Abstract: Prophylactic gonadectomy has been recommended in complete androgen insensitivity syndrome (CAIS) because of an increased risk for the development of malignant germ cell tumors in the intra-abdominal gonads. No reliable screening parameters are available to detect early (pre-)malignant changes. Because the tumor risk before puberty is very low, the timing of gonadectomy has been postponed to allow spontaneous puberty and involvement of the patients in important decisions affecting their body and health. Gonadectomy after puberty is still discussed controversially. There are difficulties in determining the absolute malignancy risk for individuals with CAIS, difficulties with hormone therapy, and lack of studies supporting different protocols. In contrast, endogenous hormone profiles show very specific features that influence bone health, psychosocial well-being, and many other aspects which still have to be investigated. For women with CAIS who wish to keep their gonads, we propose a biannual screening program which has to be evaluated in a prospective multi-center trial.

Abstract: The human chorionic gonadotropin (hCG) test represents a key step in assessing Leydig cell function in prepubertal males, but differences in terms of hCG doses, number of injections, and sequence of blood drawing are published in the literature, showing poor standardization. The few available data in healthy boys are summarized here. A recombinant hCG (rhCG) formulation might permit overcoming some controversies as well as avoid the potential biological risk related to the injection of extractive hormones. Studies in humans are scarce, but they indicate that 250 µg rhCG in a single dose may represent a useful scheme for the dynamic evaluation of Leydig cell function in children as well as in adults. The main indication for hCG testing in childhood is the investigation of 46,XY disorders of sex differentiation. The test must also be considered in order to investigate the presence of functional testicular tissue when gonadal peptide hormones cannot be measured.

Abstract: Fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) develop in the mammalian prenatal and postnatal testes, respectively. In mice, FLCs emerge in the interstitial space of the testis as early as embryonic day 12.5 and thereafter increase in number during the fetal stage. We previously established a transgenic mouse line in which FLCs are labeled with EGFP and demonstrated that the EGFP-labeled FLCs were present even in adult testes. However, the characteristics of FLCs during postnatal stages remained unclear. In the present study, a comparison of the transcriptomes of FLCs from prenatal and postnatal testes and of ALCs from adult testes revealed that FLCs gradually alter their characteristics across developmental stages and come to roughly resemble ALCs. Many cholesterogenic genes simultaneously expressed a unique alternation pattern, while many oxidative phosphorylation and β-oxidation (both mitochondrial functions) genes showed a different unique pattern. These metabolic gene expression alterations might be triggered by milieu changes, such as nutrient and oxygen supply, from the prenatal to the postnatal period.

Abstract: Hypergonadotropic hypogonadism (HH) is defined by increased gonadotropin levels in men and women. Primary ovarian failure (POF) is a form of female infertility characterized by amenorrhea, hypoestrogenism, and elevated gonadotropin levels in women under the age of 40 years. Although several genes have been associated with POF, its causative genes remain to be identified. Here, we used whole-exome sequencing (WES) to study a consanguineous family with a 46,XX girl and a 46,XY man affected by HH. All exons of both siblings and their parents were captured and massively sequenced by WES, and the candidate variant was confirmed by Sanger sequencing. A novel c.1298C>A;p.Ala433Asp missense variant of the follicle-stimulating hormone receptor (FSHR) gene was found in both affected siblings in a homozygous state and in their parents in a heterozygous state. This FSHR variant is not present in available databases (1000 Genomes and NHLBI/EVS) and Brazilian exome controls. Moreover, it is highly conserved and predicted as deleterious in all prediction sites analyzed. In conclusion, the novel homozygous FSHR variant observed in 2 siblings with HH can expand the spectrum of FSHR mutations in humans.

