🧬 PART SIX DISORDERS OF SEX DEVELOPMENT

24 CHROMOSOMAL DIFFERENCES OF SEX DEVELOPMENT CHROMOSOMAL SEX is, for the most part, congruently XX female and XY male. The XX and XY embryo are built on a fundamentally similar outline plan, and only as development proceeds do certain modifications evolve. If at any point in this sequential process some genetic instruction is faulty, inappropriate, or cannot be acted on, the direction of anatomical sexual development may proceed imperfectly or completely incongruently. In this chapter we focus on those forms in which classical and molecular cytogenetics comprise key diagnostic investigations. We provide categories for “girls and women” and “boys and men,” according to the phenotypes presented and according to the sex that the individual is regarded as being, by the individual or by the individual’s parents. We do not address the question of gender dysphoria, which is more a matter for the psyche. NOMENCLATURE These conditions are subsumed under the general heading of differences of sex development (DSDs, also known as disorders of sex development; Table 24–1). This classification also includes the sex chromosome aneuploidies Turner syndrome and Klinefelter syndrome, which are dealt with in Chapter 15. The different chromosomal categories may be indicated by reference to the sex chromosome constitution (XX or XY) and the nature of the gonad (testis, ovary, ovotestis, or dysgenetic/streak). The former expressions XX male, XY female, and hermaphrodite are now referred to as particular types of DSD.1 Genital ambiguity/intersex is simply denoted XX DSD or XY DSD, according to karyotype; clearly these are rather broad descriptors, and more precise detail might usefully be added in individual cases. With reference to male or female sex, these different levels of definition can apply: gonadal sex (ovary, testis, ovotestis, streak); anatomical/ genital sex (structure of the internal and external genital tract); karyotypic sex (46,XX, 46,XY, or other); and behavioral sex (gender identity). BIOLOGY Somewhat simplified, the fundamental plan of the reproductive tract is that bilateral gonads, arising from the genital ridge, connect with bilateral paired internal ducts 1 We retain mention of these earlier expressions in that much of the historic literature, to which the counselor may need to refer, will have used these terms. 758  DISORDERS OF SEX DEVELOPMENT (Müllerian and Wolffian) which enter a midline genital sinus, opening at the perineum. This opening is buttressed on each side by labioscrotal folds and capped above by a phallus. The basic plan of the genital ridge is laid down according to instruction from, in particular, the WT1 and NR5A1 (SF1) genes. Failure of this process results in complete gonadal agenesis. Thereafter, the direction in which gonadal development proceeds is due to the activity of a number of genes on the sexual determination pathway, with SRY playing a central role. Finally, sex differentiation takes place through hormones produced by the gonads (Baetens et al. 2019). The Key Role of the SRY and SOX9 Genes In the absence of SRY, but with input from WNT4 and RSPO1, the gonad develops into an ovary, and the duct system develops into fallopian tubes and uterus. The genital sinus remains as an opening (the vagina), flanked and surmounted by labia and clitoris. The female state results. If a Y chromosome is present—or at least that part of the Y that contains SRY, the testis-determining gene—the male direction is taken. Transient expression of the SRY gene, beginning at embryonic day 41, calls into action SOX9, which in turn stimulates the FGF9 gene; both FGF9 and SOX9 suppress WNT4, and the gonad becomes a testis. The testis, in turn, secretes hormones, of which androgen influences the genital tract to masculinize, and anti-Müllerian hormone causes regression of the female Müllerian ducts. A vas deferens forms from the duct system. The phallus enlarges. The labioscrotal folds fuse in the midline and accommodate the descending testes. The male state results. Table 24–1.  Major Genetic Categories of Differences of Sex Development (DSDs) DSD GROUP DISORDERS OF GONADAL DEVELOPMENT DISORDERS OF HORMONE SYNTHESIS OR ACTION UNCLASSIFIED DISORDERS 46,XY DSD Complete or partial gonadal dysgenesis Ovotesticular DSD Androgen biosynthesis defects Androgen insensitivity syndrome (AIS) Complex syndromic DSD 46,XX DSD Testicular DSD Ovotesticular DSD Primary ovarian insufficiency Congenital adrenal hyperplasia (CAH) Mayer-Rokitansky-Küster-Hauser syndrome Complex syndromic DSD Sex chromosomal DSD 45,X 47,XXY 45,X/46,XY mixed gonadal dysgenesis 46,XX//46,XY chimerism Source: Adapted from M Cools et al., Caring for individuals with a difference of sex development (DSD): a Consensus Statement, Nat Rev Endocrinol 14:415–429, 2018. CHROMOSOMAL DIFFERENCES OF SEX DEVELOPMENT  759 Chromosome Testing in Differences of Sex Development Classical cytogenetic testing2 is often