Abstract Background The diagnostic yield for 46,XY disorders of sex development (DSD) remains limited. Whole-genome sequencing (WGS) improves detection of both coding and non-coding variants that may be missed by routine testing. Cytochrome b5, encoded by CYB5A, is an essential co-factor for CYP17A1-mediated 17,20-lyase activity. We report on WGS on a Vietnamese family with 46,XY DSD with two siblings presenting with female external genitalia. Methods Clinical assessment and hormone profiling were conducted. WGS was conducted on peripheral blood DNA, in two affected siblings followed by variant annotation and ACMG-based classification. A minigene RNA splicing assay in HEK293 cells was used to evaluate the functional impact of the CYB5A intronic variant. Results The patient’s hormone profile showed low testosterone and estradiol. WGS identified compound-heterozygous CYB5A variants: a paternally inherited missense variant (p.Val34Glu, likely pathogenic) and a maternally inherited deep intronic deletion (c.129+862_129+863del) for which SpliceAI predicted aberrant splicing. Minigene assays confirmed that the intronic deletion creates cryptic splice sites, resulting in pseudoexon inclusion and a premature stop codon, consistent with nonsense-mediated decay. The intronic variant meets ACMG criteria for pathogenicity. Conclusion This family expands the spectrum of CYB5A -related DSD and demonstrates that compound-heterozygous variants, including deep intronic defects, can lead to a disruption in 17,20-lyase activity. These findings highlight the importance of WGS and functional assays for identifying clinically relevant non-coding variants in DSD.

Differences of sex development (DSD) represent a group of congenital conditions that affect human sex development and maturation owing to discrepancies of chromosomal, gonadal and phenotypic sex. The Chicago consensus classifies DSD as sex chromosome DSD, 46,XY DSD and 46,XX DSD, with subclassifications according to gonadal determination into testes and ovaries and hormone-dependent differentiation of Müllerian and Wolffian embryonic structures into female-typical or male-typical internal and external sex organs. DSD may occur as an isolated condition or as part of a complex syndrome. Diagnosis is based on clinical characteristics, imaging studies, hormonal measurements and genetic investigations. Management includes lifelong psychosocial support, hormonal treatments and surgical interventions that require personalization for each case as DSD encompasses a wide variety of aetiologies and presentations. This personalization must also consider individual values and preferences to ensure that clinical care is tailored to meet the unique needs and circumstances of each person, ideally provided by a care team with diverse specialities. This care involves psycho-educational counselling on the condition and its consequences, considering family and cultural norms. Additional efforts are needed to bridge gaps in knowledge related to diagnosis, management and long-term outcomes. Enhancing our understanding of the distinctions between sex and gender in societies is essential as greater awareness will inform and enrich public debates. Differences of sex development (DSD) encompass a group of rare congenital conditions characterized by atypical sex development. In this Primer, Flück and colleagues provide insights into our current understanding of rare DSD pathophysiology, its prevalence and diagnosis as well as challenges and controversies related to clinical management.

