Obstructive Azoospermia Patients Research Articles

Long-read single-cell sequencing reveals the transcriptional landscape of spermatogenesis in obstructive azoospermia and sertoli cell-only patients.

High-throughput single-cell RNA sequencing (scRNA-seq) is widely used in spermatogenesis. However, it only reveals short reads in germ and somatic cells, limiting the discovery of novel transcripts and genes. This study shows the long-read transcriptional landscape of spermatogenesis in obstructive azoospermia (OA) and Sertoli cell-only (SCO) patients. Single cells were isolated from testicular biopsies of OA and NOA patients. Cell culture was identified by comparing Pacbio long-read single-cell sequencing (OA n = 3, NOA n = 3) with short-read scRNA-seq (OA n = 6, NOA n = 6). Ten germ cell types and eight somatic cell types were classified based on known markers. Pacbio long read single-cell sequencing, short-read scRNA-seq, Polymerase Chain Reaction. A total of 130,426 long-read transcripts (100,517 novel transcripts and 29,909 known transcripts) and 49,508 long-read transcripts (26,002 novel transcripts, and 23,506 known transcripts) have been detected in OA and NOA patients, respectively. Moreover, 36,373 and 1642 new genes are identified in OA and NOA patients, respectively. Importantly, specific expressions of long-read transcripts were detected in germ and stomatic cells during normal spermatogenesis. We have identified total full-length transcripts in OA and NOA, and new genes were found. Furthermore, specific expressed full-length transcripts were detected, and the genomic structure of transcripts was mapped in different cell types. These findings may provide valuable information on human spermatogenesis and the treatment of male infertility.

An overview of CFTR mutation profiles and assisted reproductive technology outcomes in Chinesepatients with congenital obstructive azoospermia.

The cystic fibrosis transmembrane conductance regulator (CFTR) is the most common causative gene attributed to congenital obstructive azoospermia (OA). The aim of this study was to conduct an epidemiological survey of congenital OA patients, to screen for CFTR mutations, and to follow their pregnancy outcomes in assisted reproductive technology (ART). This cohort study enrolled congenital OA patients undergoing ART and whole-exome sequencing from January 2018 to September 2023. Semen parameters, sex hormones, and seminal plasma biochemistry were evaluated. CFTR mutations identified in OA patients were analyzed. In addition, the laboratory outcomes, clinical outcomes, and neonatal outcomes were compared between OA patients carrying two CFTR mutations and the others after surgical sperm extraction-intracytoplasmic sperm injection (ICSI) treatment. A total of 76 patients with congenital OA were enrolled. CFTR mutations were identified in 35 (46.1%) congenital OA patients. A total of 60 CFTR mutation sites of 27 types were identified, and 10 of them were novel. The average frequency was 1.71 (60/35) per person. The most common mutation was c.1210-11T > G (25%, 15/60). After ICSI treatment, there were no statistically significant differences in laboratory outcomes, clinical outcomes, and neonatal outcomes between OA patients carrying two CFTR mutations (n = 25) and other OA patients (n = 51). Apart from the IVS9-5T mutation, the genetic mutation pattern of CFTR in Chinese OA patients is heterogeneous, which is significantly different from that of Caucasians. Although carrying two CFTR mutations or not had no effect on the pregnancy outcomes in OA patients after ICSI, genetic counseling is still recommended for such patients.

Seminal calbindin 2 level in azoospermia and oligoasthenoteratozoospermia and its correlation with seminal and hormonal parameters.

We aimed to assess seminal calbindin 2 (CALB 2) expression in men with different semen parameters as well as its correlation with reproductive hormones in azoospermic patients and different semen parameters in oligoasthenoteratozoospermic patients. CALB 2 is also known as calretinin and 29 kDa calbindin. This prospective study was performed on 96 cases from the andrology outpatient clinic divided into 3 groups as follows: group 1 including 32 non obstructive azoospermic (NOA) patients, group 2 including 32 patients with oligoasthenoteratozoospermia (OAT), and Group 3 including normozoospermic individuals as controls. Semen analysis and estimation of seminal CALB 2 concentrations by enzyme linked immunosorbent assay (ELISA) technique were performed for all participants. Reproductive hormones were measured in nonobstructive NOA patients. The mean seminal CALB 2 level was higher in OAT patients compared to NOA patients and controls (7.8 ± 1.30 ng/ml, 7.3 ± 0.80 and 7.4 ± 1.0, respectively). Furthermore, the study had shown strong positive correlations between CALB 2 and sperm normal forms in controls and OAT patients. In contrast, there was no significant correlation between seminal CALB 2 and any of the reproductive hormones measured in NOA patients. Seminal CALB 2 may play a role in increasing the abnormal forms in OAT patients.

Comparing the clinical and singleton neonatal outcomes in male infertility patients with Oligoasthenospermia, OA, or NOA following fresh ICSI-ET using different sources of sperm.

