Deleterious variant in FAM71D cause male infertility with asthenoteratospermia.

Asthenoteratospermia is a significant cause of male infertility. FAM71D (Family with sequence similarity 71, member D), as a novel protein exclusively expressed in the testis, has been found to be associated with sperm motility. However, the association of FAM71D mutation with male infertility has yet to be examined. Here, we conducted whole-exome sequencing and identified a homozygous missense mutation c.440G > A (p. Arg147Gln) of FAM71D in an asthenoteratospermia-affected man from a consanguineous family. The FAM71D variant is extremely rare in human population genome databases and predicted to be deleterious by multiple bioinformatics tools. Semen analysis indicated decreased sperm motility and obvious morphological abnormalities in sperm cells from the FAM71D-deficient man. Immunofluorescence assays revealed that the identified FAM71D mutation had an important influence on the assembly of sperm structure-related proteins. Furthermore, intra-cytoplasmic sperm injection (ICSI) treatment performed on the infertile man with FAM71D variant achieved a satisfactory outcome. Overall, our study identified FAM71D as a novel causative gene for male infertility with asthenoteratospermia, for which ICSI treatment may be suggested to acquire good prognosis. All these findings will provide effective guidance for genetic counselling and assisted reproduction treatments of asthenoteratospermia-affected subjects.

A bi-allelic REC114 loss-of-function variant causes meiotic arrest and nonobstructive azoospermia.

Nonobstructive azoospermia (NOA), the most severe manifestation of male infertility, lacks a comprehensive understanding of its genetic etiology. Here, a bi-allelic loss-of-function variant in REC114 (c.568C > T: p.Gln190*) were identified through whole exome sequencing (WES) in a Chinese NOA patient. Testicular histopathological analysis and meiotic chromosomal spread analysis were conducted to assess the stage of spermatogenesis arrested. Co-immunoprecipitation (Co-IP) and Western blot (WB) were used to investigate the influence of variant in vitro. In addition, our results revealed that the variant resulted in truncated REC114 protein and impaired interaction with MEI4, which was essential for meiotic DNA double-strand break (DSB) formation. As far as we know, this study presents the first report that identifies REC114 as the causative gene for male infertility. Furthermore, our study demonstrated indispensability of the REC114-MEI4 complex in maintaining DSB homoeostasis, and highlighted that the disruption of the complex due to the REC114 variant may underline the mechanism of NOA.

Stepwise mini-incision microdissection testicular sperm extraction in NOA patients with a history of cryptorchidism: a case-control study.

BACKGROUND: Although the orchiopexy is recommended for cryptorchidism to preserve male fertility, non-obstructive azoospermia (NOA) may occur in adulthood. Fortunately, a great many of azoospermic men may obtain sperm by microdissection testicular sperm extraction (mTESE). Due to the potential injuries caused by testicular diagnostic biopsy and vascular damage at the time of orchidopexy, minimal invasiveness is particularly important during mTESE, aims to reduce the surgical damage and avoids secondary testicular failure. This comparative study aims to investigate the efficacy of stepwise mini-incision mTESE technique by comparison with standard mTESE in the treatment of NOA patients with a history of cryptorchidism. RESULTS: A total of 73 mTESE procedures were divided into two groups: Group 1 included 37 cases performed by stepwise mini-incision mTESE, while Group 2 included 36 cases with standard mTESE. The overall sperm retrieval rate (SRR) in the two groups was 68.5% (50/73), with no significant difference in SRR between Group 1 (78.4%, 29/37) and Group 2 (58.3%, 21/36) (P = 0.1). In addition, 46.0% of the patients (17/37) obtained sperm in the first mini-incision step in Group 1, which was also equal to an overall SRR in Group 2 (58.3%, 21/36) (P = 0.3). The operation time in Group 1 (72.6 ± 33.9 min) was significantly shorter than that in Group 2 (90.4 ± 36.4 min) (P = 0.04). Patients with an orchidopexy age no more than 10 years old had a higher SRR (79.5%, 31/39) than others (55.9%, 19/34) (P = 0.03). There were no postoperative complications including wound infection, scrotal hematoma, persistent pain, and testicular atrophy during a follow-up period of at least 6 months. CONCLUSIONS: In conclusion, our study suggests that the stepwise mini-incision mTESE could be a promising approach for sperm retrieval in NOA men with a history of cryptorchidism. While the technique may potentially reduce operation time and surgical invasiveness, further research is needed to validate these findings on a larger scale. The results also suggest that age at orchidopexy may affect SRR and have important implications for the management of cryptorchidism.

