International Journal of

Molecular Sciences

Review
Disorders of Sex Development—Novel Regulators,
Impacts on Fertility, and Options for
Fertility Preservation
Nathalia Lisboa Gomes 1,2 , Tarini Chetty 3 , Anne Jorgensen 4,5 and Rod T Mitchell 3,6, *
1 Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular (LIM/42)
da Disciplina de Endocrinologia e Metabologia do Hospital das Clínicas da Faculdade de Medicina,
Universidade de São Paulo, 05403 900 São Paulo, Brazil; [email protected]
2 Serviço de Endocrinologia da Santa Casa de Belo Horizonte, Av. Francisco Sales, 1111, 30150-221 Belo
Horizonte, Minas Gerais, Brazil
3 Department of Diabetes and Endocrinology, Royal Hospital for Sick Children, 9 Sciennes Road,
Edinburgh EH9 1LF, UK; [email protected]
4 Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Blegdamsvej 9,
2100 Copenhagen, Denmark; [email protected]
5 International Research and Research Training Centre in Endocrine Disruption of Male Reproduction and
Child Health (EDMaRC), Blegdamsvej 9, 2100 Copenhagen, Denmark
6 Medical Research Council (MRC) Centre for Reproductive Health, The University of Edinburgh, The
Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK
* Correspondence: [email protected]




Received: 28 January 2020; Accepted: 24 March 2020; Published: 26 March 2020


Abstract: Disorders (or differences) of sex development (DSD) are a heterogeneous group of
congenital conditions with variations in chromosomal, gonadal, or anatomical sex. Impaired gonadal
development is central to the pathogenesis of the majority of DSDs and therefore a clear understanding
of gonadal development is essential to comprehend the impacts of these disorders on the individual,
including impacts on future fertility. Gonadal development was traditionally considered to involve a
primary ‘male’ pathway leading to testicular development as a result of expression of a small number
of key testis-determining genes. However, it is increasingly recognized that there are several gene
networks involved in the development of the bipotential gonad towards either a testicular or ovarian
fate. This includes genes that act antagonistically to regulate gonadal development. This review will
highlight some of the novel regulators of gonadal development and how the identification of these
has enhanced understanding of gonadal development and the pathogenesis of DSD. We will also
describe the impact of DSDs on fertility and options for fertility preservation in this context.

Keywords: disorder of sex development; fertility; fertility preservation; gonads; testis; ovary;
sex determination

1. Gonadal and Germ Cell Development

Genetic sex is determined from the point of conception. In contrast, the developing gonad remains
bipotential until approximately six weeks post-conception, as until this point it can develop into an
ovary or a testis. Development of the gonad can be divided into two key phases, an initial phase with
formation of the bipotential gonad and a second phase with differentiation towards either testicular or
ovarian fate.
Bipotential gonads arise at approximately four weeks post-fertilization in the human embryo,
as paired structures known as genital ridges formed from a thickening of intermediate mesoderm
and proliferation of overlying coelomic epithelium [1]. The gonads emerge from the ventral surface

Int. J. Mol. Sci. 2020, 21, 2282; doi:10.3390/ijms21072282 www.mdpi.com/journal/ijms

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of the cranial mesonephros, which in addition give rise to the adrenal glands [2]. Each mesonephros
also contains the mesonephric duct (Wolffian duct) and paramesonephric duct (Mullerian duct),
precursors of the male and female reproductive tracts respectively [3,4]. The bipotential gonad is
initially exclusively comprised of somatic mesoderm derived cells, which are the precursors of steroid
producing testicular Leydig and ovarian theca cells and of the supporting Sertoli cells (in the testis)
and granulosa cells (in the ovary). Subsequently, by five weeks post-conception, primordial germ cells
migrate from the yolk sac to the urogenital ridge. Several genes including NR5A1 (also known as SF1),
WT1, EMX2, and LHX9 are required for the formation of the bipotential gonadal ridge in humans [5,6].
Traditionally, differentiation of the bipotential gonad into a testis or ovary was thought to be due
solely to the presence or absence of the SRY gene on the Y chromosome, with ovarian development
occurring in the absence of SRY expression. However, it is increasingly recognized that there are in
fact several gene networks involved in the complex process of gonadal differentiation towards either
ovarian or testicular fate, some of which act antagonistically on the opposite pathway.
In the XY gonad, at approximately seven weeks post conception, SRY is expressed in Sertoli cell
precursors, and can be thought of as the dominant ‘switch’ in promotion of testicular development [7].
SRY acts on SOX9, which reaches a critical threshold to drive positive regulatory loops that maintain
high levels of SO9 expression and activity independent of SRY expression [5,8]. SOX9 then initiates
differentiation of the supporting cell lineage into Sertoli cell rather than granulosa cell fate. SOX9 may
also have a role in repressing genes involved in ovarian development such as WNT4, FOXL2, and
transcription factor DMRT1 [9,10].
Sertoli cells secrete anti-Mullerian hormone (AMH), which, via bone morhogenic protein
(BMP)-like signaling pathways, promotes regression of Mullerian structures in males [11]. In
addition, Sertoli cells secrete desert hedgehog (DHH) which induces development of steroid producing
fetal Leydig cells that secrete testosterone and INSL3 from approximately eight to nine weeks of
development [12]. Testosterone promotes differentiation of the Wolffian duct into epididymis, vas
deferens, and seminal vesicles and, along with INSL3, contributes to testicular descent [13]. From around
eight weeks post-conception, dihydrotestosterone (DHT), produced primarily by enzymatic conversion
of testosterone, acts on the androgen receptor causing virilization of the external genitalia [14].
Ovarian development is an active process involving antagonistic regulatory networks that suppress
testis development. Absence of SRY expression prevents SOX9 reaching a critical threshold, and this
combined with the expression of factors such as RSPO1, WNT4, and FOXL2 that act to suppress
testicular development, results in formation of an ovary [8]. In the XX gonad, low levels of AMH and
the absence of testosterone cause Wolffian ducts to involute and Mullerian ducts to develop to form
the oviduct, uterus, and upper part of the vagina. Absence of androgens results in the development of
external female genitalia.
The presence of germ cells and their capacity to generate gametes is essential for future fertility.
During fetal development, primordial germ cells migrate into the developing gonad from approximately
five weeks gestation at which point they are termed gonocytes [15]. Gonocytes in both sexes express
pluripotency markers and undergo further development towards a spermatogonial or oogonial fate.
In males, gonocytes begin to downregulate pluripotency factors early in the second trimester leading
to the development of (pre)spermatogonia [16]. Failure of germ cells to transition from gonocyte to
spermatogonia in fetal and early postnatal life may result in the development of precursor cells for
testicular germ cell tumours [17], whilst loss of germ cells at any stage of development may impact
on fertility potential. Spermatogonia represent the germ cell population in the prepubertal testis. At
puberty, meiosis is initiated and completion of spermatogenesis results in production of a continuous
supply of spermatozoa from a self-renewing pool of spermatogonial stem cells (SSC). In contrast,
female germ cells (oogonia) enter meiosis from around 10 gestational weeks and become oocytes [18].
Importantly, germ cell survival and development in both sexes is dependent on unique interactions
with the somatic cell populations of the gonad; therefore, failure in the development of both somatic
cells and germ cells during gonad development can impact on germ cells and future fertility.
Int. J. Mol. Sci. 2020, 21, 2282 3 of 31

2. Disorders of Sex Development

Disorders (or differences) of sex development (DSD) are a heterogeneous group of congenital
conditions associated with variations of chromosomal, gonadal, or anatomical sex [19]. This diverse
group of conditions most often present in the newborn period with ambiguous genitalia or in
adolescence with atypical pubertal development. DSDs, as described in the Chicago consensus
statement, can be classified into three broad categories: sex chromosome, 46,XY, and 46,XX DSD [19].
Sex chromosome DSD encompasses differences in sex chromosome copy number including
Turner and Klinefelter syndromes. Turner syndrome (TS) is characterized by 45,X monosomy, the
presence of an abnormal X chromosome, or mosaicism of the 45,X cell line (e.g., 45,X/46,XY). Pure 45,X
monosomy is associated with a more severe phenotype of severe short stature, gonadal dysgenesis,
and dysmorphic features compared to mosaic genotypes who may enter puberty spontaneously before
developing primary ovarian failure, premature loss of oocytes, and infertility. Klinefelter syndrome is
characterized by the presence of a 47,XXY cell line and clinical features include tall stature, small testes,
gynecomastia, primary gonadal failure, progressive germ cell loss, and infertility [20]. 46,XX/46,XY
mosaicism and 46,XX/46,XY chimerism are associated with dysgenetic gonads, infertility, and a highly
variable phenotype which may present with ambiguous genitalia at birth [21].
46,XY DSD is usually characterized by ambiguous or female appearance of external genitalia with
or without the presence of Mullerian structures as a result of under virilization in utero. They can be
further subdivided into three diagnostic categories: problems with gonadal development resulting
in gonadal dysgenesis, biosynthetic defects causing impaired production of androgens (testosterone
and dihydrotestosterone), or lack of androgen action due to end organ resistance to these hormones.
The 46,XY DSDs that result in impaired gonadal development are also associated with abnormalities
of germ cell development predisposing to gonadal tumors and infertility, with the relative risk of
malignancy dependent on the specific condition [22].
46,XY complete gonadal dysgenesis (CGD), also known as Swyer syndrome, is characterized by
failure of testicular development and formation of fibrous streak gonads which lack germ cells. Lack
of production of AMH and testosterone results in a completely female appearance of the external
genitalia and well developed Mullerian structures [23]. In contrast, partial gonadal dysgenesis (PGD)
is characterized by partial testis development and can present with a spectrum of phenotypes with a
variable degree of masculinization of the external genitalia and a combination of Wolffian and Mullerian
ducts. Rarely, there can be the presence of both ovarian and testicular tissue in one individual, known
as 46,XY ovotesticular (OT) DSD.
Androgen biosynthetic defects high in the steroid pathway such as lipoid congenital adrenal
hyperplasia (CAH); caused by an abnormality in the steroidogenic acute regulator (StAR) protein) can
result in a similar biochemical picture to that of gonadal dysgenesis with low basal and stimulated
testosterone levels and low testosterone precursors. In contrast, biosynthetic blocks further down
the steroid pathway result in low testosterone levels with high steroid precursors and include
CAH variants such as 17-α hydroxylase/17,20-lyase deficiency, 3-beta-hydroxysteroid dehydrogenase,
and 17-beta-hydroxysteroid deficiencies. Disorders of androgen biosynthesis can impair germ cell
development resulting in infertility [24].
Dihydrotestosterone is formed by enzymatic reduction of testosterone by 5-α reductase via the
classical pathway [25], and by an alternative ‘backdoor pathway’ where 17α-hydoxyprogesterone
(17OHP) is converted to DHT bypassing the intermediates of testosterone and androstenedione [14].
Defects in either of these pathways can result in DHT deficiency and incomplete virilization of the
external genitalia with normal Wolffian structures. Mutations in SRD5A2 cause 5-α reductase type
2 deficiency, an autosomal recessive condition that classically presents with a female phenotype at
birth and significant virilization without breast development at puberty. Impaired spermatogenesis is
common in individuals with 5-α reductase deficiency [26].
46,XY DSD can be caused by defects in androgen action, typically due to dysfunction of the
androgen receptor (AR) [27]. DSD due to complete loss of function of the AR is termed Complete
Int. J. Mol. Sci. 2020, 21, 2282 4 of 31