To investigate clinical and singleton newborn outcomes in fresh cycles of embryo transfer after intracytoplasmic sperm injection (ICSI-ET) with diverse sperm sources (ejaculate, epididymis, and testis) in patients with Oligoasthenospermia, obstructive azoospermia (OA) or non-obstructive azoospermia (NOA). Patients who received fresh ICSI-ET for the first time at the First Affiliated Hospital of Zhengzhou University Reproductive Medicine Center between June 2011 and June 2021 were selected for this 10-year retrospective cohort analysis. After propensity score matching, only 1630 cycles were included in the investigation of ICS-ET clinical and singleton newborn outcomes in patients with Oligoasthenospermia, OA, and NOA using sperm from diverse sperm sources. After propensity score matching, our data revealed a negligible difference in baseline and cycle parameters among groups. In patients with Oligoasthenospermia and OA, different sperm sources do not appear to influence clinical pregnancy rates and live birth rates, nor do they influence newborn outcomes, such as newborn weight, premature birth rate, and neonatal sex ratio in singleton births, except for OA patients who use epididymal sperm having higher low birth weight (LBW) rates in singleton pregnancies than those who use testicular sperm. In addition, clinical pregnancy rates, live birth rates, singleton gestation birth weights, premature birth rates, and neonatal sex ratios were similar between patients with Oligoasthenospermia, OA, and NOA using testicular sperm. Regardless of the type of male infertility (Oligoasthenospermia, OA, NOA) or sperm sources (ejaculate, epididymis, testis), a successful ICSI-ET procedure can result in similar clinical and neonatal outcomes, such as clinical pregnancy rate, live birth rate, abortion rate, neonatal birth weight and sex ratio of singleton pregnancies.

Sperm immotility is associated with epididymis metabolism disorder in mice under obstructive azoospermia.

Obstructive azoospermia (OA) accounts for approximately 40% of males who suffer from azoospermia of male infertility. Currently, available treatment for OA consists of reproductive tract surgical reconstruction and sperm retrieval from the testis. However, both treatments result in low fertility compared to normal pregnancy, and the main reason remains largely unknown. Previous studies have shown that the quality of sperm retrieved from OA patients is poor compared with normal adult males but without an in-depth study. Herein, we generated a mouse OA model with vasectomy to evaluate sperm quality systematically. Our results showed that the testis had normal spermatogenesis but increased apoptotic activity in both OA patients and mice. More importantly, epididymal morphology was abnormal, with swollen epididymal tubules and vacuole-like principal cells. Especially, sperm retrieved from the epididymis of OA mice showed poor motility and low fertilization ability in vitro. Using mass spectrometry in epididymal fluid, we found differences in the expression of key proteins for sperm maturation, such as Angiotensinogen (AGT), rhophilin-associated tail protein 1 (ROPN1), NPC intracellular cholesterol transporter 2 (NPC2), and prominin 1 (PROM1). Furthermore, our results demonstrated that AGT, secreted by epididymal principal cells, could regulate sperm motility by managing PKCα expression to modify sperm phosphorylation. In conclusion, our data evaluate sperm quality systematically in OA mice and contribute to the understanding between the sperm and epididymis, which may provide novel insight into treating male infertility.

P-021 A novel SNP in HUWE1 promoter confers increased risk of NOA by affecting the RA/RARα pathway in Chinese individuals

Abstract Study question The present study aimed to investigate the role of HUWE1 in non-obstructive azoospermia (NOA) etiology and germ cell differentiation. Summary answer An SNP mutation in HUWE1 promoter region downregulates HUWE1 expression in NOA patients, the genetic polymorphisms of HUWE1 are related to the pathogenesis of NOA. What is known already HUWE1, a ubiquitin ligase, is crucial to the development of male reproductive system and embryonic development. However, the specific role of HUWE1 in regulating germ cell differentiation is unclear, and clinical evidence for the relationship between HUWE1 and the pathogenesis of male infertility is lacking. Study design, size, duration 190 NOA Chinese patients were recruited and analyzed single nucleotide polymorphisms (SNPs) in HUWE1 gene. Participants/materials, setting, methods Regulation of HUWE1 gene expression by RARα factor was evaluated by performing ChIP assay, EMSA, and siRNA-mediated RARα knockdown. We used C18-4 cells to determine whether HUWE1 participated in RA-mediated RARα signaling and performed luciferase assay, CCK-8 assay, immunofluorescence staining, RT-qPCR, and western blot analysis. The expression level of HUWE1 and RARa was evaluated in clinical samples obtained from NOA or obstructive azoospermia (OA) patients using RT-qPCR. Main results and the role of chance We explored the mechanism by which polymorphism of the HUWE1 promoter leads to spermatogenesis disorders, and focused on the identification of mutations in the SNP site of the HUWE1 regulatory sequence among patients with NOA, which may influence HUWE1 expression. The results of in vitro experiments showed that RARα binds to the HUWE1 promoter region and regulates its expression. However, the regulatory effect of RARα weakened following introduction of the mutation into the HUWE1 regulatory sequence. The expression of spermatogonial differentiation-related gene STRA8 was impaired, and γH2AX foci accumulated following inhibition of HUWE1 gene expression using siRARα. Finally, to determine whether HUWE1 expression was important in patients with NOA, its expression was verified using testicular puncture samples collected from patients with NOA and the results revealed that HUWE1 expression was abnormal (P < 0.01). In addition, RARα expression was significantly downregulated (P < 0.001). Overall, our result support the hypothesis that HUWE1 expression is induced by the RA/RARα signaling pathway and it inhibit γH2AX foci accumulation, thereby considerably promoting sperm differentiation and meiotic prophase. Limitations, reasons for caution The effect of SNPs in HUWE1 on male infertility is further studied in a wider population. Wider implications of the findings Our study of HUWE1 and RA/RARα signaling pathway provides new information on the regulatory mechanism of spermiogenesis, suggesting that SNPs in HUWE1 promoter are etiological factors in patients with NOA and providing a rationale for further drug development to target HUWE1 in these patients. Trial registration number the National Key Research and Development Program of China (2018YFC1003603)