necessary to diagnose chromosomal DSDs accurately (Figure 24–1). The presence of SRY is routinely tested with FISH or QF-PCR, and chromosome microarray is used for initial chromosome assessment, allowing confirmation of chromosomal sex and detection of copy number changes affecting known or postulated DSD genes.3 A standard microscope karyotype is undertaken to detect balanced translocations involving the sex chromosomes, and at least 30 cells should be examined in order to check for mosaicism. Molecular testing for SRY and SOX9, as well as many other specific DSD genes (e.g., NR5A1, WNT4, SOX3, ARWT1) per medium of a gene panel or exome sequencing approach, is a suitable adjunctive approach given the genotypic heterogeneity of the DSDs. Whole-genome sequencing enables the additional targeting of structural variants and regulatory elements of DSD genes in the non-coding genome, which may account for some undiagnosed DSDs (O’Connell et al. 2019; Délot and Vilain 2021). An example is a key regulatory element termed RevSex upstream of SOX9, which, when deleted, causes 46,XY DSD, and when duplicated causes 46,XX DSD (Benko et al. 2011). 2 While our focus in this book is on the chromosomal state, necessarily here we must also refer, at least in outline, to molecular analysis: the two often go hand-in-hand in DSD diagnosis. 3 Several genes in the pathways of sex development are known to have dose-dependent effects, including SOX3, SOX9, WT1, DAX1, and WNT4. Figure 24–1.  A recommended approach to genetic testing in a child with DSD. Notes: #Targeted DSD-panel may be performed if WES is not available. Source: Adapted from MA O’Connell et al., Establishing a molecular genetic diagnosis in children with differences of sex development: A clinical approach, Horm Res Paediatr 96:128-143, 2023. Courtesy A Sinclair.

760  DISORDERS OF SEX DEVELOPMENT Chromosomal Differences of Sex Development in Girls and Women XY OVARIAN DIFFERENCE OF SEXUAL DEVELOPMENT The 46,XY karyotype in an otherwise normal girl with (apparently) completely normal female anatomy is a very rare observation. A single case is reported of a child who was a compound heterozygote for mutations in the CBX2 gene, discovered only because of a discordant chromosome finding at prenatal diagnosis (Biason-Lauber et al. 2009). The internal genital tract was normal female, the gonads of normal ovarian appearance, and normal upon histology. CBX2 may have a dual role in gonad development, promoting SRY and SOX9 while repressing ovarian factors WNT4 and FOXL2 (Hart et al. 2022). XY DIFFERENCE OF SEXUAL DEVELOPMENT, COMPLETE PURE GONADAL DYSGENESIS (SWYER SYNDROME) Many cases of 46,XY gonadal dysgenesis are due to variants involving key transcription factors, along with their co-factors, that control the testicular developmental pathway (Elzaiat et al. 2022). When inherited, the rare familial forms provide a unique example of a Mendelian condition that can be inherited in an X-linked recessive, Y-linked, or sex-limited autosomal dominant or recessive mode. In the X-linked forms or autosomal forms, the XY female has a perfectly normal Y chromosome with a normal SRY testis-determining gene; the cause of the disorder is mutation in a gene (whether this be X-linked or autosomal) that affects a downstream event in testicular development. Concerning the X-linked form, rearrangements at the Xp21.2 locus that either duplicate the NR0B1 (DAX1) gene4 or disrupt its regulatory elements account for some XY gonadal dysgenesis (Barbaro et al. 2012; Meinel et al. 2023). Larger duplications result in syndromic 46,XY gonadal dysgenesis and intellectual disability, whereas small duplications cause only gonadal dysgenesis (Ledig et al. 2012). Duplications of NR0B1 have also been observed in several phenotypically normal males, indicating incomplete penetrance as observed in one male who transmitted a 1.1 Mb Xp21.2 duplication to his twin daughters (Veyt et al. 2024). He required IVF due to abnormal spermatogenesis, which may have reflected a mild manifestation of gonadal dysgenesis. Female 46,XX carriers of duplications involving NR0B1 appear to have a normal female phenotype (Veyt et al. 2024). As for the Y-linked form, there is a mutation in the SRY gene itself. In some Y-hemizygotes, the mutant gene has nevertheless been able to reach a threshold of operation that allows testis development, while in others with the same mutation it has not. Thus, for example, an XY male with a mutation in SRY may be a normal fertile man, while his XY child may be a daughter. The threshold is apparently all-or-nothing: Partial expression—that is, an intersex state—does not result (Jäger et al. 1992; Imai et al. 1999). A man may be a gonadal mosaic for an SRY deletion, as presumably was the father in Barbosa et al. (1995). Two daughters of his had XY DSD (one with gonadoblastoma) with a deletion of SRY, but he himself showed a normal SRY result; there were three other normal daughters and six normal sons. Similarly, Schmitt-Ney et al. (1995) describe two XY sisters and their XY half-sister with an SRY point mutation, whose father was shown to be mosaic for this mutation. These familial examples notwithstanding, sporadic occurrence is usual, and in ~15% of XY complete gonadal dysgenesis cases the SRY gene has a de novo mutation or rearrangement that abolishes its function of testis determination. 