DISORDERS OF SEX DEVELOPMENT (SDS): NEW CONCEPTS, AND CLINICAL MANAGEMENT

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1 DISORDERS OF SEX DEVELOPMENT (SDS): NEW CONCEPTS, AND CLINICAL MANAGEMENT Gabriela Guercio, Mariana Costanzo, Alicia Belgorosky y Marco A. Rivarola Servicio de Endocrinología, Hospital de Pediatria Garrahan, Buenos Aires, Argentina INTRODUCTION The disorders of sex development constitute a serious and complex clinical problem requiring the participation of a multidisciplinary team for adequate management of urgencies and long-term follow up In recent years, the advancement of molecular genetics has generated important advances in our knowledge of these disorders. A landmark in this subject has been the publication of a Consensus Statement, in 2006, of the Consensus Meeting on Management of Intersex Disorders (Chicago, 2005) [1], containing main discussions and recommendations. This Statement contains the following sessions: Nomenclature and Definitions, Investigations and Management of DSD, Outcome, Future Studies, Role of Supports Groups and Legal Issues. One of the recommendations is a change in the nomenclature (Table 1). TABLE 1. Changes in nomenclature proposed by the Chicago Consensus PREVIOUS NAME NEW NAME Intersex Male Pseudohermaphrodite Female Pseudohermaphrodite Trae Hermaphrodite XX male Disorders of sex development (DSD) 46,XY DSD 46,XX DSD ovotesticular DSD 46,XY complete gonadal dysgenesis 46,XX testicular DSD Mechanisms of human normal sex differentiation. A comprehensive knowledge of normal sex determination and differentiation is essential for understanding of its disorders [2]. Determination of sex chromosome constitution, 46, XY or 46,XX, defines gonad differentiation (testis or ovary, respectively). Subsequent sex differentiation defines the type of internal and external genitalia by developing of original primordial. In postnatal life, psychological differentiation is completed. Notwithstanding differences in sex chromosome constitution,

2 sex organ primordial are undistinguishable in the two sexes for several weeks. From there on gonads are differentiated (Figure 1, see Spanish version), followed by internal and external genitalia. Embryological origin of the main cellular types of the gonads are shown in Figure 2, (see Spanish version). Precursor Sertoli cells (testis) and granullosa cells (ovary) are derived from the same mesenchymal precursor cell. An initial important event is the migration of primordial germ cells from th middle line (hindgut) to urogenital ridge bilaterally, to be transformed into gonocytes. In the urogenital ridge, a steroidogenic precursor cell (to later develop into Leydig cells in the testis and theca cells in the ovary) also becomes differentiated. Finally, in the surrounding connective tissue differentiation of precursor peritubular myoid cells takes place. These cells will grow around seminiferous cords in the testis or around follicles in the ovary [3, 4]. Primordial germ cells proliferate by mitosis during migration This division is arrested when arriving to the male gonad, but it initiates meiotic division in the ovary, without completing it [3, 4]. In figure 3 (see Spanish version), main s and hormones participating in the process of sex differentiation are shown. Testicular differentiation. [1-3]. In the primitive bisexual gonad, testicular differentiation starts earlier (7 th week) than in the ovary (10 th week). Early differentiation in the bisexual primitive gonad is modulated by many transcription s, such as WT-1, Lhx9 and SF-1. The last one, encoded by a gene (NF5B) located at chromosome 9p33, is essential in adrenal, pituitary, hypothalamic and gonadal differentiation. It also regulates the expression of steroidogenic enzymes, ACTH receptor, AMH and AMH receptor [5]. The SRY gene, located in the short arm of the Y chromosome, is central in testicular differentiation. The SRY protein is expressed in Sertoli and germ cells. It is a transcription necessary to induce the cascade of events leading to testicular differentiation [6, 7]. This