Androgen Insensitivity Syndrome (CAIS), whereas mutations that retain some residual function result
in Partial Androgen Insensitivity Syndrome (PAIS). CAIS typically presents in adolescence as primary
amenorrhea despite normal breast development. Alternatively, CAIS may present in infancy with
palpable inguinal masses in an individual with a 46,XY karyotype and a typical female appearance
to the external genitalia. The phenotype in PAIS is variable depending on the degree of androgen
sensitivity and there may be associated gynecomastia due to the peripheral conversion of testosterone
to estradiol. Individuals with inactivating mutations of the LH receptor (Leydig cell hypoplasia) have
a variable appearance ranging from a completely female phenotype to a variable degree of virilization,
very similar to patients with AIS. However, breast development is not observed. Fertility is impaired
in the majority of individuals with disorders of androgen action as a result of germ cell loss and/or
failure of spermatogenesis [28].
46,XX DSD is usually characterized by ambiguous or virilized genitalia as a result of fetal exposure
to androgen excess and normal development of Mullerian structures and ovaries. Androgens may be
derived from the fetal adrenal gland such as in CAH or placental aromatase deficiency, or rarely from
exogenous sources such as transplacental passage of androgens from a maternal adrenal or ovarian
tumor. Exposure to androgens can impact fertility in 46,XX individuals with DSD despite the presence
of normal female reproductive anatomy [24]. Androgen arising from isolated testicular tissue (46,XX
testicular DSD) or from a mixed gonad of testicular and ovarian tissue (46,XX ovotesticular DSD)
occurs in a rare subgroup of 46,XX DSD. Germ cell development is impaired in these individuals with
impacts on fertility [29].

3. Novel Regulators and Mechanisms of Gonadal Development

The gonadal determination process is complex and involves numerous genes that are expressed in
a tightly regulated temporo-spatial manner in order to lead not only to gonadal differentiation, but also
its maintenance. The majority of the patients with 46,XY gonadal dysgenesis (GD), SRY negative 46,XX
ovotesticular (OT), and 46,XX testicular (T) DSD remain without a molecular diagnosis, indicating
that novel genes, genomic rearrangements, and unknown regulatory regions could be involved in
these disorders.
Recently, novel insights into the pathogenesis of human DSD have resulted from the advent of
massively parallel sequencing technologies, bioinformatics and innovative tools for the generation of
animal models, such as CRISPR/CAS9. This has increased our understanding of how novel genes, in
addition to those previously known to be associated with DSD, are involved. Also, this has identified
mechanisms and pathways dysregulated in human DSDs. The identification of the underlying
genetic basis for a specific DSD may lead to options for individualized management regarding genetic
counseling and novel therapeutic interventions, in addition to assessment and management of fertility.

3.1. The SRY/SOX Family: New Concepts in Gene Dosage and Regulation
It is known that presence of the SRY gene tips the balance to promote testis development, leading to
an up-regulation of SOX9 expression at around 6 weeks gestation in humans and 10.5 days post-coitum
in mice [7,30–32]. However, until recently, the mechanism by which SRY activates SOX9 was not
understood in detail in humans and was only partially elucidated in mice.
In mice, Sry binds synergistically with Sf1 (encoded by Nr5a1) to activate a Sox9 enhancer, known
as Testis Specific Enhancer of Sox9 (TES) and its core sequence known as TESCO [33,34]. Deletion of
TESCO or TES reduced Sox9 expression levels in XY fetal mouse gonads to 60% or 45%, respectively,
compared to wild-type testis [34]. However, loss of function of TESCO was insufficient to cause sex
reversal, indicating that TESCO is not the only Sox9 enhancer in mice [34]. In humans, no single variant
in this region had been identified [35].
Recently, Gonen and colleagues identified a novel gonadal regulatory element upstream of
Sox9, enhancer 13 (Enh13), which was shown to be essential in the initiation of mouse testicular
differentiation [36]. Deletion of Enh13 led to complete XY male-to-female sex reversal in mice.
Int. J. Mol. Sci. 2020, 21, 2282 5 of 31

Importantly, Enh13 is conserved and embedded within a 32.5-kilobase region in humans in which
deletions have been associated with XY sex reversal, suggesting that Enh13 may also be critical in
humans [36].
Since 2011, copy number variants (CNVs) located within the 2 Mb putative SOX9 upstream
regulatory region, denoted XYSR and RevSex have been increasingly identified by array comparative
genomic hybridisation (CGH) or multiplex ligation-dependant probe amplification (MLPA) in several
isolated 46,XX and 46,XY DSDs (reviewed in [37]). The refined analysis of these genomic regions has
allowed the identification of two putative enhancers 50 of SOX9, named Sex Reversal Enhancer-A
(eSR-A) and Sex Reversal Enhancer-B (eSR-B) [38]. Both of these enhancers are activated by SOX9
alone or in combination with NR5A1, but have little activation by SRY, the primary initiator of the
testis determination. This experimental data was corroborated by the fact that loss of one copy of those
elements in 46,XY DSD patients with either complete or partial gonadal dysgenesis appeared to be
sufficient to prevent upregulation or maintenance of SOX9 expression to the levels required to ensure
proper testis development. By contrast, a single additional copy of either of these enhancers promotes
the expression of SOX9 to a level that is sufficient to override the ovarian program causing testicular or
ovotesticular DSD in 46,XX individuals [38].
The identified eSR-A showed 80% sequence conservation with its orthologous mouse enhancer
Enh13 [36]. In contrast to the human enhancer, Enh13 demonstrated strong enhancer activity in response
to SF1 in combination with SOX9 and also with SRY. Thus, different SOX9 activation mechanisms may
be observed between species [38].
An additional enhancer, Alternate Long-Distance Initiator (eALDI), was also identified as an
SRY-responsive enhancer of SOX9, which is 1.4 kb upstream of human TESCO. This enhancer is
significantly activated by co-transfection with SRY+SF1 or SOX9+SF1, but not with SOX9 alone.
Therefore, eALDI appears to be the primary enhancer by which SRY and SF1 act to initiate SOX9
expression; SOX9 alone or in combination with SF1 can then upregulate expression via all three
enhancers, which may together act on the SOX9 promoter through chromatin looping events. Despite
the evidence implicating eALDI as an enhancer of SOX9, eALDI CNVs have not been identified in
DSD patients [38]. Also, the exact order in which enhancer activation occurs remains elusive given the
limitations of studying early human embryonic development.
Despite the fact that SOX9 is the only recognized SRY target gene, other SOX genes can mimic
its functions. The SRY-related HMG box 3 gene (Sox3), which is structurally very similar to Sry, is
predominantly expressed in developing neural tissue, with no function during sex determination [39,40].
However, copy number variations (CNVs) identified in SOX3 regulatory regions have been detected in
patients with 46,XX testicular and OT DSD [41,42]. Transgenic mouse models confirmed that ectopic
gonadal expression of Sox3 induces testis differentiation by upregulating Sox9 in a similar manner to
Sry, suggesting that these genes may be functionally interchangeable in sex determination [42].
Additionally, previous data suggested that Sox8 was a contributor to murine testis-determination
and maintenance of gonadal function [43,44]. SOX8 is co-expressed with NR5A1 and SOX9 in the
early stages of human testis-determination as well as in Sertoli and Leydig cells in adult men [45]. In
2018, two rearrangements involving the SOX8 locus and one missense heterozygous variant in SOX8
(c.468G>C; p.Glu156Asp) were identified in 46,XY DSD patients, and other missense variants were
associated with male infertility and ovarian insufficiency in females [45]. All these variants showed an
altered SOX8 biological activity compared to the wild-type protein.
Despite the fact that no data has been reported on fertility in these patients, a study published in
2019 was the first to link the SOX9 pathway as a possible future target for infertility intervention [46].
The identification of deleterious homozygous variants in the PPP2R3C gene (which encodes protein
phosphatase two regulatory subunit B”gamma), in four syndromic girls with 46,XY complete gonadal
dysgenesis from four unrelated families led to the genetic study of the infertile cases in the affected
families and parents who harbored the variants in heterozygous state. All three studied fathers,
including one reported as infertile, were found to have teratozoospermia with severe head, acrosomal,
Int. J. Mol. Sci. 2020, 21, 2282 6 of 31

and nuclear abnormalities following semen analysis. One mother reported oligomenorrhea and
hypomenorrhea with no abnormality in pelvic ultrasonography and another mother had menopause
at 44 years of age [46].
PPP2R3C encodes B”gamma regulatory subunit of PP2A and SOX9 is a downstream canonical
target of PP2A. SOX9 needs to be phosphorylated to SOX9-Phospho to be activated to induce
downstream pathways for testicular development. The study showed that the variants in PPP2R3C were
predicted to upregulate the catalytic function of PP2A, subsequently increasing the dephosphorylation
of phosphorylated SOX9, leading to disruption in testis development [46]. Another previous study
showed that PP2A may play an important role in testicular development and in spermatogenesis [47].
The presented data provided evidence on the key role of PPP2R3C protein not only in testicular
development, but also as a critical intracellular signaling molecule involved in spermatogenesis,
showing that heterozygous defects of this gene are associated with impaired spermatogenesis and male
infertility. The authors mention that selective pharmacologic PP2A inhibitors, such as okadaic acid, were
shown to promote fertility by increasing sperm motility, velocity, and lateral head amplitude [48,49].
Thus, these findings link specific genetic causes of human DSD with novel therapeutic targets of
human infertility.