RéSUMé: CONTEXTE: Bien que l'orchidopexie soit recommandée en cas de cryptorchidie afin de préserver la fertilité masculine, une azoospermie non obstructive (NOA) peut survenir à l'âge adulte. Heureusement, un grand nombre d'hommes azoospermiques peuvent obtenir des spermatozoïdes lors d'une extraction de spermatozoïdes testiculaire par microdissection (mTESE). En raison des potentielles lésions causées par la biopsie diagnostique testiculaire et des lésions vasculaires survenant au moment de l'orchidopexie, une approche minimalement  invasive est particulièrement importante pendant la mTESE;  elle vise à réduire les dommages chirurgicaux et à éviter une insuffisance testiculaire secondaire. La présente étude comparative a pour but d' étudier l'efficacité de la mTESE par mini-incision par étapes en comparaison avec la mTESE standard dans le traitement des patients NOA qui ont des antécédents de cryptorchidie. RéSULTATS: Au total, 73 procédures de mTESE ont été divisées en deux groupes: le Groupe 1 comprenait 37 cas effectués avec la mTESE par mini-incision par étapes, tandis que le Groupe 2 comprenait 36 cas réalisés par la mTESE standard. Le taux global de récupération de spermatozoïdes (SRR) dans les deux groupes était de 68, 5% (50/73), sans différence significative de SRR entre le Groupe 1 (78, 4%, 29/37) et le Groupe 2 (58, 3%, 21/36) (P = 0,1). De plus, 46% des patients (17/37) ont obtenu des spermatozoïdes lors de la première étape de mini-incision dans le Groupe 1, ce qui était identique au SRR global dans le Groupe 2 (58%, 21/36) (P = 0,3). Le temps opératoire du Groupe 1 (72, 6 ± 34 min) était significativement plus court que celui du Groupe 2 (90, 4 ± 36 min) (P = 0,04). Les patients dont l'orchidopexie avait été réalisée au plus tard à l'âge de 10 ans avaient un SRR plus élevé (79, 5%, 31/39) que les autres (55, 9%, 19/34) (P = 0,03). Il n'y a pas eu de complications postopératoires, que ce soit infection de la plaie, hématome scrotal, douleur persistante, ou atrophie testiculaire pendant une période de suivi d'au moins 6 mois. CONCLUSIONS: Notre étude suggère que la mTESE par mini-incision par étapes pourrait être une approche prometteuse pour la récupération de spermatozoïdes chez les hommes NOA ayant des antécédents de cryptorchidie. Bien que la technique puisse potentiellement réduire le temps d'opération et le caractère invasif du geste chirurgical, des recherches supplémentaires sont nécessaires pour valider ces résultats à plus grande échelle. Les résultats suggèrent également que l'âge à l'orchidopexie peut affecter le SRR et avoir des implications importantes pour la prise en charge de la cryptorchidie.

A homozygous frameshift variant in SYCP2 caused meiotic arrest and non-obstructive azoospermia.