The Dahl salt-sensitive (SS) rat is a model of hypertension that replicates important subclinical features of human salt sensitivity, including blood pressure (BP) changes and end-organ damage in response to a high salt (HS) diet challenge. In rats, multiple sex determining region Y ( Sry ) gene paralogues encoding for SRY protein crucial for male-typical development, are located on the Y chromosome, obscuring our understanding the relative contributions of X-chromosome gene dosage to male hypertension phenotypes in this model. To investigate the impact of X-chromosome complement on male salt-sensitive hypertension, a transgene harboring the Sry1, Sry4a, and Sry3c genes was inserted into the Dahl SS strain on an autosome. Natural mating of XY SryTg males results in male offspring with XY, XX SryTg , or XY SryTg genotypes. We hypothesized that this model can be used to segregate the role of X-chromosome gene dosage in the male hypertensive phenotypes of the SS rat. Male rats of each genotype were challenged with an 8.0% NaCl diet beginning at ten weeks of age for two weeks. Body weight was tracked, and urinalysis and kidney histology were assessed at the end of the study. While no differences in bodyweight between XY and XX SryTg males were observed, XY SryTg were larger than XX SryTg gonadal males from 4-12 weeks of age (week seven body weight: XY=208±8, XX SryTg =183±3, XY SryTg =219±4 g). Albumin and protein excretion trended lower in XY SryTg but were not significantly different by genotype (UAE: XY=274±60, XX SryTg =246±35, XY SryTg =162±18 mg/day; UPE: XY=531±75, XX SryTg =447±46, XY SryTg =362±29 mg/day). No significant differences in heart or kidney weight, or medullary protein casting were observed between male genotype (heart: XY=1.32±0.05, XX SryTg =1.24±0.03, XY SryTg =1.34±0.03 g; kidney: XY=1.91±0.11, XX SryTg =1.83±0.06; XY SryTg =1.87±0.06 g; protein casting: XY=4.24±0.45, XX SryTg =4.01±0.49, XY SryTg =4.38±0.47 %). In summary, X-chromosome complement did not impact indications of heart or renal injury in response to high salt diet in intact male SS rats. Funded by NIH R01 OD030496 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background We have generated a rat model similar to the Four Core Genotypes mouse model, allowing comparison of XX and XY rats with the same type of gonad. The model detects novel sex chromosome effects (XX vs. XY) that contribute to sex differences in any rat phenotype. Methods XY rats were produced with an autosomal transgene of Sry , the testis-determining factor gene, which were fathers of XX and XY progeny with testes. In other rats, CRISPR-Cas9 technology was used to remove Y chromosome factors that initiate testis differentiation, producing fertile XY gonadal females that have XX and XY progeny with ovaries. These groups can be compared to detect sex differences caused by sex chromosome complement (XX vs. XY) and/or by gonadal hormones (rats with testes vs. ovaries). Results We have measured numerous phenotypes to characterize this model, including gonadal histology, breeding performance, anogenital distance, levels of reproductive hormones, body and organ weights, and central nervous system sexual dimorphisms. Serum testosterone levels were comparable in adult XX and XY gonadal males. Numerous phenotypes previously found to be sexually differentiated by the action of gonadal hormones were found to be similar in XX and XY rats with the same type of gonad, suggesting that XX and XY rats with the same type of gonad have comparable levels of gonadal hormones at various stages of development. Conclusion The results establish a powerful new model to discriminate sex chromosome and gonadal hormone effects that cause sexual differences in rat physiology and disease.

Male sex, early life chemical exposure and the brain aromatase enzyme have been implicated in autism spectrum disorder (ASD). In the Barwon Infant Study birth cohort (n = 1074), higher prenatal maternal bisphenol A (BPA) levels are associated with higher ASD symptoms at age 2 and diagnosis at age 9 only in males with low aromatase genetic pathway activity scores. Higher prenatal BPA levels are predictive of higher cord blood methylation across the CYP19A1 brain promoter I.f region (P = 0.009) and aromatase gene methylation mediates (P = 0.01) the link between higher prenatal BPA and brain-derived neurotrophic factor methylation, with independent cohort replication. BPA suppressed aromatase expression in vitro and in vivo. Male mice exposed to mid-gestation BPA or with aromatase knockout have ASD-like behaviors with structural and functional brain changes. 10-hydroxy-2-decenoic acid (10HDA), an estrogenic fatty acid alleviated these features and reversed detrimental neurodevelopmental gene expression. Here we demonstrate that prenatal BPA exposure is associated with impaired brain aromatase function and ASD-related behaviors and brain abnormalities in males that may be reversible through postnatal 10HDA intervention. Prenatal bisphenol A exposure is associated with an increased risk of ASD in boys through a mechanism involving aromatase suppression. These resulting ASD-related behaviors and brain abnormalities may be reversed through postnatal intervention with 10HDA in mice.

[This corrects the article DOI: 10.3389/fcell.2024.1337714.].

