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Spermatogenesis is precisely controlled by sophisticated gene expression programs and is driven by epigenetic reprogramming, including histone modification alterations and histone-to-protamine transition. Nuclear receptor binding SET domain protein 2 (Nsd2) is the predominant histone methyltransferase catalyzing H3K36me2 and its role in male germ cell development remains elusive. Here, we report that NSD2 protein is abundant in spermatogenic cells. Conditional loss of Nsd2 in postnatal germ cells impaired fertility owing to apoptosis of spermatocytes and aberrant spermiogenesis. Nsd2 deficiency results in dysregulation of thousands of genes and remarkable reduction of both H3K36me2 and H3K36me3 in spermatogenic cells, with H3K36me2 occupancy correlating positively with expression of germline genes. Nsd2 deficiency leads to H4K16ac elevation in spermatogenic cells, probably through interaction between NSD2 and PSMA8, which regulates acetylated histone degradation. We further reveal that Nsd2 deficiency impairs EP300-induced H4K5/8ac, recognized by BRDT to mediate the eviction of histones. Accordingly, histones are largely retained in Nsd2-deficient spermatozoa. In addition, Nsd2 deficiency enhances expression of protamine genes, leading to increased protamine proteins in Nsd2-deficient spermatozoa. Our findings thus reveal a previously unappreciated role of the Nsd2-dependent chromatin remodeling during spermatogenesis and provide clues to the molecular mechanisms in epigenetic abnormalities impacting male reproductive health.

Sperm DNA injury is one of the common causes of male infertility. Folic acid deficiency would increase the methylation level of the important genes, including those involved in DNA double-strand break (DSB) repair pathway. In the early stages, we analysed the correlation between seminal plasma folic acid concentration and semen parameters in 157 infertility patients and 91 sperm donor volunteers, and found that there was a significant negative correlation between seminal folic acid concentration and sperm DNA Fragmentation Index (DFI; r = -0.495, p 0.01). Then through reduced representation bisulphite sequencing, global DNA methylation of sperm of patients in the low folic acid group and the high folic acid group was analysed, it was found that the methylation level in Rad54 promoter region increased in the folic acid deficiency group compared with the normal folic acid group. Meanwhile, the results of animal model and spermatocyte line (GC-2) also found that folic acid deficiency can increase the methylation level in Rad54 promoter region, increased sperm DFI in mice, increased the expression of γ-H2AX, that is, DNA injury marker protein, and increased sensitivity of GC-2 to external damage and stimulation. The study indicates that the expression of Rad54 is downregulated by folic acid deficiency via DNA methylation. This may be one of the mechanisms of sperm DNA damage caused by folate deficiency.

Otogelin-like protein (encoded by Otogl) was highly structural similar to the gelforming mucin proteins. Although human OTOG mutations have been linked to deafness, the biological function of OTOGL in male germ cell development remains enigmatic. In screening 336 patients with non-obstructive azoospermia (NOA), OTOGL displays the high mutant ratio (13.99 %). Then, we examined the expression of OTOGL in developing mouse testes. Otogl mRNA and protein are continually expressed in postnatal developing testes from postnatal day 0 (P0) testes to P21 testes exhibiting a decreased trend with the age growth. We thus generated a global Otogl knockout mouse (KO) model using the CRISPR/Cas9 technology; however, Otogl KO mice displayed normal development and fertility. Further histological analysis of Otogl knockout mouse testes revealed that all types of spermatogenic cells are present in Otogl KO seminiferous tubules. Together, our study suggested that OTOGL is nonessential for male germ cell development and spermatogenesis.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe type of teratozoospermia. In this study, whole-exome sequencing was conducted on 55 patients with MMAF, and biallelic mutations of CFAP58 were identified in two patients. The variants are rare and pathogenic, and CFAP58 was absent in the CFAP58-mutated sperm. The F037/II:1 couple benefited from intracytoplasmic sperm injection (ICSI). This study further indicated that CFAP58 is a pathogenic gene associated with MMAF and ICSI is an effective treatment.