Genetic causation for the majority of non-obstructive azoospermia (NOA) remains unclear. Mutations in synaptonemal complex (SC)-associated genes could cause meiotic arrest and NOA. Previous studies showed that heterozygous truncating variants in SYCP2 encoding a protein essential for SC formation, are associated with non-obstructive azoospermia and severe oligozoospermia. Herein, we showed a homozygous loss-of-function variant in SYCP2 (c.2689_2690insT) in an NOA-affected patient. And this variant was inherited from heterozygous parental carriers by natural reproduction. HE, IF, and meiotic chromosomal spread analyses demonstrated that spermatogenesis was arrested at the zygotene stage in the proband with NOA. Thus, this study revealed that SYCP2 associated with NOA segregates in an autosomal recessive inheritance pattern, rather than an autosomal dominant pattern. Furthermore, our study expanded the knowledge of variants in SYCP2 and provided new insight into understanding the genetic etiology of NOA.

Identification of a missense variant of MND1 in meiotic arrest and non-obstructive azoospermia.

Meiotic arrest is a common pathologic phenotype of non-obstructive azoospermia (NOA), yet its genetic causes require further investigation. Meiotic nuclear divisions 1 (MND1) has been proved to be indispensable for meiotic recombination in many species. To date, only one variant of MND1 has been reported associated with primary ovarian insufficiency (POI), yet there has been no report of variants in MND1 associated with NOA. Herein, we identified a rare homozygous missense variant (NM_032117:c.G507C:p.W169C) of MND1 in two NOA-affected patients from one Chinese family. Histological analysis and immunohistochemistry demonstrated meiotic arrest at zygotene-like stage in prophase I and lack of spermatozoa in the proband's seminiferous tubules. In silico modeling demonstrated that this variant might cause possible conformational change in the leucine zippers 3 with capping helices (LZ3wCH) domain of MND1-HOP2 complex. Altogether, our study demonstrated that the MND1 variant (c.G507C) is likely responsible for human meiotic arrest and NOA. And our study provides new insights into the genetic etiology of NOA and mechanisms of homologous recombination repair in male meiosis.

Efficacy of stepwise mini-incision microdissection testicular sperm extraction for nonobstructive azoospermia with varied etiologies.

Stepwise mini-incision microdissection testicular sperm extraction (mTESE) is a procedure that attempts to minimize testicular damage. However, the mini-incision approach may vary in patients with different etiologies. Here, we performed a retrospective analysis of 665 men with nonobstructive azoospermia (NOA) who underwent stepwise mini-incision mTESE (Group 1) and 365 men who underwent standard mTESE (Group 2). The results showed that the operation time (mean ± standard deviation) for patients with successful sperm retrieval in Group 1 (64.0 ± 26.6 min) was significantly shorter than that in Group 2 (80.2 ± 31.3 min), with P <0.001. The total sperm retrieval rate (SRR) was 23.1% in our study, and there was no significant difference between Group 1 and Group 2 ( P >0.05), even when the etiologies of NOA were taken into consideration. The results of consecutive multivariate logistic regression analysis (odds ratio [OR]: 0.57; 95% confidence interval [CI]: 0.38-0.87; P =0.009) and receiver operating characteristic (ROC) analysis (area under the ROC curve [AUC]=0.628) showed that preoperative anti-Müllerian hormone (AMH) level in idiopathic NOA patients was a potential predictor for surgical outcomes after initial three small incisions made in the equatorial region without sperm examined under an operating microscope (Steps 2-4). In conclusion, stepwise mini-incision mTESE is a useful technique for NOA patients, with comparable SRR, less surgical invasiveness, and shorter operation time compared with the standard approach. Low AMH levels may predict successful sperm retrieval in idiopathic patients even after a failed initial mini-incision procedure.