process however has multiple stimulating and inhibiting s which, when altered, generate different types of testicular dysgenesis. SOX9, ATRX, FGF9/FGFR2, Gata4/Fog2, Pod1, Vanine, Nexine, DHH, DHH receptor, Patched2, NGF, HGF, PDGF, Pod1, Pdgfr, M33, and Ir/Igfr1/Irr have been described as participating s [1-3]. Mechanisms of differentiation of non germ cells are summarized in figure 4 (see Spanish version). Promordial testis differentiation is induced by SRY through SOX9 action. SOX9 gene, located in chromosome 17q24, transcribes a transcription protein [7]. Another protein, DAX-1 would have inhibitory effects on SF-1 and SOX9. DAX-1 gene is locatedin chromosome Xp21. On the other hand, another protein, Wnt4, a growth, stimulates DAX-1 and inhibits Leydig cell differentiation [7, 8]. These modulators of testicular differentiation are dose dependent, i. e., both deficiency as well as excess of stimulation or inhibition determines final effect. Recent advances in molecular genetics have help to understand many aspects of this complex differentiation process, but many aspects remain without definition. Testis development implies the differentiation of fetal Leydig, peritubular myioid and pre-sertoli cells. The latter surround seminiferous cords containing germ cells. Fetal Leydig cells secrete testosterone, necessary for male gonaduct and male external genitalia differentiation; and Insl3 (insulinlike3 peptide necessary for the first stage of testicular descent [9]. Moreover, Sertoli cells secrete AMH necessary for regression of ipsi-lateral Müller ducts, preventing then differentiation of Fallopian ducts an the uterus [10]. The main players of the process of testis differentiation are shown in Figura 5 (see Spanish version Ovarian differentiation. [1-4] It has been proponed that the delay in ovarian differentiation depends on chromosome X double dose. However, little molecular information is available on the initial stages of ovarian differentiation. The most remarkable characteristic of ovarian development is the exponential increment in mitoses of germ cells. Early in embryonic life, around the 11 th week, 46,XX gonocytes mature into oogonia and star meiotic division in intrauterine life (in contrast with spermatogonia which star meiosis at puberty).

3 However, ovarian meiotic division is not completed, being arrested at the diplotene prophase of the first meiotic division. Proliferation of follicles start at week 16 th, but different from testis, endocrine activity i scarce during fetal life since estrogens are not necessary for differentiation of internal nor external genitalia. Expression of aromatase, FSH and LH receptors are late in prenatal life. This suggests that ovarian development is fetal gonadotropin independent, as indicated by normal ovarian development in the anencephalus fetus and in LH receptor inactivating mutations. Wnt4 is required for ovarian differentiation including germ cells. Germ cells are necessary for follicle formation. FOXL2, CBX2 and RSPO1 are s necessary for follicular development and for suppression of testicular development in 46,XX embryos [1-4]. See Figure 6, Spanish version. Defects in many of the s involved in the process of sex determination and differentiation are responsible for different types of gonadal dysgenesis, both in humans and in mouse models (Table 2). Differentiation of Gonaducts. Regardless of chromosome constitution, in the absence of functional testes, Müllerian ducts are differentiated, under Wnt4 stimulation, in fallopian ducts, uterus and the upper third of the vagina. As mentioned earlier, Sertoli cells of the fetal testis secrete AMH to inhibit ipsilateral Müller ducts, probabl by local diffusion, between the 9 th and the 12 th weeks. This effect is mediated by AMH specific membrane receptor. On the other hand, Fetal Leydig cells secrete testosterone to induce ipsilateral differentiation of Wolff ducts into epididymis and vas deferens. Differentiation of External Genitalia. Similarly, regardless of chromosome constitution, in the absence of functional testes