4 Loss-of-function variants in NR0B1 give rise to a different condition, X-linked congenital adrenal hypoplasia with hypogonadotropic hypogonadism. CHROMOSOMAL DIFFERENCES OF SEX DEVELOPMENT  761 The first autosomal gene for an XY DSD to be discovered was DHH, at 12q12. Homozygosity for this gene was identified in three of six apparently non-consanguineous Mexican-mestizo women with XY DSD, two of whom, not known to be related, had the same mutation (Canto et al. 2004). A number of autosomal dominant XY gonadal dysgenesis loci have since been identified, of which the major contributors are NR5A1 (SF1), MAP3K1, and DHX37 (Elzaiat et al. 2022). The gonad in the autosomal form of XY DSD is dysgenetic and is seen as a “streak.” The genital tract does not virilize. Familial “ovarian” malignancy was a notable observation in a sibship of three XY women (the karyotype presumed in two who had died at ages 19 and 20 years) described in Kempe et al. (2002). XY DIFFERENCE OF SEXUAL DEVELOPMENT, COMPLETE ANDROGEN INSENSITIVITY SYNDROME This is a Mendelian condition in which the locus happens to be on the X chromosome. In this condition, the defect lies further down the developmental path. The gonad becomes a testis and produces testosterone, but the genital tract, internal and external, is resistant to the effects of androgen. The inheritance is X-linked recessive, and the locus is the androgen receptor gene at Xq12. The individual appears externally very much as a female, but there is amenorrhea, and pubic and axillary hair is absent. Internally the vagina is short, and the uterus and tubes are represented only by remnants; the testes may be in the inguinal canal. Malignancy of the gonad, gonadoblastoma or dysgerminoma, is less of a concern than in Swyer syndrome, and is seen in only 1%–2% of patients with complete androgen insensitivity, although a greater risk, 15%, applies in partial insensitivity (Delli Paoli et al. 2023). One example is on record in which, in a sense, the X-linkage was directly visible to the cytogeneticist; that is, the X chromosome was abnormal, including the region containing the androgen receptor locus. An affected aunt and niece had the karyotype 46,Y,inv(X)(q11.2q27) and the connecting mother was 46,X,inv(X)(q11.2q27) (Xu et al. 2003). A unique case is that of androgen insensitivity due to uniparental disomy X in a woman with the XXY karyotype (Uehara et al. 1999a). Chromosomal Differences of Sex Development in Boys and Men XX TESTICULAR DIFFERENCE OF SEXUAL DEVELOPMENT Most males with 46,XX testicular DSD (“XX males” in former parlance) arise from the presence of Yp material (rarely visible cytogenetically) on one of the X chromosomes, from occult XX/XXY mosaicism or from the inappropriate activity of a gene that is normally switched on only in response to a Y-originating genetic instruction. In approximately three-fourths of cases, the SRY gene is present, typically the consequence of an abnormal exchange between the X and Y during meiosis I in gametogenesis in the father and thus clearly a sporadic event. These cases are referred to as SRY + XX males, or SRY + XX testicular DSD. Délot and Vilain (2022) provide a full review. The phenotype in males with SRY + XX testicular DSD shares with Klinefelter syndrome the phenotype of hypergonadotropic hypogonadism and near universal sterility, but differs in that height is normal and intelligence unimpaired (Ferguson-Smith et al. 1990). Margarit et al. (1998) describe six SRY + cases due to translocation of Yp material

762  DISORDERS OF SEX DEVELOPMENT to Xp22.3, in whom different Y breakpoints could be identified but whose clinical phenotypes were very similar: normal intelligence, normal stature, and testicular atrophy with azoöspermia. In these SRY + cases, a more accurate cytogenetic designation would be 46,X,der(X)t(X;Y)—or more fully 46,X,der(X)t(X;Y)(p22.3;p11.2), though the exchange is not usually visible on standard cytogenetics—and so there is reference to this entity also in the section on the X;Y translocation (Chapter 6). Rare cases are known of a male with XX testicular DSD in whom the SRY gene had been translocated onto a terminal arm of an autosome (Dauwerse et al. 2006; Queralt et al. 2008). Males with XX testicular DSD and having no SRY gene are denoted SRY– (Abalı and Guran 2024). The fact of male development being able to proceed (to some extent, at least) despite the lack of SRY product presumably reflects an inappropriate activation of the testis-determining cascade in an otherwise normal 46,XX embryo, either as a