nmol/l (153ng/dl)
for bioavailabletestosterone60.
Epidemiology of hormonal dysfunction

Hypoandrogenism is a common and often correctable
cause of male infertility, but attempts to correlate the two
conditions have not been definitive. Sussman etal.20 performed a single-institution retrospective analysis of 120
men who presented with infertility. The authors reported
that 45% of men with ASD, 42.9% of men with oligozoo
spermia, and 35.3% of men with adequate bulk semen
parameters were diagnosed with hypoandrogenism.
However, consistent correlations between serum testosterone or hypoandrogenic symptoms and bulk seminal parameters and testicular pathology have not been
observed6163. Overall, the most reliable association has
been observed between clinical presentation with ASD
and hypoandrogenism the incidence among men
with ASD ranges from 4547%64.
The ratios of testosterone to gonadotropins or
oestradiol have also been found to be associated with
male infertility. A large prospective cohort study of 357
men with idiopathic infertility and 318 fertile controls
found that infertile men had significantly reduced total
testosterone, free testosterone, testosterone:LH ratio, and
testosterone:oestradiol ratios (all P<0.001)65. In small
retrospective cohort study, Pavlovich etal.66 noted a concordance between infertility, bulk seminal parameters,
and a diminished ratio of testosterone:oestradiol. They
defined an abnormal ratio as <10, coincident with the
20th percentile cut-off point among fertile men. Such a
diminished ratio of testosterone:oestradiol is observed in
obese men with relatively increased frequency, perhaps
owing to increased peripheral aromatization67.
Androgens in spermatogenesis
Intratesticular androgen microenvironment
Understanding the relationship between our traditional
tools for determining androgen status and the intra
testicular environment is vitally important. Research
using testicular aspirations from fertile men has confirmed that testosterone is the predominant intratesticular androgenic hormone68. The concentration of
intratesticular testosterone is 60,000ng/dl, much higher
than the average male serum testosterone concentration of 500ng/dl. Intratesticular testosterone concentration exceeded that of SHBG, signalling that most
intratesticular testosterone is likely to be in the bioavailable form68. Infact, Jarow etal.69 employed a novel
androgen-bioactivity assay based on modified androgen
receptor avidity and showed that 70% of total intratesticular testosterone is bioactive, which is a surrogate
for but not identical to bioavailable testosterone69.
Most importantly, the authors found that serum total-
testosterone concentration correlated poorly with intratesticular testosterone concentration (P=0.38). Based on
data from their novel assay, serum bioactive androgens
are not highly correlated with intratesticular bioactive
androgen levels (r=0.46, P=0.03). Interestingly, concentration of SHBG which might reduce the bioavailability of androgens in the testes and in serum were
strongly correlated, failing to explain the considerable
disparity between serum and intratesticular bioactive
androgen concentrations68,69. Unfortunately, serum

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bioavailable testosterone or free testosterone values
were not compared using the bioactive androgen assay,
so the clinical significance of data from this novel assay
remains uncertain.
Roth etal.70 performed a similar experiment by analysing testicular aspirations from fertile men. Contrary
to the findings of Jarow and colleagues68, the authors
found that serum testosterone and intratesticular testosterone, drawn at the same time, did correlate strongly
(r=0.67, P=0.03). Interestingly, they also found signifi
cant intrasubject variability in intratesticular testosterone levels which correlated very strongly with serum
LH (r=0.87, P=001). They hypothesized that the wellknown pulsatility of LH secretion could reflect similarly
pulsatile secretion of intratesticular testosterone. Further
evidence for the pulsatile secretion of intratesticular testosterone was provided by a study of testicular testosterone and oestradiol concentrations in cannulated gonadal
veins of men with varicoceles. Sampling every 15minutes revealed hourly testosterone pulses (35 pulses
per hour) with a mean amplitude of 17642ng/ml, a
6.6fold increase from the nadir71. However, currently,
the effect of intratesticular testosterone pulsatility on
spermatogenesis is unknown.
Several studies of infertile men and intratesticular androgen status have been performed. In one such
study, investigators found that intratesticular testosterone significantly correlated with increased serum
LH (r=0.67; P<0.001) and reduced testicular volume (P<0.001)72. Interestingly, Marie and colleagues73
found that infertile men with normal-range FSH had
elevated levels of intratesticular testosterone (P<0.01)
and intratesticular oestradiol (P<0.05), and a decreased
intratesticular testosterone:oestradiol ratio (P<0.05)
when compared with fertile controls. Conversely,
Shinjo etal.74 noted similar intratesticular testosterone
concentrations between men with ASD who underwent
a successful first microsurgical testicular sperm extraction (mTESE) and men who underwent an unsuccessful
surgery. However, men who were treated with salvage
exogenous human chorionic gonadotropin (hCG) and
underwent a successful second mTESE were more likely
to have lower baseline intratesticular testosterone levels
than men who failed salvage therapy, indicating a role for
targeted hormone therapy in this population.
The relationship between intratesticular testosterone and the clinical presentation of infertility remains
unclear, but researchers have also sought correlations
between testicular pathology for examining the degree
of spermatogenic dysfunction and intratesticular testosterone concentrations. Lardone etal.75 performed
a prospective study of 261 men with oligozoospermia
or azoospermia, including 68 men with obstructive
azoospermia who served as controls. The investigators
found that testicular pathological specimens with worsening degrees of spermatogenic dysfunction actually
contained increasing concentrations of intratesticular
testosterone relative to controls. Strong correlations
were also observed between worsening testicular spermatogenic pathology, larger Leydig cell clusters, and
decreased serum testosterone:LH ratio. These results
are consistent with the high prevalence of dysfunctional