P-712 Transcriptomic Prediction of Spermatogenesis in the Ejaculates of Nonobstructive Azoospermic Men and Genomic Characterization of their Embryo Developmental Competence

Abstract Study question Can the genomic assessment of ejaculates from azoospermic men predict successful sperm extraction at testicular biopsy (TESE) and provide information on their reproductive potential? Summary answer RNAseq on the ejaculates of NOA men identified transcriptomic predictors of successful TESE outcome, while DNAseq evidenced gene-related mechanistics linked to reproductive performance. What is known already The most puzzling form of azoospermia is the testicular type. The chances of successful surgical sperm retrieval for these cases can be unpredictable, even when a benign histopathology has been obtained. The ability to identify individuals with complete loss of spermatogenesis at the seminiferous tubule level would help eliminate anesthesia and surgical risks, as well as financial and emotional distress. We propose to noninvasively assess the ejaculates from these men to predict successful TESE sperm retrieval. We utilize DNAseq to assess the impact of germline mutations on the embryo developmental competence of the surgically retrieved gametes. Study design, size, duration Over 4 years, we recruited men with normal peripheral karyotypes and azoospermia (n = 23), confirmed by absence of spermatozoa in extensive semen analyses. We performed epigenetic assessments on their ejaculates to compare transcriptomic profiles according to whether spermatozoa were successfully retrieved after TESE ((+)Sperm,n=11), or not ((-)Sperm,n=12). We then performed DNAseq (IRB 1006101085) on the testicular spermatozoa retrieved, to assess their ability to generate a pregnancy (fertile) or not (infertile), while controlling for maternal age. Participants/materials, setting, methods RNA was isolated from seminal fluid for epigenetic assessment (21,855 genes). A > 1 absolute log2fold change and P<0.0005 were considered significant. Transcriptomic profiles were compared between the (-)Sperm and (+)Sperm cohorts. For genetic assessment, surgically retrieved spermatozoal DNA from the (+)Sperm and obstructive azoospermic (OA) control (n = 19) cohorts were extracted and amplified. Gene mutation profiles, detected by CLC Genomic Server 9.0, were assessed for the (+)Sperm cohort according to ICSI outcomes, compared to the OA-control. Main results and the role of chance Transcriptomic analyses on seminal plasma from the 23 NOA men identified 8 imbalanced genes, involved in spermatogenesis (TMCO5B,C10orf62,SMKR1,SPZ1), sperm function (NEU1,TPTE2), and testis development (TRPC1,IGSF11-AS1). TPTE2 was almost completely expressed in 81.8%(9/11) of the (+)Sperm cohort (paternal age, 37.1±6yrs), while IGSF11-AS1 was underexpressed in all 12 (-)Sperm men (paternal age, 34.3±5yrs). Both genes are implicated in spermatogenic defects and are normally highly expressed in testis. Interestingly, NEU1, localized on the sperm acrosome and crucial for sperm capacitation, was exclusively underexpressed in (-)Sperm while overexpressed in (+)Sperm cohorts. Genomic analyses of spermatozoal DNA from the (+)Sperm cohort was compared to a matched control of OA patients (paternal age, 36.8±7yrs). Spermatozoa from the study cohort displayed mutations on genes involved in RNA transcription (POLR2L), apoptosis (AP5M1), and spermiogenic functions (AP1S2,AP1G2,APOE), while these genes were unaffected in the OA-control. When we compared sperm genetic profiles of the successful TESE cohort (maternal age, 36.8±2yrs, paternal age, 37.1±6yrs) according to ICSI outcome, we found that spermatozoa from the 8 fertile patients, all with term pregnancies, displayed consistent mild mutations on MPIG6B (stem cell lineage differentiation) while gametes from their 3 infertile counterparts carried severe mutations on genes (MBD5,CCAR1,PMEPA1,POLK,REC8,REPIN1,MAPRE3,ARL4C) involved in early embryonic development, and therefore failed to achieve embryo implantation. Limitations, reasons for caution Although we predicted, by transcriptomic analyses of ejaculates from NOA men, the presence of spermatozoa and characterized their embryo developmental competence through genetic profiling, these preliminary results need validation in a larger study cohort. Moreover, although controlled for, a confounding female factor cannot be undoubtfully excluded. Wider implications of the findings Epigenetic assessment of seminal fluid from NOA men can noninvasively identify biomarkers capable of predicting complete loss of spermatogenesis, sparing these patients from unnecessary surgery. Gene profiling can identify gametes capable of supporting term pregnancies, laying the groundwork for precision medicine in male infertility screening. Trial registration number n/a

A novel SNP in HUWE1 promoter confers increased risk of NOA by affecting the RA/RARα pathway in Chinese individuals.