sporadic stochastic event or due to a particular genetic predisposition. Variants in at least a dozen genes encoding transcription factors and signaling molecules have been shown to cause 46,XX male DSD, their actions mediated either by decreased expression of genes involved in ovarian development (e.g., WNT4, RSPO1, NR2F2) or through overexpression of pro-testicular genes (e.g., SOX9, SOX3, SOX10, DMRT1). Overexpression of pro-testicular genes is frequently the result of gene duplication or of different copy number variants in the upstream promotor regions, and these may be detectable by microarray. A detailed review is provided by Ferrari et al. (2024). Three cases are reported of males with 47,XXX chromosomes. In one well-studied example, the man was mildly intellectually disabled, with gynecomastia and hypogenitalism, and severe testicular atrophy on biopsy (Ogata et al. 2001). One X of the three was positive for SRY. In addition to an Xp-Yp interchange in paternal gametogenesis that produced the SRY-positive X chromosome, a coincidental maternal nondisjunction was responsible for a disomic X ovum. Thus, the combination at fertilization was XX(mat) + der(X)t(X;Y)(pat), giving 47,XX,der(X)t(X;Y) and appearing karyotypically as “47,XXX.” XX testicular DSD can be diagnosed prenatally, following the recognition that the chromosomal and ultrasonographic anatomical genders do not match, an observation that has become more frequent since the widespread adoption of noninvasive prenatal testing (Shen et al. 2023a). 45,X MALE We refer to this rare condition on p. 178. Most (quite possibly all) “45,X males” have, in fact, a molecular translocation of the SRY gene to an autosome or to the X chromosome (and might therefore be thought of as a type of Y;autosome or X;Y translocation). In some, the underlying constitution might actually be an X/XY mosaicism. Y ISOCHROMOSOMES A Y isochromosome, idic(Y)(q11) in mosaic state with a 45,X line is a rare observation in individuals presenting with a disorder of sex development5 (Manotas et al. 2020). The isochromosome presumably arises in paternal gametogenesis, with loss in an early mitosis of the embryo, to produce the 45,X line. 5 The usual presentation with the idic(Yq) is infertility in an otherwise normal male (p. 481). CHROMOSOMAL DIFFERENCES OF SEX DEVELOPMENT  763 Ovotesticular Differences of Sex Development The term hermaphroditism, of classical Greek derivation,6 has lost favor among those so diagnosed; and the qualifiers “true” and “pseudo” were always somewhat arcane. These days, we speak of ovotesticular DSD. As that descriptor indicates, the defining feature is that the gonads comprise both ovarian and testicular elements. There may be a testis and an ovary (Figure 24–2), or one or both may be an ovotestis. The most common karyotype is 46,XX (thus XX ovotesticular DSD), seen in 60%; one-third have mosaicism with one cell line that includes Y chromosomal sequences, mostly 46,XX/46,XY; a few are 46,XY, and other more rare forms are known (Syryn et al. 2023). Ovotesticular DSD often presents with variation in genital appearance of the newborn such that sex cannot be determined from phenotype alone. 46,XX Most 46,XX ovotesticular cases test negative on peripheral blood analysis for the SRY gene (Grinspon and Rey 2016). In some, there may be cryptic mosaicism within the gonad itself, with an island or islands of tissue containing the SRY gene or mosaic variants affecting other sex-determining genes (Ortenberg et al. 2002; Queipo et al. 2002). In terms of genetic causes, there is considerable overlap with 46,XX testicular DSD. In fact, it may be that 46,XX testicular DSD and 46,XX ovotesticular DSD are fundamentally the same genetic entity,7 with ovotesticular DSD representing an intermediate or incomplete form of testicular DSD (Délot and Vilain 2022). A (relatively) common cause in these cases is duplication of the RevSex element that influences the activity of SOX9 as noted above, this locus lying upstream of SOX9 within 17q24.3 (Croft et al. 2018). Incomplete feminization has been described in several XX patients with duplications Figure 24–2.  Ovotesticular DSD, surgical investigation in a newborn. At upper right, a fallopian tube has been delivered through the incision, its fimbriated opening (arrow) clearly evident, with an ovary (arrowhead) adjacent. At center, the forceps are retracting the prepuce of the phallus. At lower left, a testis has been delivered through the incision in the hemi-scrotum. The karyotype was mosaic XX/XY (or possibly chimeric XX//XY). 6 In Greek mythology, Hermaphroditus was the son of Aphrodite and Hermes. He was a handsome youth with whom the Naiad nymph Salmacis fell in love and prayed to be united with forever. The gods answered her prayer and merged their two bodies into one androgynous person. 7 Délot and Vilain emphasize that despite shared genetic origins, the two conditions have very different complications and management.