3.2. NR5A1 (SF1) and WT1: Old Genes, New Mechanisms

As an important regulator of adrenal function, the first NR5A1 homozygous mutations
were identified in two patients with primary adrenal insufficiency and 46,XY complete gonadal
dysgenesis [50,51]. After the identification of a patient with partial gonadal dysgenesis without adrenal
insufficiency [52], an increasing number of heterozygous variants in this gene were identified in
patients with a range of 46,XY DSD phenotypes without adrenal insufficiency, including patients with
complete and partial GD, isolated hypospadias, and bilateral anorchia [53,54]. Thus, NR5A1 variants
are considered one of the most common causes of 46,XY DSD [55]. In addition, NR5A1 variants are a
recognized cause of isolated male infertility. This includes reports of men with azoospermia or severe
oligozoospermia (with and without previous cryptorchidism), some whom also had low testosterone
and elevated gonadotropins [56].
However, the precise mechanism by which NR5A1 action fails and leads to DSD is not fully
understood, nor has an explanation been provided for the wide-ranging phenotypes associated with
different and, in some cases identical, NR5A1 variants. This is despite numerous studies attempting to
resolve this question by analyzing defective NR5A1 function at steroidogenic target promoter (such as
CYP11A1, CYP17A1, and CYP19A1) or sex differentiation genes (AMH and INSL3) [57].
A recent analysis of TESCO activation together with NR5A1 nuclear localization showed
correlation, at least for most of the studied cases, with the phenotypic severity. This indicates
that defective TESCO/SOX9 activation may account for the high phenotypic variability in patients
with 46,XY DSD harboring deleterious variants in NR5A1 [58]. Other studies have suggested that this
variability is due to digenic/oligogenic inheritance as rare variants identified in additional DSD genes
in several patients with NR5A1 mutations [59–62].
In 46,XX individuals, NR5A1 variants were previously only associated with ovarian
insufficiency [53,54]. However, in 2016, a single recurrent heterozygous NR5A1 variant (p.Arg92Trp)
was identified in patients with 46,XX testicular and OT DSD by three independent groups [1,63,64].
Subsequently, additional cases have been reported [54], all without evidence of adrenal insufficiency.
A different homozygous variant involving the same amino acid (p.Arg92Gln) was identified in
two patients with adrenal insufficiency, a 46,XX girl [65] and a 46,XY GD individual [51], while
this same variant was identified in a heterozygous state in a 46,XX OT individual without adrenal
insufficiency [66]. A third heterozygous NR5A1 mutation (p.Ala260Val), was recently described in a
46,XX OT DSD individual [67].
The wide phenotypic variability observed among 46,XX and 46,XY DSD individuals, with and
without adrenal insufficiency, has been explored using in vitro assays. During normal ovarian
Int. J. Mol. Sci. 2020, 21, 2282 7 of 31

development, NR5A1 and β-catenin form a complex that upregulates the Nr0b1 gene, which is involved
in SOX9 repression in 46,XX individuals. The NR5A1 p.Arg92Trp and p.Ala260Val variants impede the
action of the NR5A1/β-catenin complex interaction and thus undo the NR0B1-mediated repression of
SOX9 probably leading to a disruption in ovarian determination [1,63,64,67]. In 46,XY individuals, the
p.Arg92Trp variant reduced activation of several minimal promoters (AMH, CYP11A1) and enhancers
(SOX9, TESCO) involved in testis development, thus explaining the GD phenotype [1]. This variant also
exhibited partial loss of DNA binding and transcriptional activity, explaining the adrenal insufficiency
phenotype when transmitted in homozygous state [51]. However, it remains unclear why there are
46,XX and 46,XY individuals who are asymptomatic and fertile carriers of these variants [1,51,63].
WT1 deleterious variants were previously only associated with 46,XY DSD in Denys-Drash and
Frasier syndromes. In recent years, the involvement of WT1 in XX gonadal development has been
demonstrated by the description of two deleterious heterozygous variants in two 46,XX patients with
premature ovarian insufficiency (POI) [68] without kidney disease, and also in a patient with POI
and adult-onset focal segmental glomerulosclerosis [69]. Recently, a de novo frameshift variant of
WT1 was identified in a girl with 46,XX testicular DSD, c.1453_1456del, p.(Arg485Glyfs*14). Structural
protein remodeling suggests an increased activation of target genes, mainly NR5A1. However, these
assumptions remain to be experimentally validated [70].

3.3. PBX1 and CBX2: Gene Interactions that Promote Testis Development
The TALE homeodomain of Pre-B-Cell Leukemia Transcription Factor 1 (Pbx1) is known to play
an important role in mouse adrenal and urogenital development [71]. At E14.5, Pbx1−/− mice exhibit
severely impaired testis development associated with markedly decreased cell proliferation in the
genital ridge [71].
In humans, deleterious variants involving PBX1 were previously associated with congenital
anomalies of the kidney and urinary tract, but not with DSD [72,73]. PBX1 has been implicated
in human testicular development after the identification of a single de novo heterozygous variant
(p.Arg235Gln) in a 46,XY GD patient with normal kidneys and radiocubital synostosis [74]. This
variant is located in a highly conserved TALE homeodomain of the protein, as opposed to previously
described variants that are located in the consensus splice site, potentially explaining the different
phenotype [72,73]. In vitro studies of cellular location showed that the mutated p.Arg235Gln PBX1
protein was unable to correctly localize into the nucleus and also had an impaired biological activity,
as its physical interaction with two proteins known to be involved in testis determination (EMX2 and
CBX2) were abolished [74]. This data suggests that specific variants located in the TALE homeodomain
of PBX1 are a novel cause of human 46,XY DSD.
CBX2 isoform 1 is thought to lie upstream of SRY gene expression in the human sex development
cascade based on the findings in mouse models. Cbx2 (M33) knockout mice have hypoplastic gonads
in both sexes and, in the XY mice, the expression of Sry and Sox9 is reduced [75,76]. Additionally, it has
been shown that forced expression of Sry or Sox9 in Cbx2 XY KO mice could rescue their sex reversal,
although they present with smaller gonads compared to wild-type mice [76]. This data suggests that
Cbx2 regulates Sry expression in gonadal development and might also influence gonadal size.
In humans, compound heterozygous CBX2.1 variants were identified in a 46,XY DSD patient with
complete gonadal dygenesis and histologically normal ovaries, resembling the knock-out (KO) XY
mice phenotype [77]. Functional studies demonstrated that these variants do not bind to, or adequately
regulate the expression of, target genes important for gonadal development, such as NR5A1 [77].
A more recent in vitro study showed that Cbx2.1 also mediates repression of bivalent ovary
determining genes, such as the downstream Wnt signaler Lef1 in mice [78]. In addition, functional
analysis revealed that CBX2.1 is upstream of genes contributing to ovarian function including
folliculogenesis and steroidogenesis and it also regulates genes associated with POI, such as POF1B,
BMP15, and HOXA13, suggesting that CBX2.1 is essential for gonad formation in both sexes [79].
Int. J. Mol. Sci. 2020, 21, 2282 8 of 31

Another CBX2 isoform (CBX2.2) was implicated in the aetiology of 46,XY GD after the identification
of deleterious heterozygous variants in two patients with 46,XY partial GD. These CBX2.2 variants
failed to regulate the expression of genes essential for gonad development, primarily EMX2 [80]. CBX2
isoforms are a cause of 46,XY GD and to date no information about fertility potential has been reported.

3.4. NR2F2: A “Pro-Ovary and Anti-Testis” Gene

The orphan nuclear receptor NR2F2 gene, which encodes the transcription factor chicken
ovalbumin upstream promoter transcription factor 2 (COUP-TF2), has been described as a “pro-ovary
and anti-testis” gene following the identification of two frameshift variants in three syndromic
46,XX children, one with ovarian dysgenesis and the other with OT DSD, associated with congenital
heart disease and variable somatic anomalies including blepharophimosis-ptosis-epicanthus inversus
syndrome (BPES) [81]. A 3 Mb deletion containing the NR2F2 gene was also described in an
individual with 46,XX OT DSD with a similar phenotype [82]. BPES in usually caused by heterozygous
loss-of-function FOXL2 variants with or without ovarian dysgenesis. The precise mechanism by
which COUP-TF2 defects leads to 46,XX DSD has not been elucidated. However, at the initiation of
ovarian development, FOXL2 and COUP-TF2 appear to be mutually exclusive at the cellular level,
with distinct location in the somatic and stromal cells of the fetal ovary, respectively [81]. COUP-TF2 is
expressed at the same time as WT1 in early gonadal embryogenesis [83] and it regulates negatively
the expression of the pro-testis Sox9 gene in the osteogenic mesenchyme. Hence, it is hypothesized
that testis development in these individuals with NR2F2 mutations is driven by SOX9 activation via
WT1 [82].
In addition, COUP-TF2 is responsible for eliminating the male reproductive tract in female
embryos through its activation in the Wolffian duct mesenchyme, independently of the absence of
androgens [83]. In 46,XY individuals, COUP-TF2 is expressed in Leydig cells from the adult population
and is known to bind to DNA sequences similar to NR5A1 elements. Both of them regulate Star [84]
and Insl3 [85] gene expression in Leydig cells. Recently, it was shown that not only is NR5A1 capable
of activating Amhr2 gene expression in Leydig cells, but also COUP-TF2, independently of NR5A1
activation [86]. No NR2F2 variants havebeen described in 46,XY individuals. However, in mouse
models, inactivation of Nr2f2 during prepubertal stages of male sexual development results in infertility,
hypogonadism, and a block in spermatogenesis due to a failure of progenitor Leydig cells to mature
and produce sufficient testosterone [86].