Leydig cell micronodules among infertile men owing to
LH overstimulation, which might ultimately account for
the higher intratesticular testosterone concentrations.
Indeed, permissive growth of larger Leydig cell clusters might be secondary to smaller seminiferous tubule
volume in infertile men76. The importance of Leydig
cell dysfunction is illustrated by the weak hormonal
responses to hCG therapy an exogenous form of LH
among men with spermatogenic dysfunction compared with controls48. Thus, intratesticular testosterone
undoubtedly has a vital role in spermatogenesis, but the
reality of this relationship is difficult to interpret based
on traditional serum androgen assays.
The protein insulin-like 3 (INSL3) is specifically
secreted by mature Leydig cells and is, therefore, a very
promising biomarker of intratesticular androgen status77.
Roth etal.78 performed a prospective, randomized, nonblinded study of healthy men who underwent treatment
with the GnRH antagonist acycline to induce a hypo
androgenic state. The men were subsequently treated
with increasing doses of hCG. Testicular aspirations
and serum assays confirmed that INSL3 was strongly
correlated with intratesticular testosterone both during
the induced hypoandrogenic state and after subsequent
treatment with hCG (r=0.79, P<0.001). Other potential biomarkers, including inhibin B and anti-Mllerian
hormone, were not correlated with intratesticular testosterone. Despite these promising results, INSL3 has yet to
be employed in routine clinical practice as a biomarker of
Leydig cell function and intratesticular androgen status.
Sertoli-cell-mediated spermatogenesis is controlled
by multiple paracrine factors including transforming
growth factors and , epidermal growth factor, and
fibroblast-like growth factors and local steroidogenic
factors secreted from Leydig cells79. Data from mouse
models has shown that spermatogenesis persists until
the intratesticular testosterone level decreases below
75% of baseline concentration70. However, the gradient of intratesticular testosterone concentrations across
which human spermatogenesis is impaired remains to
be elucidated. Further evidence for the hormonal basis
of spermatogenesis is provided by experiments on cultured germ cells from men with pathological signs of
late-phase and early-phase spermatogenic maturation
arrest. Increasing doses of testosterone and FSH enabled
progression of germ cells from these men to spermatids
invitro, which were amenable to intracytoplasmic sperm
injection80. Testicular immunohistochemical staining
for the androgen receptor (AR) in infertile men isolated
their distribution to Sertoli cells only81. AR expression
in Sertoli cells and adequate intratesticular testosterone concentrations are both necessary for completion
of germ cell meiosis and proliferation of spermatogonia82. Specifically, Sertoli cell ARknockout in mice has
been shown to result in early spermatogenic maturation
arrest82,83. Inadequate intratesticular testosterone concentrations are associated with abnormal blood-testis
barrier integrity and abnormal spermatid adhesion
in mice84. Experiments with mouse hypoandrogenic,
Sertoli-cell-specific ARknockout models have further
REVIEWS
demonstrated that testosterone supplementation might
only induce spermatogenesis in the presence of a transgenic AR85,86. Finally, analyses of rat models of androgen
suppression (testosterone implants, oestradiol implants,
and FSH antibody) have revealed that the level of spermatogenic dysfunction is at the final stage of spermiation, owing to derangements in cell-cell adhesions
between spermatids and the Sertoli cell84,87.
Attempts to clinically correlate AR distribution
and function with the degree of infertility have been
unsuccessful thus far. Kato etal.81 discovered higher AR
density in men with ASD than in men with obstructive
azoospermia. Surprisingly, no correlation was found
between AR density and testicular volume, serum testosterone levels, serum gonadotropin levels, or intratesticular testosterone concentration. Furthermore,
exogenous FSH triggered an increase in AR density
while hCG therapy had no effect, underscoring the
importance of FSH-dependent Sertoli cell AR expression. Thus, gonadotropin monotherapy with hCG
would be inadequate in the setting of hypogonadotropic
hypoandrogenism. Attempts to associate the degree of
spermatogenic dysfunction and bulk seminal param
eters with polyglutamine polymorphisms in the AR have
yielded conflicting results, with some studies identifying
correlations and others not8891. Thus, FSH-dependent
AR expression is a necessary component for complete
spermatogenesis; however, accurate functional assays of
AR activation and, therefore, true androgen status, are
sorely lacking in clinical practice.
Hormonal therapy for male reproduction