The ubiquitin ligase HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 is essential for the establishment and maintenance of spermatogonia. However, the role of HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 in regulating germ cell differentiation remains unclear, and clinical evidence linking HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 to male infertility pathogenesis is lacking. This study aims to investigate the role of HUWE1 in germ cell differentiation and the mechanism by which a HUWE1 single nucleotide polymorphism increases male infertility risk. We analyzed HUWE1 single nucleotide polymorphisms in 190 non-obstructive azoospermia patients of Han Chinese descent. We evaluated HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 regulation by retinoic acid receptor alpha using chromatin immunoprecipitation assays, electrophoretic mobility shift assays, and siRNA-mediated RARα knockdown. Using C18-4 spermatogonial cells, we determined whether HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 participated in retinoic acid-mediated retinoic acid receptor alpha signaling. We performed luciferase assays, cell counting kit-8 assays, immunofluorescence, quantitative real-time polymerase chain reaction, and western blotting. We quantified HUWE1 and retinoic acid receptor alpha in testicular biopsies from non-obstructive azoospermia and obstructive azoospermia patients using quantitative real-time polymerase chain reaction and immunofluorescence. Three HUWE1 single nucleotide polymorphisms were significantly associated with spermatogenic failure in 190 non-obstructive azoospermia patients; one (rs34492591) was in the HUWE1 promoter. Retinoic acid receptor alpha regulates HUWE1 gene expression by binding to its promoter. HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 participates in retinoic acid/retinoic acid receptor alpha signaling pathway and regulates the expression of germ cell differentiation genes STRA8 and SCP3 to inhibit cell proliferation and reduce γH2AX accumulation. Notably, significantly lower levels of HUWE1 and RARα were detected in testicular biopsy samples from non-obstructive azoospermia patients. An HUWE1 promoter single nucleotide polymorphism significantly downregulates its expression in non-obstructive azoospermia patients. Mechanistically, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 regulates germ cell differentiation during meiotic prophase through its participation in retinoic acid/retinoic acid receptor alpha signaling and subsequent modulation of γH2AX. Taken together, these results strongly suggest that the genetic polymorphisms of HUWE1 are closely related to spermatogenesis and non-obstructive azoospermia pathogenesis.

A Modified Transversal Two-Suture Microsurgical Intussusception Vasoepididymostomy for the Treatment of Epididymal Obstructive Azoospermia

Introduction: We have developed a modified vasoepididymostomy procedure, namely “fenestrated” transversal two-suture microsurgical intussusception vasoepididymostomy. This study aimed to investigate the therapeutic efficacy and outcome of this fenestrated vasoepididymostomy for epididymal obstructive azoospermia (OA). Methods: Microsurgical two-suture transversal intussusception vasoepididymostomy was performed using our modified fenestration technique in 64 OA patients due to epididymal obstruction at our hospital. Fenestration means making an opening on the epididymal tubule wall. The edges of the epididymal tubule “window” were stitched transversally (two stitches) using the two double-armed 9–0 atraumatic sutures. The epididymal tubule was anastomosed to the lumen of the vas deferens. The patency rate and pregnancy rate were assessed. Results: Of the 64 OA patients, 45 received bilateral microsurgical two-suture transversal intussusception vasoepididymostomy, while 19 underwent unilateral microsurgical two-suture transversal intussusception vasoepididymostomy. All of the patients were followed up after the operation. The follow-up period ranged from 4 to 54 months. Among 45 cases of bilateral surgery, the patency rate was 88.89% (40/45), and the natural pregnancy rate was 28.89% (13/45). After the patency was confirmed postoperatively, 3 cases had recurrent OA, of which 2 cases had return of sperm to the ejaculate by oral antibiotics and scrotal self-massage. As for the 19 cases of unilateral microsurgery, the patency rate was 68.42% (13/19), and the natural pregnancy rate was 21.05% (4/19). Conclusion: The fenestrated transversal two-suture microsurgical intussusception vasoepididymostomy can achieve a good patency rate in OA patients and did not increase the difficulty and duration of the procedure.

Joint analysis of m6A and mRNA expression profiles in the testes of idiopathic nonobstructive azoospermia patients.