Abstract: Complete androgen insensitivity syndrome (CAIS) is due to complete androgen resistance in androgen-dependent tissues. Since androgens are involved in growth, development, and mass maintenance of the skeleton, bone health may be a relevant clinical issue for improving quality of life of women living with CAIS. Bone mineral density (BMD) in women with CAIS and intact gonads has been reported in a normal range, although exceptions are known showing a low BMD mainly at the lumbar level. In women with CAIS and removed gonads, BMD is usually reduced at both the lumbar spine and femoral neck. However, the fracture risk remains largely unknown. In women with CAIS, hormonal replacement therapy may improve BMD, but it does not normalize it. Several factors may be operative (e.g., loss of AR signaling at the bone level, gonadal removal, and age at surgery [before or after attainment of the peak bone mass], inadequate sex steroid replacement therapy, poor compliance with hormonal treatment, high serum FSH levels, lack of testicular protein hormones after gonadal removal), but they are poorly evaluated. In conclusion, the maintenance of testes may represent a strategy to improve bone health in women with CAIS, but a strict follow-up to monitor the cancer risk is mandatory mainly from their 20s onwards. Optimal sex steroid substitutive therapy in adolescence and adulthood is a key factor to improve BMD status in women with CAIS and removed gonads, but conclusive data on optimal management are lacking.

Abstract: 45,X/46,XY mosaicism is a rare chromosomal abnormality and probably underdiagnosed. Although clinical and genetic analyses have been performed in some disorders of sexual development, there have been few studies focusing on the phenotype and genetic details of 45,X/46,XY mosaicism, especially in the Chinese population. The aim of this study was to describe the experience of our service in relation to 16 cases with 45,X/46,XY mosaicism. The age at the first evaluation of the patients ranged from 43 days to 30 years. Eight patients were reared as female and 8 as male. The main reasons for examination were primary amenorrhea, sterility, or ambiguous genitalia. Short stature was more common in female than in male patients. Two patients accepted gonadectomy due to tumor risk and none presented gonadal malignancy. The SRY gene was amplified positively in all of the patients. AZF gene microdeletions were present in 6 of 8 male patients, and all adult male patients had no sperm. No correlation has been found between clinical manifestations and the proportion of mosaic cells in peripheral blood. Our observations may permit a better management of people with 45,X/46,XY mosaicism.

Abstract: R-spondin proteins are secreted agonists of canonical WNT/β-catenin signaling. Homozygous RSPO1 mutations cause a syndrome of 46,XX disorder of sexual development (DSD), palmoplantar keratoderma (PPK), and predisposition to squamous cell carcinoma. We report exome sequencing data of two 46,XX siblings, one with testicular DSD and the other with suspected ovotesticular DSD. Both have PPK and hearing impairment and carried a novel homozygous mutation c.332G>A (p.Cys111Tyr) located in the highly conserved furin-like cysteine-rich domain-2 (FU-CRD2). Cysteines in the FU-CRDs are strictly conserved, indicating their functional importance in WNT signaling through interaction with the leucine-rich repeat-containing G-protein-coupled receptors. This is the first RSPO1 missense mutation reported in association with human disease.

Abstract: Despite widespread temperature-dependent sex determination (TSD) in reptiles, it is still unclear how the molecular network responds to temperature variation and drives the sexual fate. Profiling of sex-related genes is the first step in understanding the sex determination system in reptiles. In this study, we cloned the full-length coding sequences of Cyp19a1, Foxl2, Rspo1, Sf1, and Sox9 in an Asian freshwater turtle (Mauremys reevesii) with TSD and identified the expression patterns of these genes and Dmrt1 at different incubation temperatures to understand their roles in urogenital development. Our results showed that Cyp19a1, Foxl2, and Rspo1 were expressed in the adrenal-kidney-gonadal complex at a high level in females, while Sf1 and Dmrt1 were highly expressed in males. In addition, Foxl2 and Rspo1 showed sex-dimorphic expression in the presumed early thermosensitive period (TSP), Dmrt1 was upregulated at the beginning of the presumed TSP, and Sox9 did not show sex-dimorphic expression until the end of the presumed TSP. These results suggest that Foxl2 and Rspo1 are probably upstream genes involved in female sex determination and that Dmrt1 may be a key factor in male sex determination. Therefore, our study provides a solid foundation for further investigations on the molecular mechanism underlying sex determination in M. reevesii.