STUDY QUESTION Whether the testis-specific extracellular vesicle (EV) long noncoding RNAs (lncRNAs) in seminal plasma could be utilized to predict the presence of testicular spermatozoa in nonobstructive azoospermia (NOA) patients? SUMMARY ANSWER Our findings indicate that the panel based on seminal plasma EV lncRNAs was a sensitive and specific method in predicting the presence of testicular spermatozoa and may improve clinical decision-making of NOA. WHAT IS KNOWN ALREADY The adoption of sperm retrieval techniques, especially microdissection testicular sperm extraction (mTESE), in combination with ICSI has revolutionized treatment for NOA. However, there are no precise and noninvasive methods for predicting whether there are testicular spermatozoa in NOA patients before mTESE. STUDY DESIGN, SIZE, DURATION RNA sequencing was performed on seminal plasma EVs from 6 normozoospermic men who underwent IVF due to female factor and 5 idiopathic NOA patients who failed to obtain testicular spermatozoa by mTESE and were diagnosed as having Sertoli cell-only syndrome by postoperative pathology. A biomarker panel of lncRNAs was constructed and verified in 96 NOA patients who underwent mTESE. Decision-making process was established based on the panel in seminal plasma EVs from 45 normozoospermia samples, 43 oligozoospermia samples, 62 cryptozoospermia samples, 96 NOA samples. PARTICIPANTS/MATERIALS, SETTING, METHODS RNA sequencing was done to examine altered profiles of EV lncRNAs in seminal plasma. Furthermore, a panel consisting of EV lncRNAs was established and evaluated in training set and validation sets. MAIN RESULTS AND THE ROLE OF CHANCE A panel consisting of nine differentially expressed testis-specific lncRNAs, including LOC100505685, SPATA42, CCDC37-DT, GABRG3-AS1, LOC440934, LOC101929088 (XR_927561.2), LOC101929088 (XR_001745218.1), LINC00343 and LINC00301, was established in the training set and the AUC was 0.986. Furthermore, the AUC in the validation set was 0.960. Importantly, the panel had a unique advantage when compared with models based on serum hormones from the same group of NOA cases (AUC, 0.970 vs 0.723; 0.959 vs 0.687, respectively). According to the panel of lncRNAs, a decision-making process was established, that is when the score of an NOA case exceeds 0.532, sperm retrieval surgery may be recommended. LIMITATIONS, REASONS FOR CAUTION In the future, the sample size needs to be further expanded. Meanwhile, the regulatory functions and mechanism of lncRNAs in spermatogenesis also need to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS When the score of our panel is below 0.532, subjecting the NOA patients to ineffective surgical interventions may not be recommended due to poor sperm retrieval rate. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (81871110, 81971314 and 81971759); the Guangdong Special Support Plan-Science and Technology Innovation Youth Top Talents Project (2016TQ03R444); the Science and Technology Planning Project of Guangdong Province (2016B030230001 and 201707010394); the Key Scientific and Technological Program of Guangzhou City (201604020189); the Pearl River S&T Nova Program of Guangzhou (201806010089); the Transformation of Scientific and Technological Achievements Project of Sun Yat-sen University (80000-18843235) and the Youth Teacher Training Project of Sun Yat-sen University (17ykpy68 and 18ykpy09). There are no competing interests related to this study. TRIAL REGISTRATION NUMBER N/A.

Objective. To explore the risk of Ureaplasma urealyticum (UU) affecting sperm quality. Methods. Prospective cross-sectional study was conducted. In total, 340 semen samples were collected. According to whether they were infected with UU, the samples were divided into the UU-positive group (observation group) and UU-negative group (control group). The patients with UU-positive were followed up to obtain treatment and collected the semen again after treatment. The semen characteristics and sperm parameters were detected and compared, and the relationship of UU and the sperm quality was analyzed by mathematical models. Results. There were 104 UU-positive semen samples in all, with an overall infection rate of 30.6%, which was highest in 31 to 40-year-old men, and over 40-year-old men were the lowest. The pH, PR, VCL, VSL, and STR in the observation group were significantly lower than those in the control group (all P < 0.001 ), while SV, NP, and WOB were significantly higher (all P < 0.001 ). After treatment, the pH, VSL, LIN, WOB, and STR in the observation group were significantly higher than before (all P < 0.001 ), while SV and VCL were significantly lower (all P < 0.001 ). UU infection was closely correlated with pH, PR, NP, VCL, VSL, WOB, and STR. During the treatment, pH, PR, VSL, WOB, and STR increased, but NP and VCL decreased. 7 major factors that would affect SQ were extracted, of which VAP, LIN, and UU were the first three main factors. The risk of SQ declining after UU infection increased nearly twice with the change of PR and VCL and increased 0.08 times with STR. Conclusion. UU may approximately double the risk of altering the sperm’s curvilinear movement rate and straightness to affect the sperm quality.