3.5. FGF and WNT Signaling: Antagonistic Pathways in Gonadal Sex Differentiation
The sex-specific differentiation of gonads is controlled through a combination of signaling factors
promoting either male or female differentiation and antagonism of the opposite pathway (reviewed
in [87]). Studies in mice have elegantly demonstrated that FGF9 and WNT4 act antagonistically on the
female and male promoting pathways, respectively [88]. The loss of either Fgf9 or Fgfr2 in XY gonads
results in elevated expression of Wnt4 and subsequently in complete male-to-female sex reversal.
However, when the female-promoting factor Wnt4 is simultaneously ablated, testicular differentiation
of the gonad is promoted, suggesting that the primary role of FGF9/FGFR2 signaling is the repression
of female-promoting genes [89]. Interestingly, the relationship between these two signaling pathways
does not appear to be completely symmetrical since loss of Fgf9 in XX Wnt4−/− gonads do not rescue
the observed partial female-to-male sex-reversal [89].
Consistent with a conserved role for FGF9/FGFR2 signaling in human gonadal development,
a heterozygous missense mutation (c.1025G>C, p.Cys342Ser) in the FGFR2c gene was associated
with complete 46,XY gonadal dysgenesis with a female phenotype [90]. No information about
fertility potential was reported for the patient with this specific mutation who was gonadectomized
at age 15 years due to bilateral dysgerminoma. Additional antagonistic interactions between testis-
and ovary-promoting pathways have been identified in mice. FGFR2c and FOXL2 display such
an antagonistic relationship based on the finding of complete sex reversal in XY Fgfr2c−/− gonads,
Int. J. Mol. Sci. 2020, 21, 2282 9 of 31

but rescue of the gonadal sex reversal when Foxl2 was simultaneously ablated, thereby suggesting
that testicular differentiation involves FGFR2c-mediated repression of the FOXL2-driven promotion
of ovarian differentiation [90]. Moreover, antagonistic interactions between Sox9/Rspo1 [91] and
Sox9/β-catenin [92] have been reported in mice, with testicular differentiation observed in both
Sox9/Rspo1 and Sox9/β-catenin double knockouts. These findings confirm that male and female gonadal
sex differentiation are both induced by distinct genetic pathways and that repression of the opposite
pathway is essential to ensure normal sex-specific gonadal development.
In human fetal gonads, WNT4 expression does not appear to be sex-specific or show temporal
fluctuations, whereas RSPO1 expression is ovary-specific [93,94]. Nevertheless, both WNT4 and
RSPO1 are important in the promotion of ovarian development in humans based on evidence from
patients with mutations in these genes. Loss-of-function mutations in WNT4 have been identified
in 46,XX individuals that are virilized and lack Müllerian structures [95,96], while loss-of-function
mutations in RSPO1 leads to 46,XX DSD with complete sex-reversal [97], or 46,XX ovotesticular DSD,
although no information about fertility was reported in these patients [98]. Conversely, duplication
of chromosome 1p31-p35 (which contains both WNT4 and RSPO1) has been reported to cause 46,XY
male-to-female sex reversal, suggesting that genes important in human gonadal sex differentiation
may be dosage-sensitive [99]. However, neither WNT4 nor RSPO1 were duplicated in another more
recent case of male-to-female sex reversal with partial duplication of 1p, thereby suggesting that other
genes in this region may contribute or indeed be the cause of the observed gonadal phenotype [100].
Interestingly, the WNT signaling antagonist ZNRF3, which is also a direct target of RSPO1-mediated
inhibition, was recently shown to be required for testicular differentiation in mice, with XY mice lacking
Znrf3 exhibiting complete or partial gonadal sex reversal [101]. In accordance, three human ZNRF3
variants were identified in rare cases of 46,XY female DSD, thereby identifying a testis-determining
function for ZNRF3 in humans and suggesting an antagonistic relationship between ZNRF3 and
RSPO1 also in human gonadal sex differentiation [101]. In the five patients with ZNRF3 variants and
46,XY DSD varying degrees of gonadal dysgenesis was reported, but no information about fertility
was included [101].
Following initiation of the female promoting pathway by WNT4/RSPO1/β-catenin signaling,
granulosa cell fate and ovarian development is enforced by expression of the transcription factor
FOXL2 in mice [102–106]. It is likely that the WNT4/RSPO1/β-catenin signaling pathway contributes
to the upregulation of FOXL2 in granulosa cells, which in human fetal ovaries are expressed in a
sub-population of the somatic cells from around 10 weeks post-conception [107] although the mechanism
through which this may be mediated is not understood. In humans, an autosomal dominant mutation
in FOXL2 has been associated with premature ovarian failure, but not sex reversal [108,109], suggesting
that also in humans FOXL2 is not essential for the initial establishment of the granulosa cell population.

4. Maintenance of Sex-Specific Somatic Cell Lineages

Importantly, the carefully regulated balance between the promotion of testicular or ovarian
differentiation and simultaneous suppression of the opposite pathway during embryonic and fetal
development is not the final sex-fate decision. Genetic studies in mice have shown that sex-specific
gonadal fates must be actively maintained in adulthood, emphasized by the continued requirement to
maintain Sertoli or granulosa somatic cell fate.

4.1. FOXL2: Maintenance of Female Fate

Studies in Foxl2 null mice have demonstrated numerous effects on ovarian function [104,106],
including upregulation of Sox9 after birth, which suggests a continuous requirement for FOXL2
to maintain granulosa cell identity throughout development and maturation [103]. In accordance,
conditional deletion of Foxl2 in adult ovarian follicles of mice resulted in upregulation of testis-specific
genes, including Sox9 thereby demonstrating reprogramming of granulosa cells into Sertoli cell-like
cells [105]. Additionally, steroid hormone production was altered in females with ablated Foxl2, with
Int. J. Mol. Sci. 2020, 21, 2282 10 of 31

testosterone levels comparable to those of normal XY littermates, thereby suggesting reprogramming

of theca cells towards a Leydig cell-like fate [105].
Additional detailed analyses demonstrated that Foxl2 interacts with estrogen receptors through
TESCO, the gonad-specific enhancer of SOX9, thereby suppressing Sox9 expression and reprogramming
of granulosa cells in the adult mouse ovary [105,110]. Conversely, transgenic gonadal expression
of Foxl2 in XY mice resulted in ovotestis-like gonads at 13.5 dpc with disrupted tubular structures
and reduced AMH expression [111]. In accordance, male-to-female sex reversal was observed in XY
Fgfr2c−/− gonads in which upregulation of Foxl2 was reported [112].
Consistent with the role of RSPO1/WNT4/β-catenin and FOXL2 in the promotion and maintenance
of ovarian fate, the combined loss of Foxl2 and Wnt4, or Foxl2 and Rspo1 results in female-to-male sex
reversal that occurs earlier and with a more severe phenotype than sex reversal resulting from loss
of Foxl2, Wnt4, or Rspo1 alone [111,113]. Together, these studies indicate that FOXL2 is required for
the maintenance of the granulosa cell lineage in ovaries and suppression of the testicular pathway
by preventing trans-differentiation of granulosa cells into Sertoli cells, thereby emphasizing that
maintenance of the ovarian phenotype continues throughout life.

4.2. DMRT1: Maintenance of Male Fate

Consistent with the concept of continued maintenance of sex-specific gonadal fate, studies in mice
have identified DMRT1 as an essential factor responsible for maintaining the Sertoli cell lineage and
testicular fate. Dmrt1−/− XY mice are born with testes [114], but these undergo abnormal differentiation
during postnatal development with loss of Sox9 expression and upregulation of Foxl2 [115]. In
accordance, conditional loss of Dmrt1 in adult testis results in upregulation of FOXL2 expression,
reprogramming of Sertoli cells into granulosa cells, and presence of theca-like cells and germ cells
that appeared feminized [115]. This suggests that in mice Dmrt1 is essential to maintain testis
determination and antagonize female promoting factors (mainly FOXL2) throughout life. Conversely,
induced expression of Dmrt1 in XX gonads was recently reported to reprogram sex-specific gonadal
differentiation and promote testicular development in mice [10,116].
Ectopic expression of Dmrt1 in the ovary resulted in downregulation of the female sex-maintenance
gene Foxl2, reprogramming of granulosa cells to Sertoli-like cells, and formation of structures resembling
male seminiferous cords [10]. In accordance, an independent study found that overexpression of Dmrt1
in XX gonads was indeed sufficient to promote testicular differentiation [116]. Additionally, these
transgenic XX gonads had typical testicular vasculature, reduced expression of Foxl2, and induction of
Sox9 expression. Also, fetal Leydig-like cells and non-meiotic germ cells were found in the transgenic
XX gonads, but in this model formation of seminiferous cords was not observed [116]. Together
these results suggest that ectopic expression of Dmrt1 in ovaries is sufficient to promote testicular
differentiation in mice even though it is dispensable for the initial establishment of Sertoli cell fate
during fetal life.
In humans, loss of DMRT1 has been reported in relation to deletions of chromosome 9p24,
which results in varying degrees of 46,XY gonadal dysgenesis [117–120], suggesting that DMRT1
may contribute to the maintenance of male fate in human supporting cells. Whilst it is likely that
cases of 46,XY DSD in individuals with deletions of chromosome 9p24 are due to loss of DMRT1, it
cannot be excluded that other genes in this region contribute, or indeed cause, the observed gonadal
phenotypes [121–123]. In accordance with the former hypothesis, knockdown of DMRT1 expression in
a human fetal testis ex vivo model induced focal testicular dysgenesis and expression of FOXL2 in a
small sub-population of supporting cells in which SOX9 expression was lost [124].

4.3. DHX37: A Novel Participant of Gonadal Development and Maintenance

Variants of the DEAH-box helicase 37 (DHX37) gene have recently been identified as an important
cause of 46,XY GD, especially for embryonic testicular regression syndrome (ETRS). DHX37 is an
RNA-helicase which has a role of DHX37 in ribosome biogenesis [125] and is expressed in the somatic
Int. J. Mol. Sci. 2020, 21, 2282 11 of 31