Objective To investigate the role of a potential SOX9 target gene, Tyro3 , along with its family members, Axl and Mertk (TAM family) in mouse testis development. Design Experimental laboratory study. Setting Research institute units. Subject(s) Embryonic day (E)11.5 Swiss mouse gonads for ex vivo gonad culture; Tyro3 knockout mouse embryos. Intervention(s) E11.5 Swiss mouse gonads were cultured in hanging droplets of 30 µL DMEM medium supplemented with 10% FBS and 1% antibiotic-antimycotic. A pair of gonads were treated with 20 μM of BMS-777607 or 30 μM of LDC1267 and an equivalent volume of the vehicle control DMSO. Main Outcome Measure(s) Immunofluorescence to measure morphological changes of ex vivo cultured gonads and in vivo Tyro3 mouse testes; qRT-PCR to measure gene expressions. Result(s) Inhibition of the TAM family in E11.5 ex vivo cultured male mouse gonads led to reduced germ cell numbers caused by reduced proliferation and increased apoptosis of the germ cells. Tyro3 knockout mice exhibited reduced expression levels of the germ cell genes Ddx4 , Dazl and Pou5f1 and increased expression levels of the Sertoli cell genes Sox9 and Amh at E12.5. However, by E14.5, the expression of Ddx4 , Dazl , Sox9 and Amh had returned to normal levels in Tyro3 knockout testes. Tyro3 knockout testes displayed normal morphology and structures during fetal testis development. Conclusion(s) TAM family members have redundant roles in regulating germ cell development during early testis development. Attestation Statement Data Sharing Statement N/A Capsule Inhibition of the TAM family led to loss of germ cells in fetal gonads and deletion of Tyro3 alone disturbed gene expressions of germ cells and Sertoli cells. ### Competing Interest Statement The authors have declared no competing interest.

Subjects in medical research have predominantly been male (1). Women experience 50-75% more adverse drug responses (2) resulting in withdrawn medications (3). While sex differences in metabolism, disease and treatment response are increasingly recognised, sex-informed medicine is lagging. In 2016, USAs National Institutes of Health (NIH) formulated the Sex as a Biological Variable policy (4), stating that grant recipients must consider sex in experimental design, planning, analysis and reporting of their findings. Australian data is lacking on the inclusion of both males and females as well as appropriate analysis of sex differences. We analysed the 219 Medical Journal of Australia (MJA) research articles over 2019-2023 (Box 1). We tallied when; i) both males and females were included in the study, ii) sex differences were reported and/or considered, and iii) the analysis was appropriate to support sex-related claims. We found that articles published in MJA are including males and females, however testing of sex differences is uncommon and appropriate statistical analysis is lacking. We hope that this article will bring attention to this fundamental issue and improve future efforts to investigate sex differences. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research received financial support from the National Health and Medical Research Council Program Grant 2002426 and Fellowship APP1154870 awarded to VH. Additional support was provided by the Australian Government Research Training Program Scholarship for JR. We also acknowledge funding through the Victorian Governments Operational Infrastructure Support Program. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: We used only published, openly available research articles that reported human data from the Medical Journal of Australia journal. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

ATR-X ( al pha t halassemia, m ental r etardation, X -linked) syndrome is a severe developmental disorder affecting males caused by mutations in the chromatin remodelling gene ATRX . Genital abnormalities in affected boys include hypospadias and ambiguous genitalia, and patients show small poorly formed testes with only a few seminiferous tubules. Our mouse model recapitulated these testicular defects when Atrx was specifically deleted in Sertoli cells (Sc Atrx KO). Sc Atrx KO mice develop small testes with fewer and discontinuous tubules due to G2/M arrest and apoptosis of Sertoli cells. Here, we investigated the mechanism underlying the Sertoli cell defects in ATR-X syndrome. In healthy male control mice, Sertoli cell nuclei contain a single novel “GATA4 PML nuclear body (NB)” that strongly expresses the transcription factor GATA4, as well as ATRX and its binding partner DAXX. The GATA4 PML NB co-localizes with heterochromatin protein HP1α and PH3 (a marker of chromosome condensation), and with the short arm of the Y chromosome (Yp). In contrast, Sc Atrx KO Sertoli cells contain a single giant GATA4 PML NB, frequently associated with DNA double-strand breaks in G2/M-arrested Sertoli cells that underwent apoptosis. HP1α and PH3 were absent from the giant GATA4 foci suggesting a local failure in heterochromatin formation and chromosome condensation. Our data indicate that in Sertoli cells, ATRX protects a chromosomal region of Yp from DNA damage, probably during replication stress, and thus protects Sertoli cells from cell death. We discuss Y chromosome damage as a novel mechanism for testicular failure and the potential role of GATA4 during this process. Disclosure Summary The authors have nothing to disclose.