Abstract: The presence of a Y chromosome in patients with Turner syndrome (TS) is a risk factor for the development of gonadal tumor and/or virilization. With conventional cytogenetic analysis, some cells containing a Y chromosome can be missed. The aim of this study was to determine the presence and incidence of Y chromosome-derived material in TS patients using PCR and the markers SRY, DYZ1, DYZ3, DYS132, ZFY, and TSPY. Fifty-five TS patients (aged 5.5-26.75 years) were analyzed. A total of 17/55 (30.9%) were Y-positive, but only 7/17 had a Y chromosome in their karyotype and underwent gonadectomy. In 2 of these patients (28.6%), histopathologic examination revealed gonadoblastoma and dysgerminoma, respectively. In 8 patients in the studied group (8/55; 14.5%), the TSPY gene was detected, and the SRY gene (or a fragment) was identified in 9(3)/55 patients. No coding region mutations were observed in these SRY-positive patients. In conclusion, we have shown a high prevalence of Y chromosomal material in TS. Y markers were also observed in patients who had no Y chromosome in their karyotype, and PCR is very precise in detecting the presence of genetic material from the Y chromosome. Further follow-up of these Y-positive TS patients is mandatory.

Abstract: In most mammals, the sex of an individual is genetically determined by the Y chromosome-specific SRY gene. The presence of at least one functional copy of this gene determines the development of the primordial gonads into testes. However, testicular tissue does develop in the absence of SRY, albeit rarely, which is the case in testicular XX (SRY-negative) disorder of sex development (DSD). This condition is very important for studying the process of sexual determination because it allows the identification of genetic factors that are able to promote the male developmental pathway in the absence of SRY and thereby enables a better understanding of this process. Until now, this condition has been identified in various animal species but has never been reported in cat. In this study, we describe the first case of an XX (SRY-negative) DSD cat. The cat possesses a tortoiseshell coat associated with male-like external genitalia, including normal scrotum with 2 palpably normal testicles. Histological analysis confirmed the presence of the testes, and cytogenetic and genetic analyses showed a female karyotype associated with the absence of the SRY gene. Finally, sequencing of the RSPO1 gene revealed no mutation, and FISH analysis of the SOX9 locus did not reveal any large abnormalities.

Abstract: Androgen insensitivity syndrome (AIS) is a hereditary condition in patients with a 46,XY karyotype in which loss-of-function mutations of the androgen receptor (AR) gene are responsible for defects in virilization. The aim of this study was to investigate the consequences of the lack of AR activity on germ cell survival and the degree of testicular development reached by these patients by analyzing gonadal tissue from patients with AIS prior to Sertoli cell maturation at puberty. Twenty-three gonads from 13 patients with AIS were assessed and compared to 18 testes from 17 subjects without endocrine disorders. The study of the gonadal structure using conventional microscopy and the ultrastructural characteristics of remnant germ cells using electron microscopy, combined with the immunohistochemical analysis of specific germ cell markers (MAGE-A4 for premeiotic germ cells and of OCT3/4 for gonocytes), enabled us to carry out a thorough investigation of germ cell life in an androgen-insensitive microenvironment throughout prepuberty until young adulthood. Here, we show that germ cell degeneration starts very early, with a marked decrease in number after only 2 years of life, and we demonstrate the permanence of gonocytes in AIS testis samples until puberty, describing 2 different populations. Additionally, our results provide further evidence for the importance of AR signaling in peritubular myoid cells during prepuberty to maintain Sertoli and spermatogonial cell health and survival.

Abstract: Anti-mullerian hormone (AMH) is produced by Sertoli cells and signals through 2 transmembrane receptors (AMHR), specific types I and II, leading to regression of mullerian ducts during fetal male sex differentiation. Mutations in AMH and AMHR2 lead to the persistence of mullerian ducts in males which is transmitted in a recessive pattern. Here, we report 2 Egyptian DSD (disorder of sex development) patients reared as males who presented with bilateral cryptorchidism and otherwise normal male external genitalia and who both had a 46,XY karyotype. The first patient presented at the age of 2 years. Laparoscopic surgery revealed a uterus and fallopian tubes with the presence of 2 gonads, and biopsy and pathology revealed prepubertal testicular tissue showing small-sized tubules with mostly Sertoli cells and very few spermatogonia, edematous stroma, and no detectable ovarian tissue. The second patient presented at the age of 3 years. Laparoscopic surgery revealed a uterus and fallopian tubes, and serum AMH was very low (0.1 ng/mL). Molecular studies revealed a novel missense mutation in the AMHR2 gene in the first patient (c.767A>C; p.H256P) and a novel frameshift mutation in the AMH gene in the second patient (c.203delC; p.L70Cfs*7). We conclude that persistent mullerian ducts should be included in the differential diagnosis of cryptorchidism.