Selective oestrogen receptor modulators
Selective oestrogen receptor modulators (SERMs) have
been a used in the treatment of male infertility for over
3decades, albeit off-label (BOX2). Clomiphene citrate is the
most well-described and widely used SERM92. The mechanism of action is via competitive inhibition of hypothalamic oestrogen receptors, which leads to increased
anterior pituitary gonadotropin release and subsequent
endogenous testosterone production93. Clomiphene citrate is a racaemic mixture of cis-isomers (zuclomiphene)
and trans-isomers (enclomiphene citrate), has a half-life
of approximately 5days, and has both antagonistic and
agonistic effects on oestrogen receptors. However, the
unwanted agonistic effects are largely mediated by
the zuclomiphene isomer. Enclomiphene citrate has a
more desirable anti-oestrogen profile and a shorter halflife than clomiphene citrate and has been tested in phaseII
trials94,95. However, these studies have not examined
men with infertility. Hopefully, future studies will assess
whether enclomiphene citrate could be a useful addition
to the available medications for men with infertility.
Adverse effects of SERMs are generally mild and
include irritability, but can manifest as rare visual disturbances, presumably owing to pituitary hyperplasia93,96.
Hyperestrogenism is a possible consequence of treatment, but we routinely monitor for its development
after treatment begins9799. Rare case reports exist of
paradoxical hormonal effects after treatment with clomiphene citrate including decreasing serum androgen
levels and even the induction of azoospermia; however,

the reasons for these effects are unknown100,101. Finally,
despite demonstrated anti-oestrogenic effects, SERMs
are not associated with osteoporosis, and might actually
improve bone mineral density102.
Data from several studies have examined the effects
of clomiphene citrate on androgen status. Original
dosing regimens were cyclical and evolved to pulsatile
every-other-day dosing to replicate the physiology of
LH secretion92,103. The only Level 1a evidence provided
for the use of clomiphene citrate in the treatment of
hypoandrogenism employed a 50mg every-other-day
dose regime. Significant increases in total testosterone
(P<0.01), free testosterone (P<0.01), and gonadotropins (LH P<0.01 and FSH P<0.05) were noted after
2months of treatment104. Further studies of 50mg
every other day (the authors preferred starting dosage) and 25mg daily showed statistically significantly
(P<0.001) improvements in serum testosterone97,105. In
a single-centre retrospective cohort study of 76 hypoandrogenic men treated with a starting dosage of 25mg
every other day, investigators found that only 64% of
patients eventually achieved adequate androgen status after titration to total testosterone >400ng/dl98.
Improvements in hypoandrogenic symptoms, as quantified using validated questionnaires have ranged from
6091% 98,106. Predictors of an adequate hormonal
response have included younger age (age 3050years),
testicular volume <14ml, and pretreatment LH levels
>6IU/l98,105. Interestingly, case reports indicate that
clomiphene citrate might even be effective in men
with idiopathic hypogonadotropic hypogonadism, but
not in men with true panhypopituitarism, who only
respond to gonadotropin therapy33,99,107. Semen ana
lysis after any hormonal therapy should be delayed by
3months to allow the spermatogenic cycle to establish
and the sperm to transit the epididymis. Our practice
is to monitor patients on infertility-related hormonal
therapy with periodic serum PSA and complete bloodcount assays, after the standard surveillance practices
for exogenous t estosterone replacement therapy.
Box 2 | Hormonal therapy
Selective oestrogen receptor modulators
Clomiphene citrate
Tamoxifen citrate
Enclomiphene citrate
Aromatase inhibitors
Anastrozole
Testolactone
Letrozole
Gonadotropin therapy:recombinant or purified
urine-derived
Follicle stimulating hormone
Human chorionic gonadotropic (luteinizing
hormone-analogue)
Urine-derived
Human menopausal gonadotropin

REVIEWS
Table 1 | Prospective trials of empirical therapy with selective oestrogen receptor modulators
Author and Trial design
publication
year
Treatment
Baseline male
factor
Effect of treatment on semen

analysis
Pregnancy rate
Foss etal.103
1972
RCT, double-blind,
CC 100mg daily for
placebo-controlled, three 10day periods
cross-over
successively versus
placebo
n=114
Idiopathic
infertility
No change in sperm concentration
NR
Ronnberg
etal.151
1980
RCT, double-blind,
CC 50mg daily
placebo-controlled, versus placebo for
cross-over
34months (3months
washout)
n=30
Euandrogenic
infertile men
CC: sperm concentration 13.8 28.7
CC: n=3
Placebo: n=1
Abel etal.152
1982
RCT, non-blind,
non-placebocontrolled
CC 50mg daily for