Background Disorders/differences of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. With overlapping phenotypes and multiple genes involved, poor diagnostic yields are achieved for many of these conditions. The current DSD diagnostic regimen can be augmented by investigating transcriptome/proteome in vivo, but it is hampered by the unavailability of affected gonadal tissue at the relevant developmental stage. We try to mitigate this limitation by reprogramming readily available skin tissue-derived dermal fibroblasts into Sertoli cells (SC), which could then be deployed for different diagnostic strategies. SCs form the target cell type of choice because they act like an organizing center of embryonic gonadal development and many DSD arise when these developmental processes go awry. Methods We employed a computational predictive algorithm for cell conversions called Mogrify to predict the transcription factors (TFs) required for direct reprogramming of human dermal fibroblasts into SCs. We established trans-differentiation culture conditions where stable transgenic expression of these TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli like cells (SLCs) were validated for SC phenotype using several approaches. Results SLCs exhibited Sertoli-like morphological and cellular properties as revealed by morphometry and xCelligence cell behavior assays. They also showed Sertoli-specific expression of molecular markers such as SOX9, PTGDS, BMP4, or DMRT1 as revealed by IF imaging, RNAseq and qPCR. The SLC transcriptome shared about two thirds of its differentially expressed genes with a human adult SC transcriptome and expressed markers typical of embryonic SCs. Notably, SLCs lacked expression of most markers of other gonadal cell types such as Leydig, germ, peritubular myoid or granulosa cells. Conclusions The trans-differentiation method was applied to a variety of commercially available 46, XY fibroblasts derived from patients with DSD and to a 46, XX cell line. The DSD SLCs displayed altered levels of trans-differentiation in comparison to normal 46, XY-derived SLCs, thus showcasing the robustness of this new trans-differentiation model. Future applications could include using the SLCs to improve definitive diagnosis of DSD in patients with variants of unknown significance.

SOX9 is a key transcription factor for testis determination and development. Mutations in and around the SOX9 gene contribute to Differences/Disorders of Sex Development (DSD). However, a substantial proportion of DSD patients lack a definitive genetic diagnosis. SOX9 target genes are potentially DSD-causative genes, yet only a limited subset of these genes has been investigated during testis development. We hypothesize that SOX9 target genes play an integral role in testis development and could potentially be causative genes in DSD. In this study, we describe a novel testicular target gene of SOX9, Trpc3. Trpc3 exhibits high expression levels in the SOX9-expressing male Sertoli cells compared to female granulosa cells in mouse fetal gonads between embryonic day 11.5 (E11.5) and E13.5. In XY Sox9 knockout gonads, Trpc3 expression is markedly downregulated. Moreover, culture of E11.5 XY mouse gonads with TRPC3 inhibitor Pyr3 resulted in decreased germ cell numbers caused by reduced germ cell proliferation. Trpc3 is also expressed in endothelial cells and Pyr3-treated E11.5 XY mouse gonads showed a loss of the coelomic blood vessel due to increased apoptosis of endothelial cells. In the human testicular cell line NT2/D1, TRPC3 promotes cell proliferation and controls cell morphology, as observed by xCELLigence and HoloMonitor real-time analysis. In summary, our study suggests that SOX9 positively regulates Trpc3 in mouse testes and TRPC3 may mediate SOX9 function during Sertoli, germ and endothelial cell development.

According to twin studies, there is a heritable contribution to gender incongruence, but the genetic mechanisms of this are unknown. Recent efforts to identify an aetiology of gender incongruence have focused on the hypothesis that sex hormones establish gender identity through influencing the development of neuroanatomy. Candidate gene studies that have sought to elucidate whether polymorphisms in sex steroidogenesis genes are overrepresented in transgender populations have been equivocal. A systematic search for case-control genetic association studies in transgender populations was conducted. Mean (+SD) or allele frequencies were extracted and combined quantitatively in random effects meta-analysis, summarised as standardised mean difference for continuous alleles or odds ratios for allele frequencies. Eight studies were included in the analysis. These studies spanned polymorphisms in five genes; the CAG repeat in androgen receptor ( AR ), the TA repeat in estrogen receptor 1 ( ESR1 ), the CA repeat in estrogen receptor 2 ( ESR2 ), the TTTA repeat in cytochrome P450 family 19 subfamily A member 1 ( CYP19 ), and the T>C SNP in cytochrome P450 family 17 subfamily A member 1 ( CYP17 ). Pooled estimates indicated that transgender women have a significant overrepresentation of short ESR1 alleles compared to cisgender men (OR = 1.23, 95% CI: 1.06, 1.44, p = 0.0089). This may contribute an increased likelihood of developing gender incongruence amongst natal males. Future investigations into gender incongruence should use genome-wide methods.