cell lineage of the mouse and human (7–12 weeks of gestation) gonad early during testis determination
and development [126]. Co-expression with Sox9 in a proportion of cells indicates the presence of
DHX37 in Sertoli cells [126], whilst it is also expressed in Leydig cell cytoplasm and in germ cells at
different stages of maturation (27 and 33 weeks of gestation) and, in adult human testes, the protein is
mainlyInt. J.localized in21,
Mol. Sci. 2020, spermatogonia
x FOR PEER REVIEW [126,127]. 11 of 31
Four heterozygous missense rare variants classified as pathogenic or likely pathogenic in DHX37
Four heterozygous missense rare variants classified as pathogenic or likely pathogenic in DHX37
have been reported in 11 patients from five families and in six sporadic cases with gonadal dysgenesis
have been reported in 11 patients from five families and in six sporadic cases with gonadal dysgenesis
and also ETRS [127]. Seven different missense heterozygous DHX37 variants were also identified in 13
and also ETRS [127]. Seven different missense heterozygous DHX37 variants were also identified in
children from another cohort with similar phenotypes [126]. All the identified variants are clustered in
13 children from another cohort with similar phenotypes [126]. All the identified variants are
two highly
clustered conserved
in two highly functional
conserveddomains and were
functional domains specifically
and wereassociated
specificallywith gonadal
associated withdysgenesis
gonadal and
ETRSdysgenesis
in both cohorts.
and ETRS Segregation analysis
in both cohorts. of the DHX37
Segregation analysisvariants displayed
of the DHX37 a sex-limited
variants displayed aautosomal
sex-
dominant
limitedpattern,
autosomal mostly
dominant de pattern,
novo ormostlymaternally
de novoinherited andinherited
or maternally paternally and inherited in one family.
paternally inherited
in one family. The
The p.Arg308Gln, p.Arg308Gln,
p.Arg674Trp, andp.Arg674Trp,
p.Thr304Metand were p.Thr304Met were the DHX37
the most common most commonvariants DHX37
identified.
variants identified. In both cohorts, the frequency of DHX37 deleterious
In both cohorts, the frequency of DHX37 deleterious variants were similar to the NR5A1 variants, variants were similar to the
accounting for 14% and 11% of the 46,XY GD patients, respectively [126,127]. In a third cohort ofa46,XY
NR5A1 variants, accounting for 14% and 11% of the 46,XY GD patients, respectively [126,127]. In
DSDthirdadultcohort
women, of 46,XY DSD adult women, DHX37 variants accounted for 15.4% of the partially
DHX37 variants accounted for 15.4% of the partially virilized individuals [128].
virilized individuals [128].
The fact that the same variant could be observed in individuals with gonadal dysgenesis and
The fact that the same variant could be observed in individuals with gonadal dysgenesis and
others withwith
others ETRSETRS reinforces that that
reinforces thesethese
phenotypes
phenotypesare part of the
are part of same clinical
the same spectrum
clinical and and
spectrum indicates
that indicates
this genethat is important
this gene isnot only for
important notgonadal
only fordetermination, but alsobut
gonadal determination, itsalso
maintenance.
its maintenance.
Despite fact that the precise mechanism by which DHX37
Despite the fact that the precise mechanism by which DHX37 variants leadlead
the variants to GDto remains
GD remainsto be to be
elucidated,
elucidated, the thepresent
present genetic
geneticevidence
evidencehighlights ribosomopathies
highlights ribosomopathies as as a novel
a novel mechanism
mechanism for DSD.
for DSD.
Regarding
Regarding fertility
fertility issues,
issues, curiously,
curiously, inin oneofofthe
one thereported
reportedfamilies,
families, there
there is one fertile
fertile father
father who
who harbored
harbored the recurrent the recurrent
p.Arg308Glnp.Arg308Gln
DHX37DHX37 deleterious
deleterious variant,
variant, resembling
resembling NR5A1NR5A1 variants
variants [127].
[127].
Current understanding of the timeline and genetic interactions that are required for mammalian
Current understanding of the timeline and genetic interactions that are required for mammalian
sex determination and differentiation are summarized in Figure 1.
sex determination and differentiation are summarized in Figure 1.

Figure
Figure 1. Timeline
1. Timeline of of genesinvolved
genes involvedin
in gonadal
gonadal development.
development. Genes
Genesshown are known
shown to play
are known to aplay
role a role
in sex-specific gonadal development in human and mice. Testis-related genes (blue) and
in sex-specific gonadal development in human and mice. Testis-related genes (blue) and ovary-related ovary-
related genes (pink) represent a regulatory pathway which leads to Sertoli and granulosa cell
genes (pink) represent a regulatory pathway which leads to Sertoli and granulosa cell development,
development, respectively. The orange arrows represent an antagonistic action. Interactions that are
respectively. The orange arrows represent an antagonistic action. Interactions that are postulated but
postulated but unproven are indicated with (?). The ectopic gonadal expression of Sox3 (represented
unproven are indicated with (?). The ectopic gonadal expression of Sox3 (represented by red dotted
by red dotted line) induces testis differentiation by upregulating Sox9 in a similar way to Sry.
line) induces testis differentiation by upregulating Sox9 in a similar way to Sry.
5. Impact of DSD on Fertility and Options for Fertility Preservation
The impact of DSD on fertility will depend on several factors. The key determinant of fertility
potential will be gonadal development and function, and ultimately whether the individual has
ovarian tissue with viable oocytes, or a testis capable of producing functional sperm. Whilst fertility
Int. J. Mol. Sci. 2020, 21, 2282 12 of 31

5. Impact of DSD on Fertility and Options for Fertility Preservation

The impact of DSD on fertility will depend on several factors. The key determinant of fertility
potential will be gonadal development and function, and ultimately whether the individual has
ovarian tissue with viable oocytes, or a testis capable of producing functional sperm. Whilst fertility
preservation options are well established for many patient groups such as those receiving gonadotoxic
treatment for cancer [129], there are a number of important additional considerations for those with
DSD that must be evaluated on an individual patient basis (Table 1).

Table 1. Key considerations for fertility and fertility preservation in DSD.

Consideration Management Implications for Fertility

- Natural fertility not possible
Gonadectomy - Gametes may be obtained at surgery
- Gonadal tissue may be obtained at surgery
Malignancy risk
- Natural fertility may be possible
Conservative - gametes or gonadal tissue may be obtained
in adulthood
- Gamete/tissue retrieval only possibleprior
Progressive germ cell loss Fertility preservation
to loss
Gonads/gametes - Gametes may be obtained and stored
incongruent to sex of Fertility counseling - Gonadal tissue may be cryopreserved
rearing
Transmission of genetic - Specific diagnosis/variant may influence
Genetic/fertility
abnormality to pregnancy outcomes
counseling
subsequent generations - Potential risk of recurrence in offspring
- Potential for carrying a fetus to term may
Presence/absence of Radiology/surgical be limited
Mullerian structures assessment - Absence of birth canal will prevent
vaginal delivery

5.1. Sex Chromosome DSD

The relationship between DSD and impaired fertility is well established, especially for the two
most common sex chromosome DSDs. For individuals with Turner syndrome (45,X) and Klinefelter
syndrome (47,XXY), primary gonadal failure is common. For both disorders, pubertal delay with
hypergonadotrophic hypogonadism is frequent, resulting in sex steroid deficiency and infertility.

5.1.1. Turner Syndrome

Turner syndrome (TS) affects 1:2000 girls and the majority (95–98%) of individuals with TS are
infertile [130]. For most individuals with TS, primary ovarian failure occurs during prepuberty and
spontaneous menarche has been reported to occur in only 20% (95/480) of cases in one large cohort of
patients [131]. Spontaneous pregnancy is rare in individuals with TS, occurring in only 2–5%, with
an increased rate of fetal anomalies and pregnancy loss also reported [132]. Infertility results from
an accelerated loss of oocytes which begins in fetal life and progresses during postnatal life [130]. A
complete 45,X karyotype has been reported to be predictive of ovaries without presence of follicles,
whereas a mosaic karyotype and spontaneous menarche increase the likelihood of follicles being
present [133]. In a cohort of 15 individuals (aged 5–22 years) with TS, follicles were identified in 9 (60%)
and in the majority of these cases (7/9) the follicle counts were within the 95% confidence interval for
the normal population. All were mosaic, except for a 5-year-old girl with a 45,X karyotype. However,
despite the presence of follicles in the ovaries of the majority of those TS cases, the morphology of
the follicles was frequently abnormal, suggesting that the functional capacity of these oocytes may be
impaired [133].
Int. J. Mol. Sci. 2020, 21, 2282 13 of 31

For post-pubertal patients it may be possible to perform ovarian stimulation and oocyte retrieval;
however, this is entirely dependent on the presence follicles from which oocytes can be retrieved.
For the majority of individuals with TS it is not possible to preserve fertility and in these cases
counseling regarding fertility should be offered and egg/embryo donation or adoption considered
where appropriate.
Whilst ovarian tissue cryopreservation (OTC) has been shown to be a successful strategy for
fertility preservation in young females at risk of infertility due to planned gonadotoxic therapies,
it is not clear whether this is a suitable option for individuals with Turner syndrome. Although
spontaneous menarche, mosaic karyotype, detectable AMH, and normal follicle stimulating hormone
(FSH) may offer some prediction about the presence of follicles, they do not predict the functional
capacity of the oocytes within the cryopreserved tissue [133]. Given the abnormal morphology of the
follicles, fertility restoration by re-transplantation of cryopreserved ovarian tissue is unlikely to be
successful and therefore subsequent use of cryopreserved ovarian tissue would likely require ex vivo
oocyte maturation and artificial reproductive technologies. To date, there have been no reports of
restoration of fertility using cryopreserved ovarian tissues from individuals with TS.

5.1.2. Klinefelter Syndrome

Klinefelter syndrome (KS) is common, occurring in 1:600 live births. KS is associated with
hypogonadism and infertility with fewer than half of adult men reported to have spermatozoa
present in the ejaculate, although residual foci with spermatogenesis may be present in some
individuals with apparent azoospermia [134]. The testicular phenotype of KS involves a progressive
loss of spermatogonial stem cells (SSC) beginning in prepuberty, with testicular fibrosis occurring in
(peri)pubertal and adult patients [135]. A meta-analysis of the presence of spermatogonia in individuals
with KS demonstrated spermatogonia in the testes of all fetal/infantile samples, 83% of those obtained
from prepubertal patients, and in 40–50% of adolescent/adult individuals [20].
Options for fertility preservation in individuals with KS are dependent on the age of the patient.
For post-pubertal patients, the primary goal is to obtain viable spermatozoa that can be stored for
future use in artificial reproductive technologies. This involves surgical intervention to retrieve sperm
using testicular sperm extraction (TESE). According to a recent systematic review and meta-analysis,
TESE has been shown to result in successful retrieval of sperm in ~40% of cases [134]. Interestingly,
clinical and biochemical parameters, such as age, testis volume, and hormone status at baseline, were
not predictive of successful sperm retrieval [134]. A total of 29 studies in this meta-analysis reported
fertility outcomes using sperm retrieved by TESE with pregnancy and live-birth rate of 43% [134].
For prepubertal boys, it is not possible to obtain sperm and therefore the only option is to
cryopreserve and store testicular tissue with the aim of preserving SSCs that can be used to generate
sperm for future use in artificial reproductive technologies. Whilst testicular tissues are increasingly
being stored from prepubertal boys due to receive gonadotoxic therapies, there are no proven clinical
methods to generate sperm from these tissues [136]. Furthermore, the underlying testicular and
germ cell abnormalities in individuals with KS may result in additional challenges for the use of this
approach for fertility preservation. Therefore, this approach has recently been called into question [135]
and testicular tissue cryopreservation in prepubertal patients should only be considered as part of an
ethically approved research study.
Options for fertility preservation in 45,X and 47,XXY are summarized in Figure 2.
Int. J. Mol. Sci. 2020, 21, 2282 14 of 31
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 31