Bi-allelic MEI1 variants cause meiosis arrest and non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is characterized by the failure of sperm production due to testicular disorders and represents the most severe form of male infertility. Growing evidences have indicated that gene defects could be the potential cause of NOA via genome-wide sequencing approaches. Here, bi-allelic deleterious variants in meiosis inhibitor protein 1 (MEI1) were identified by whole-exome sequencing in four Chinese patients with NOA. Testicular pathologic analysis and immunohistochemical staining revealed that spermatogenesis is arrested at spermatocyte stage, with defective programmed DNA double-strand breaks (DSBs) homoeostasis and meiotic chromosome synapsis in patients carrying the variants. In addition, our results showed that one missense variant (c.G186C) reduced the expression of MEI1 and one frameshift variant (c.251delT) led to truncated proteins of MEI1 in in vitro. Furthermore, the missense variant (c.T1585A) was assumed to affect the interaction between MEI1 and its partners via bioinformatic analysis. Collectively, our findings provide direct genetic and functional evidences that bi-allelic variants in MEI1 could cause defective DSBs homoeostasis and meiotic chromosome synapsis, which subsequently lead to meiosis arrest and male infertility. Thus, our study deepens our knowledge of the role of MEI1 in male fertility and provides a novel insight to understand the genetic aetiology of NOA.

Vasectomy reversal in China during the recent decade: insights from a multicenter retrospective investigation.

To date, there is little information about the demography of vasectomy reversal (VR) patients or the factors currently influencing VR effectiveness in China, especially after the universal two-child policy was released in 2015. In this research, demographic data and perioperative medical records of VR patients were extracted from seven major hospitals in different provinces or municipalities of China. Meanwhile, a telephone survey of the patients was conducted to collect follow-up information. Eventually, 448 VR cases from the past 13 years were included. The results were analyzed by stratified comparison to investigate factors that can influence postoperative vas deferens patency and pregnancy rate. Appropriately statistical methods were used, and all of the protocols were approved by the Ethics Committees of the institutes in this research. The results showed that the annual operation volume of VR quadrupled after the two-child policy was implemented. Nonmicrosurgery and a long duration of vasectomy were significantly associated with a lower patency rate. A follow-up survey showed that the general postoperative pregnancy rate was 27.2%. For female partners over the age of 35 years, the postoperative pregnancy rate showed a more severe decline, but only 35.5% of them had been given a fertility examination before their husbands' VR surgery. Our work revealed that more patients in China have been demanding VR in recent years. High-quality microsurgery and a short duration of vasectomy are crucial for restoring patency by VR. Clinical andrologists should perform a preoperative fertility evaluation of the patients' female partners.

Clinical features and microsurgical reconstruction of congenital unilateral absence of the vas deferens with obstructive azoospermia: a tertiary care center experience.

Patients with congenital unilateral absence of the vas deferens (CUAVD) manifest diverse symptoms from normospermia to azoospermia. Treatment for CUAVD patients with obstructive azoospermia (OA) is complicated, and there is a lack of relevant reports. In this study, we describe the clinical features and evaluate the treatments and outcomes of CUAVD patients with OA. From December 2015 to December 2020, 33 patients were diagnosed as CUAVD with OA in Shanghai General Hospital (Shanghai, China). Patient information, ultrasound findings, semen analysis, hormone profiles, and treatment information were collected, and the clinical outcomes were evaluated. Of 33 patients, 29 patients were retrospectively analyzed. Vasoepididymostomy (VE) or cross VE was performed in 12 patients, the patency rate was 41.7% (5/12), and natural pregnancy was achieved in one of the patients. The other 17 patients underwent testicular sperm extraction as the distal vas deferens (contralateral side) was obstructed. These findings showed that VE or cross VE remains an alternative treatment for CUAVD patients with OA, even with a relatively low rate of patency and natural pregnancy.

The Efficacy and Safety of Thrice vs Twice per Week Low-Intensity Pulsed Ultrasound Therapy for Erectile Dysfunction: A Randomized Clinical Trial.