Abstract: 17α-hydroxylase/17,20-lyase deficiency is a rare form of congenital adrenal hyperplasia caused by mutations in CYP17A1 . Two phenotypic female sisters, aged 17 and 15 years and with

Abstract: There are only 2 patients with 47,XXY karyotype and androgen receptor (AR) gene mutation reported in the literature, and both are diagnosed as complete androgen insensitivity syndrome (CAIS). We report a 22-year-old female with 47,XXY karyotype and atypical external genitalia. Sequencing of AR revealed the heterozygous p.Asn849Lys*32 mutation, and extensive X chromosome microsatellite analysis showed homozygosity for Xp and heterozygosity for Xq, suggesting partial X maternal isodisomy. Partial androgen insensitivity syndrome (PAIS) developed in this case, probably because of the presence of the heterozygous AR mutation and random X- inactivation of the healthy allele. This is the first report of a female patient with 47,XXY karyotype and PAIS phenotype.

Abstract: Gonadoblastoma and malignant transformations thereof can occur in females with Turner syndrome (TS) and Y chromosomal material. However, in females with TS and no Y chromosomal material, this is rarely seen. We report a female with an apparent 45,X karyotype (in blood and tumor) who was diagnosed with a metastatic embryonal carcinoma. Exome sequencing of blood and the tumor was done, and no Y chromosomal material was detected, while predicted deleterious mutations in KIT (likely driver), AKT1, and ZNF358 were identified in the tumor. The patient was treated with chemotherapy (first-line: cisplatin, etoposide, and bleomycin; second-line: paclitaxel and gemcitabine), and after that surgical debulking was performed. She is currently well and without signs of relapse. We conclude that embryonal carcinoma can apparently occur in 45,X TS without signs of Y chromosomal material.

Abstract: Impaired androgen activity induces defective sexual differentiation of the male reproductive tract, including hypospadias, an abnormal formation of the penile urethra. Androgen signaling in the urethral mesenchyme cells (UMCs) plays essential roles in driving dimorphic urethral development. However, cellular events for sexual differentiation remain virtually unknown. In this study, histological analyses, fluorescent staining, and transmission electron microscopy (TEM) were performed to reveal the cellular dimorphisms of UMCs. F-actin dynamics and migratory behaviors of UMCs were further analyzed by time-lapse imaging. We observed a prominent accumulation of F-actin with poorly assembled extracellular matrix (ECM) in female UMCs. In contrast, thin fibrils of F-actin co-aligning with the ECM through membrane receptors were identified in male UMCs. Processes for dimorphic F-actin assemblies were temporally identified during an androgen-regulated masculinization programming window and spatially distributed in several embryonic reproductive tissues. Stage-dependent modulation of the F-actin sexual patterns by androgen in UMCs was also demonstrated by time-lapse analysis. Moreover, androgen regulates coordinated migration of UMCs. These results suggest that androgen signaling regulates the assembly of F-actin from cytoplasmic accumulation to membranous fibrils. Such alteration appears to promote the ECM assembly and the mobility of UMCs, contributing to male type genital organogenesis.

Abstract: The aim of the current report is to provide guidance relevant to psychological evaluation for healthcare providers and researchers working in the field of disorders of sexual development (DSD). In doing so, we give careful consideration to methodological issues and limitations that may influence the utility of investigations. For example, rarity and heterogeneity of DSD conditions restrict sample sizes when conducting evaluations aimed at establishing condition-specific psychological outcomes. At the same time, the potential for stigmatization by virtue of conducting psychological evaluations is particularly high given the fundamental contribution of sex and gender to one's sense of self and integrity. This article will provide basic theory for psychological evaluation as well as give a review of specific measures that can be employed for clinical purposes depending on a variety of parameters, including life stage of the patient and goal(s) of the evaluation. Care providers and service users may benefit from guidance in coping with the difficulties inherent in having and/or treating DSD. The potential for identification with the patient with DSD is higher than in other domains of medicine because sexual and gender identities are fundamental to all humans and are continually evolving from a sociological perspective.