25days 5days off
versus vitaminC for
6months
n=179
Infertile men
NR
CC: 17%
VitaminC 13%
RCT, placebo
controlled,
non-double-blinded
(or undescribed
blinding)
CC 25mg (n=11) or
50mg daily (n=18)
versus placebo (n=7)
versus mesterolone
(n=12) versus
pentoxifylline (n=11)
versus testosterone
enanthate (n=15) for
6months
n=46
Euandrogenic
idiopathic
oligozoospermia
CC 25mg daily versus

placebo
n=23
Euandrogenic
idiopathic
oligozoospermia
Wang
etal.153
1983
Sokol
etal.154
1988
RCT, double-blind,
placebo-controlled
million/ml*
Placebo: sperm concentration
unchanged
(Baseline FSH not

predictive of response)
Sperm concentration:
CC 25mg daily: 8.87.2
15.314.8*
CC 50mg daily: 7.17.2
10.911.5*
Placebo: 8.24.4
8.35.6million/ml
Mesterolone: 8.39.0 10.314.8
Pentoxifylline: 10.34.3
11.15.8 (NS)
Testosterone enanthate:
suppression of spermatogenesis
(no statistical analysis)
CC: 25mg daily (36.4%)

CC: 50mg daily (22.2%)
Placebo: 0%
Mesterolone: 0%
Pentoxifylline: 0%
Testosterone
enanthate: 0%
Mean change count (million):

CC 37.35116.52
Placebo 26.0043.54
CC: 9.09%
Placebo: 44.44%
(No statistical test

performed)
Mean change motility (%):

CC 0.4615.66
Placebo 6.809.17
Mean change sperm
penetration (%):
CC 0.090.13
Placebo 0.040.14;
Check
etal.155
1989
RCT, non-blind,
non-placebo-controlled
CC 25mg daily for

25days 5days off
versus vitamin C for
8months
n=100
Idiopathic
infertility
(normal semen
parameters)
No effect on bulk semen parameters

parameters or hamster ova
penetration test
CC: 58%
VitaminC: 16%*
WHO156
1992
RCT, double-blind
CC 25mg daily versus

placebo for 6months
n=191
Idiopathic
abnormal semen
parameters
No significant changes
CC: 8.1%
Placebo: 11.7%
Patankar
etal.157
2007
Open-label
CC 25mg daily for

25days 5days off
n=25
Group 1: Motile sperm
Severe
concentration: 1.740.25
oligozoospermia
3.920.83million/ml*
(Group 1)
Group 2: Motile sperm
n=40, moderate
concentration 8.270.4
oligozoospermia
10.050.56million/ml
(Group 2)
NR
Ghanem
etal.158
2010
RCT, double-blind,
placebo-controlled
CC 25mg daily and

vitaminE versus
placebo for 6months
n=30
CC and
vitaminE
n=30
Placebo
Idiopathic OAT
CC: 36.7%
Placebo: 13.3%*
Count (million)
CC: 10.24.14 1815*
Placebo: 11.37.13 128.6
Motility (%)
CC: 3319 3421
Placebo: 3018 2416
Abnormal forms (%)
CC: 4116 3818
Placebo: 4115 5114
REVIEWS
Table 1 (cont.) | Prospective trials of empirical therapy with selective oestrogen receptor modulators
Author and Trial design
publication
year
Treatment
Baseline male
factor
Effect of treatment on semen

analysis
Pregnancy rate
Moradi
etal.159
2010
RCT
lcarnitine versus
CC 25mg daily for
3months
n=52
Idiopathic
infertility
Sperm concentration
improvement* (no difference from
lcarnitine)
Motility improvement* (greater
improvement than lcarnitine)
Morphology improvement*
(greater improvement than
lcarnitine)
NR
ElSheikh
etal.160
2015
RCT
VitaminE versus CC
25mg daily versus
vitaminE+CC for
6months
n=90 (1:1:1)
Idiopathic OAT
Sperm concentration:
VitaminE: no change
CC: improvement*
VitaminE+CC: improvement
NR
Sperm motility:
VitaminE: improvement*
CC: improvement*
VitaminE+CC: improvement*
CC, clomiphene citrate; FSH, follicle-stimulating hormone; NR, not reported; OAT, oligoasthenoteratozoospermia; RCT, randomized controlled trial *Statistically
significant.
Empirical treatment with SERMs is the most-studied

method of hormonal therapy for idiopathic male-factor
infertility and many prospective trials have been performed (TABLE1). In fact, many studies of SERM use
explicitly exclude men with hypoandrogenism and/or
abnormal gonadotropin levels. A meta-analysis of RCTs
of SERM therapy by Chua etal.96 showed improved
pregnancy rates after treatment (OR 2.42, 95% CI 1.47
3.94; P=0.0004). However, the authors tempered their
findings after identifying considerable sources of bias
among the cited studies, including high-risk randomization practices including inadequate practices related
to blinding, allocation concealment, or sequence generation. A systematic analysis of prospective trials with
clomiphene citrate therapy alone failed to discern sufficient evidence for use of empirical therapy108. Finally, the
authors of a Cochrane review similarly concluded that
insufficient evidence exists to support the empirical use
of SERMs in men with idiopathic oligosathenoteratozoospermia109. Despite these reports, results of a survey
of urologists published in 2012 found that up to twothirds prescribe empirical therapy for male infertility
in the USA110. The treatment of certain types of female
anovulation includes screening for FSH receptor polymorphisms before therapy with clomiphene citrate to
predict success, whereas clomiphene citrate treatment of
infertile men include no such reliable predictors of treatment response111. However, normal range pretreatment
FSH is a predictor of response to gonadotropin therapy,
no such evidence exists for clomiphene citrate.
Unlike empirical therapy, the use of targeted SERM
therapy for a specific hormonal abnormality suffers from a lack of supporting trials prospective or
otherwise. One retrospective study of 17 men with
idiopathic oligozoospermia found that pretreatment
gonadotropin and total-testosterone levels did not
correlate with an improvement in semen parameters112. Whitten etal.33 retrospectively examined men
with adult-onset hypogonadotropic hypoandrogenism