or circulatin androgens of another origin, external genitalia conformation is feminine. Circulating testosterone reaches external genitalia and the prostrate to induce masculinization between the 8 th and 13 th week. However, testosterone acts as a pre-hormone and it needs to be converted into dihydrotestoterone, by 5α-reductase enzyme, to induce complete masculinization. This mechanism requires ligand binding to the androgen receptor to be able to stimulate specific genes. The androgen receptor has 8 exons and it is located in chromosome Xq The protein has 919 aa and the characteristic structure of steroid hormone receptors. This receptor is a ligand-dependent transcription with higher affinity for dihydrotestosterone than for testosterone. The androgen receptor-dihydrotestosterone complex binds DNA as homodimer to activate transcription of androgen dependent genes. Testicular descent into the scrotum. Testicular descent into the scrotum, which takes place during the third trimester of pregnancy, completes sex differentiation in males. This process can be divided in two stages: 1, trans-abdominal migration and 2, inguino-scrotal descent. Trans-abdominal migration is facilitated by the cranial suspensory ligament (induced by testosterone) which keeps the testis fixed to the posterior abdominal wall, and by the gubernaculums testis, stimulated by the insulin-like 3 peptide (Insl3), which attaches the inferior testicular pole to bottom of the scrotum. Insl3 acts through the so-called GREAT (G proteincoupled receptor affecting testis descent) receptor. During stage 1, testes are taken to the internal inguina orifice. The final descent to the scrotum is mediated by the gubernaculum retraction stimulated by testosterone. Prenatal and/or perinatal brain sexualization. There is considerably controversy regarding a possible effect of androgens on pre or perinatal brain programming of male sexual identity in adult life. For analysis it is convenient to consider separately sexual behavior and sexual identity [12]. Information arising from experimental animals, as well as data collected from patients disorders of sex development point out to role for prenatal programming in sexual behavior and identity. In our opinion, this is a to be considered, but not a decisive one for sex assignment.

4 Table 2. Gene Function Abnormality in the murine model Abnormal Human Phenotype WT1 Urogenital ridge blockade Denys Drash, WARG, Frasier SF1 Lhx9 Urogenital ridge blockade Urogenital ridge blockade Variable testicular dysgenesis with and without adrenal insufficiency Emx2 Urogenital ridge blockade CBX2 Gonadal dysgenesis Gata4/Fog2 co Sry inhibition, SRY SOX9, XX sex reversal (GF) Camptomyelic dysplasia, XX sex reversal (GF) Sox8 with Sox9 hypofunction Fgf9 Signaling molecule DAX1 Nuclear co- Cord formation and Espermatogenesis impairment. XY sex reversal Hypogonadism and adrenal hypoplasia. XX sex reversal (GF) Pod1 DHH Pdgfr Arx ATRX Pax2 DMRT1 WNT4 RSPO1 Signaling molecule Receptor Helicase Signaling molecule Growth Leydig cell differentiation impairment Mesonephric cell differentiation impairment Abnormal testicular differentiation Mesonephric cell differentiation impairment Absence of Sertoli and germ cells Agenesia de ductos mullerianos, XX sexr reversal XY Dysgenesis with or without polyneuropathy. X-linked Lisencephalia, XY DSD Alpha-thalassemia, X-linked mental retardation, XY DSD (GF) XX sex reversal Gene defects described refer to loss-of-function mutations, unless indicated (GF: gain of function mutation). Modified from Wilhelm D et al, Sex Determination and Gonadal Development in Mammals, Physiol Rev 87:1 28, 2007 (11)