Figure
Figure Options
2. 2. Optionsfor
forfertility
fertilitypreservation
preservation in
in sex chromosome
chromosomeDSD DSD(aneuploid).
(aneuploid).* Mostly
* Mostly mosaics.
mosaics. ** **
Postpubertal.# #Potential
Postpubertal. Potentialforforgonadal
gonadal tissue
tissue cryopreservation
cryopreservationbased
basedononpublished
published literature indicating
literature indicating
presence
presence of germ
of germ +/− tissue
cellscells +/− tissue cryopreservation;
cryopreservation; however,
however, there
there are are no of
no reports reports of successful
successful restoration
restoration of fertility and this should be considered within the context of an ethically
of fertility and this should be considered within the context of an ethically approved research study. approved
research study.
5.1.3. 45,X/46,XY
5.1.3.
The45,X/46,XY
phenotype of individuals with 45,X/46,XY depends on the proportion and distribution of
each sex chromosome
The phenotype complement
of individuals (45,X
withor45,X/46,XY
46,XY) within the body
depends on thetissues. As a result,
proportion the appearance
and distribution of
of each
45,X/46,XY can range from
sex chromosome that of 45,X
complement (Turner
(45,X syndrome)
or 46,XY) withinfemale to a phenotypic
the body tissues. Asmale. 45,X/46,XY
a result, the
appearance
is the most commonof 45,X/46,XY
cause ofcanmixed
rangegonadal
from that of 45,X (Turner
dysgenesis syndrome)
in which the gonads female
aretoa combination
a phenotypic of
male. 45,X/46,XY
a streak gonad andisathe most common
contralateral cause
testis of mixed
[137]. gonadal
For those dysgenesis
individuals in which
raised the gonads
as male, are a
spontaneous
combination
puberty usually ofoccurs,
a streakalthough
gonad and a contralateral
testosterone maytestis [137]. Fortothose
be required individuals
complete puberty raised
[137].as Fertility
male,
in spontaneous
individuals puberty usually occurs,
with 45,X/46,XY althoughon
is dependent testosterone
the potentialmayforbe sperm
required to complete
production in puberty
the testis,
[137]. Fertility in individuals with 45,X/46,XY is dependent on the potential for
although the testis tissue is frequently dysgenetic. Whilst it is generally considered that individualssperm production in
the testis, although the testis tissue is frequently dysgenetic. Whilst it is generally
with 45,X/46,XY are infertile, there are isolated case reports of fertility in both males and females. A considered that
individuals
single case of with 45,X/46,XY
successful sperm areextraction
infertile, there
wasare isolatedincase
reported reportswith
a patient of fertility in both males
azoospermia [138].andThis
females. A single case of successful sperm extraction was reported in a patient with azoospermia
individual had a 50:50 split in mosaicism and 96% of the retrieved sperm were aneuploid. Sperm
[138]. This individual had a 50:50 split in mosaicism and 96% of the retrieved sperm were aneuploid.
were cryopreserved from this patient, but no information about the functionality of the sperm was
Sperm were cryopreserved from this patient, but no information about the functionality of the sperm
reported [138]. A recent study in a relatively large (n = 63) cohort of males with 45,X/46,XY mosaicism
was reported [138]. A recent study in a relatively large (n = 63) cohort of males with 45,X/46,XY
showed that the majority (79%) entered puberty spontaneously, but 39% of all individuals in the cohort
mosaicism showed that the majority (79%) entered puberty spontaneously, but 39% of all individuals
required testosterone supplementation at some stage during follow-up [139]. Whilst the gonadal
in the cohort required testosterone supplementation at some stage during follow-up [139]. Whilst the
phenotype and histology
gonadal phenotype was highly
and histology wasvariable, the majority
highly variable, of patients
the majority had dysgenetic
of patients had dysgenetictestes. In the
testes.
majority of postpubertal patients no evidence of spermatogenesis was found,
In the majority of postpubertal patients no evidence of spermatogenesis was found, although 25% although 25% had focal
areas with spermatogenesis. Seventeen patients had semen analyzed and live spermatozoa were
Int. J. Mol. Sci. 2020, 21, 2282 15 of 31
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 31

present in 17.6%
had focal (3/17),
areas with albeit with lowSeventeen
spermatogenesis. total number of sperm
patients [139].
had semen Spontaneous
analyzed and livepregnancies
spermatozoa have
been
werereported in17.6%
present in females with
(3/17), mosaicism
albeit with low[140]. Remarkably,
total number of sperma spontaneous pregnancy
[139]. Spontaneous has been
pregnancies
have been
reported in reported
a female in females
with with mosaicism
a predominantly [140].
46,XY Remarkably,
karyotype, a spontaneous
including pregnancy
in the gonad has been
(95% 46,XY) [141].
Areported in a of
recent study female with a predominantly
44 individuals 46,XY karyotype,
with Turner syndrome including
and presence of Yinchromosome
the gonad (95% 46,XY)
material found
[141]. A recent
spontaneous study in
menarche of 2/26
44 individuals withwith
(7.6%) of those Turner syndromekaryotype,
a 45,X/46,XY and presence of Y chromosome
although the potential for
material found spontaneous menarche in 2/26
fertility in these individuals was not reported [142]. (7.6%) of those with a 45,X/46,XY karyotype, although
the For
potential for fertility in these individuals was not reported [142].
males the options are similar to those described for KS. However, an additional consideration
For males those
in all individuals the options are similar karyotype
with a 45,X/46,XY to those is described for KS.
the increased riskHowever,
of gonadalan additionalthat
malignancy
consideration in all individuals those with a 45,X/46,XY karyotype is the increased risk of gonadal
may necessitate gonadectomy [22].
malignancy that may necessitate gonadectomy [22].
5.1.4. 45,XX/46,XY
5.1.4. 45,XX/46,XY
45,XX/46,XY DSD may arise as a result of mosaicism or true chimerism [21,143]. Phenotypes range
45,XX/46,XY DSD may arise as a result of mosaicism or true chimerism [21,143]. Phenotypes
from typical male to typical female. Gonads in individuals with 46,XX/46,XY DSD may include testicular
range from typical male to typical female. Gonads in individuals with 46,XX/46,XY DSD may include
and ovarian tissues. In phenotypic females, spontaneous pregnancies have been reported [144]. For
testicular and ovarian tissues. In phenotypic females, spontaneous pregnancies have been reported
males, azoospermia is considered almost universal, although there are reports of successful TESE and
[144]. For males, azoospermia is considered almost universal, although there are reports of successful
subsequent pregnancies using the cryopreserved sperm [145,146] and a case of an azoospermic male
TESE and subsequent pregnancies using the cryopreserved sperm [145,146] and a case of an
whose sperm count
azoospermic became
male whose normal
sperm after
count Mullerian
became normalstructures had been
after Mullerian removed
structures had[143]. These cases
been removed
indicated that fertility preservation may be possible in some 46,XX/46,XY individuals
[143]. These cases indicated that fertility preservation may be possible in some 46,XX/46,XY using semen
cryopreservation or TESE.
individuals using semen cryopreservation or TESE.
Options
Optionsfor
forfertility
fertilitypreservation
preservation in 46,XX/46,XY and
in 46,XX/46,XY and 46X/46,XY
46X/46,XYDSD
DSDarearesummarized
summarizedininFigure
Figure 3.
3.

Figure 3. Options for fertility preservation in sex chromosome DSD (mixed gonadal dysgenesis).
Figure 3. Options for fertility preservation in sex chromosome DSD (mixed gonadal dysgenesis). *
* Usually dysgenetic. ** May be possible for IVF. *** Rare case reports. # Potential for gonadal
Usually dysgenetic. ** May be possible for IVF. *** Rare case reports. # Potential for gonadal tissue
tissue cryopreservation based on published literature indicating presence of germ cells +/− tissue
cryopreservation based on published literature indicating presence of germ cells +/− tissue
cryopreservation; however, there are no reports of successful restoration of fertility and this should be
cryopreservation; however, there are no reports of successful restoration of fertility and this should
considered within the context of an ethically approved research study.
be considered within the context of an ethically approved research study.
Int. J. Mol. Sci. 2020, 21, 2282 16 of 31

5.2. 46,XX DSD

5.2.1. Disorders of Androgen Production or Action

The majority of 46,XX DSD are due to excess androgen production. Most commonly this results
from CAH due to 21-hydroxylase deficiency. The primary pathology is due to overproduction of
adrenal androgens, as opposed to a failure of normal ovarian development. As a result, XX individuals
with 21-hydroxylase CAH may be fertile [132]. However, women with CAH are reported to have
lower pregnancy rates when compared to age-matched controls [147]. Live-birth rates of 33–50%
have been reported in those with ‘simple virilizing’ forms of CAH [24]. However, the likelihood of
fertility is significantly lower in those with severe ‘salt-wasting’ forms of CAH in whom live-birth
rates of 0–10% have been reported [24]. In non-classical forms of CAH due to partial enzyme
deficiencies, live-birth rates are higher (63–90%) than classical CAH and are similar to age-matched
controls [24]. In more rare forms of 46,XX CAH that may also affect gonadal steroid production, fertility
is rarely described, other than in few case reports of a spontaneous pregnancy, e.g., in an individual
with 3β-hydroxysteroid dehydrogenase deficiency [148], and successful in vitro fertilization (IVF) in
individuals with 17α-hydroxylase deficiency [149–151], and another with congenital lipoid adrenal
hyperplasia [152]. In CAH, infertility may occur as a result of anovulation, menstrual irregularities,
thickening of cervical mucus, and anatomical factors [132]. In addition, there are important psychosocial
factors that impact on fertility in individuals with CAH, which may include the lack of a steady
relationship or a reduction in those trying for a pregnancy compared to the general population [147].
Overall, the data indicate that the likelihood of fertility is associated with disease severity and disease
control. Therefore, the mainstay of treatment involves optimizing androgen levels with appropriate
steroid therapy [132].