and found that clomiphene citrate therapy effectively
increased gonadotropin levels, serum testosterone, and
bulk semen parameters in three of four men, the partners of two of these three men went on to achieve pregnancies. Finally, Helo etal.113 performed a randomized,
double-blind, non-placebo-controlled trial of 26 men
with eugonadotropic hypoandrogenism and idiopathic
infertility, men with cryptozoospermia were excluded.
The men were treated with either 1mg daily anastrozole
or 25mg daily clomiphene citrate. As expected, anastrozole significantly improved the ratio of total testosterone:oestrogen compared with clomiphene citrate
(P<0.05). However, semen parameters did not improve
significantly after 3months of treatment with either
modality and pregnancy outcomes were not provided.
Treatment of hypergonadotropic hypogonadism with
SERMs could be attempted, but a reproducible cut-off
parameter for LH, above which therapy is futile, is yet
to be found.
Aromatase inhibitors
Unlike the SERMs, targeted therapy with aromatase inhibitors has been repeatedly studied.
Aromataseinhibitors include the steroidal agent testolactone, the nonsteroidal third-generation agent
letrozole, and fourth-generation nonsteroidal agent,
anastrozole, which has the highest relative aromatase
enzyme selectivity (BOX 2) . Targeted treatment is
directed toward correction of a diminished testosterone:oestrogen ratio114,115. Cavallini etal.116 performed a
double-blind, placebo-controlled RCT of targeted letrozole therapy for 6months in men with azozoospermia
or cryptozoospermia with a testosterone:oestrogen ratio
<10. Their results demonstrated significant improvements in bulk seminal parameters compared with placebo including resolution of all instances of ASD (sperm
concentration P<0.01, sperm percentage motility

REVIEWS
P<0.01). No spontaneous pregnancies were reported in
either group. Two notable single-institution retrospective series of men with oligozoospermia and testosterone:oestrogen ratio <10 have been published66,117. Of the
men with pretreatment and post-treatment semen analyses, anastrozole and testolactone yielded similar overall
improvements in bulk seminal parameters. Anastrozole
improved the testosterone:oestrogen ratio to a greater
extent, but men with Klinefelter syndrome responded
more readily to testolactone. Similarly, Gregoriou
etal.115 prospectively studied the relative effects of
6months of therapy with either anastrozole (1.0mg
daily) or letrozole (2.5mg daily) in 29 infertile men
with testosterone:oestrogen ratio <10. Semen parameters improved in >70% of patients in both treatment
arms. Saylam etal.67 examined 27 infertile men with
a testosterone:oestrogen ratio <10 who were treated
prospectively with 2.5mg letrozole for 6months. These
authors also observed significant overall improvements
in the testosterone:oestrogen ratio (P=0.001) and all
bulk seminal parameters evaluated (P<0.05). Thus, the
evidence for targeted therapy with aromatase inhibitors
is particularly promising.
Gonadotropin therapy
Therapeutic gonadotropin preparations have been
used clinically for the past 100years. These treatments
evolved from animal-based preparations to human
urine extracts then to highly purified recombinant
formulations. Purified injectable human menopausal
gonadotropin (hMG) contains bioactive FSH and LH
in 75IU formulations, with up to 70% gonadotropin
purity (BOX2). Two recombinant formulations of FSH
are commercially available, GonalF (Merck KGaA,
Germany) and Puregon (N.V. Organon, Netherlands),
which are notable for their higher purity than hMG.
Finally, hCG is available in a recombinant or urine-
derived formulation118,119. The relatively lower purity
of hMG than recombinant forms of FSH could be concerning for female patients with infertility in whom
the ratio of LH to FSH is vitally important for achieving
controlled ovarian stimulation such differences are
of negligible importance for infertile men given that the
ratio of LH to FSH does not play a clinically relevant
role in spermatogenesis. The lower cost of urine-derived
formulations compared with recombinant formulations
might, ultimately, prove to be the more important driver
of treatment choice.
Similar to SERM treatment, empirical gonadotropin
therapy has been used to treat idiopathic male infertility. Caroppo etal.120 performed a prospective open-label
trial of 33 men with idiopathic oligoasthenoteratozoospermia treated with 150IU of recombinant FSH for
3months. The authors observed significant improvements in all bulk seminal parameters after treatment,
but not in the untreated control group (P<0.05 for
sperm count and motile sperm. P<0.01 for percent
normal morphology, and P<0.001 for percent viable sperm). Of note, the baseline serum FSH level for
patients in the experimental arm was 9.686.05IU/l,
which reflects a population of men with normal range
FSH. In a Cochrane review, Attia etal.121 examined