5 Postnatal activation of the testis (minipuberty). In the 70s, Forest et al. [13] described that serum concentration of testosterone has a transient strong increment during the second and third months of postnatal life, to decrease later to very low values. Recently, increments in serum concentration of inhibin B and AMH [14], two Sertoli cell secreted products, have also been reported to be elevated. The effects of this postnatal activation of the testis are not well understood. Experimental evidence in monkeys suggests that this activation would have important effects for adult sex life, for instance, for brain maturation that is very active at this age. Postnatal gonadotrophic activation is also seen in females. DEFINITIONS AND NOMENCLATURE Ambiguous genitalia are referred to those genital disorders that alter the complete female or male phenotype. Sex discordance refers to a dissociation between chromosomal sex (XX, XY), gonadal sex (testis, ovary), gonaducts and external genitalia (masculine, feminine). It can co-exist or not with ambiguous genitalia, for instance, in complete androgen insensitivity syndrome where external genitalia are completely feminine. Traditionally, terms such as intersex and ambiguous genitalia, has been mainly used to define genital anomalies in newborns. Furthermore, due to the complexity of these disorders multiple classifications have been used. Some terms, such as pseudohermaphroditism, hermaphroditism, intersex, are controversial. They might be perceived as pejorative, confusing and stigmatizing for parents and patients [15]. For these reason, the Chicago Consensus for the management of DSD discussed the more controversial and critical points providing recommendations and remarking aspects requiring future research [16, 17]. However, some of the new recommendations are difficult to implement. When possible, terms should be descriptive and precise (i. e., androgen insensitivity syndrome), it should reflex genetic etiology and accommodate phenotypic variations. As a goal, terminology should be unanimously utilized by professionals and patients. The study of the karyotype is necessary for using the new classification. An approximation to etiologic diagnosis is central for the process of decision making. However, decisions might be difficult and under the pressure of urgency in the context of time-consuming diagnostic studies. Recent advances in molecular diagnosis have helped in the understanding many of these disorders. However, a precise molecular diagnosis is reached in less than 20% of DSD [16]. Even though defining of karyotype is essential in many cases, sometime, as in congenital adrenal hyperplasia (CAH), clinical information and hormone determinations are enough to make a diagnosis. Moreover, since some patients with CAH have a life-threatening sodium loss, this is the most urgent diagnosis to be made. In this context, the neonate weight curve is an important clinical information. In all newborns with ambiguous genitalia and no palpable gonads, serum electrolytes and 17-hydroxyprogesterone are urgent determinations. It has to be kept in mind that serum electrolytes might be normal during the first days of life and that the upper limit of normal serum 17-hydroxyprogestrerone is higher than later in life, particularly in premature birth. Later studies will confirm the presence of a CIP21B gene mutation. SPECIFIC CLINICAL EXAMINATION

6 It is important to interrogate about possible affected members in the family: siblings with ambiguous genitalia, male precocious puberty or sudden and unexplained death early in life (possibility of acute adrenal insufficiency), or other affected members in the mother s family, such as amenorrheic aunts or grand aunts (androgen insensitivity syndrome). Questioning about pregnancy is also important: ingestion of androgenic steroids or signs of virilization in the mother during pregnancy. After birth, general health, feeding and thriving of the child should be recorded. Table 3. Classification of Disorders of sex development, DSD, as proposed by the Consensus Statement [1]. Sex Chromosome DSD DSD 46,XY DSD 46,XX 45,X (Turner Syndrome and variants) 47,XXY (Klinefelter Syndrome and variants) 45,X/46,XY (mixed gonadal dysgenesis, ovotesticular DSD) 46,XX/46,XY (chimeric, ovotesticular DSD ) Disorders of gonadal (testicular) development: 1) Complete gonadal dysgenesis (Swyer Syndrome) 2) Parcial gonadal dysgenesis 3) Gonadal regression 4) Ovotesticular DSD 5) CBX2 gene