5.2.2. 46,XX Testicular DSD

46,XX testicular DSD is characterized by the presence of testicular tissue despite the absence
of the Y chromosome. The majority of these individuals have a translocation of the SRY gene,
accounting for 80% of those with non-syndromic 46,XX testicular DSD with phenotypically male
external genitalia [153]. Despite the presence of the SRY gene, impaired spermatogenesis in these
individuals may be due to the lack of other Y chromosome genes that are known to be important
for fertility, such as the azoospermia (AZF) region [154]. The phenotype in individuals with 46,XX
testicular DSD may resemble KS and infertility with azoospermia is reported to be universal in this
population [29,137].
Options for fertility preservation in 46,XX DSD are summarized in Figure 4.
Int. J. Mol. Sci. 2020, 21, 2282 17 of 31

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 17 of 31

Figure 4. Options
Figure for fertility
4. Options preservation
for fertility in 46,XX
preservation in DSD.
46,XX* DSD.
Optimise therapy for
* Optimise CAH. for
therapy ** Postpubertal.
CAH. **
# Potential for gonadal tissue cryopreservation based on published literature indicating presence of
Postpubertal. # Potential for gonadal tissue cryopreservation based on published literature indicating
germ cells +/−
presence of tissue
germ cryopreservation; however, there are
cells +/− tissue cryopreservation; no reports
however, of successful
there restoration
are no reports of fertility
of successful
and this should be considered within the context of an ethically approved research study.
restoration of fertility and this should be considered within the context of an ethically approved
research study.
5.3. 46,XY DSD
5.3.46. ,XY DSD
5.3.1. Gonadal Dysgenesis
5.3.1. Gonadal
Complete Dysgenesis
gonadal dysgenesis in 46,XY individuals results from mutations in key testis-determining
genes inComplete
a phenotypic female
gonadal with internal
dysgenesis in 46,XYMullerian structures
individuals and from
results bilateral streak gonads.
mutations in key The lack
testis-
of either testicular or ovarian tissue means that it is not possible to obtain spermatozoa
determining genes in a phenotypic female with internal Mullerian structures and bilateral streak or oocytes from
these individuals
gonads. The lackandoftherefore infertilityoris ovarian
either testicular universal. However,
tissue meansthe presence
that it is notof possible
Mulleriantostructures
obtain
means that pregnancy
spermatozoa or oocytesmay be these
from possible using donor
individuals eggs or infertility
and therefore embryos is and IVF, as illustrated
universal. However, the by a
recent report
presence of of successful
Mullerian pregnancies
structures meansinthat two sisters, with
pregnancy mayabe
healthy live
possible birth
using in one
donor and
eggs oran ongoing
embryos
pregnancy
and IVF, inas the other [155].
illustrated by a recent report of successful pregnancies in two sisters, with a healthy live
Individuals
birth in one and with 46,XY partial
an ongoing gonadal
pregnancy dysgenesis
in the (PGD) present with variable genital ambiguity
other [155].
Individuals
and varying degrees with 46,XY partial
of testicular gonadal
dysgenesis dysgenesis
or streak gonads(PGD)
[156].present with variable
Whilst severe genital
oligozoospermia
ambiguity and varying degrees of testicular dysgenesis or streak gonads
has been reported in a long-term follow-up study of males with PGD [156], for phenotypic males [156]. Whilst severe
with
mild abnormalities of gonadal development or external genitalia (e.g., hypospadias), fertilityfor
oligozoospermia has been reported in a long-term follow-up study of males with PGD [156], may
be phenotypic
possible. males with mild abnormalities of gonadal development or external genitalia (e.g.,
hypospadias),
Options for fertility
fertilitymay be possible.
preservation in 46,XY gonadal dysgenesis DSD are summarized in Figure 5.
Options for fertility preservation in 46,XY gonadal dysgenesis DSD are summarized in Figure 5.
Int. J. Mol. Sci. 2020, 21, 2282 18 of 31
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 18 of 31

5. Options for fertility

Figure 5. fertility preservation
preservation inin 46,XY
46,XY DSD
DSD with
with gonadal
gonadaldysgenesis.
dysgenesis. GD—gonadal
GD—gonadal
dysgenesis. ** Often
Often dysgenetic.
dysgenetic.****MayMaybebepossible
possibleforformilder phenotypes.# Potential
milderphenotypes. # Potentialforforgonadal
gonadal
tissuecryopreservation
tissue cryopreservation based
based on on published
published literature
literature indicating
indicating presence
presence of germ germ+/−
of cells cells +/−
tissue
cryopreservation; however, there
tissue cryopreservation; are nothere
however, reportsareofno
successful
reportsrestoration of fertility
of successful and thisofshould
restoration be
fertility
considered within the context of an ethically approved research study.
and this should be considered within the context of an ethically approved research study.
5.3.2. Disorders of Androgen Production
5.3.2. Disorders of Androgen Production
46,XY males with 21-hydoxylase CAH are reported to have reduced fertility and semen
46,XY males with 21-hydoxylase CAH are reported to have reduced fertility and semen
parameters [24,157]. Fertility appears to be largely dependent on good control of excess androgens
parameters [24,157]. Fertility appears to be largely dependent on good control of excess androgens
and avoidance of over-treatment with steroids [24]. Testicular adrenal rest tumors (TART) can occur in
and avoidance of over-treatment with steroids [24]. Testicular adrenal rest tumors (TART) can occur
individuals with CAH. Whilst failure to suppress excess adrenal androgen production can promote their
in individuals with CAH. Whilst failure to suppress excess adrenal androgen production can
development, it is also recognized that TARTs can occur in some patients who are well-controlled [24].
promote their development, it is also recognized that TARTs can occur in some patients who are well-
The presence of bilateral TARTs with pressure and destruction of the testicular tissue may also result in
controlled [24]. The presence of bilateral TARTs with pressure and destruction of the testicular tissue
primary gonadal failure [24]. Hypogonadotrophic hypogonadism may occur in males with CAH as a
may also result in primary gonadal failure [24]. Hypogonadotrophic hypogonadism may occur in
consequence of the elevated circulating androgens. Similar to females with CAH, optimal control of
males with CAH as a consequence of the elevated circulating androgens. Similar to females with
adrenal androgen production represents the main approach to preserving fertility potential. Recovery
CAH, optimal control of adrenal androgen production represents the main approach to preserving
of a normal sperm count in an azoospermic man following treatment with a long-acting glucocorticoid
fertility potential. Recovery of a normal sperm count in an azoospermic man following treatment
was recently reported [158], although the benefits and potential risks of this form of therapy remain
with a long-acting glucocorticoid was recently reported [158], although the benefits and potential
unclear [24].
risks of this form of therapy remain unclear [24].
For other disorders of steroidogenic enzymes in 46,XY individuals, mutations may affect gonadal
For other disorders of steroidogenic enzymes in 46,XY individuals, mutations may affect
steroidogenesis. These disorders result in varying degrees of sex-reversal often with delayed puberty
gonadal steroidogenesis. These disorders result in varying degrees of sex-reversal often with delayed
and infertility [137]. Fertility and paternity are rarely reported in these patients, although there is some
puberty and infertility [137]. Fertility and paternity are rarely reported in these patients, although
evidence to suggest that non-classic forms in which there is partial enzyme activity, and the individual
there is some evidence to suggest that non-classic forms in which there is partial enzyme activity, and
is raised as male, may have normal puberty and fertility [137,159].
the individual is raised as male, may have normal puberty and fertility [137,159].
Mutations in the LH receptor resulting in reduced testicular androgen production will result in
Mutations in the LH receptor resulting in reduced testicular androgen production will result in
similar effects. For those with complete forms, phenotypic appearance is often female and therefore
similar effects. For those with complete forms, phenotypic appearance is often female and therefore
sex-of-rearing is usually female. In such cases, pubertal induction with estrogen is required and
sex-of-rearing is usually female. In such cases, pubertal induction with estrogen is required and
gonadectomy is often performed due to increased risk of gonadal malignancy. Whilst individuals with
gonadectomy is often performed due to increased risk of gonadal malignancy. Whilst individuals
Leydig cell hypoplasia are considered to be azoospermic, a case-report has described an individual
with Leydig cell hypoplasia are considered to be azoospermic, a case-report has described an
individual with an inactivating mutation of the LH receptor from whom sperm were successfully
obtained by TESE after treatment with hCG [160]. The cryopreserved sperm were used for intra-
Int. J. Mol. Sci. 2020, 21, 2282 19 of 31

with an inactivating mutation of the LH receptor from whom sperm were successfully obtained by
TESE
Int. after
J. Mol. Sci.treatment withPEER
2020, 21, x FOR hCGREVIEW
[160]. The cryopreserved sperm were used for intra-cytoplasmic19sperm of 31
injection (ICSI). resulting in a successful pregnancy and live birth [160]. Mutations in 5-α-reductase
cytoplasmic
gene results sperm injection
in failure (ICSI).
to convert resulting intoaDHT.
testosterone successful pregnancy is
The phenotype and live birth
variable and[160]. Mutations
can range from
in 5-α-reductase
ambiguous genetoresults
genitalia typicalin failure
female to convert testosterone
appearance. to DHT. The
Gender reassignment fromphenotype
female to maleis variable and
may occur
can
as arange
resultfrom ambiguous
of virilization at genitalia to typical
puberty [137]. female appearance.
Spontaneous fertility isGender
believed reassignment
to be rare infrom female
individuals
to
withmale may occur as
5-α-reductase a result of
deficiency asvirilization
a result of at pubertyabnormalities
frequent [137]. Spontaneous fertility is believed
in spermatogenesis to be
and semen
rare in individuals with 5-α-reductase deficiency as a result of frequent
parameters, including increased semen viscosity and reduced semen volume [26]. However, successful abnormalities in
spermatogenesis
pregnancies and births and semen parameters,
have been reportedincluding
followingincreased semen
intra-uterine viscosity and
insemination reduced
(IUI) or ICSIsemen
using
volume
semen samples[26]. However,
obtainedsuccessful pregnancies
from patients [26,161].and births have been reported following intra-uterine
insemination (IUI)preservation
For fertility or ICSI usingin semen
youngsamples
malesobtained from patients
with disorders [26,161]. production, semen
of androgen
For fertility may
cryopreservation preservation in young
be considered, whilstmales withwith
for those disorders of androgen
azoospermia, TESE mayproduction,
be an optionsemen to
cryopreservation
obtain sperm for assisted may be considered,
reproductivewhilst for those
technologies with[24,137],
(ART) azoospermia, TESE
although themay be an
success option
rates basedto
obtain sperm for
on individual assistedorreproductive
disorders technologies
specific mutations (ART)
are often [24,137], although the success rates based
unknown.
on individual
Options for disorders
fertilityor preservation
specific mutations are often
in 46,XY DSDunknown.
due to impaired androgen production are
Options for
summarized in Figure 6. fertility preservation in 46,XY DSD due to impaired androgen production are
summarized in Figure 6.