the effectiveness of empirical FSH therapy through
six RCTs four of which were placebo-controlled
including 456 participants. The authors observed a significantly higher spontaneous pregnancy rate among
the partners of men treated with gonadotropins than of
those men who received no treatment or placebo (OR
4.94, 95% CI 2.1311.4, based on moderate-quality
evidence). Results of a meta-analysis published in 2015
echoed these findings, indicating an improved spontaneous pregnancy rate (OR 4.5, 95% CI 2.179.33),
regardless of the assisted-reproduction technique
employed. However, the authors cited the considerable
heterogeneity among published treatment algorithms,
which would serve to undermine the conclusions of
the meta-analysis122. Retrospective analysis of empirical treatment of idiopathic male infertility with injectable hMG with or without hCG showed improved
sperm concentration in 25% of treated patients, with
reduced baseline FSH and advanced sperm maturation
on testicular biopsy being predictive of success123,124.
Appreciating that men with oligozoospermia and
normal-range FSH, specifically without evidence of
maturation-arrest pathology, are the best candidates
for FSH gonadotropin therapy is vital125,126. Empirical
therapy with hCG monotherapy has not been thoroughly investigated, which is surprising given the limi
ted success hCG monotherapy has achieved in patients
with hypogonadotropichypoandrogenism.
Effective treatment of either congenital or acquired
hypogonadotropic hypoandrogenism (which often
presents with ASD) with gonadotropin therapy has
been well documented in a variety of retrospective and
multicentre prospective open-label trials. Historically,
pulsatile GnRH administration via a subcutaneous
pump was used to induce spermatogenesis in men with
hypogonadotropic hypoandrogenism with isolated
hypothalamic dysfunction. However, GnRH therapy
is now infrequently used, owing to its complexity and
equivalent results being obtained with simpler injectable gonadotropin therapy35,110,127,128. Apart from in
rare cases of isolated FSH or LH deficiency (known as
Pasqualini syndrome) the combination of recombinant
or urine-derived hCG and recombinant FSH or hMG
is necessary to induce spermatogenesis. Studies have
demonstrated that dual therapy with FSH or hMG and
hCG are necessary in patients with a congenital presentation, specifically indicating that a leadin period of
hCG monotreatment is only sufficient to induce spermatogenesis in 6170% of patients129131. However, dual
therapy with FSH or hMG and hCG for at least 6months
successfully induced spermatogenesis in 7594% of
patients128,130,132135. Baseline FSH was, unfortunately,
not a reliable indicator of the need to add hMG to hCG
monotherapy129. However, after induction of spermato
genesis with dual therapy, monotherapy with hCG
might successfully maintain semen density for up to
1year136. In fact, 10% of incidences (whether congenital
or acquired) resolve spontaneously after treatment cessation137. Riskfactors for treatment failure include cryptorchidism and smaller testicular volume (<4ml)130,132,135.
REVIEWS
Initial hormonal evaluation of infertile male
(total testosterone, FSH, LH, oestradiol, albumin and SHBG)
Yes
Currently treated with exogenous

testosterone replacement therapy?
Is bioavailable testosterone
<155 ng/dl?
Yes
No
Evaluate for other causes

of infertility
No
Stop therapy. Repeat lab

assessment in 24 weeks
Are gonadotropins undetectable

(hypogonadotropic hypoandrogenism)?
Yes
Evaluate and treat underlying

medical conditions. If persistent,
treat with hCG + hMG and/or FSH
No
Aromatase inhibitor
therapy
Yes
Is oestradiol >50 pg/dl?

If not, is the testosterone:
oestradiol ratio <10?
No
Clomiphene citrate therapy
Yes
After 2 weeks of therapy, is

oestradiol >50 pg/dl? If not,
is the testosterone:oestradiol
ratio <10?
No
Is bioavailable
testosterone adequate?
Yes
Check semen analysis,
total testosterone, PSA
and CBC in 4 months
No
Is FSH <1.5 baseline?

Yes
Stop clomiphene citrate.
Start hCG + hMG or rFSH
No
Add hCG therapy
Figure 2 | A simple algorithm for diagnosing and treating hormonal dysfunction in the setting of infertility to
assist clinicians in the decision-making process. CBC, complete blood count; FSH, follicle-stimulating
hormone;
hCG,
Nature Reviews
| Urology
human chorionic gonadotropin; hMG, human menopausal gonadotropin; LH, luteinizing hormone; r, recombinant; SHBG,
sex hormone-binding globulin.
Finally, most relevant studies have evaluated men with