def. (ovaries + fem. ext. gen.) Disorders of androgen synthesis or action: 1) Androgen biosynthesis defects ( 17-hydroxylase, 5αRD2, StAR protein, 3β-HSD, 17β-HSD) 2) Defects in androgen actions ( CAIS, PAIS) 3) Defects in LH receptor (Leydig cell hypoplasia) 4) Defects in AMH or AMH receptor (Persistence Müllerian ducts syndrome) Other (cloacal extrophy, severe hypospadias) Disorders of gonadal (ovarian) development: 1) Ovotesticular DSD 2) Testicular DSD (SRY +, duplication of SOX9), 46,XX males. Def. gen 3) Gonadal dysgenesis Androgen excess: 1) Fetal (Defects in 21-hydroxilase, or 11-hydroxylase) 2) Fetoplacental (deficiencia de aromatasa, POR [P450 oxidoreductasa]) 3) Maternal (luteoma, exogenous androgens, etc) Other (cloacal extrophy, vaginal atresia, other)

7 Physical examination of genitalia should follow a systematic order. Starting from the middle line, characteristics of the phallus, size (length, diameter), curvature, urethral opening, erections should be registered; as well as of the scrotum and/or labia majora (labio-scrotal folds); genital openings and genital skin pigmentation. Finally, a careful palpation looking for gonads including inguinal canals is of paramount importance. The location, size, and firmness of the two gonads, or the absence of them should be registered. The neonatal findings suggesting a DSD are the following [16, 17]: - Overt of genital ambiguity. - Apparent female genitalia with an enlarged clitoris, posterior labial fusion, or an inguinal or labial mass. - Apparent male genitalia with bilateral undescended testes, micropenis, isolated perineal hypospadias. - A family history of DSD - A discordance between genital appearance ans pre-natal karyotype. In newborns, complete masculinization without palpable gonads (Figure 8, see Spanish version) and complete feminization with bilateral hernias require a karyotype to discard complete masculinization in 46,XX CAH and complete feminization in 46,XY CAIS, respectively. In general, initial studies are karyotype withdetection of SRY by FISH, serum electrolytes, serum 17-hydroxyprogesterone, gonadotropins, testosterone, AMH [16-18], and pelvic US. Depending on initial diagnostic orientation, other hormonal determinations (eg., serum DHT), and hcg or ACTH tests are useful. Occasionally, laparoscopy and gonadal biopsy are necessary for a precise diagnosis. Finally, a gene analysis can provide the etiologic diagnostic. CLASSIFICATION OF DSD Since the last Chicago Convention the recommended classification is based on sex chromosome constitution. This is better than a classification based on gonad characteristics because frequently there is no gonadal biopsy available. On this basis, there are 3 main categories: 1, 46,XX DSD, 2, 46,XY DSD, 3 Abnormal Sex chromosome DSD (Table 3 and Figure 8, Spanish version). MANAGEMENT OF DSD A newborn with ambiguous genitalia is a classical urgency in pediatric endocrinology. A fluid communication between the medical team and the family is essential in these circumstances. A strict confidentiality and respect for family privacy in the context of a great anxiety and suffering are also important. An institutional medical should take over management in these cases. Unfortunately, the specialist is not the first physician in charge, but initial management and disclosure of information are important in these critical problem. Decision about sex becomes urgent after the first examination. Since in many instances this is not clear and it requires additional studies, parents need to make an effort to be patient and it is important that they find support for the management of these situations. The medical team should be careful with the use of words, such as testes or ovaries or a define sex before enough information is gathered. Frequently, the geneticist informs the karyotype in terms of feminine or masculine instead of 46,XX or 46,XY, which might generate confusion among family members. Psychological damage should be minimized. Finally, it is important to remember that adrenal insufficiency has to be ruled out [16-18].