Rare coding variants have been proven to be one of the significant factors contributing to spermatogenic failure in patients with non-obstructive azoospermia (NOA) and severe oligospermia (SO). To delineate the molecular characteristics of idiopathic NOA and SO, we performed whole-exome sequencing of 314 unrelated patients of Chinese Han origin and verified our findings by comparing to 400 fertile controls. We detected six pathogenic/likely pathogenic variants and four variants of unknown significance, in genes known to cause NOA/SO, and 9 of which had not been earlier reported. Additionally, we identified 20 novel NOA candidate genes affecting 25 patients. Among them, five (BRDT, CHD5, MCM9, MLH3 and ZFX) were considered as strong candidates based on the evidence obtained from murine functional studies and human single-cell (sc)RNA-sequencing data. These genetic findings provide insight into the aetiology of human NOA/SO and pave the way for further functional analysis and molecular diagnosis of male infertility.

The majority of circular RNAs (circRNAs) spliced from coding genes contain open reading frames (ORFs) and thus, have protein coding potential. However, it remains unknown what regulates the biogenesis of these ORF-containing circRNAs, whether they are actually translated into proteins and what functions they play in specific physiological contexts. Here, we report that a large number of circRNAs are synthesized with increasing abundance when late pachytene spermatocytes develop into round and then elongating spermatids during murine spermatogenesis. For a subset of circRNAs, the back splicing appears to occur mostly at m6A-enriched sites, which are usually located around the start and stop codons in linear mRNAs. Consequently, approximately a half of these male germ cell circRNAs contain large ORFs with m6A-modified start codons in their junctions, features that have been recently shown to be associated with protein-coding potential. Hundreds of peptides encoded by the junction sequences of these circRNAs were detected using liquid chromatography coupled with mass spectrometry, suggesting that these circRNAs can indeed be translated into proteins in both developing (spermatocytes and spermatids) and mature (spermatozoa) male germ cells. The present study discovered not only a novel role of m6A in the biogenesis of coding circRNAs, but also a potential mechanism to ensure stable and long-lasting protein production in the absence of linear mRNAs, i.e., through production of circRNAs containing large ORFs and m6A-modified start codons in junction sequences.

BACKGROUND: Because of focal spermatogenesis in some nonobstructive azoospermia (NOA) patients, testicular spermatozoa can be retrieved by microdissection testicular sperm extraction (micro-TESE) for intracytoplasmic sperm injection (ICSI) to achieve successful fertilization. Currently, testicular biopsy is widely performed for the prognosis of micro-TESE; however, it might miss foci with active spermatogenesis because of the ‘blind manner’ of puncture, highlighting the needs for biomarkers that could indicate actual spermatogenesis conditions in the testis. Thus, we screened microRNAs in the seminal plasma for potential biomarkers to provide a non-invasive and reliable preoperative assessment for micro-TESE. METHODS: We screened the seminal plasma microRNAs from NOA patients with and without sperm retrieval (n=6 in each group) together with fertile men (n=6) by RNA sequencing, and the selected microRNAs were validated by quantitative polymerase chain reaction (qPCR). Next, a predictive model was established by performing ordered logistic regression using the qPCR data of 56 specimens, and the predictive accuracy of this model was evaluated using 40 more specimens in a blind manner. RESULTS: Four microRNAs (hsa-miR-34b-3p, hsa-miR-34c-3p, hsa-miR-3065-3p, and hsa-miR-4446-3p) were identified as biomarkers, and the predictive model Logit = 2.0881+ 0.13448 mir-34b-3p + 0.58679 mir-34c-3p + 0.15636 mir-3065-3p + 0.09523 mir-4446-3p was established by machine learning. The model provided a high predictive accuracy (AUC =0.927). CONCLUSIONS: We developed a predictive model with high accuracy for micro-TESE, with which NOA patients might obtain accurate assessment of spermatogenesis conditions in testes before surgery.