Figure 6. Options for fertility preservation in 46,XY DSD associated with impaired androgen production.
Figure 6. Options for fertility preservation in 46,XY DSD associated with impaired androgen
* Optimise therapy for CAH. # Potential for gonadal tissue cryopreservation based on published
production. * Optimise therapy for CAH. # Potential for gonadal tissue cryopreservation based
literature indicating presence of germ cells +/− tissue cryopreservation; however, there are no reports
on published
of successful literature
restoration indicating
of fertility presence
and this shouldof
be germ cellswithin
considered +/− tissue cryopreservation;
the context of an ethically
however, there are
approved research study. no reports of successful restoration of fertility and this should be
considered within the context of an ethically approved research study.
5.3.3. Disorders of Androgen Action
5.3.3. Disorders of Androgen Action
Androgen Insensitivity Syndrome
Androgen Insensitivity
Individuals Syndrome
with CAIS are born with a female phenotype and are usually not diagnosed until
childhood when they present
Individuals with CAIS are with an with
born inguinal hernia,
a female which includes
phenotype a gonad,
and are usuallyor later
not with primary
diagnosed until
childhood when they present with an inguinal hernia, which includes a gonad, or later with primary
amenorrhoea. Mullerian structures are not present, precluding patients with CAIS from carrying a
pregnancy. The gonads are composed of testicular tissue, although the histology is highly abnormal
with a rapid decline in germ cell number beginning in infancy [28]. Spermatogonia are present in the
gonads of ~30% of adults with CAIS but the majority of tubules are Sertoli-cell-only [87]. Presence of
Int. J. Mol. Sci. 2020, 21, 2282 20 of 31

amenorrhoea. Mullerian structures are not present, precluding patients with CAIS from carrying a
pregnancy. The gonads are composed of testicular tissue, although the histology is highly abnormal
with a rapid decline in germ cell number beginning in infancy [28]. Spermatogonia are present in the
gonads
Int. J. Mol.of ~30%
Sci. of adults
2020, 21, withREVIEW
x FOR PEER CAIS but the majority of tubules are Sertoli-cell-only [87]. Presence 20 of of
31
spermatozoa has not been described in the testis of an individual with CAIS and therefore the potential
spermatozoa
for fertility (e.g.,hasusing
not been
ICSI) described in the testis of an individual with CAIS and therefore the
is not possible.
potential for fertility (e.g.,
The phenotype of PAISusing ICSI) isvariable,
is highly not possible.
ranging from ambiguous genitalia in severe cases
The phenotype of PAIS is highly variable,
to cryptorchidism, hypospadias, and infertility in classical ranging from cases.
ambiguous Whilst genitalia in severe
subfertility cases to
is generally
cryptorchidism,
considered hypospadias,
universal and infertility
for individuals with PAISinraised
classical cases.spontaneous
as male, Whilst subfertility is generally
natural fertility was
considered
reported in universal
one case [162]for individuals
and following withstimulation
PAIS raised as male,
with high-dosespontaneous
testosteronenatural
andfertility
subsequentwas
reported in one case [162] and following stimulation with
ICSI [163]. In other cases of subfertility, IVF may also be possible [137]. high-dose testosterone and subsequent
ICSI For[163]. In other
46,XY cases
females of subfertility,
with disorders of IVF may also
androgen be possible
action, there are[137].
no options for fertility preservation
due to Forthe46,XY
lack females
of viablewith gametesdisorders of androgen
and Mullerian action, there
structures. For the aremajority
no options for fertility
of males, sperm
preservation
retrieval is notduean to the lack
option of viable gametes
as spermatogenesis andpresent
is not Mullerian structures.
in these For the majority
testes [28,137]. Therefore,of males,
future
sperm retrieval
strategies is not an
for fertility option as spermatogenesis
preservation is not are
in these individuals present
likelyin to
these testes on
depend [28,137]. Therefore,
the presence of
future strategies for fertility preservation in these individuals are likely
spermatogonia within the gonad. Such an approach would require testicular tissue cryopreservation to depend on the presence of
spermatogonia
and subsequent within
developmentthe gonad. Such an approach
of spermatozoa from the would
storedrequire testicular
tissue using tissue
in vitro and cryopreservation
transplantation
and subsequent
techniques, which development
are being developed of spermatozoa
as a strategyfrom the stored
for fertility tissue using
preservation in vitrofacing
in individuals and
transplantation techniques, which are being developed as a strategy
gonadotoxic therapies [136]. However, additional considerations for these individuals include for fertility preservation in
individuals abnormality
underlying facing gonadotoxic
of the testistherapies
that may[136].
preventHowever,
development additional considerations
of spermatozoa [28,164]forandthese
the
individuals include underlying abnormality
risk of gonadal malignancy for those with AIS [28,165]. of the testis that may prevent development of
spermatozoa
Options for [28,164] and
fertility the risk of gonadal
preservation in 46,XYmalignancy
DSD due to for those with
impaired AIS [28,165].
androgen action are summarized
Options
in Figure 7. for fertility preservation in 46,XY DSD due to impaired androgen action are
summarized in Figure 7.

Figure 7. Options for fertility preservation in 46,XY DSD associated with impaired androgen
Figure 7.
action. Options
* Single forreport.
case fertility**preservation
Single case in 46,XY(high-dose
report DSD associated with impaired
testosterone). androgen
# Potential action.
for gonadal
* Single
tissue case report. ** based
cryopreservation Single oncase report (high-dose
published testosterone).
literature indicating # Potential
presence for cells
of germ gonadal tissue
+/− tissue
cryopreservation based on published literature indicating presence of germ cells +/−
cryopreservation; however, there are no reports of successful restoration of fertility and this should be tissue
cryopreservation;
considered within however,
the contextthere
of anare no reports
ethically of successful
approved researchrestoration
study. of fertility and this should
be considered within the context of an ethically approved research study.

5.4. Ovotesticular DSD

Ovotesticular DSD represents a unique group of DSD in which there is presence of ovarian and
testicular tissues in individuals with a 46,XX, 46,XY, or 46,XX/46,XY karyotype. The underlying
Int. J. Mol. Sci. 2020, 21, 2282 21 of 31

5.4. Ovotesticular DSD

Ovotesticular DSD represents a unique group of DSD in which there is presence of ovarian and
testicular tissues in individuals with a 46,XX, 46,XY, or 46,XX/46,XY karyotype. The underlying genetic
causes and associated phenotypes are variable, as are the internal and external genitalia. As a result, the
potential for fertility is largely dependent on gonadal pathology, reproductive tissues, and whether the
individual is raised as male or female. The ovarian component of the gonad tends to have a relatively
normal histology, whereas the testicular component is frequently dysgenetic, with limited germ cells
and rare spermatozoa [137]. Whilst spontaneous pregnancies have been reported in 11 females with
ovotesticular DSD [166], only one successful paternity has been described in a 46,XX/46,XY male with
ovotesticular DSD, after TESE and ICSI [145].
For phenotypic males, fertility preservation options are similar to those described for individuals
with 46,XY GD DSD. For phenotypic females with ovotesticular DSD who are unable to achieve a
natural pregnancy, oocyte retrieval for ART may be possible, although this will also depend on the
presence and anatomy of Mullerian structures and the potential for the individual to carry a pregnancy
to term. Recent advances in uterine transplant have increased the possibilities for individuals with
DSD who lack Mullerian structures. Whilst this has primarily been applied to a small number of
individuals with uterine factor infertility e.g., Mayer–Rokitansky–Küster–Hauser syndrome [167], in
the future this may be applied to other cases in which Mullerian structures are not present.

6. Fertility Preservation in Prepubertal Individuals with DSD—Ovarian or Testicular Tissue

Cryopreservation
Over recent years there has been a growing interest in fertility preservation in prepubertal patients
from whom it is not possible to obtain sperm or eggs. This has largely been applied to children due
to receive gonadotoxic treatment for cancer [168]. For these patients, the only option available is to
cryopreserve and store gonadal tissue with the aim of preserving germ cells that can be used to generate
gametes for ART or for re-transplantation to restore fertility [169]. Whilst ovarian cryopreservation and
re-transplantation has successfully restored fertility in females, there are no proven clinical methods to
restore fertility using prepubertal testis tissues [136].
Cryopreservation of gonadal tissues from young individuals with DSD has recently been reported
in 10 patients undergoing gonadectomy for a variety of DSD diagnoses [170]. This study demonstrated
the presence of germ cells in 5/10 patients and 4 of these elected for tissue cryopreservation. This
included four females (two individuals with mixed gonadal dysgenesis, one with PAIS and 1 with OT
DSD) and one male (with partial gonadal dysgenesis). Of the 5 females with no germ cells present,
three had mixed gonadal dysgenesis (Turner Syndrome with Y-chromosome material), whilst the other
two had complete gonadal dysgenesis and tissue was not cryopreserved in these individuals [170].
Important considerations for future development of such an approach will be to consider the potential
for obtaining and differentiating viable germ cells, optimal timing of gonadectomy in relation to the
risk of gonadal malignancy balanced against the likelihood of successful restoration of fertility using
the tissue. Furthermore, the application of such an approach to patients in whom gonadectomy is not
clinically indicated must be carefully considered from an ethical perspective with consideration of the
clinical risks and benefits of surgery. Whilst gonadal tissue cryopreservation may ultimately lead to
new strategies for fertility preservation in DSD, this approach should currently be considered only in
the context of an ethically approved research study.

7. Conclusions
Significant progress has been made in understanding the mechanisms that underlie the
development of DSD in humans. The complexity of gene interactions that drive development
of the bipotential gonad to either a testis or an ovary are increasingly recognized. In many cases, this
involves a delicate balance between ‘promoting’, ‘inhibiting’ and ‘antagonistic’ signaling pathways,
which results in a wide variation in clinical phenotype for individuals with DSD. Despite the fact that
Int. J. Mol. Sci. 2020, 21, 2282 22 of 31

fertility is often impaired in DSD, established options for fertility preservation are limited and there are
important ethical and practical considerations for the development of future fertility therapies in this
population. Future research should focus on developing safe and effective strategies that may preserve
fertility potential in individuals with DSD.

Funding: R.T.M. is supported by a UKRI Future Leader Fellowship (MR/S017151/1). This work was undertaken
in the MRC Centre for Reproductive Health funded by the grant MR/N022556/1.
Acknowledgments: The authors would like to thank Ronnie Grant for his assistance with the illustrations.
Conflicts of Interest: The authors declare no conflict of interest.

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