During sex determination in the mouse, fibroblast growth factor 9 signals through the fibroblast growth factor receptor 2c isoform (FGFR2c) to trigger Sertoli cell and testis development from 11.5 days post coitum (dpc). In the XX gonad, the FOXL2 and WNT4/RSPO1 pathways drive granulosa cell and ovarian development. The function of FGFR2 in the developing ovary, and whether FGFR2 is required in the testis after sex determination, is not clear. In fetal mouse gonads from 12.5 dpc, FGFR2 shows sexually dimorphic expression. In XX gonads, FGFR2c is coexpressed with FOXL2 in pregranulosa cells, whereas XY gonads show FGFR2b expression in germ cells. Deletion of Fgfr2c in XX mice led to a marked decrease/absence of germ cells by 13.5 dpc in the ovary. This indicates that FGFR2c in the somatic pregranulosa cells is required for the maintenance of germ cells. Surprisingly, on the Fgfr2c-/- background, the germ cell phenotype could be rescued by ablation of Foxl2, suggesting a novel mechanism whereby FGFR2 and FOXL2 act antagonistically during germ cell development. Consistent with low/absent FGFR2 expression in the Sertoli cells of 12.5 and 13.5 dpc XY gonads, XY AMH:Cre; Fgfr2flox/flox mice showed normal testis morphology and structures during fetal development and in adulthood. Thus, FGFR2 is not essential for maintaining Sertoli cell fate after sex determination. Combined, these data show that FGFR2 is not necessary for Sertoli cell function after sex determination but does play an important role in the ovary.

Sex development relies on the sex-specific action of gene networks to differentiate the bipotential gonads of the growing fetus into testis or ovaries, followed by the differentiation of internal and external genitalia depending on the presence or absence of hormones. Differences in sex development (DSD) arise from congenital alterations during any of these processes, and are classified depending on sex chromosomal constitution as sex chromosome DSD, 46,XY DSD or 46,XX DSD. Understanding the genetics and embryology of typical and atypical sex development is essential for diagnosing, treating and managing DSD. Advances have been made in understanding the genetic causes of DSD over the past 10 years, especially for 46,XY DSD. Additional information is required to better understand ovarian and female development and to identify further genetic causes of 46,XX DSD, besides congenital adrenal hyperplasia. Ongoing research is focused on the discovery of further genes related to typical and atypical sex development and, therefore, on improving diagnosis of DSD.

The transcription factor SOX9 is essential for the development of multiple organs including bone, testis, heart, lung, pancreas, intestine and nervous system. Mutations in the human SOX9 gene led to campomelic dysplasia, a haploinsufficiency disorder with several skeletal malformations frequently accompanied by 46, XY sex reversal. The mechanisms underlying the diverse SOX9 functions during organ development including its post-translational modifications, the availability of binding partners, and tissue-specific accessibility to target gene chromatin. Here we summarize the expression, activities, and downstream target genes of SOX9 in molecular genetic pathways essential for organ development, maintenance, and function. We also provide an insight into understanding the mechanisms that regulate the versatile roles of SOX9 in different organs.

46,XY gonadal dysgenesis (GD) is a Disorder/Difference of Sex Development (DSD) that can present with phenotypes ranging from ambiguous genitalia to complete male-to-female sex reversal. Around 50% of 46,XY DSD cases receive a molecular diagnosis. In mice, Fibroblast growth factor 9 (FGF9) is an important component of the male sex-determining pathway. Two FGF9 variants reported to date disrupt testis development in mice, but not in humans. Here, we describe a female patient with 46,XY GD harbouring the rare FGF9 variant (missense mutation), NM_002010.2:c.583G > A;p.(Asp195Asn) (D195N). By biochemical and cell-based approaches, the D195N variant disrupts FGF9 protein homodimerisation and FGF9-heparin-binding, and reduces both Sertoli cell proliferation and Wnt4 repression. XY Fgf9D195N/D195N foetal mice show a transient disruption of testicular cord development, while XY Fgf9D195N/- foetal mice show partial male-to-female gonadal sex reversal. In the general population, the D195N variant occurs at an allele frequency of 2.4 × 10-5 , suggesting an oligogenic basis for the patient's DSD. Exome analysis of the patient reveals several known and novel variants in genes expressed in human foetal Sertoli cells at the time of sex determination. Taken together, our results indicate that disruption of FGF9 homodimerization impairs testis determination in mice and, potentially, also in humans in combination with other variants.