Growing evidence has indicated that epigenetic factors might be associated with the pathophysiology of idiopathic nonobstructive azoospermia (iNOA). As the most common RNA modification, N6-methyladenosine (m6A) methylation has recently attracted more attention in the regulation of spermatogenesis; however, its role in the mechanisms of iNOA is still unclear. To determine the differential expression of mRNA and m6A methylation status in the testes of iNOA patients. Testes tissues from diagnosed iNOA and controlled obstructive azoospermia (OA) patients were collected and grouped according to the histological examinations. Total RNA was isolated and quantified by an m6A RNA Methylation Quantification Kit. The expression level of mRNAs was detected by qRT-PCR analysis. Differentially expressed m6A genes were analyzed using the human ArrayStar m6A epitranscriptomic microarray, and bioinformatics analyses were applied. A total of 36 iNOA and 8 controlled patients were included. The global expression of m6A in the iNOA group was significantly decreased. A dosage relationship was observed between the m6A decline and the degree of impaired spermatogenesis, with the successive process of normal spermatogeneis, hypospermatogenesis (HP), maturation arrest (MA), and Sertoli cell-only syndrome (SO). Four down-expressed genes (BDNF, TMEM38B, RPL3L, and C22orf42) displayed significantly lower expression of m6A methylation. Additionally, they also showed a gradually down-expressed tendency in the three groups (OA, HP, SO/MA groups). Moreover, m6A reader EIF3A was approved to have differential expression through microarrays analysis, which was consistent with the result from the qRT-PCR test. The m6A expression was gradually downregulated in the testes tissue from iNOA patients in accordance with the degree of spermatogenic dysfunction. The determined differential expression of mRNA and m6A methylation status may represent potentially novel molecular targets for the mechanism study of iNOA in the epigenetic level, which could benefit the understanding of the pathophysiology of iNOA.

A novel testis-enriched gene, Samd4a, regulates spermatogenesis as a spermatid-specific factor.

Spermatogenesis is the highly orchestrated process involving expression of a series of testicular genes. Testis-enriched genes are critical for cellular processes during spermatogenesis whose disruption leads to impaired spermatogenesis and male infertility. Nevertheless, among poorly investigated testicular genes are the mouse Samd4a and human SAMD4A which were identified in the current study as novel testis-enriched genes through transcriptomic analyses. In particular, as orthologous alternative splicing isoforms, mouse Samd4a E-form and human SAMD4AC-form containing the SAM domain were specific to testes. Western blot analyses revealed that the murine SAMD4AE-form was predominantly found in the testis. Analyses on GEO2R and single-cell RNA-seq datasets revealed that the Samd4a/SAMD4A expression was enriched in spermatids among various types of cells in adult testes. To investigate in vivo functions of Samd4a, Samd4a knockout mice were generated using the CRISPR/Cas9 system. The Samd4a deficiency resulted in lower testis weight, absence of elongated spermatids, and an increased number of apoptotic cells. Profiling of gene expression in human testis samples revealed that the SAMD4A expression was comparable between obstructive azoospermia patients and normal controls, but significantly lowered in nonobstructive azoospermia (NOA) patients. Among three subgroups of NOA, pre-meiotic arrest (NOA-pre), meiotic arrest (NOA-mei), and post-meiotic arrest (NOA-post), expression level of SAMD4A was higher in the NOA-post than the NOA-mei, but there was no difference between the NOA-pre and NOA-mei. The current studies demonstrated spermatid stage-specific expression of Samd4a/SAMD4A, and impairment of the late stages of spermatogenesis by disruption of the mouse Samd4a gene. These data suggest that Samd4a/SAMD4A plays an essential role in normal spermatogenesis, and SAMD4A, as a spermatid specific marker, can be used for subcategorizing NOA patients. Further understanding the molecular role of SAMD4A will advance our knowledge on genetic regulations in male infertility.

Factors predicting the outcome of percutaneous epididymal sperm aspiration in men with obstructive azoospermia

To report on the live birth rates (LBRs) following percutaneous epididymal sperm aspiration (PESA) in men with obstructive azoospermia (OA) and factors affecting treatment outcome which is under reported in the literature. This is a multicenter study that was conducted in Egypt including all couples undergoing intra cytoplasmic sperm injection (ICSI) for OA using PESA-derived sperms. Men were subdivided according to aetiology into congenital, iatrogenic and idiopathic groups. Fertilization, pregnancy and LBRs were determined and compared in each group. The longitudinal LBR, crude and expected cumulative delivery rates (CCDR, ECDR) were calculated. Multiple logistic regression analysis was used to determine significant associations between maternal, paternal and ICSI factors with successful live births. Ninety couples were included in the study. Viable sperm for ICSI was retrieved in 89 men (98.9%). A total of 155 ICSI cycles with 17 frozen embryo transfers resulted in 81 pregnan-cies and 55 live births. After 5 cycles, the longitudinal LBR, CCDR and ECDR were 30%, 57.3% and 88.6% respectively. Maternal age and number of fertilized eggs were the only fac-tors significantly affecting LBRs. PESA is a minimally invasive procedure for secur-ing viable sperm for ICSI in OA men, with high cumulative delivery rates. Maternal age and number of fertilized eggs are the only factors that significantly affecting LBR. The contempo-rary longitudinal and cumulative LBRs provide objective out-come data to counsel OA patients undergoing fertility treat-ments.