764  DISORDERS OF SEX DEVELOPMENT of chromosome 22, a region now recognized to include the SOX10 locus at 22q13.1 (Sreenivasan et al. 2022). A child with one gonad a testis and the other an ovary is described in Aleck et al. (1999); the karyotype in this child, who had presented with ambiguous genitalia, was 46,XX,rec(22)dup(22q) inv(22)(p13q13.1)mat. Rare familial cases of 46,XX ovotesticular DSD may reflect a mutation, whether autosomal or X-linked (such as RSPO1 or NR5A1) that induces the testis developmental cascade to proceed post-SRY stage (Domenice et al. 2016). Autosomal dominant variants might be transmitted through an unaffected 46,XY father, an example being duplication of SOX9 enhancers (Cox et al. 2011). Intrafamilial phenotypic variability and incomplete penetrance expressivity is a feature in some affected families (Syryn et al. 2023). Slaney et al. (1998) describe the case of four 46,XX cousins with abnormal sexual differentiation. Three had 46,XX ovotesticular DSD, and one was a 46,XX male. The putative testis-development gene had been transmitted through two mothers. Affected distant relatives due to a familial X;Y translocation are noted on p. 180. 46,XY XY ovotesticular DSD is rare indeed. The basis may be molecular rather than cytogenetic. For example, one non-mosaic 46,XY case had a post-zygotic mutation in SRY with SRY+/SRY– gonadal mosaicism (Braun et al. 1993). Presumably the SRY + line was responsible for the testicular elements in the gonad, and the SRY– line for the ovarian elements. XX/XY MOSAICISM The mosaic XX/XY ovotesticular DSD state more usually results due to the fusion of twin XX and XY embryos (XX//XY chimerism).8 Strain et al. (1998) reported a notable example of iatrogenic ovotesticular DSD which followed in vitro fertilization, presumably due to an XX and an XY embryo fusing; Malan et al. (2007) reached a similar conclusion in a case diagnosed prenatally and which could be referred to as “tetragametic chimerism.” A further theoretical route is from the post-zygotic loss of the X and of the Y in separate cells of an initially 47,XXY conception (Niu et al. 2002). Hercent et al. (2019) reported the case of a 26-year-old infertile male who was initially diagnosed as a XX SRY- male based on karyotype and absence of SRY in blood. Yet against expectation, a small number of sperm were obtained, and FISH studies showed equal numbers of X- and Y-bearing sperm, indicating that these gametes originated from normal XY cells. X/XY, XXY/XX, AND OTHER MOSAICISMS Other ovotesticular DSD mosaicisms include X/XY, XXY/XX, and X/X,idic(Yq). In the case in Röpke et al. (2007), a baby girl presenting with clitoral hypertrophy typed 46,XY on blood, but analysis of the removed dysgenetic gonads revealed X/XY mosaicism. On histological examination, the gonads contained testicular and ovarian elements. It was logically congruent that the XY state was observed more in the testicular component of the gonad, while cells with only an X chromosome predominated in the ovarian fraction. Kanaka-Gantenbein et al. (2007) report a boy, regarded as normal except for an undescended left testis, who presented as a 13-year-old with a left scrotal hemorrhage. In 8 Fusion of zygotes with the same (XX/XX or XY/XY) chromosomal gender results in typical and fertile female or male individuals, and may only come to light if a SNP array is done for another reason. CHROMOSOMAL DIFFERENCES OF SEX DEVELOPMENT  765 fact, the undescended gonad was an ovary that had actually ovulated, and presumably this had been the cause of the bleed. There was a left hemi-uterus and fallopian tube; the testis, on the right, was dysgenetic. On both blood and testicular biopsy, the karyotype was 47,XXY/46,XX. As expected, given the presence of a male gonad albeit an imperfect one, SRY and AZF loci were present. Becker and Akhavan (2016) report an infant presenting with genital ambiguity in whom “the family made an initial gender assignment of female until patient preference could be elicited.” The karyotype was 45,X[90]/ 46,X,idic(Yq)[10]; the isodicentric Y, with two copies of Yp, showed two copies of SRY. On laparoscopy, a normal-appearing uterus was seen; on the right, the gonad was an ovotestis and on the left, the gonad comprised only a “streak” (Figure 24–3). Prophylactic gonadectomy was done. An extraordinary case is the family described in Haines et al. (2015), in which a phenotypically normal mother had a child with ovotesticular DSD, initially karyotyping as 46,XX, and mother and child were both subsequently shown to be heterozygous for an insertional translocation, 46,X,ins(X;1)(q27;q25.2q25.3). A 770 kb segment of chromosome 1 at q25.2q25.3 was translocated