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Environmental pollution has emerged as a global concern due to its detrimental effects on human health. One of the critical aspects of this concern is the impact of environmental pollution on sperm quality in males. Male factor infertility accounts for approximately 40%- 50% of all infertility cases. Nonobstructive azoospermia (NOA) is the most severe type of male infertility. Human umbilical cord mesenchymal stem cell (hUCMSC) exosomes enhance proliferation and migration, playing crucial roles in tissue and organ injury repair. However, whether hUCMSC exosomes impacting on NOA caused by chemotherapeutic agents remains unknown. This study aimed to explore the functional restoration and mechanism of hUCMSC exosomes on busulfan-induced injury in GC-1 spg cells and ICR mouse testes. Our results revealed that hUCMSC exosomes effectively promoted the proliferation and migration of busulfan-treated GC-1 spg cells. Additionally, oxidative stress and apoptosis were significantly reduced when hUCMSC exosomes were treated. Furthermore, the injection of hUCMSC exosomes into the testes of ICR mice treated with busulfan upregulated the expression of mouse germ cell-specific genes, such as vasa, miwi, Stra8 and Dazl. Moreover, the expression of cellular junction- and cytoskeleton-related genes, including connexin 43, ICAM-1, β-catenin and androgen receptor (AR), was increased in the testicular tissues treated with exosomes. Western blot analysis demonstrated significant downregulation of apoptosis-associated proteins, such as bax and caspase-3, and upregulation of bcl-2 in the mouse testicular tissues injected with hUCMSC exosomes. Further, the spermatogenesis in the experimental group of mice injected with exosomes showed partial restoration of spermatogenesis compared to the busulfan-treated group. Collectively, these findings provide evidence for the potential clinical applications of hUCMSC exosomes in cell repair and open up new avenues for the clinical treatment of NOA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m 6 A) plays an essential role in regulating various biological processes. However, the function of m 6 A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m 6 A methyltransferase METTL3 or the m 6 A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m 6 A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m 6 A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m 6 A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m 6 A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m 6 A modification in restricting L1 retrotransposition., (© 2024. The Author(s).)

Background: Differential diagnosis of men with subtypes of non-obstructive azoospermia (NOA) is important for their treatment. Many genes are transcripted during meiosis. We hypothesized that some of these genes can be detected in cell-free seminal message RNA (mRNAs) (cfs-mRNA) and be developed as non-invasive biomarkers for diagnosing NOA subtypes., Objective: To screen cfs-mRNA to diagnose the completion of meiosis and predict successful sperm retrieval (SR) in men with NOA., Materials and Methods: NOA patients who visited our institutes from September 2018 to December 2020 for testicular histopathological diagnosis (n = 109) or testicular SR (n = 92) were screened for participation in the study. Microarray and real-time quantitative polymerase chain reaction were used in five stages to obtain candidate cfs-mRNAs for comparisons between patients with early maturation arrest (eMA, meiosis not completed) and late MA or hypospermatogenesis (meiosis completed) and between NOA patients with successful SR and SR failure., Results: Twelve cfs-mRNAs were selected based on this comparison between men with eMA and hypospermatogenesis and their gene expression and function information. Of these, AKAP1, BOLL, TCP11 and SETX predominantly derived from testes and germ cells were proposed as candidate cfs-mRNAs. Further quantification in men with NOA demonstrated significantly higher levels of BOLL cfs-mRNA (p < 0.0001) in men with late MA or hypospermatogenesis (n = 23), compared with men with eMA (n = 51); and significantly higher levels (p < 0.0001) in patients with successful SR (n = 44) when compared with patients with SR failure (n = 37). Interestingly, with a similar cutoff value, BOLL cfs-mRNA showed high sensitivity and specificity in diagnosing late MA and hypospermatogenesis (>404 copies/ml) and predicting successful SR (>415 copies/ml). Correlation of BOLL mRNA levels was observed in paired semen and testicular tissues., Discussion and Conclusions: We propose that BOLL cfs-mRNA is a promising non-invasive marker for diagnosing the completion of meiosis and predicting successful testicular SR in men with NOA., (© 2022 American Society of Andrology and European Academy of Andrology.)