Does ICSI outcome in obstructive azoospermia differ according to the origin of retrieved spermatozoa or the cause of epididymal obstruction? A comparative study

PurposeTo determine whether ICSI outcomes are affected by sperm source or genital tract inflammatory status.MethodsA retrospective cohort study was conducted in all consecutive obstructive azoospermia patients who underwent testicular sperm aspiration (TESA) or percutaneous epididymal sperm aspiration (PESA) and ICSI between February 1, 2017, and December 31, 2020. Couples were excluded if they were diagnosed with monogenic disease, abnormal karyotype, or had female uterine malformation. The primary objective was to determine whether ICSI outcomes are affected by the use of testicular or epididymal spermatozoa, and the secondary objective was to explore the effect of granulocyte elastase on ICSI outcomes using epididymal spermatozoa.ResultsCompared with TESA, inflammatory and non-inflammatory PESA patients exhibited a better high-quality embryo rate, with significant differences among the three groups (49.43 vs. 55.39% and 56.03%; odds ratio, 6.345 and 6.631; 95% confidence interval, 0.340–12.350, and 1.712–11.550; P = 0.038 and P = 0.008, respectively). The fertilization rate, clinical pregnancy rate, live birth delivery rate, and congenital anomaly birth rate were similar in patients who underwent TESA or PESA (with or without inflammation).ConclusionsThe high-quality embryo rate in PESA patients was higher than that in TESA patients. After successful pregnancy, ICSI outcomes did not differ between patients with obstructive azoospermia who experienced TESA or PESA and those with or without genital tract inflammation.

Is There an Increased Incidence of Aneuploid Embryos in Cases of Obstructive and Non-Obstructive Azoospermia?

Background: Current evidence supports that males with low spermatozoa concentrations, as well as obstructive azoospermia (OA) or non-obstructive azoospermia (NOA), present with high rates of aneuploid spermatozoa. This issue could potentially lead to an increased number of aneuploid embryos in subsequent ICSI cycles using surgically retrieved spermatozoa, however, azoospermia is not routinely considered to be a clinical indication for pre-implantation genetic testing for aneuploidies (PGT-A). Aim: To identify the incidence of aneuploid embryos in PGT-A cycles that used testicular spermatozoa extracted through surgical sperm retrieval (SSR). In addition, whether aneuploidy rates differed between embryos fertilised with SSR compared with ejaculated spermatozoa. Method: A retrospective study of OA and NOA patients who underwent SSR between March 2017 and September 2020 and had PGT-A cycles. Next-generation sequencing was employed to detect embryo aneuploidies. Data were compared and analysed using a chi-square test with PGT-A cycles using ejaculated spermatozoa within the same time-period. Results: A total of 57 males underwent SSR, leading to 68 cycles that included the use of PGT-A. A total of 152 embryos with conclusive results identified there to be 57.2% euploid embryos (87/152) and 38.2% aneuploid embryos (58/152) in the SSR group. In comparison, the ejaculated spermatozoa group resulted in 59.2% (2115/3677) euploid embryos and 35.7% (1312/3677) aneuploid embryos. The incidence of aneuploid embryos (38.2%) in the SSR group was not statistically significant [Odds ratio: 1.11 (0.79-1.56) p=0.53]. Importantly, there were no differences for mean maternal age for both study groups (SSR female partners: 36.7 years; ejaculated spermatozoa female partners: 37.1 years; p>0.1). Conclusion: The current outcomes indicate there is not an increased incidence of aneuploid embryos in PGT-A cycles of men who underwent to SSR compared with men using ejaculated spermatozoa.

Seminal plasma extracellular vesicles tRF-Val-AAC-010 can serve as a predictive factor of successful microdissection testicular sperm extraction in patients with non-obstructive azoospermia

BackgroundThere is a lack of biomarkers for distinguishing non-obstructive azoospermia (NOA) patients with successful sperm retrieval (Sp+) from those with failed sperm retrieval (Sp-). This study aimed to determine the potential of extracellular vesicles tRNA-derived small RNA (tsRNA) as a novel non-invasive biomarker for successful sperm retrieval by microdissection testicular sperm extraction (mTESE).MethodsThe study included 18 patients with NOA with successful sperm retrieval (Sp+) and 23 patients with NOA with failed sperm retrieval (Sp-), 15 obstructive azoospermia (OA) patients, 5 idiopathic oligospermia (IO) patients, and 12 healthy people. Seminal plasma extracellular vesicles tsRNA levels were used in a two-stage case-control study (screened by tsRNA sequencing on Illumina NextSeq instrument and validated by qRT-PCR). The bioinformatic analysis was performed to determine the role of tsRNA in the pathogenesis of non-obstructive azoospermia.ResultsTwo tsRNAs (tRF-Val-AAC-010: AUC = 0.96, specificity = 80%, sensitivity = 95%; tRF-Pro-AGG-003: AUC = 0.96, specificity = 87%, sensitivity = 95%) were found to have high predictive accuracy for distinguishing the origin of azoospermia. In addition, the extracellular vesicles tRF-Val-AAC-010 resulted in high predictive ability (AUC = 0.89, sensitivity = 72%, specificity = 91%, P < 0.0001) in predicting the presence of sperm in non-obstructive azoospermia undergoing mTESE. Finally, bioinformatic analysis revealed that tRF-Val-AAC-010 were involved in spermatogenesis.ConclusionsThis study identified that the extracellular vesicles tRF-Val-AAC-010 and tRF-Pro-AGG-003 are biomarkers for the diagnosis of non-obstructive azoospermia, and that tRF-Val-AAC-010 as a potential non-invasive biomarker for predicting the presence of sperm in non-obstructive azoospermia testicular tissue.