8 The newborn with ambiguous genitalia is frequently referred to a high complexity Health Center. Depending on local Institutional organization, members of the DSD Team include a pediatric endocrinologist, a geneticist, a surgeon with experience in pediatric genital malformations, a pediatric urologist, a psychologist, an image specialist, a pediatrician and a lawyer with experience in legal sex assignment. In our group, the pediatric endocrinologist coordinates the group and it interacts with family members to discuss decisions. LEGAL AND SOCIAL SEX ASSINGMENT Due to the complexity of the causes of DSD, briefly discussed above, in many instances it is necessary to assign sex without a precise etiologic diagnosis. However, an approximation to diagnosis is essential. The medical team in charge of sex assignment needs to have rules for functioning. Decisions should be made by consensus, after group discussions. In case of disagreements, documents should be added to clinical charts, and opinions and justifications for decisions should be written down and signed. Surgeons have strong legal responsibilities, therefore, their points of view are essential. Clinical follow up is in the hands of the pediatric endocrinologist and the surgeon. Psychological support is often needed for long periods of time. Infants have the right to benefit from appropriate decisions, taken at the appropriate time, on their behalf, by their parents. The Medical Team recommends but parents or patients, depending on age, decide. Three age periods can be distinguished in the problem of sex assignment: 1. The newborn period, extended to early infancy, when parents decide 2. From 2 to 10 years of age, when sex re-assignment is not recommended 3. From 11 years of age up to late adolescence or young adulthood, when patients decide. When sex assignment becomes necessary, particularly in newborns, the aim is to choose that sex that will allow the best future functional adaptation that better goes along with the biological sex. Decisions might be difficult and controversial because in some cases there is no good solution. In general recommendations in infants are based on: 1) Etiologic diagnosis (if available, molecular diagnosis), diagnosis of type of DSD is useful because disorder evolution partially depends on etiology, known from both personal experiences and scientific publications. 2) Development of external genitalia and potential future sex function, 3) Possibility of surgical correction, 4) Development of internal genitalia, and fertility potential, 5) Paternal acceptance, 6) Psychological evaluation of parents and family, including social environment. Prenatal programming of central nervous system by androgens has little influence on decisions. Understanding the social and cultural background of the family is crucial for a fruitful relationship between parents and doctors. However, it is sometimes difficult to coordinate (or avoid) delivery of information to parents by different members of the team. Special efforts should be made by the informing professional to ensure appropriate understanding by the family of the situation, including chromosomal constitution, gene function, heredity, gonadal development, as well as external and internal genitalia differentiation. This often requires a previous explanation of normal biological development. Words should be selected carefully avoiding misunderstandings and offensive interpretations. It is advisable to frequently stop explanations in order to ask parents (or patients) to explain in their own words what they have understood so far.

9 Moreover, during follow up patients will go through all stages of growth and development (from infancy to adulthood) and information has to be delivered according to patient mental developing and understanding. The medical team has to be permanently alert with new developments that might arise along many years, and to give answers to questions, which are frequently, influenced by a high emotional impact. Corrective surgery arises many questions: Feminization surgery. In the correction of clitoromegalia and urogenital sinus it is important to preserve erectile function. Frequently postnatal surgery is a first step requiring some degree of vaginoplastia in later life. Vaginal dilatation is not recommended before full estrogenization and imminent sexual activity. Masculinization surgery. Urethral and ventral cord reconstruction might be facilitated with a reasonable use of testosterone. Re-operation because of fistulae is common. It is important to recognize that there are serious difficulties for penile reconstruction. Gonadectomy. Dysgenetic gonads of 46,XY DSD subjects have high risk of developing gonadoblastoma and are frequently removed. Therefore, scrotal gonads of subjects raised as males might be retained, but require periodic check up with clinical examination and US imaging for early detection of abnormal masses. The age of gonadectomy in CAIS is matter of debate. Since tumor development in these gonad is rare before puberty, it has been recommended to postpone gonadectomy to late adolescence to allow for spontaneous breast development. However, the psychological burden of carrying two testes for a girl has to be considered and time of gonadectomy should be discussed with the parents (and the patient after a certain age). IN SUMMARY, PATIENTS WITH DSD SHOULD RECIEVE LONG-TERM CARE PROVIDED BY MULTIDISCIPLINARY TEAMS IN CENTERS OF EXCELLENCE WITH AMPLE EXPERIENCE IN THIS CLINICAL MANAGEMNET. However, members of any team should be aware that errors can be made, therefore, decisions should be carefully thought, and discussed, before final recommendation. REFERENCES See Spanish version.