Purpose: Male infertility is a multifactorial syndrome encompassing a wide variety of disorders. A previous Chinese genome-wide single-nucleotide polymorphism (SNP) association studies have identified four SNPs (rs12097821 in PRMT6 gene, rs2477686 in PEX10 gene, rs6080550 in SIRPA-SIRPG, and rs10842262 in SOX5 gene) as being significantly associated with risk factors for nonobstructive azoospermia (NOA). However, the results were not fully repeated in later studies, which calls for further investigations., Methods: We here performed a case-control study in a central Chinese population to explore the association between the four SNPs and male infertility, which included 631 infertile men (NOA and oligozoospermia) and 720 healthy fertile men. The genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism and confirmed by sequencing., Results: The results showed that rs12097821 and rs10842262 were strongly associated with the risk of NOA but not total male infertility or oligozoospermia, while rs2477686 and rs6080550 were not associated with the risk of total male infertility, NOA, or oligozoospermia. To improve the statistical strength, a meta-analysis was conducted. The results suggested that rs2477686, rs6080550, and rs10842262 were significantly associated with male infertility, especially with NOA, while rs12097821 was only found to be associated with total male infertility., Conclusions: Collectively, the rs2477686, rs6080550, and rs10842262 may indeed be the genetic risk factors for NOA, which requires further investigation using larger independent sets of samples in different ethnic populations.

Objective: To explore the feasibility of quantification of testicular DNA methylation from cell-free seminal DNA (cfsDNA), and analyze promoter methylation alterations in men with idiopathic nonobstructive azoospermia (NOA)., Design: Comparison between testicular DNA and paired cfsDNA, and among NOA patients with different testicular phenotypes., Setting: Academic research institute and andrology practice., Patient(s): Eighty-eight idiopathic NOA patients with different testicular phenotypes and 24 normozoospermic men., Intervention(s): Testicular biopsies and semen analysis., Main Outcome Measure(s): Five testis-specific methylated promoters were selected. Promoter methylation was quantified using MethyLight in testicular DNA and paired cfsDNA, and the mRNA level was determined by real-time quantitative polymerase chain reaction., Result(s): Correlations of methylation of the selected five promoters between testicular DNA and paired cfsDNA were observed; and promoter methylation was negatively related to the messenger RNA level in testis. The cfsDNA methylation of these promoters showed different dynamic changes among the subtypes of NOA and normozoospermia. Among them, CCNA1 and DMRT1 promoter methylation in the hypospermatogenesis group was higher than in other groups and showed diagnostic potential for the patient with hypospermatogenesis., Conclusion(s): Cell-free seminal DNA could be a novel, noninvasive biomarker for the detection of testicular epigenetic aberrations. Epigenetic information in cfsDNA related to spermatogenesis may serve to predict successful testicular sperm retrieval in NOA patients., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Cell-free seminal mRNA (cfs-mRNA) contains testis-specific transcripts from bilateral testes. This study determined the presence of DEAD box polypeptide 4 (DDX4) in cfs-mRNA to identify and characterize the incidence of Sertoli cell-only (SCO) syndrome in men with non-obstructive azoospermia (NOA). DDX4 cfs-mRNA was determined in 315 men with NOA, and compared with testicular samples obtained by microdissection from 19 NOA patients. Karyotype and azoospermia factor microdeletion analysis were performed, and clinical features were evaluated. Negative DDX4 cfs-mRNA suggestive of SCO was found in 13.7% of NOA patients, with a similar incidence in NOA men with known genetic causes and those without known genetic causes. DDX4 cfs-mRNA was absent in 44% of SCO cases diagnosed by testicular histopathology, but present in all patients presenting with maturation arrest or hypospermatogenesis. Furthermore, 84.2% of NOA men with DDX4 cfs-positive mRNA had a DDX4-positive testicular sample. In NOA men without genetic causes, SCO patients discriminated by negative DDX4 cfs-mRNA showed different clinical features when compared with non-SCO cases. These results suggest that the evaluation of DDX4 cfs-mRNA is more accurate than testicular histopathology in discriminating SCO, and also permits the identification of a specific group of NOA men with distinct clinical features., (Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Y-chromosomal microdeletion (YCM) serves as an important genetic factor in non-obstructive azoospermia (NOA). Multiplex polymerase chain reaction (PCR) is routinely used to detect YCMs by tracing sequence-tagged sites (STSs) in the Y chromosome. Here we introduce a novel methodology in which we sequence 1,787 (post-filtering) STSs distributed across the entire male-specific Y chromosome (MSY) in parallel to uncover known and novel YCMs. We validated this approach with 766 Chinese men with NOA and 683 ethnically matched healthy individuals and detected 481 and 98 STSs that were deleted in the NOA and control group, representing a substantial portion of novel YCMs which significantly influenced the functions of spermatogenic genes. The NOA patients tended to carry more and rarer deletions that were enriched in nearby intragenic regions. Haplogroup O2* was revealed to be a protective lineage for NOA, in which the enrichment of b1/b3 deletion in haplogroup C was also observed. In summary, our work provides a new high-resolution portrait of deletions in the Y chromosome.