into the X chromosome, at Xq27, and this site is only 82 kb distant from the SOX3 gene. SOX3 may, in certain circumstances, have an SRY-like influence; and in this case, the inserted chromatin may have induced, or allowed, inappropriate SOX3 activity, with its ectopic expression in one gonad (but not the other) producing a testis. Possibly, the mother’s typical femaleness may have reflected a favorable X-inactivation. A somewhat similar circumstance is recorded in Ohnesorg et al. (2017), the case of a teenage male presenting with testicular pain, the gonad in fact proving to be an ovotestis. The karyotype was 46,XX, and SRY–, but upon multiplex ligation-dependent probe analysis (MLPA) he had a de novo ~300 kb duplication which included the upstream regulatory region for SOX9, at chr17:71.3-71.6 Mb. Figure 24–3.  Gonadal observations at laparoscopy in an infant with an ovotesticular difference of sex development, having the karyotype 45,X/46,X,idic(Yq). The bilobar-appearing gonad on the child’s right (a) is in part testicular (arrow) and in part ovarian (arrowhead). The gonad on the left (b) is a fibrotic “streak” (the curved structure in mid-field). Source: From REN Becker and A Akhavan, Prophylactic bilateral gonadectomy for ovotesticular disorder of sex development in a patient with mosaic 45,X/46,X,idic(Y)q11.222 karyotype, Urol Case Rep 5:13–16, 2016. Courtesy REN Becker, and with the permission of Elsevier.

766  DISORDERS OF SEX DEVELOPMENT In most ovotesticular DSD, the molecular etiology remains unknown. In some, the basis of the defect may be a sporadic, inappropriate activation of the testicular developmental cascade in part of the gonadal tissue during its embryonic formation, as a stochastic event. Other genetic mechanisms postulated to play a role are non-coding variants, structural variants, epigenetic variation, and oligogenic inheritance (Délot and Vilain 2021). It is a curious and unexplained fact that ovotesticular DSD (mostly with a 46,XX karyotype) is far more common in the South African Black population than in Europeans (Wiersma 2004; Ganie et al. 2017). A small number of pregnancies have been reported in women with ovotesticular DSD, both spontaneous and with assistance of IVF (Stancampiano et al. 2024). With regard to 46,XY ovotesticular DSD, Zayed et al. (2008) reported a man who had had surgery for removal of an intra-abdominal testicular seminoma which included ovarian elements. At the same operation, a uterus and tubes were identified and removed. A few years later he underwent testicular aspiration of the remaining gonad, which yielded sperm: These were used for intracytoplasmic sperm injection, and eventually a pregnancy resulted in the birth of a normal daughter. A similar case was reported by Sugawara et al. (2012). Mixed Gonadal Dysgenesis Mixed gonadal dysgenesis (MGD) is a phenotype that borders upon that of ovotesticular DSD. One gonad may be a streak (as the case in Figure 24–3b), and the other of apparently testicular form. The typical karyotype is 45,X/46,XY mosaicism; the body build and external genital phenotype can range very considerably from near-normal male, through ambiguity, to Turner-like female (Peng et al. 2024). Some with 46,XY on peripheral blood may show X/XY on analysis of the gonad, as Nishina-Uchida et al. (2015) show in an infant presenting as female with clitoromegaly and a gonad in the right labium majus. The removed gonad contained normal-appearing and abnormal testicular elements, undifferentiated gonadal tissue, Wolffian and Müllerian derivatives, and included nests of gonadoblastoma; 46,XY and 45,X cells could be demonstrated. The other gonad was a streak. Rare Differences of Sex Development with Extragonadal Defects A number of rare conditions exist in which sex reversal coexists with other physical differences, and in some, intellectual disability; the SOX loci have a notable role (Sreenivasan et al. 2022). One of the less rare of these conditions is campomelia (long bone bowing) with sex reversal. The usual cause is a mutation within the SOX9 gene (at 17q24.3q25.1), this being one of the genes operating on the sexual differentiation pathway and which also influences limb bud mesenchymal development (Wagner et al. 1994). A cytogenetic form of this syndrome is seen in ~5% of affected individuals, who have an apparently balanced translocation disrupting the SOX9 locus at 17q24.3q25.1 (Figure 3–18). Concerning SOX10, whose locus is at 22q13.1, Falah et al. (2017) describe a child presenting with Hirschsprung disease and Waardenburg syndrome type IV, and going on to develop a peripheral and central demyelination, who had male external