BACKGROUND: A recent sham-controlled clinical study has shown that low-intensity pulsed ultrasound twice per week can safely and effectively treat patients with mild-to-moderate erectile dysfunction (ED). However, large-scale clinical trials are needed to verify its efficacy and safety and determine a reasonable treatment interval. AIM: To study whether low-intensity pulsed ultrasound therapy thrice per week is non-inferior to twice per week in patients with mild-to-moderate ED. METHODS: A randomized, open-label, parallel-group, non-inferiority clinical trial was conducted in 7 hospitals in China. A total of 323 patients with mild-to-moderate ED were randomized (1:1) into thrice per week (3/W) and twice per week (2/W) groups. Low-intensity pulsed ultrasound was applied on each side of the penis for 16 sessions. OUTCOMES: The primary outcome was response rate using the minimal clinically important difference in the International Index of Erectile Function (IIEF-EF) score at week 12. Secondary outcomes included Erection Hardness Score (EHS), Sexual Encounter Profile, Global Assessment Question, and Self Esteem and Relationship Questionnaire. RESULTS: Response rates in 3/W and 2/W groups were 62.0% and 62.5%, respectively. Treatment effect in the 3/W group was noninferior to that of the 2/W group, with rate difference lower bound of -0.01% [95% confidence interval -0.11 to 0.10%] within the acceptable margin (-14.0%). No significant difference was found among secondary outcomes. IIEF-EF score showed a significant increase from baseline in the 3/W group (16.8 to 20.7) and 2/W group (17.8 to 21.7), and the percentage of patients with EHS ≥3 increased in the 3/W (54.9% to 84.0%) and 2/W (59.5% to 83.5%) groups. There was no significant difference in response rate between the 2 groups after controlling for strata factors and homogeneous tests. No treatment-related adverse events were reported. CLINICAL IMPLICATIONS: Low-intensity pulsed ultrasound therapy displays similar efficacy and safety for mild-to-moderate ED when administered thrice or twice per week for 16 sessions. This study provides two options to suit patients' needs. STRENGTHS & LIMITATIONS: This is a large-sample, randomized, controlled, noninferiority trial study. Short-term follow-up and mostly younger patients are the main limitations. CONCLUSION: Low-intensity pulsed ultrasound therapy thrice and twice per week showed equivalent therapeutic effects and safety for mild-to-moderate ED in a young and generally healthy population. This therapy warrants further investigation of its potential value in rehabilitation of ED. Chen, H., Li Z., Li X., et al. The Efficacy and Safety of Thrice vs Twice per Week Low-Intensity Pulsed Ultrasound Therapy for Erectile Dysfunction: A Randomized Clinical Trial. J Sex Med 2022;19:1536-1545.

Single-cell transcriptome atlas of the human corpus cavernosum.

The corpus cavernosum is the most important structure for penile erection, and its dysfunction causes many physiological and psychological problems. However, its cellular heterogeneity and signalling networks at the molecular level are poorly understood because of limited access to samples. Here, we profile 64,993 human cavernosal single-cell transcriptomes from three males with normal erection and five organic erectile dysfunction patients. Cell communication analysis reveals that cavernosal fibroblasts are central to the paracrine signalling network and regulate microenvironmental homeostasis. Combining with immunohistochemical staining, we reveal the cellular heterogeneity and describe a detailed spatial distribution map for each fibroblast, smooth muscle and endothelial subcluster in the corpus cavernosum. Furthermore, comparative analysis and related functional experiments identify candidate regulatory signalling pathways in the pathological process. Our study provides an insight into the human corpus cavernosum microenvironment and a reference for potential erectile dysfunction therapies.

Novel copy number variations within SYCE1 caused meiotic arrest and non-obstructive azoospermia.

BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe disease in male infertility, but the genetic causes for majority of NOA remain unknown. METHODS: Two Chinese NOA-affected patients were recruited to identify the genetic causal factor of infertility. Whole-exome sequencing (WES) was conducted in the two patients with NOA. Sanger sequencing and CNV array were used to ascertain the WES results. Hematoxylin and eosin (H&E) staining and immunofluorescence (IF) were carried out to evaluate the stage of spermatogenesis arrested in the affected cases. RESULTS: Novel heterozygous deletion (LOH) within SYCE1 (seq[GRCh37] del(10)(10q26.3)chr10:g.135111754_135427143del) and heterozygous loss of function (LoF) variant in SYCE1 (NM_001143763: c.689_690 del:p.F230fs) were identified in one NOA-affected patient. While homozygous deletion within SYCE1 (seq[GRCh37] del(10)(10q26.3)chr10:g.135340247_135379115del) was detected in the other patient with meiotic arrest. H&E and IF staining demonstrated that the spermatogenesis was arrested at pachytene stage in the two patients with NOA, suggesting these two novel CNVs within SYCE1 could lead to meiotic defect and NOA. CONCLUSIONS: We identified that two novel CNVs within SYCE1 are associated with meiotic arrest and male infertility. Thus, our study expands the knowledge of variants in SYCE1 and provides a new insight to understand the genetic etiologies of NOA.

Novel bi-allelic variants in KASH5 are associated with meiotic arrest and non-obstructive azoospermia.

KASH5 is an essential component of the LINC (linker of the nucleoskeleton and cytoskeleton) complex that regulates chromosome movements and nuclear envelope (NE) remodeling in mouse spermatocytes during meiosis prophase I, but its expression and function in human cells, as well as its association with male infertility are largely unknown. In this study, a novel heterozygous copy number variation (CNV) (seq [GRCh37] del(19) (19q13.33) chr19: g.49894043-49903011del) and a heterozygous loss of function variant (NM_144688: c.979_980del: p.R327Sfs*21) in human KASH5 were identified in a non-obstructive azoospermia (NOA)-affected patient and in his infertile sister by whole-exome sequencing and CNV array. Spermatogenesis in the proband was arrested at zygotene-like stage with a deficiency in homolog pairing and synapsis. KASH5 protein expression in human spermatocytes was evaluated and reported first in this study. Single-cell RNA sequencing demonstrated that the LINC complex and associated genes in human and mouse shared a similar expression pattern, indicating a conserved mechanism in the regulation of chromosome movements and NE remodeling. Kash5 knockout mouse displayed similar phenotypes, including a meiotic arrest at a zygotene-like stage and impaired pairing and synapsis. Collectively, we have identified novel rare variants within human KASH5 in patients with NOA and meiosis arrest. Our study expands the knowledge of KASH5 and associated proteins in regulating human meiosis prophase I progress and provides new insight into the genetic etiology of NOA.

Preliminary experience with 3D digital image microscope system on the treatment of varicocele.

The three-dimension digital image microscope system (3D-DIM) with a better ergonomic design and equipment characteristics can contribute to the achievement of good results during microsurgery. In this study, the safety and efficiency of 3D-DIM assisted varicocelectomy was evaluated. From July 2019 to November 2019, fifteen cases with varicocele (20 sides of varicocele in total) were included, seven cases underwent 3D-DIM-assisted modified microsurgical subinguinal varicocelectomy, and eight cases underwent modified microsurgical subinguinal varicocelectomy under standard operating microscope (SOM). The mean operative time of 3D-DIM group (67 ± 12.3 min) was a little longer than that of SOM group (55 ± 12.9 min) (p < 0.05). There was no significant difference between the two groups in the number of internal spermatic arteries, internal spermatic vein, lymphatics, gubernacular vein, external spermatic vein and post-operation complications. The 3D-DIM showed a significant difference in image definition for nurse (p < 0.01) and in doctor-nurse cooperation (p < 0.05) over SOM. The 3D-DIM with better ergonomic design and image definition can be applied to perform microsurgical subinguinal varicocelectomy, and could improve the surgeon's fatigue and doctor-nurse cooperation. We believe that the 3D-DIM would be widely used in the field of male infertility microsurgery in the near future.