Non-obstructive azoospermia (NOA), a severe form of male infertility, is often suspected to be linked to currently undefined genetic abnormalities. To explore the genetic basis of this condition, we successfully sequenced ~650 infertility-related genes in 757 NOA patients and 709 fertile males. We evaluated the contributions of rare variants to the etiology of NOA by identifying individual genes showing nominal associations and testing the genetic burden of a given biological process as a whole. We found a significant excess of rare, non-silent variants in genes that are key epigenetic regulators of spermatogenesis, such as BRWD1, DNMT1, DNMT3B, RNF17, UBR2, USP1 and USP26, in NOA patients (P = 5.5 × 10(-7)), corresponding to a carrier frequency of 22.5% of patients and 13.7% of controls (P = 1.4 × 10(-5)). An accumulation of low-frequency variants was also identified in additional epigenetic genes (BRDT and MTHFR). Our study suggested the potential associations of genetic defects in genes that are epigenetic regulators with spermatogenic failure in human.

Background: Cell-free seminal mRNA (cfs-mRNA) exists in human ejaculate at high concentrations and with high stability, and contains many tissue-specific transcripts secreted from the male reproductive system. Owing to the sensitivity of RNA technology, cfs-mRNAs are ideal candidates for non-invasive biomarkers of physiopathological conditions. This study applied cfs-mRNA in identifying the presence of either germ cells or complete obstruction in men with azoospermia., Methods: RT-PCR was performed to amplify the germ cell-specific (DDX4), seminal vesicle-specific (SEMG1) and prostate-specific (TGM4) mRNAs from cfs-mRNAs, which were isolated from the seminal plasma of men with non-obstructive azoospermia (NOA) or obstructive azoospermia (OA). The 39 patients with NOA, diagnosed by testicular biopsy, included 8 men with maturation arrest (MA), 3 men with incomplete sertoli cell only (iSCO) syndrome and 28 men with complete SCO (cSCO). The 29 patients with OA, confirmed by the presence of sperm in the testis or epididymis, included 8 men with congenital bilateral absence of the vas deferens (CBAVD) and 21 men with non-CBAVD. Healthy individuals and vasectomized men were enrolled as controls., Results: TGM4 was detected in all participants. Consistent with their diagnosis, DDX4 was detected in all patients with MA or iSCO but was absent in most cases of cSCO (n = 21, 75.0%) or non-CBAVD (n = 18, 85.7%), and in all men with vasectomy or CBAVD. The presence of DDX4 in the other seven men with cSCO and three patients with non-CBAVD suggests the presence of germ cells in the testis, and incomplete obstruction, respectively. SEMG1 was undetectable in three patients with CBAVD with bilateral absence of the seminal vesicles, and in two non-CBAVD cases with low ejaculate volume., Conclusions: These results suggest that, with high sensitivity and representativity, cfs-mRNA could be non-invasive biomarkers for identifying the presence of germ cells or complete obstruction in azoospermia.