CHROMOSOMAL DIFFERENCES OF SEX DEVELOPMENT  767 genitalia with intrascrotal gonads, the only genital abnormality being a first-degree hypospadias. He had a chromosome 22 duplication, 46,XX,dup(22)(q11.2q13), and was SRY-negative. Another syndrome is WT1 disorder, caused by heterozygous loss of WT1 and characterized by 46,XY DSD, nephrotic syndrome, and high risk of Wilms tumor.9 Otherwise, very many chromosomal conditions include a genital abnormality as part of an overall malformation syndrome. GENETIC COUNSELING XY Difference of Sexual Development, Complete Gonadal Dysgenesis (Swyer Syndrome) FAMILIAL/INHERITED CASES As noted above, the rare familial forms of XY gonadal dysgenesis provide an example of a Mendelian condition that can be inherited in a Y-linked, X-linked recessive, or sex-limited autosomal dominant or recessive mode (Table 24–2). Careful assessment of the pedigree may provide clues to the mode of inheritance, noting that phenotypic sex may not be concordant with chromosome sex and that expression of the DSD may be limited to the XY state. An individual with a sex-limited autosomal dominant difference of testicular development may have inherited the gene variant from their mother, or less commonly their mildly affected father. Autosomal recessive inheritance would be improbable in a multigenerational family tree, while on the other hand this mode would be strongly supported in a single affected sibship with more than one affected and in the setting of parental consanguinity. In the multigenerational scenario, a clear interpretation of autosomal versus X-linkage may not always be possible. The risk to the female carrier (as judged by position in the pedigree) to have an affected child would be a simple 25% if the X chromosome is implicated, but not readily calculable if a partially penetrant autosomal gene is the cause. As noted above, more DSD genes are coming to be identified, with gene interrogation “panels” being developed (O’Connell et al. 2023), and genetic counseling will be better underpinned as this knowledge evolves. Although the XY female phenotype is close to that of a normal female (but of course associated with infertility) some couples may want to consider prenatal or preimplantation diagnosis. The use of cytogenetics or NIPT (XY chromosomes) and ultrasound morphology (female external genitalia) Table 24–2.  Inheritance of Non-Syndromic 46,XY Gonadal Dysgenesis MODE OF INHERITANCE EXAMPLES Sex-limited autosomal recessive DHH Sex-limited autosomal dominant DMRT1, DHX37, MAP3K1, NR5A1 Y-linked SRY X-linked Xp21.2 duplications involving NR0B1 (DAX1) Source: Adapted from L Mohnach et al., Nonsyndromic disorders of testicular development overview, GeneReviews, Seattle: University of Washington, 2022. 9 In WAGR syndrome, WT1 is deleted in a contiguous gene deletion that also includes PAX6 (p. 416). 768  DISORDERS OF SEX DEVELOPMENT would allow detection of the condition; naturally, diagnosis could be precise if a DSD gene were identified. The Y-linked form is recognized by the demonstration of an SRY mutation carried by the XY girl and her XY father. This circumstance would allow the counselor the rare opportunity to apply principles of Y-linked inheritance with incomplete penetrance to risk estimation. Mutational analysis of the SRY gene (including deletion detection) may provide the basis for carrier detection and prenatal or preimplantation diagnosis. SPORADIC CASES Advice on the recurrence risk in the sporadic case is less straightforward. If a de novo SRY mutation is demonstrated, only paternal testicular mosaicism—which, as noted earlier, has been observed—could imply an increased risk for recurrence. An autosomal recessive or X-linked form may be identifiable on DSD panel testing. Again, failing the knowledge of a specific gene, prenatal diagnosis by chromosomal/ultrasound gender discordance should be feasible. For the XY woman herself, assisted conception is possible if a uterus is present, and a handful of successful pregnancy outcomes using donated oöcytes have been reported. (Hosseinirad et al. 2021). ASPECTS OF MANAGEMENT Couples electing not to consider prenatal diagnosis (or to continue a pregnancy in which a positive diagnosis has been made) should know of the importance of two particular factors in managing these girls (Jorgensen et al. 2010). First, the psychosexual orientation of these individuals is female (Babu and Shah 2021). But with secondary sexual characteristics developing incompletely and infertility being invariable, their self-image is vulnerable. In discussing the condition with parents, the counselor should note the importance of using language that reinforces their view of themselves as girls and women, and the