Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is characterised by aplasia of the uterus, cervix and upper part of the vagina. The genetic aetiology remains incompletely understood. We performed gene-level and gene set-level burden analyses based on exome sequencing/genome sequencing data from 727 probands with MRKHS and 2504 female control individuals. Single-cell RNA sequencing (scRNA-seq) was performed on human and mouse embryonic metanephros at different developmental stages. Genetic and transcriptomic data were integrated to prioritise suboptimal genetic signals, identify relevant cell types and determine key developmental stages. Potential digenic inheritance was assessed and prioritised using coexpression patterns from scRNA-seq data. We identified known MRKHS genes (PAX8, BMP7, GREB1L) and novel candidates (PAN2, AGPAT2) with exome-wide significance. Enriched biological processes included cell apoptosis and mesenchymal-to-epithelial transition. In human embryos, MRKHS-associated genes were enriched in the uterine epithelium at eight gestational weeks (w8) and Wolffian duct epithelium at w11, supporting the biological relevance of burden signals. We detected 992 digenic combinations in MRKHS, with three achieving exome-wide significance (CPSF3L/CYP2A7, AICDA/NOS1, EVC2/KANK1). Our study reveals both established and novel genetic contributors to MRKHS, links them to specific embryonic cell types and stages, and highlights potential digenic inheritance patterns. Integrating genetic burden and single-cell transcriptomic data provides new insights into the complex molecular mechanisms underlying MRKHS.

Classic Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency is typically diagnosed in early life. We report a 46,XX completely virilized 46,XX patient who was diagnosed with classic CAH at the age of 73 years. He was under follow-up for prostate hyperplasia and referred after the finding of giant bilateral adrenal myelolipomas. He presented with hormonal values initially interpreted as suggestive of hypogonadotropic hypogonadism, prompting further biochemical and genetic analysis. Next-generation sequencing identified heterozygous variants in X-linked genes, uncovering a 46,XX difference of sex development (DSD). Then, CYP21A2 molecular analysis revealed compound heterozygosity for two pathogenic variants (p.I173N, p.R357W), confirming simple virilizing CAH. The patient's reticent attitude contributed to the diagnostic delay. However, this unique case reveals the challenges generated by the paraurethral glands hyperplasia - mimicking a prostate due to prolonged untreated hyperandrogenism - as well as the repeated failure to recognize Müllerian remnants on imaging and the critical issues related to diagnostic communication.

<p>Background: 46,XX testicular/ovotesticular disorders of sexual development (T/OT DSD) are infrequent congenital conditions characterized by the presence of functional ovarian and testicular or only testicular parenchyma. The aim of the study was to retrospectively describe clinical, hormonal, and genetic characteristics of 29 patients with 46,XX T/OT DSD (2000-2023), focusing on gonadal function, hormonal production, and long-term follow-up. Most patients (n = 25, 86.2%) presented with atypical genitalia that suggested DSD. Median age at first assessment was 0.38 years. Sex assignment was male in 21 patients without reports of discordant gender identity. Sex assignment was recommended before expert evaluation and without adequate studies in 64% of those patients with atypical genitalia (16/25). The median external masculinization score was 8 (range 4-12). During mini-puberty, LH, testosterone, AMH, and the LH/FSH ratio were above the female reference range and no different from the normal male reference range. Spontaneous puberty was observed in one female and 10 male-assigned subjects. Among the latter, pubertal virilization occurred with signs of hypergonadotropic hypogonadism and gynecomastia. Molecular studies identified the underlying mechanism in 7 patients: SRY gene was identified in two, WT1 gene variations were detected in three others, and 2 syndromic patients harbored complex chromosomal rearrangements. Our findings underscore the clinical and biochemical variability in 46,XX T/OT DSD. Expert evaluation and accurate diagnostic work-up are essential prior to sex assignment and to prevent misdiagnosis and inappropriate treatments. Mini-puberty was characterized by a masculinized pattern of gonadotropin secretion. The potential for functional male pubertal development should be taken into account when making sex assignment decisions. </p>.