Abstract: Recently, a heterozygous missense mutation in NR5A1, p.R92W, was identified as a cause of 46,XX testicular/ovo-testicular disorders of sexual development (DSD). We report a sibling pair with 46,XX DSD due to an NR5A1 mutation with distinct phenotypes, including external and internal genitalia and gonads, for whom different rearing sexes were selected. Thus, the phenotypes of p.R92W vary, even within a family. The father of the patients showed oligozoospermia with the p.R92W mutation, suggesting that in 46,XY individuals, the mutation would cause various gonadal phenotypes. We review and discuss the general role of the R92W mutation in sexual development.

Abstract: Testicular feminization, an earlier term coined for describing a syndrome resulting from failure of masculinization of target organs by androgen secretions during embryo development, has been well documented not only in humans but also in the domestic horse. The pathology, actually referred to as androgen insensitivity syndrome (AIS), has been proposed to follow an X-linked recessive pattern of inheritance in some horse breeds already investigated. Affected individuals are characterized by a female phenotype but with a stallion genotype of 64,XY SRY+ constitution. We identified a Warmblood horse pedigree segregating AIS, where the molecular analyses of the androgen receptor gene in the family provided evidences that a 25-bp deletion of the DNA-binding domain is causative of this equine syndrome.

Abstract: Scientific knowledge to understand the biological basis of sex development was prompted by the observation of variants different from the 2 most frequent body types, and this became one of the fields first studied by modern pediatric endocrinology. The clinical observation was supported by professionals working in different areas of laboratory sciences which led to the description of adrenal and gonadal steroidogenesis, the enzymes involved, and the different deficiencies. Steroid hormone measurements evolved from colorimetry to radioimmunoassay (RIA) and automated immunoassays, although gas and liquid chromatography coupled to mass spectrometry are now the gold standard techniques for steroid measurements. Peptide hormones and growth factors were purified, and their measurement evolved from RIA to automated immunoassays. Hormone action mechanisms were described, and their specific receptors were characterized and assayed in experimental materials and in patient tissues and cell cultures. The discovery of the genetic basis for variant sex developments began with the description of the sex chromosomes. Molecular technology allowed cloning of genes coding for the different proteins involved in sex determination and development. Experimental animal models aided in verifying the roles of proteins and also suggested new genes to be investigated. New candidate genes continue to be described based on experimental models and on next-generation sequencing of patient DNAs.

Abstract: In teleosts, elevated temperature during embryogenesis can act on germline cell development, which in turn plays a role for sexual fate. In Nile tilapia, a species with high-temperature-induced masculinization, little is known about the effects of increased temperature on gonadal development in non-masculinized females. The aim of the present work was to investigate persistent effects on the germline of genetically female (XX) Nile tilapia reared at normal (28°C) or elevated temperature (36°C) during the critical time of gonadal sex differentiation at 10 to 20 days post fertilization. Non-sex-reversed females were compared to control females to determine persistent effects of temperature on subsequent ovarian development using histological approaches. Germline stem cells were identified using the germline marker Vasa in combination with the proliferation marker PCNA. Vasa- and PCNA-positive germline stem cells were found in ovaries of both high-temperature-treated and control females. In both groups, ovarian germline stem cells were located at the germinal epithelium of the ovigerous lamellae. Although no detrimental effects of high temperature on gonadal development in female Nile tilapia were observed, implications on the reproductive fitness caused by elevated temperature need to be investigated in greater depth.

Abstract: Ovotesticular or testicular disorder of sexual development in dogs with female karyotype and lack of SRY (XX DSD) is a common sexual anomaly diagnosed in numerous breeds. The molecular background, however, remains unclear, and epigenetic mechanisms, including DNA methylation, have not been studied. The aim of our study was comparative methylation analysis of CpG islands in promoters of candidate genes for XX DSD: SOX9, SOX3, and WNT4. Methylation studies were performed on DNA extracted from formalin-fixed/paraffin-embedded or frozen gonads from 2 dogs with ovotesticular and 2 dogs with testicular XX DSD as well as control females (n = 4) and males (n = 2). Bisulfite-converted DNA was used for CpG methylation analysis using quantitative pyrosequencing. Promoter regions of SOX9 and WNT4 showed similar CpG methylation in each group, ranging from 0 to 5.5% and from 39 to 74%, respectively. The SOX3 promoter showed significantly higher methylation in the ovotesticular XX DSD cases and the testicular XX DSD and control males, suggesting that SOX3 methylation may play a role in canine XX DSD pathogenesis.