The influence of varicocele and microsurgical varicocelectomy on semen quality remains unclear. No previous study has investigated the relationship between semen metabolism and the abnormalities in reproductive function caused by varicocele. Here, we used the non-targeted and targeted metabolic analysis to investigate the different metabolites in seminal plasma within normal, varicocele, and varicocelectomy groups. We clearly showed that varicocele significantly affects sperm metabolism, and microsurgical varicocelectomy can reverse this metabolic abnormality. Moreover, we characterized the landscape of three dipeptides in the seminal plasma of patients with varicocele that have not been identified previously in human tissues or biofluids. Interestingly, the levels of these three dipeptides decreased after microsurgical varicocelectomy coincident with an improvement in semen quality. Western blotting confirmed the downregulation of DPEP3 (dipeptidase 3) in the varicocele group and the upregulation of DPEP3 in the varicocelectomy group. Furthermore, we found that eight metabolites may be helpful to distinguish varicocele patients from normal subjects. Our results can be applied to earlier diagnosis or to predict the outcome of microsurgery for varicocele.

Background: Depression is a potential factor affecting semen quality. However, the relationship between depression and semen quality remains to be fully elucidated. We investigated the association between depression and semen quality in men during the COVID-19 pandemic.Methods: Cross‐sectional, participants completed a questionnaire assessing lifestyle factors the Patient Health Questionnaire-9 (PHQ-9) was used to evaluate the psychological depression status of subjects. Semen quality assessment implements the requirements of the fifth edition of the World Health Organization (WHO )manual ,multivariable logistic regression analysis and linear regression analysis were conducted to assess the associations between depression and semen quality.Results: Of the 896 participants, 173 were depressed (19.30%). A Linear regression model was fitted to assess the strength and significant level of the association between depressive symptoms and semen quality. After adjusting for potential confounders, depression was significantly associated with 9.27 (95% confidence interval [CI] 3.39, 15.15), 0.57(95% CI 0.27, 0.87), 62.20 (95% CI 32.93, 91.48), and 12.96 (95% CI 10.55, 15.37) reduction in sperm concentration, sperm volume, total sperm count, and progressive motility, respectively. Which are consistent with the logistic regression analysis results. The semen concentration and total sperm count were linearly correlated with depression scores, whereas the semen volume and progressive motility of sperm were nonlinearly correlated with depression scores.Conclusions: Depression were associated with lower levels of semen concentration, semen volume, total sperm count, and progressive motility, which could affect male reproductive health.

Objectives: There is no report about association of anxiety on sperm quality during the COVID-19 epidemic. Purpose of this study was to investigate the relationship between anxiety and sperm quality during the COVID-19 outbreak in China.Methods: 896 sperm donors from 7 sperm banks in China were investigated, and passed the screening for sperm donation between 23 January 2020 and 8 June 2020. Semen quality analysis follows the standards of the WHO Fifth Edition Human Semen Examination and Processing Manual. Anxiety symptoms were assessed by the Generalized Anxiety Disorder Scal-7(GAD-7). Logistic regression and linear regression were used to identify the association between anxiety level and sperm quality.Results: Participants with anxiety status (N = 155; 17.3%) had lower sperm concentration (56.81 ± 32.70 vs. 47.21 ± 30.76 [106/mL]), sperm volume (3.58 ± 1.65 vs. 3.04 ± 1.69 [mL], P < 0.001), total sperm count (7.29 ± 1.23 vs. 6.56 ± 1.63 [106], P < 0.001), and progressive motility (50.86 ± 12.45 vs. 38.50 ± 17.00 [%], P < 0.001) than those without anxiety. Linear regression showed that anxiety symptoms was significantly associated with 8.13 (95% confidence interval [CI]: 2.51, 13.75), 0.49 (0.20, 0.79) ,51.05 (23.43, 78.68) and 12.15 (95% CI: 23.43, 78.68), reduction in sperm concentration, semen volume, and total sperm count and progressive motility respectively.Conclusions: This study revealed that anxiety symptoms were negatively associated with semen quality, and interaction was discovered between debt stress and anxiety.