46,XX testicular/ovotesticular differences/disorders of sexual development (TDSD/OTDSD) are rare in childhood and exhibit marked distinctions compared to those in adulthood. This study aimed to summarize the clinical characteristics and outcomes of 46,XX TDSD/OTDSD in childhood. The sexual development characteristics, hormone profiles, chromosomal analysis, fluorescence in situ hybridization analysis (FISH) sex-determining region Y (SRY) analysis (peripheral blood and tissues), molecular genetic etiology, gonadal pathology, risk of gonadal tumors, and assigned gender of 52 patients were collected and analyzed. The median age at initial presentation was 18 months, and external masculinization score(EMS) within the range of 3 < EMS ≤ 6 was more prevalent. There were no statistical differences in hormone levels [luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T)] between the different age groups. Among the 52 children, 4 showed positive SRY in peripheral blood, whereas none of the 8 children exhibited positive SRY in tissue samples. A total of 29 children underwent whole exome sequencing (WES) and copy number variant (CNV) analysis, but no genetic variants were identified. A total of 47 children underwent gonadal biopsy and showed no evidence of tumors. However, immunohistochemical analysis revealed that 2 of 16 children were OCT3/4 positive. The most frequent type of gonadal pathology (17/47) was bilateral seminiferous tubules. After the assessment, gender assignment was revised in six cases: five individuals originally assigned as female at birth were reassigned as male, while one individual assigned as male was changed to female. In seven cases, the gender of rearing remained undetermined pending further longitudinal psychosocial assessment. Among the female-reared cohort, three children were more than 11 years old. As a result of undergoing bilateral gonadectomy at an early age, the patients were unable to spontaneously enter puberty. However, given their short stature, they are receiving growth hormone (GH) treatment and have not yet received sufficient sex hormone replacement therapy (HRT). Among the male-reared cohort, seven children had entered puberty. The average age at puberty onset was 12 ± 0.87 years, the average testicular volume was 5.14 ± 1.57 mL, the mean basal LH level was 6.44 ± 4.19 IU/L, the mean basal FSH level was 13.18 ± 10.22 IU/L, and the mean basal T was 3.40 ± 1.63 nmol/L. Compared to adults, children with 46,XX testicular/ovotesticular DSD were very different. SRY-negative children were predominant and tended to have more severe external genital abnormalities during childhood. Peripheral blood or tissue SRY mosaicism was not a prevalent cause and the intricate genetic pathways behind these cases were unknown. There were no statistical differences in hormone levels (LH, FSH, and T) between the different age groups. The assigned gender is mainly male, and the incidence of gonadal tumor risk markers was modest. During adolescence, their testosterone levels could normalize despite elevated FSH and LH levels.

Perrault syndrome is a heterogeneous phenotype that generally encompasses the findings of sensorineural hearing loss in both 46,XX and 46,XY individuals and varying degrees of abnormal ovarian function in 46,XX individuals. In this case report, we present two brothers with LARS2-related Perrault syndrome who have undervirilization. In addition to bilateral profound sensorineural hearing loss, both brothers had bilateral undescended testes that required surgical intervention. In addition, the younger affected brother had hypospadias with chordee. Quad exome sequencing on both brothers was consistent with 46,XY and revealed the same biallelic pathogenic variants in LARS2, a gene known to be associated with Perrault syndrome. No other variants were reported on exome analysis. To date, undervirilization in 46,XY individuals who have Perrault syndrome has only rarely been reported, although the number of reported males with Perrault syndrome continues to be small. Pathogenic variants in LARS2 have been found to lead to ovarian dysgenesis in 46,XX individuals and complete infertility due to failure to produce germ cells in Caenorhabditis elegans ( C . elegans ), indicating that LARS2 is expressed in gonadal tissue and can impact gonadal development. Undervirilization in affected males is likely an underrecognized component of the LARS2-related Perrault syndrome. In this article, we suggest that LARS2 be included in the differential diagnosis of both 46,XX and 46,XY individuals with DSD conditions and should be considered in 46,XY individuals with hearing loss and evidence of undervirilization.