Bi-allelic SPATA22 variants cause premature ovarian insufficiency and nonobstructive azoospermia due to meiotic arrest.

The genetic causes of idiopathic premature ovarian insufficiency (POI) and nonobstructive azoospermia (NOA) remain unclear. We performed whole-exome sequencing (WES) in members of a consanguineous family with two POI and two NOA patients to screen for potential pathogenic variants for familial POI and NOA. And a homozygous variant in SPATA22 (c.400C>T:p.R134X) was identified. Histological analysis and spermatocyte spreading assay demonstrated that the spermatogenesis was arrested at a zygotene-like stage in the proband with NOA. The candidate gene was further screened in the in-house WES database of idiopathic POI-affected patients. One additional compound heterozygous variant in SPATA22 (c.900+1G>A and c.31C>T:p.R11X) was found in one patient with sporadic POI and validated by minigene assay. Thus, this is the first report identifying SPATA22 as the causative gene for human POI. Combined with the observations in the familial patient with NOA, our findings highlighted the essential role of meiotic HR genes in gametogenesis and gonadal function maintenance.

Novel bi-allelic MSH4 variants causes meiotic arrest and non-obstructive azoospermia.

BACKGROUND: Non-obstructive azoospermia (NOA) is one of the most severe type in male infertility, and the genetic causes of NOA with meiotic arrest remain elusive. METHODS: Four Chinese families with NOA participated in the study. We performed whole-exome sequencing (WES) for the four NOA-affected patients in four pedigrees. The candidate causative gene was further verified by Sanger sequencing. Hematoxylin and eosin staining (H&E) and immunohistochemistry (IHC) were carried out to evaluate the stage of spermatogenesis arrested in the patients with NOA. RESULTS: We identified two novel homozygous frameshift mutations of MSH4 and two novel compound heterozygous variants in MSH4 in four pedigrees with NOA. Homozygous loss of function (LoF) variants in MSH4 was identified in the NOA-affected patient (P9359) in a consanguineous Chinese family (NM_002440.4: c.805_812del: p.V269Qfs*15) and one patient with NOA (P21504) in another Chinese family (NM_002440.4: c.2220_2223del:p.K741Rfs*2). Also, compound heterozygous variants in MSH4 were identified in two NOA-affected siblings (P9517 and P9517B) (NM_002440.4: c.G1950A: p.W650X and c.2179delG: p.D727Mfs*11), and the patient with NOA (P9540) (NM_002440.4: c.G244A: p.G82S and c.670delT: p.L224Cfs*3). Histological analysis demonstrated lack of spermatozoa in seminiferous tubules of all patients and IHC showed the spermatogenesis arrested at the meiotic prophase I stage. Consistent with the autosomal recessive mode of inheritance, all of these mutations were inherited from heterozygous parental carriers. CONCLUSIONS: We identified that six novel mutations in MSH4 responsible for meiotic arrest and NOA. And these results provide researchers with a new insight to understand the genetic etiology of NOA and to identify new loci for genetic counselling of NOA.

Low-Intensity Pulsed Ultrasound Alleviates Human Testicular Leydig Cell Senescence In Vitro.

Aging has a significant negative impact on human testicular function; steroidogenesis is gradually impaired, and testosterone replacement therapy still has many risks. Low-intensity pulsed ultrasound (LIPUS) has been used as a novel non-invasive treatment for male erectile dysfunction and other fields, and has been shown to increase testosterone levels in animal models. Testosterone is synthesized and secreted by Leydig cells (LCs), and the serum testosterone level decreases after aging due to the LCs senescence. However, the effect of LIPUS on human senescent LCs has not been reported. In this study, human senescent LCs were isolated and stimulated with different energy intensities in vitro, and cell morphology, cell apoptosis, cell proliferation, cell senescence levels, lipid droplet number, testosterone and INSL3 secretion levels were tested and analyzed. Quantitative Polymerase Chain Reaction (QPCR) and Western Blot were performed to compare cell senescence characteristics and the expression profile of key pathways of testosterone secretion, and transcriptome analysis was performed to explore the signaling pathways of LCs alteration after LIPUS stimulation. It was safe and effective to stimulate LCs with the 75 mW/cm2 energy of LIPUS in vitro, which not only improved the senescence phenotype, but also effectively enhanced the secretory function of LCs in vitro, and increased the expression of key pathways of the testosterone synthesis pathway. These results suggest that LIPUS could be used as a novel treatment to human senescent LCs with decreased testosterone secretion levels in vitro.