P-034 Rate of testicular histology failure in predicting successful testicular sperm extraction in Non-Obstructive Azoospermia

Abstract Study question Does testicular histology predict successful or unsuccessful TEsticular Sperm Extraction (TESE) in Non-Obstructive Azoospermia (NOA) patients? Summary answer Testicular histology failed to predict successful TESE in 1 of 3 NOA patients. What is known already The management of patients with Non-Obstructive Azoospermia (NOA) involves TEsticular Sperm Extraction (TESE) combined with IntraCytoplasmic Sperm Injection (ICSI). Sperm retrieval is successful in up to 50% of men with NOA; however, there is no single clinical finding or investigation that can accurately predict a positive outcome. Previous studies have concluded that testicular histology is the best predictor of a successful or unsuccessful TESE. Study design, size, duration This is a retrospective study of 525 patients who underwent TESE between January 2018 and December 2020 in Humanitas Fertility Center. Participants/materials, setting, methods The cohort was dived in five groups: 287 NOA, 95 necrozoospermia, 18 anejaculation, 1 testicular trauma and 124 Obstructive Azoospermia (OA) patients. Main results and the role of chance Sperm was retrieved and cryopreserved in 218/287 patients with NOA (75,95%), 55/95 in necrozoospermia (57,89%), 18/18 anejaculation (100%), 1/1 testicular trauma (100%) and 120/124 in OA patients (96,77%). Interesting that, when we compare sperm recovery data through TESE with the histological assessment (HA) of corresponding testicular biopsy, we found discordance against HA. In particular we retrieved spermatozoa in 218 NOA patients but in 74 of them HA failed to detect them (33,94%). Moreover HA failed to detect spermatozoa in 8/55 (14,54%) in case of necrozoospermia, 1/18 (5,55%) in anejaculation and 10/120 (8,33%) in OA patients. Limitations, reasons for caution Testis's heterogeneity is a limitation of the study. Wider implications of the findings We found a discordance when HA is compared with sperm recovery data of corresponding testicular biopsy. This data suggest that in case of testicular diagnostics histology in 33,94% of NOA cases there is a wrong diagnosis and 1 of 3 patients it doesn’t retrieve and cryopreserve spermatozoa when it could. Trial registration number N A

RNF144B stimulates the proliferation and inhibits the apoptosis of human spermatogonial stem cells via the FCER2/NOTCH2/HES1 pathway and its abnormality is associated with azoospermia.

Studies on gene regulation and signaling transduction pathways of human spermatogonial stem cells (SSCs) are of the utmost significance for unveiling molecular mechanisms underlying human spermatogenesis and gene therapy of male infertility. We have demonstrated, for the first time, that RNF144B stimulated cell proliferation and inhibited the apoptosis of human SSCs. The target of RNF144B was identified as FCER2 by RNA sequencing. We revealed that RNF144B interacted with FCER2 by immunoprecipitation. Consistently, overexpression of FCER2 reversed the phenotype of proliferation and apoptosis of human SSCs caused by RNF144B knockdown. Interestingly, FCER2 pulled down N2ICD (NOTCH2 intracellular domain), while N2ICD could bind to FCER2 in human SSCs. The levels of NOTCH2, FCER2, HES1, and HEY1 were reduced by RNF144B siRNA in human SSCs. Significantly, RNF144B was expressed at a lower level in nonobstructive azoospermia (NOA) patients than in the obstructive azoospermia (OA) patients with normal spermatogenesis, and 52 patients with heterozygous mutations of RNF144B were detected in 1,000 NOA patients. These results implicate that RNF144B promotes the proliferation of human SSCs and suppresses their apoptosis via the FCER2/NOTCH2/HES1 pathway and that the abnormality of RNF144B is associated with spermatogenesis failure. This study thus provides novel molecular mechanisms regulating the fate determinations of human SSCs, and it offers new biomarkers for the diagnosis and treatment of male infertility.

Comparison of pregnancy and neonatal outcomes of intracytoplasmic sperm injection performed with frozen versus fresh testicular sperm.