Abstract: With each new microarray or RNA-seq experiment, massive quantities of transcriptomic information are generated with the purpose to produce a list of candidate genes for functional analyses. Yet an effective strategy remains elusive to prioritize the genes on these candidate lists. In this review, we outline a prioritizing strategy by taking a step back from the bench and leveraging the rich range of public databases. This in silico approach provides an economical, less biased, and more effective solution. We discuss the publicly available online resources that can be used to answer a range of questions about a gene. Is the gene of interest expressed in the system of interest (using expression databases)? Where else is this gene expressed (using added-value transcriptomic resources)? What pathways and processes is the gene involved in (using enriched gene pathway analysis and mouse knockout databases)? Is this gene correlated with human diseases (using human disease variant databases)? Using mouse fetal testis as an example, our strategies identified 298 genes annotated as expressed in the fetal testis. We cross-referenced these genes to existing microarray data and narrowed the list down to cell-type-specific candidates (35 for Sertoli cells, 11 for Leydig cells, and 25 for germ cells). Our strategies can be customized so that they allow researchers to effectively and confidently prioritize genes for functional analysis.

Abstract: We report the case of 2 sisters (46,XX) born from consanguineous Moroccan parents. Both sisters had normal female genitalia, but within 2 weeks after birth, they presented with a severe salt-wasting crisis. Hormonal investigations suggested the diagnosis of congenital adrenal hyperplasia, which was confirmed by subsequent molecular analysis to be caused by 3β-hydroxysteroid dehydrogenase type 2 deficiency. Here, we discuss the main features like onset, possible complications, genetics, and replacement therapy of this rare disease.

Abstract: Complete gonadal dysgenesis (CGD) is characterized by an incomplete differentiation of the genital organs in a patient with a 46,XY karyotype. It is induced by mutations in the sex-determining region Y (SRY) gene which plays a key role in testis-determining pathways. The aim of this study was to investigate the possible pathogenic nature of a novel SRY mutation (p.Y127H) identified in a 46,XY female patient. To determine the effect of this mutation on SRY function, we studied its impact on DNA interaction by electrophoretic mobility shift assays. Since tyrosine 127 is close to the C-terminal nuclear localization signal of SRY, we conducted HA-SRY protein expression to observe the impact of the mutation on the nuclear import function in transfected cells. Our results showed that the Y127H mutation nearly abolishes the DNA-binding capacity of SRY and strongly impairs the nuclear localization of the mutated protein. Together with a previously described mutation analyzed in parallel in this paper (p.Y127C), our results highlight this tyrosine residue as a crucial structural determinant of the high mobility group box domain. This is the first report to explain the molecular mechanism of the Y127H mutation causing sex reversal and gives new insights for clinical practice to benefit patients with disorders of sex development.

Abstract: Sex determination and sexual differentiation pathways are highly conserved between marsupials and eutherians. There are 2 different pathways of prostaglandin D2 (PGD2) synthesis: prostaglandin D synthase (PTGDS) and haematopoietic prostaglandin D synthase (HPGDS). PGD2 regulates the subcellular localization of SOX9 during gonadal sexual differentiation. To investigate the function of PGD2 in the tammar gonad, we cultured undifferentiated male gonads in the presence of the HPGDS inhibitor HQL-79 and female gonads with exogenous PGD2 to mimic activation of the PTGDS-PGD2 pathway. Tammar PTGDS and HPGDS have only 50% similarity with mouse and human orthologues, but functional domains are conserved. The expression of SOX9 was unchanged by the treatments in cultured gonads, but its subcellular localization was markedly affected. SOX9 remained cytoplasmic in the Sertoli cells of testes treated with HQL-79. Treated testes developed a thickened ovary-like surface epithelium. In contrast, SOX9 became nuclear in the granulosa cells of developing ovaries treated with PGD2 and the surface epithelium was thin, as in testes. These results demonstrate that PGD2 regulates the subcellular localization of SOX9 and subsequent gonadal development in the developing marsupial gonads, as it does in mice, and that it must have been an ancestral mechanism.