counselor should avoid using such terms as “genetic male.” It may be explained to them, beginning in simple terms in childhood, that a genetic factor prevented their ovaries from developing normally (Goodall 1991). The essential components of psychosocial care to families affected by DSD have been detailed in a consensus statement (Cools et al. 2018). As mentioned above, pregnancy may be achievable with in vitro fertilization using a donor ovum. Second, there may be an important risk of neoplastic change in the dysgenetic gonad. A gonadoblastoma arises in 15%–35% of XY gonadal dysgenesis (Looijenga et al. 2007). The gonadoblastoma itself is noninvasive, but it is often associated with malignant elements, most commonly dysgerminoma, which do invade. Thus, and given that the gonad does not usefully contribute in terms of hormone production, early (first decade) gonadectomy is advisable. These and other aspects of management are rehearsed in detail in Jorgensen et al. (2010) and Alhomaidah et al. (2017). Considerable publicity in 2009 concerning an athlete who recorded extraordinary times in women’s running races at an international meeting put in sharp focus the question of how such people are to be regarded. The unfortunate woman’s internal genital state (which apparently included testicular elements) became the subject of public speculation and then of public documentation. The Athletics Federation resolved the issue with some wisdom and imaginativeness, acknowledging her “unfair”

CHROMOSOMAL DIFFERENCES OF SEX DEVELOPMENT  769 but entirely innocent physical advantage as a runner in allowing her to keep her gold medal, but also awarding a gold medal to the second-placed athlete. XY Difference of Sexual Development, Complete Androgen Insensitivity Syndrome This condition is inherited as an X-linked recessive trait, and the risk of recurrence follows classic Mendelian principles. The carrier may be identified and preimplantation/ prenatal diagnosis accomplished by molecular analysis of the androgen receptor gene. While complete androgen insensitivity typically has a consistent phenotype within families, incomplete androgen insensitivity is often seen with an intrafamilial variability (Boehmer et al. 2001). Issues relating to prenatal diagnosis are discussed in Morel et al. (1994), who also make the interesting but unsurprising point that incomplete forms imply a worse burden than the complete form, with partially virilized males (known as Reifenstein syndrome) having “considerable psychological distress and poor function in their adult life.” Similar considerations with respect to gender orientation in the XY girl, as discussed in the preceding section, apply to complete androgen insensitivity; but for those with partial androgen insensitivity, considerably more gender fluidity is reported (Delli Paoli et al. 2023). The risk for neoplastic change in the gonad is less, in the vicinity of 1%–2%, in the case of complete androgen insensitivity syndrome. Thus, gonadectomy may reasonably be delayed to allow spontaneous pubertal feminization (Jorgensen et al. 2010), although regular clinical and imaging checks would be advisable. Lacking a uterus, pregnancy is not possible.10 XX Testicular Difference of Sexual Development Many XX testicular DSD boys are not diagnosed until after childhood, by which time the parents are likely to have completed their family. Some cases may be recognized prenatally following discordant chromosomal and ultrasonographic sex. Nearly all SRY + cases have a de novo translocation of SRY to an X chromosome, with very low chance of recurrence. Two very rare exceptions are these: first, if the father has SRY translocated onto an autosome, or second, if he has an extra copy of SRY translocated onto the X chromosome (in addition to that on the Y). These possibilities can be excluded by performing karyotype and SRY FISH in the father. Concerning the rare case of the SRY– XX male, genetic testing may allow cases that would carry a high recurrence risk to be identified. Duplications or rearrangements involving SOX9 may be transmitted from a clinically unaffected mother, and in rare instances from an anatomically typical father (Cox et al. 2011). Heterozygous variants in NR5A1 are often inherited from XX mothers (Baetens et al. 2017).11 If prenatal diagnosis 10 Uterine transplantation is a procedure that offers a potential pathway to pregnancy that has resulted in live birth, but remains highly experimental and not without risk (Barragan-Wolff et al. 2024). 11 A specific NR5A1 missense variant, p.Arg92Trp, has been reported to cause 46,XX testicular DSD with reduced penetrance and to be transmitted from both mothers and fathers.