BackgroundIt remains controversial whether there is a difference in the prognosis of intracytoplasmic sperm injection (ICSI) using frozen or fresh testicular sperm in patients with obstructive azoospermia (OA). Moreover, in the available studies, few have tracked neonatal outcomes. This study aimed to compare the pregnancy and neonatal outcomes of ICSI using cryopreserved sperm versus fresh sperm collected by testicular sperm aspiration (TESA).MethodsA total of 317 OA patients treated with ICSI in a university affiliated hospital from January 2016 to December 2020 were included in this study. The participants were divided into two groups according to the type of sperm used for ICSI: frozen sperm group (n=154) and fresh sperm group (n=163). The pregnancy and neonatal outcomes of the two groups were compared.ResultsThe data produced by this study showed no significant statistical difference in the 2 pronuclei (2PN) fertilization rate, 2PN cleavage rate, high-quality blastocyst rate, and the average number of transferred embryos in the frozen and fresh sperm groups. Similarly, no difference was found in implantation rate, clinical pregnancy rate, multiple pregnancy rate, miscarriage rate, premature delivery rate, live birth rate, and gender ratio at birth (P>0.05). The average newborn birth weight was similar in both groups (2,932.61±728.40 vs. 3,100.32±515.64 g, respectively) (P>0.05). A higher incidence of low birthweight (LBW) newborns was found in the frozen sperm group (20.91% vs. 8.49%) (P<0.05). Multiple logistic regression analysis showed that LBW is related to single or twin pregnancies (P<0.01), but not sperm (frozen or fresh) (P>0.05). We further analyzed the twin and single pregnancies in the two groups separately, and found that the incidences of LBW were both similar (P>0.05). There was no difference in the Apgar scores at 1 min and 5 min after birth between the two groups (P>0.05).ConclusionsThe use of frozen testicular sperm by TESA was efficient for men with OA. There were similar pregnancy and neonatal outcomes following TESA-ICSI using frozen or fresh sperm in this retrospective study. Prospective investigations are needed for further validation.

Vasal vessel‐sparing microsurgical single‐armed vasoepididymostomy to epididymal obstructive azoospermia: A retrospective control study

This study aimed to evaluate the efficacy and safety of vasal vessel-sparing modified single-armed 2-suture longitudinal intussusception vasoepididymostomy (SA-LIVE) to epididymal obstructive azoospermia patients. Forty consecutive epididymal obstructive azoospermia cases, who underwent microsurgical vasoepididymostomy in Shanghai General Hospital from January 2019 to October 2019, were included in this study. Twenty cases underwent SA-LIVE (group A), and 20 cases underwent vasal vessel-sparing SA-LIVE (group B). Until March 2021, the mean follow-up period was 16.9±4.1 (12-23) months. The overall patency rate was 82.5%, and 80% and 85% for group A and group B respectively. The mean time to achieve patency was 4.11±2.74months. The overall natural pregnancy rate was 51.5%(17/33) at the mean follow-up of 16.9months. The natural pregnancy rate was 50.0% for group A and 52.9% for group B (p>.05). At the time of 6months post-operation, the patency rate was 70% for group A and 80% for group B (p=.465); the natural pregnancy rate was 0% for group A and 31.3% for group B (p=.022). Vasal vessel-sparing SA-LIVE is safe and effective to achieve favourable patency and pregnancy rates. Preserving vasal vessel would improve natural pregnancy rate at a very early stage.

Diagnostic value of transrectal combined scrotal ultrasonography in acquired obstructive azoospermia

ObjectiveTo investigate the transrectal and scrotal ultrasonographic features of acquired obstructive azoospermia and evaluate the role of ultrasonography in the location diagnosis of acquired obstructive azoospermia patients. MethodsRetrospectively analysis of 92 cases of acquired obstructive azoospermia in recent years. All the patients underwent transrectal and scrotal ultrasonography. The ultrasonographic features were observed of testis, epididymis, scrotal segment of vas deferens, seminal vesicle, ejaculatory duct and prostate. Eighty cases with normal semen were taken as control group. ResultsAmong the 92 cases of acquired obstructive azoospermia, 28 cases were prostate midline cyst, 32 cases were stone or calcification of the ejaculation duct and 21 cases were chronic seminal vesicle inflammation, which were found through transrectal ultrasonography; 27 cases were vas deferens dilation, 30 cases had abnormal mass of epididymis tail, 31 cases were epididymis thickness with reticular change, 8 cases were cystic dilatation of rete testis, which were found through scrotal ultrasonography. Compared with the control group, ultrasound examination increased the detection rate of distal seminal duct lesions and epididymal lesions in acquired obstructive azoospermia patients (p<.01). Comparison of testicular volume between case group and control group did not reveal significant difference (p>.05). The thickness of the head, body and tail of epididymis in case group was significantly bigger than that in control group (p<.01). ConclusionTransrectal and scrotal ultrasonography can find lesions in different parts of the seminal passage. Acquired obstructive azoospermia patients have increased rate of distal seminal duct lesions, epididymal lesions and epididymis volume. Transrectal and scrotum ultrasonography can provide reliable imaging evidence for the location diagnosis of acquired obstructive azoospermia.