Otogelin-like protein (encoded by Otogl) was highly structural similar to the gelforming mucin proteins. Although human OTOG mutations have been linked to deafness, the biological function of OTOGL in male germ cell development remains enigmatic. In screening 336 patients with non-obstructive azoospermia (NOA), OTOGL displays the high mutant ratio (13.99 %). Then, we examined the expression of OTOGL in developing mouse testes. Otogl mRNA and protein are continually expressed in postnatal developing testes from postnatal day 0 (P0) testes to P21 testes exhibiting a decreased trend with the age growth. We thus generated a global Otogl knockout mouse (KO) model using the CRISPR/Cas9 technology; however, Otogl KO mice displayed normal development and fertility. Further histological analysis of Otogl knockout mouse testes revealed that all types of spermatogenic cells are present in Otogl KO seminiferous tubules. Together, our study suggested that OTOGL is nonessential for male germ cell development and spermatogenesis.

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Asthenoteratospermia is an important cause of male infertility. Here, we report two infertile patients with severe asthenoteratospermia accompanied by new genetic abnormality. Whole-exome sequencing and bioinformatics analysis suggested that compound heterozygous mutations in DNAH8 (MIM:603337) may be responsible for multiple morphological abnormalities of the sperm flagella (MMAF). Immunofluorescence assay showed that DNAH8 protein expression was significantly decreased in the sperm tail of the patients, and electron microscopy exhibited an abnormal flagellum ultrastructure, while clinical pregnancy could be achieved by intracytoplasmic sperm injection. Therefore, the compound heterozygous mutations in the DNAH8 gene may be responsible for MMAF.

Of the numerous health benefits of garlic, the anticancer effect is probably the most noticeable. Observations over the past years have shown that the consumption of garlic in the diet provides strong protection against cancer risk. Previous studies involving garlic phytochemicals have usually focused on the cancer chemopreventive properties, but there is little published evidence showing its therapeutic potential in cancer treatment. In view of the multitargeted carcinoma actions and lack of severe toxicity, some components of garlic are likely to play vital roles in the selective killing of cancer cells. However, the rational design of experimental studies and clinical trials are required to verify this concept. This paper discusses the promises and pitfalls of garlic for the treatment of cancer.

Background: Exposure to residential greenness has been associated with benefits on certain reproductive health outcomes. However, its potential benefits on semen quality remain unknown. Objectives: To quantitatively explore the association between exposure to residential greenness and semen quality. Methods: We investigated 9142 sperm donation volunteers who underwent 38,682 semen examinations at Guangdong provincial human sperm bank in China during 2016–2019. Exposure to residential greenness was assessed using mean daily Normalized Difference Vegetation Index (NDVI) at each subject’s residential address with a 400 m buffer during 0–90 days before each semen collection. Multivariate linear mixed models and linear regression models were used to assess the association between exposure to residential greenness and semen quality. Results: An interquartile range increase in exposure to residential greenness was significantly associated with a 0.034 (95% confidence interval [CI]: 0.005, 0.063) ml, 4.06 (95% CI: 0.76, 7.37) × 106, and 0.32% (95% CI: 0.22%, 0.41%) increase in semen volume, total sperm number, and normal forms, respectively; similar trends were observed across quartiles of exposure to residential greenness (all p-values for liner trend

The majority of circular RNAs (circRNAs) spliced from coding genes contain open reading frames (ORFs) and thus, have protein coding potential. However, it remains unknown what regulates the biogenesis of these ORF-containing circRNAs, whether they are actually translated into proteins and what functions they play in specific physiological contexts. Here, we report that a large number of circRNAs are synthesized with increasing abundance when late pachytene spermatocytes develop into round and then elongating spermatids during murine spermatogenesis. For a subset of circRNAs, the back splicing appears to occur mostly at m(6)A-enriched sites, which are usually located around the start and stop codons in linear mRNAs. Consequently, approximately a half of these male germ cell circRNAs contain large ORFs with m(6)A-modified start codons in their junctions, features that have been recently shown to be associated with protein-coding potential. Hundreds of peptides encoded by the junction sequences of these circRNAs were detected using liquid chromatography coupled with mass spectrometry, suggesting that these circRNAs can indeed be translated into proteins in both developing (spermatocytes and spermatids) and mature (spermatozoa) male germ cells. The present study discovered not only a novel role of m(6)A in the biogenesis of coding circRNAs, but also a potential mechanism to ensure stable and long-lasting protein production in the absence of linear mRNAs, i.e., through production of circRNAs containing large ORFs and m(6)A-modified start codons in junction sequences.