undetectable or very-low gonadotropin levels. The subcategory of men with hypoandrogenism and normal
gonadotropin levels remains largelyunexamined.
Fertility treatment of iatrogenic hypogonadotropic
hypoandrogenism caused by exogenous testosterone
therapy or anabolic steroid use has become an increasingly complicated and prevalent clinical scenario.
Recovery of spermatogenesis after cessation of exogen
ous hormonal supplementation usually occurs within
6months, but can take several years138. Rare cases of
resolution of azoospermia after 5years of abstinence
from exogenous therapy have been documented139.
However, reports on the duration of exogenous therapy
and its effect on the potential for the recovery of fertility
are sparse. Several empirical treatment regimens exist to
successfully reverse the effects of exogenous therapy on
fertility, including injectable hCG treatment combined
with SERM therapy, have been presented140. Interestingly,
experiments involving men treated with exogenous testosterone (which induces hypogonadotropic hypoandrogenism and azoospermia) have shown that treatment
with either FSH or hCG might be sufficient to induce
normal spermatogenesis141,142.
The optimum recovery regimen and whether or
not treatment offers any advantage over watchful waiting for recovery of spermatogenesis remain unknown;
however a simple algorithm for diagnosing and treating
hormonal dysfunction in the setting of infertility would
assist clinicians in the decision-making process (FIG.2).
Hormonal therapy for ASD

In addition to initiation of spermatogenesis in men
with hypogonadotropic hypoandrogenism, SSR rates
among men with ASD are an ideal outcome measure
for quantifying the effectiveness of hormonal therapy.
Retrospective analyses of this scenario have yielded
disparate results. In a large single-institution cohort of

REVIEWS
1,054 men, targeted hormonal therapy for hypoandrogenic men with ASD did not yield any differences in
SSR rates, pregnancy rates, or live birth rates compared
with rates in untreated men143. However, a sub-analysis
of this same cohort revealed that SSR rates in men with
non-mosaic Klinefelter syndrome treated with hormonal therapy (mostly aromatase inhibitors with or
without exogenous testosterone supplementation) were
significantly higher for men who experienced adequate
improvements in serum androgen levels than in men
who did not respond (77% versus 55%; P=0.05)144,145.
Shiraishi etal.146 studied men who had an ineffective
initial mTESE and were subsequently treated with hormonal therapy. The treated men experienced a 21% SSR
rate compared with a 0% rate among untreated men.
Their treatment regimen consisted of high dosage hCG
(5,000IU three times per week for 3months), with
recombinant FSH added to the treatment regimen only if
serum FSH levels decreased from baseline. Interestingly,
53% of men experienced such a decrease in FSH from
baseline secondary to supraphysiologic serum testosterone, which serves as an important warning to monitor
such FSH levels if high-dose hCG monotherapy is pursued. Furthermore, men with severe hypoandrogenism
(total testosterone <200ng/dl) were excluded from their
study, which limits the applicability of their results146.
None of these retrospective studies included power
analyses to evaluate their results, which undermines the
relevance of the study findings.
Several prospective uncontrolled trials have been
performed to explore this important clinical presentation. Hussein etal.147 conducted a trial of clomiphene
citrate therapy in 42 men with ASD, of which 43% had
hypoandrogenism based on serum total testosterone
levels. After treatment for a mean duration of 5months,
64% of men developed sperm in their ejaculate and all
remaining men underwent successful mTESE. Post-hoc
analyses of preoperative testicular pathology indicated
that early maturation arrest was a negative prognostic
indicator of the development of ejaculated sperm, after
excluding men with Sertoli-cell-only syndrome (SCOS)
pathology from enrolment in the study. This group
performed a second controlled open-label prospective
trial of clomiphene citrate therapy before mTESE in
496 men. Men who failed to achieve post-treatment
FSH levels 50% greater than baseline were treated with
additional injectable gonadotropin therapy. Importantly,
the authors noted significantly higher SSR rates in the
experimental arms than in the untreated control arm
(61.7% versus 33.6%, P<0.001)101. Aydos etal.148 similarly
1.
2.
3.
Crowe,S.J., Cushing,H. & Homans,J.

inExperimental Hypophysectomy 127167
(JohnHopkins Press, 1910).
Hg,A., Mafm,Y., Aboulghar,M., Fj,B. & Jg,S.
Gonadotrophin-releasing hormone antagonists
for assisted reproductive technology.
CochraneDatabase Syst. Rev. 5, CD001750
(2011).
Palomba,S., Falbo,A. & La Sala,G.B. Metformin
andgonadotropins for ovulation induction in patients
with polycystic ovary syndrome: a systematic review
with meta-analysis of randomized controlled trials.
Reprod.Biol. Endocrinol. 12, 3 (2014).
demonstrated that men with idiopathic ASD treated with

