Meiotic maturation failure in primary ovarian insufficiency: insights from a bovine model.

PURPOSE: Oocytes from women presenting primary ovarian insufficiency (POI) generate viable embryos at a lower rate than non-POI women, but the mechanisms responsible for the lower oocyte quality remain elusive. Due to the scarcity of human oocytes for research, animal models provide a promising way forward. We aimed at investigating the molecular events characterizing final maturation in POI oocytes in a well-defined POI-like bovine model. METHODS: Single-cell RNA-sequencing of bovine control and POI-like, GV, and MII oocytes (n = 5 per group) was performed. DEseq2 was used to identify differentially expressed genes. Further, a Gene set enrichment analysis and a transcriptomic meta-analysis between bovine and human oocytes were performed. RESULTS: In control cows, we found 2223 differentially expressed genes between the GV and MII stages. Specifically, the affected genes were related to RNA processing and transport, protein synthesis, organelle remodeling and reorganization, and metabolism. The meta-analysis with a set of young human oocytes at different maturation stages revealed 315 conserved genes through the GV-MII transition in cows and humans, mostly related to meiotic progression and cell cycle. Gene expression analysis between GV and MII of POI-like oocytes showed no differences in terms of differentially expressed genes, pointing towards a substantial failure to properly remodel the transcriptome in the POI model, and with the clustering analysis indicating that the cow's genetic background had a higher impact than the oocyte's maturation stage. CONCLUSION: Overall, we have identified and characterized a valuable animal model of POI, paving the way to identifying new molecular mechanisms involved in POI.

Age-associated increased stiffness of the ovarian microenvironment impairs follicle development and oocyte quality and rapidly alters follicle gene expression.

In humans, aging triggers cellular and tissue deterioration, and the female reproductive system is the first to show signs of decline. Reproductive aging is associated with decreased ovarian reserve, decreased quality of the remaining oocytes, and decreased production of the ovarian hormones estrogen and progesterone. With aging, both mouse and human ovaries become pro-fibrotic and stiff. However, whether stiffness directly impairs ovarian function, folliculogenesis, and oocyte quality is unknown. To answer this question, we cultured mouse follicles in alginate gels that mimicked the stiffness of reproductively young and old ovaries. Follicles cultured in stiff hydrogels exhibited decreased survival and growth, decreased granulosa cell viability and estradiol synthesis, and decreased oocyte quality. We also observed a reduction in the number of granulosa cell-oocyte transzonal projections. RNA sequencing revealed early changes in the follicle transcriptome in response to stiffness. Follicles cultured in a stiff environment had lower expression of genes related to follicle development and greater expression of genes related to inflammation and extracellular matrix remodeling than follicles cultured in a soft environment. Altogether, our findings suggest that ovarian stiffness directly modulates folliculogenesis and contributes to the progressive decline in oocyte quantity and quality observed in women of advanced maternal age.

Specific processing of meiosis-related transcript is linked to final maturation in human oocytes.

Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.

Genetic dissection of spermatogenic arrest through exome analysis: clinical implications for the management of azoospermic men.

PURPOSE: Azoospermia affects 1% of men and it can be the consequence of spermatogenic maturation arrest (MA). Although the etiology of MA is likely to be of genetic origin, only 13 genes have been reported as recurrent potential causes of MA. METHODS: Exome sequencing in 147 selected MA patients (discovery cohort and two validation cohorts). RESULTS: We found strong evidence for five novel genes likely responsible for MA (ADAD2, TERB1, SHOC1, MSH4, and RAD21L1), for which mouse knockout (KO) models are concordant with the human phenotype. Four of them were validated in the two independent MA cohorts. In addition, nine patients carried pathogenic variants in seven previously reported genes-TEX14, DMRT1, TEX11, SYCE1, MEIOB, MEI1, and STAG3-allowing to upgrade the clinical significance of these genes for diagnostic purposes. Our meiotic studies provide novel insight into the functional consequences of the variants, supporting their pathogenic role. CONCLUSION: Our findings contribute substantially to the development of a pre-testicular sperm extraction (TESE) prognostic gene panel. If properly validated, the genetic diagnosis of complete MA prior to surgical interventions is clinically relevant. Wider implications include the understanding of potential genetic links between nonobstructive azoospermia (NOA) and cancer predisposition, and between NOA and premature ovarian failure.

Short anogenital distance is associated with testicular germ cell tumour development.

BACKGROUND: Testicular germ cell tumour is a multifactorial disease in which various genetic and environmental factors play a role. Testicular germ cell tumour is part of the testicular dysgenesis syndrome which includes also cryptorchidism, hypospadias, oligo/azoospermia and short anogenital distance. OBJECTIVES: The primary objective was to examine anogenital distance in testicular germ cell tumour cases and healthy fertile controls. The secondary objective was to assess the (CAG)n polymorphism of the Androgen Receptor gene in relationship with anogenital distances and testicular germ cell tumour development. MATERIAL AND METHODS: 156 testicular germ cell tumour patients and 110 tumour-free normozoospermic controls of Spanish origin. All subjects underwent full andrological workup (including semen and hormone analysis) and genetic analysis (Androgen Receptor (CAG)n). The main outcome measures were the anopenile distance (AGDap), the anoscrotal distance (AGDas) and AR(CAG)n. RESULT: We observed significantly shorter anogenital distances in the group of testicular germ cell tumour patients in respect to controls (P < .001) independently from sperm count and testis histology. Threshold values, applicable only to our cohort, were calculated for anogenital distances with the best sensitivity and specificity. Subjects with AGDap and AGDas below threshold showed a significantly increased risk for testicular germ cell tumour (OR = 4.97, 95% CI = 2.01-12.33, P = .001 and OR = 4.11, 95% CI = 1.89-8.92, P ≤ .001, respectively). No significant correlation was observed between AR(CAG)n polymorphism and anogenital distances. The median values of the AR(CAG)n were similar between cases and controls, excluding a major role for this polymorphism in the etiopathogenesis of these testicular dysgenesis syndrome components. CONCLUSIONS: Ours is the first study focusing on anogenital distances in testicular germ cell tumour patients. We identified short anogenital distances (which is a surrogate biomarker of androgen action during foetal life) as a significant risk factor for this disease. After further validation of our preliminary data, anogenital distance measurement could become part of testicular germ cell tumour screening in order to better define those individuals who would benefit from long-term active follow-up.