Studies have explored the assisted reproductive technology (ART) outcomes of Y-chromosome azoospermia factor c (AZFc) microdeletions, but the effect of sperm source on intracytoplasmic sperm injection (ICSI) remains unknown. To determine the ART results of ICSI using testicular sperm and ejaculated sperm from males with AZFc microdeletions, we searched Embase, Web of Science, and PubMed to conduct a systematic review and meta-analysis. The first meta-analysis results for 106 cycles in five studies showed no significant differences in the live birth rate between the testicular sperm group and the ejaculated sperm group (risk ratio: 0.97, 95% confidence interval [CI]: 0.73–1.28, P = 0.82). The second meta-analysis of 106 cycles in five studies showed no difference in the abortion rate between the testicular sperm group and ejaculated sperm group (risk ratio: 1.06, 95% CI: 0.54–2.06, P = 0.87). The third meta-analysis of 386 cycles in seven studies showed no significant difference in clinical pregnancy rates between the testicular sperm group and the ejaculated sperm group (risk ratio: 1.24, 95% CI: 0.66–2.34, P = 0.50). Inevitable heterogeneity weakened our results. However, our results indicated that testicular sperm and ejaculated sperm yield similar ART outcomes, representing a meaningful result for clinical treatment. More properly designed studies are needed to further confirm our conclusions.

To explore the biological characteristics of the recombinant zona pellucida 3 (ZP3) peptides of rhuZP3a 22,176 and rhuZP3b 177,348 , we examined whether rhuZP3a 22,176 or rhuZP3b 177,348 trigger the acrosome reaction (AR) of human spermatozoa and we investigated the underlying mechanism. The assessment of AR was performed using chlortetracycline staining. The intracellular free calcium concentration ((Ca 2+ )i) in Fura-2/AM- loaded human sperm was monitored with a spectrofluorophotometer. We found that rhuZP3a 22,176 and rhuZP3b 177,348 were capable of eliciting AR at different concentrations. With the addition of either peptide, the (Ca 2+ )i level was raised to a peak with or without a plateau. The AR could be inhibited by pertussis toxin (PTX), EGTA, and pimozide (a T-type calcium channel blocker), whereas verapamil was less effective in this regard. The results of the present study suggest that peptides rhuZP3a 22,176 and rhuZP3b 177,348 have a role similar to human ZP3, and that the mechanism of the response to the peptides involves influx of calcium, the G protein pathway, and a T- type calcium channel.

To explore the biological characteristics of the recombinant zona pellucida 3 (ZP3) peptides of rhuZP3a22 approximately 176 and rhuZP3b177 approximately 348, we examined whether rhuZP3a22 approximately 176 or rhuZP3b177 approximately 348 trigger the acrosome reaction (AR) of human spermatozoa and we investigated the underlying mechanism. The assessment of AR was performed using chlortetracycline staining. The intracellular free calcium concentration ([Ca2+]i) in Fura-2/AM-loaded human sperm was monitored with a spectrofluorophotometer. We found that rhuZP3a22 approximately 176 and rhuZP3b177 approximately 348 were capable of eliciting AR at different concentrations. With the addition of either peptide, the [Ca2+]i level was raised to a peak with or without a plateau. The AR could be inhibited by pertussis toxin (PTX), EGTA, and pimozide (a T-type calcium channel blocker), whereas verapamil was less effective in this regard. The results of the present study suggest that peptides rhuZP3a22 approximately 176 and rhuZP3b177 approximately 348 have a role similar to human ZP3, and that the mechanism of the response to the peptides involves influx of calcium, the G protein pathway, and a T-type calcium channel.