Abstract: risk of testicular malignancy has been reported in adulthood and seldom also before puberty [Hughes et al., 2012; Achus and Quint, 2015]. Obviously, adequate hormone replacement therapy must be started from adolescence onward in the girls operated on before puberty to assure the development of secondary sexual characteristics as well as in females who undergo gonadectomy during or after adolescence to complete or maintain female phenotypic features [Bertelloni et al., 2011], while intact gonads permit well-being from endogenous hormones and avoid lifelong medical therapy. Recommendations for gonadal removal in women living with CAIS vary in literature. The “Consensus statement on management of intersex disorders” stated that “the testes in patients with CAIS ..... should be removed to prevent malignancy in adulthood. The availability of oestrogen replacement therapy allows for the option of early removal at the time of diagnosis which also takes care of the associated hernia, psychological problems with the presence of testes and the malignancy risk” [Hughes et al., 2006]. Indeed, literature indications range from “the testes should be removed as soon as they are discovered” [Donahoue, 2016] to “support leaving the testes in until after puberty has been completed and then either removing them ..... or instituting a careful monitoring process for the early detection of seminoma” The complete androgen insensitivity syndrome (CAIS) is due to inactivating mutations or deletions in the androgen receptor gene, completely abolishing the androgenic effects on target tissues before and after birth [Hughes et al., 2012]. These individuals present with a female phenotype, but a male karyotype and hormonal active testes [Hughes et al., 2012]. Diagnosis may be suspected in a prepubertal girl presenting with inguinal hernia containing male gonads or in an adolescent with a female habitus and normal breast development but absent or scanty growth of pubic and axillary hair, and primary amenorrhea [Audi et al., 2010; Hughes et al., 2012]. Today, a mismatch between the genital appearance at birth and the prenatal karyotype can be an additional feature for diagnosis [Audi et al., 2010]. In females with CAIS, the androgens produced by the testes are converted into estrogens with the action of aromatase. In the absence of androgen action, this mechanism permits the development of female secondary sexual characteristics and all the other estrogen actions [Audi et al., 2010; Hughes et al., 2012], including the effects on bone maturation and mineralization [Almeida et al., 2017]. CAIS was clinically characterized in the 1950s [Morris, 1953]. From then on, a long-lasting debate regarding the need and the timing of gonadal removal in these women started. The debate aroused mainly because an increased Accepted: March 29, 2017 by M. Schmid Published online: June 14, 2017

Abstract: Embryonic gonad sex in many reptilian species is determined by the incubation temperature of the egg, a differentiation process known as temperature-dependent sex determination (TSD). Incubation at the pivotal temperature (PvT) results in approximately an equal number of offspring of both sexes. We investigated the potential contribution of genetic variations that drives the gonadal differentiation into testes or ovaries under this temperature in the red-eared slider turtle (Trachemys scripta). Four male and 4 female hatchlings of eggs that had been incubated at the PvT were examined for polymorphisms at an approximately 23-kb region of the aromatase (cyp19a1) gene. By aligning the red-eared slider aromatase gene to a reference genome of the western painted turtle (Chrysemys picta bellii), we discovered 22 exonic and 1,268 intronic polymorphisms. Of these, 12 (55%) exonic polymorphisms were unique to the individuals of the red-eared slider; 10 were synonymous and 2 were nonsynonymous changes. We found no pattern in these genetic variants as well as intronic variants that are consistently different between male and female hatchlings at the PvT. Overall, our study suggests that polymorphisms within the aromatase gene - at least by themselves - do not constrain the gonad sex differentiation in embryos developed at the PvT.

Abstract: N-Nitrosodiethylamine (DEN), a well-known hepatocarcinogen, is found in certain food products as such or as a metabolic byproduct. This study investigated the effects of DEN on sexual development, gam