recombinant FSH for 3months responded with significantly higher SSR rates than untreated controls (64%
versus 33%; P<0.01). The authors found that men with
pretreatment pathology consistent with hypospermatogenesis responded to treatment, but men with pathology indicative of SCOS or maturation-arrest did not. In
a small trial cohort of men with late-maturation arrest
pathology who failed first mTESE, 19.2% of patients
treated with recombinant FSH had a return of sperm to
the ejaculate149. One important lesson from these studies
is the promising effectiveness of empirical hormonal
therapy for men with ASD, specifically improving SSR
rates during mTESE. Interestingly, while testicular biopsy
is unnecessary from a purely diagnostic perspective,
the result of such a biopsy could help in predicting the
success of perioperative hormonal therapy. Finally,
reproductive endocrinologists employ the clomiphene
citrate challenge test, based on post-treatment FSH levels, to determine fertility potential and the potential usefulness of that medication150. Hussein and colleagues101
have initiated such an algorithmic approach to hormonal
therapy, but further research is necessary to join our colleagues who treat female fertility and are truly employing
precision medicine.
Conclusions
Treatment of male infertility has evolved to encompass
physicians who routinely address complex hormonal
therapy in addition to surgery. Our knowledge of the
genetic aspects of male fertility has moved forward,
but research into hormonal therapy for men has largely
relied on heterogeneous treatment regimens with unreliable outcomes. The information garnered from such
studies is important, although it pales in comparison
to the level of evidence available to our colleagues in
female reproductive endocrinology. Investigations
into female infertility benefit from reliance on objective, verifiable outcomes such as ovulation, biochemical pregnancy, and clinical pregnancy. Meanwhile, the
male counterpart has been hampered by the necessary
dependence on bulk seminal parameters, which are
notoriously poor predictors of fertility potential. Perhaps
the only truly reliable semen analysis is one indicating
azoospermia and that is where the most exciting clinical outcomes research has focused. The performance
of multi-institutional, high-quality trials with sufficient
enrolment numbers and meaningful and reliable outcomes, and the development of reference databases are
vital to best treat ourpatients.
Greenberg,S.H., Lipshultz,L.I. & Wein,A.J.

Experience with 425 subfertile male patients. J.Urol.
119, 507510 (1978).
5. Lee,R., Li,P.S., Goldstein,M., Schattman,G.
&Schlegel,P.N. A decision analysis of treatments
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Competing interests
C.N. is on the American Urological Association Journal

of Urology Urological Survey Editorial Committee is
coeditor-inchief of Fertility and Sterility, founder of NexHand
and Chief Technology Officer, he is supported by a Ferring
research grant support. M.K. declares no competing
interests.
Author contributions
M.K. wrote the article, both authors researched data for the
article, discussed the content and reviewed and edited
themanuscript before submission.
Review criteria
We performed a search of the PubMed database. We employed

search criteria relevant to the diagnosis and treatment of hormonal dysfunction in the setting of male infertility. Search
terms included infertility, azoospermia, oligozoospermia,
gonadotropin therapy, clomiphene citrate, anastrozole,
human chorionic gonadotropin, human menopausal gonadotropin, empiric therapy, and intratesticular. Reference
lists from the reviewed manuscripts were also included for
finalreview.


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DiagDiagnosis and Treatment of Infertilityrelated Male Hormonal Dysfunctionnosis and Treatment of Infertilityrelated Male Hormonal Dysfunction

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REVIEWS

Diagnosis and treatment of infertilityrelated male hormonal dysfunction

Abstract | Treatment of infertility-related hormonal dysfunction in men requires an understanding

Division of Urology, Brigham

gonadotropin-releasing hormone (GnRH) agonists with

NATURE REVIEWS | UROLOGY

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to male infertility currently exists in the USA. Indeed,

2 | ADVANCE ONLINE PUBLICATION

Figure 1 | The hypothalamicpituitarygonadal axis. Secretion of the anterior pituitary

Hormonal biomarkers to determine fertility potential

Associated clinical conditions

NATURE REVIEWS | UROLOGY

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onset of hypogonadotropic hypoandrogenism 33,34

serum prolactin levels >250ng/ml. However, MRI

Determination of androgen status

4 | ADVANCE ONLINE PUBLICATION

Epidemiology of hormonal dysfunction

NATURE REVIEWS | UROLOGY

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are consistent with the high prevalence of dysfunctional

6 | ADVANCE ONLINE PUBLICATION

Hormonal therapy for male reproduction

levels and even the induction of azoospermia; however,

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Effect of treatment on semen

No change in sperm concentration

CC: sperm concentration 13.8 28.7

CC 50mg daily for

CC 25mg daily versus

(Baseline FSH not

CC: 25mg daily (36.4%)

Mean change count (million):

(No statistical test

Mean change motility (%):

CC 25mg daily for

No effect on bulk semen parameters

CC 25mg daily versus

CC 25mg daily for

CC 25mg daily and

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Effect of treatment on semen

Empirical treatment with SERMs is the most-studied

with adult-onset hypogonadotropic hypoandrogenism

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FSH. In a Cochrane review, Attia etal.121 examined

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Currently treated with exogenous

Evaluate for other causes

Stop therapy. Repeat lab

Are gonadotropins undetectable

Evaluate and treat underlying

Is oestradiol >50 pg/dl?

After 2 weeks of therapy, is

Is FSH <1.5 baseline?

Finally, most relevant studies have evaluated men with

Hormonal therapy for ASD

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Crowe,S.J., Cushing,H. & Homans,J.

demonstrated that men with idiopathic ASD treated with