Deleterious variants in TAF7L cause human oligoasthenoteratozoospermia and its impairing histone to protamine exchange inducing reduced in vitro fertilization.

Introduction: Oligoasthenoteratozoospermia (OAT) is a major cause of infertility in males. Only a few pathogenic genes of OAT have been clearly identified till now. A large number of OAT-affected cases remain largely unknown. Methods: Here, Whole-exome sequencing (WES) in 725 idiopathic OAT patients was performed. Ejaculated spermatozoa by OAT patients were microinjected into mouse oocytes to estimate fertilization potential. Diff-quick staining and transmission electron microscopy were performed to evaluate sperm morphology and ultrastructure. The protein expression level and localization In vitro were detected by Western Blotting and Immunocytochemistry. Results: We identified four X-linked hemizygous deleterious variants of TAF7L-namely, c.1301_1302del;(p.V434Afs*5), c.699G>T;(p.R233S), c.508delA; (p. T170fs), c.719dupA;(p.K240fs) -in five probands. Intracytoplasmic sperm injection (ICSI) were carried out in M1, M2-1and M3 patient's wife. However only M1 patient's wife became pregnant after embryo transfer. In vitro study demonstrated significantly reduced fertilization ability in patient with TAF7L mutation. The TAF7L mutation let to abnormal sperm head and impaired histone-to protamine exchange. Variant 719dupA (p. K240fs) resulted in producing a truncated TAF7L protein and localized massively within the nucleus. In addition, TAF7L expression were not able to be detected due to variants c.1301_1302del (p. V434Afs*5) and c.508delA (p. T170fs) In vitro. Conclusion: Our findings support that TAF7L is one of pathogenic genes of OAT and deleterious mutations in TAF7L may cause impaired histone-to-protamine affected the chromatin compaction of sperm head.

Novel Hemizygous Mutations of TEX11 Cause Meiotic Arrest and Non-obstructive Azoospermia in Chinese Han Population.

Testis-expressed gene 11 (TEX11) mutation has been associated with non-obstructive azoospermia (NOA) and meiotic arrest. An analogous mutation of TEX11 in the mouse impairs meiosis and can be rescued by in vitro expansion of SSCs and gene therapy. However, a lack of genetic screening of a large cohort of Asian patients (including pedigree analysis) and proper functional evaluation limit the clinical application of TEX11 mutation screening. Thus, we performed whole-exome sequencing (WES) in 479 patients with NOA and identified three novel mutations (two splicing mutations and one missense mutation) in TEX11 in three pairs of siblings from three families and four novel pathogenic mutations (three frameshift mutations and a non-sense mutation) of TEX11 in four sporadic NOA-affected cases. Novel variants among family members were segregated by disease phenotype, and all the seven mutations were predicted to be pathogenic. Histological analysis showed that three patients with TEX11 mutations underwent meiotic arrest. The four mutations that resulted in protein truncations and defective meiosis-specific sporulation domain SPO22 were validated by Western blot. In total, we find seven of 479 patients of NOA (1.5%) carrying TEX11 mutations. Our study expands the knowledge of mutations of TEX11 gene in Asian patients with NOA. The high prevalence and X-linked inherited mode indicated that TEX11 might be included in genetic screening panels for the clinical evaluation of patients with NOA.