TEX13B is essential for metabolic reprogramming during germ cell differentiation.

STUDY QUESTION: What is the functional significance of Tex13b in male germ cell development and differentiation? SUMMARY ANSWER: Tex13b regulates male germ cell differentiation by metabolic reprogramming during spermatogenesis. WHAT IS KNOWN ALREADY: Studies in mice and humans suggest that TEX13B is a transcription factor and is exclusively expressed in germ cells. STUDY DESIGN, SIZE, DURATION: We sequenced the coding regions of TEX13B in 628 infertile men and 427 ethnically matched fertile control men. Further, to identify the molecular function of Tex13b, we created a Tex13b knockout and conditional overexpression system in GC-1spg (hereafter, GC-1) cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Our recent exome sequencing study identified novel candidate genes for male infertility. TEX13B was found to be one of the potential candidates, hence we explored the role of TEX13B in male infertility within a large infertile case-control cohort. We performed functional analyses of Tex13b in a GC-1 cell line using CRISPR-Cas9. We differentially labelled the cell proteins by stable isotope labelling of amino acids in cell culture (SILAC) and performed mass spectrometry-based whole-cell proteomics to identify the differential protein regulation in knockout cells compared to wild-type cells. We found that Tex13b knockout leads to downregulation of the OXPHOS complexes and upregulation of glycolysis genes, which was further validated by western blotting. These results were further confirmed by respirometry analysis in Tex13b knockout cells. Further, we also performed a conditional overexpression of TEX13B in GC-1 cells and studied the expression of OXPHOS complex proteins by western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a rare variant, rs775429506 (p.Gly237Glu), exclusively in two non-obstructive-azoospermia (NOA) men, that may genetically predispose these men for infertility. Further, we demonstrated that Tex13b functions in the transcription regulation of OXPHOS complexes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We examined the function of Tex13b in GC-1 in vitro by knocking out and conditional overexpression, for understanding the function of Tex13b in germ cells. Unfortunately, this could not be replicated in either an animal model or in patient-derived tissue due to the non-availability of an animal model or patient's testis biopsies. WIDER IMPLICATIONS OF THE FINDINGS: This study identified that Tex13b plays an important role in male germ cell development and differentiation. The findings of this study would be useful for screening infertile males with spermatogenic failure and counselling them before the implementation of assisted reproduction technique(s). STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the Council of Scientific and Industrial Research (CSIR) under the network project (BSC0101 and MLP0113) and SERB, the Department of Science and Technology, Government of India (J C Bose Fellowship: JCB/2019/000027). The authors do not have any competing interest.

Exome sequencing and functional analyses revealed CETN1 variants leads to impaired cell division and male fertility.

Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1-5' UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.

Does Choosing Microfluidics for Sperm Sorting Offer an Advantage to Improve Clinical Pregnancies in Donor Egg Recipients?

Background: Microfluidics (MF), an advanced sperm sorting technology results in the extraction of spermatozoa with higher DNA integrity and lower DNA damage compared to existing conventional sperm sorting methods. Aims: The aim of the present study is to assess the efficiency of MF and to isolate the best spermatozoa for intracytoplasmic sperm injection (ICSI) over the density gradient (DG) technique. Study Setting and Design: We recruited couples who choose the oocyte donation programme for this study to eliminate confounding factors associated with oocyte quality. Materials and Methods: Sperm was processed by MF (n = 180) and DG (n = 151). ICSI was performed and positive pregnancy, miscarriage and clinical pregnancy rates were compared. Statistical Analysis: All variables were analysed using Graph Pad Prism 5. The unpaired two-tailed t-test was used to assess the significance. A value of P < 0.05 was considered statistically significant. Results: There was no significant difference in pregnancy rates between the groups. However, a clear demarcation is seen in terms of clinical pregnancy rates, where the DG group achieved higher clinical pregnancies (91.7%) compared to the MF group (80.7%). Further, we compared miscarriage rates and biochemical pregnancies, and found a significantly higher miscarriage and biochemical pregnancy rate in the MF group (14.5% and 4%, respectively) compared to the DG group (6% and 1%, respectively). Conclusions: Based on the available literature, we anticipated a higher clinical pregnancy rate with MF compared with conventional processing. Our results show MF does not have any add-on positive effect on clinical pregnancy rate.

The interchangeability of two assays for the measurement of anti-Müllerian hormone when personalizing the dose of FSH in in-vitro fertilization cycles.

OBJECTIVE: Study the interchangeability of Roche Elecsys and Beckman Coulter Access anti-Müllerian Hormone (AMH) assays to select the gonadotropin starting dose in IVF cycles. METHODS: Patients' AMH was measured using both Elecsys and Access automated assays. AMH values were then used to calculate the FSH starting dose. The main outcome is the percentage of women that would have been stratified to a different dose of gonadotropin due to differences in AMH values from the two tests. RESULTS: The Access assay systematically gives higher values compared with the Elecsys assay (slope = 0.88). For Follitropin Alfa, the difference in starting dose was > 15% in 2/113 patients, when Access AMH was used instead of Elecsys. For Follitropin Delta the difference in the starting dose was >15% in 21/113 patients when using Access AMH. When considering women with high ovarian reserve, only 4/51 would have received a Follitropin Delta dose that exceeded a 15% difference using Access AMH as a substitute for the Elecsys value. CONCLUSIONS: The use of the Roche Elecsys or Beckman Coulter Access leads to modest differences in AMH values, which seem to little affect the calibration of FSH dose used for ovarian stimulation.

Timely Identification of Pregnancy in Noncommunicating Horn of Unicornuate Uterus by Three-Dimensional Transvaginal Ultrasonography.

Pregnancy in the rudimentary horn of a unicornuate uterus is uncommon and needs to be diagnosed at early stages to avoid uterine rupture to avert the high morbidity and mortality. In this case report, we discussed the advantage of three-dimensional transvaginal ultrasonography (3D TV-USG) in assessing the early pregnancy in the noncommunicating rudimentary horn of uterus. A 23-year-old woman approached us for routine pregnancy scan. The location of 5-week pregnancy was confirmed in the right noncommunicating horn of a unicornuate uterus by 3D TV-USG. She has undergone laparohysteroscopy, and excision of a gravid rudimentary horn was done. After an interval of 6 months, the patient received fertility treatment and conceived consequently. Although magnetic resonance imaging (MRI) is an excellent way of diagnosing uterine anomalies, the procedure is expensive, time-consuming, and not widely available. 3D USG is less expensive and more readily accessible for early diagnosis of uterine anomalies, particularly in health-care centers where MRI is not readily available or affordable.