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

BACKGROUND: 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. OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Rapid cryopreservation of small quantities of human spermatozoa by a self-prepared cryoprotectant without animal component.

Cryopreservation of small quantities of human spermatozoa whilst maintaining adequate post-thawing motility has been found an essential challenge for male fertility preservation. Therefore, the study used an effective, and convenient rapid-freezing method to freeze small amounts of human spermatozoa by adding self-prepared cryoprotectant (SPC) without animal component. In the feasibility experiment, no significant differences in progressive motility, normal sperm morphology, vitality or DNA fragmentation index between the conventional slow freezing and rapid freezing were realised. The present study prospectively analysed the effects of sperm freezing and resuscitation in 175 patients with severe oligozoospermia (sperm concentration <1 × 106 /ml). We observed the 120 severe oligozoospermia specimens had a mean recovery rate of 60.19% ± 10.43% and a mean cryosurvival rate of 68.0% ± 9.17%. In addition, 55 cryptozoospermia specimens were analysed. The small-volume cryopreservation showed advantages. The total sperm recovery, motility recovery and sperm loss rates were 98.48%, 50.17% and 1.52% respectively. In short, the SPC is safe and effective, and can be used to rapidly freeze severe oligozoospermia specimens. That is useful for successful sperm freezing whilst avoiding the risk of azoospermia in the later stages and promoting comprehensive fertility preservation.

Author Correction: Loss-of-function mutations in QRICH2 cause male infertility with multiple morphological abnormalities of the sperm flagella.

In the original version of this Article, the Acknowledgements section omitted the National Key Research and Development Program of China (SQ2018YFC1003603).

CFTR mutation compromises spermatogenesis by enhancing miR-15b maturation and suppressing its regulatory target CDC25A†.

MicroRNAs (miRNAs) have recently been shown to be important for spermatogenesis; both DROSHA and Dicer1 KO mice exhibit infertility due to abnormal miRNA expression. However, the roles of individual miRNAs in spermatogenesis remain elusive. Here we demonstrated that miR-15b, a member of the miR-15/16 family, is primarily expressed in testis. A miR-15b transgenic mouse model was constructed to investigate the role of miR-15b in spermatogenesis. Impaired spermatogenesis was observed in miR-15b transgenic mice, suggesting that appropriate expression of miR-15b is vital for spermatogenesis. Furthermore, we demonstrated that overexpression of miR-15b reduced CDC25A gene post-transcriptional activity by targeting the 3'-UTR region of CDC25A, thus regulating spermatogenesis. In vitro results further demonstrated that a mutation in CFTR could affect the interaction between Ago2 with Dicer1 and that Dicer1 activity regulates miR-15b expression. We extended our study to azoospermia patients and found that infertile patients have a significantly higher level of miR-15b in semen and plasma samples. Taken together, we propose that CFTR regulation of miR-15b could be involved in the post-transcriptional regulation of CDC25A in mammalian testis and that miR-15b is important for spermatogenesis.

Loss-of-function mutations in QRICH2 cause male infertility with multiple morphological abnormalities of the sperm flagella.

Aberrant sperm flagella impair sperm motility and cause male infertility, yet the genes which have been identified in multiple morphological abnormalities of the flagella (MMAF) can only explain the pathogenic mechanisms of MMAF in a small number of cases. Here, we identify and functionally characterize homozygous loss-of-function mutations of QRICH2 in two infertile males with MMAF from two consanguineous families. Remarkably, Qrich2 knock-out (KO) male mice constructed by CRISPR-Cas9 technology present MMAF phenotypes and sterility. To elucidate the mechanisms of Qrich2 functioning in sperm flagellar formation, we perform proteomic analysis on the testes of KO and wild-type mice. Furthermore, in vitro experiments indicate that QRICH2 is involved in sperm flagellar development through stabilizing and enhancing the expression of proteins related to flagellar development. Our findings strongly suggest that the genetic mutations of human QRICH2 can lead to male infertility with MMAF and that QRICH2 is essential for sperm flagellar formation.

[Advantages of cryopreservation technology for rare human sperm].

OBJECTIVE: To search for an effective method for cryopreservation of rare human sperm (RHS) by comparing the effect of RHS cryopreservation technology with that of conventional cryopreservation technology on post-thaw sperm from patients with severe oligozoospermia. METHODS: Semen samples obtained from 82 patients with severe oligozoospermia were preserved by RHS cryopreservation technology, and another 24 samples cryopreserved by conventional technology, the former divided into groups A (sperm concentration < 1×106/ml, n = 54) and B (1×106/ml ≤ sperm concentration < 5×106/ml, n = 28), and the latter included in group C (sperm concentration < 15×106 /ml, n = 24). The survival rate of post-thaw sperm and recovery rate of progressively motile sperm (PMS) were compared among the three groups. RESULTS: The survival rate of post-thaw sperm was significantly higher in groups A and B than in C (ï¼»62.8 ± 18.7ï¼½% and ï¼»61.9 ± 17.2ï¼½% vs ï¼»50.7 ± 13.5ï¼½%, P < 0.05), and so was the recovery rate of PMS (ï¼»68.7 ± 18.4ï¼½% and ï¼»70.7 ± 15.5ï¼½% vs ï¼»29.2 ± 12.4ï¼½% , P < 0.05), but there were no statistically significant differences between groups A and B in either of the two parameters (P > 0.05). CONCLUSIONS: The cryopreservation technology for rare human sperm may yield relatively stable post-thaw results and deserves a wide clinical application in preserving male fertility.

Sperm gamma-aminobutyric acid type A receptor delta subunit (GABRD) and its interaction with purinergic P2X2 receptors in progesterone-induced acrosome reaction and male fertility.

The mechanism underlying the non-genomic action of progesterone in sperm functions and related Ca2+ mobilisation remains elusive. Herein we report the expression of gamma-aminobutyric acid type A receptor delta subunit (GABRD) in human and rodent sperm and its involvement in mediating the progesterone-induced acrosome reaction. GABRD was localised in the sperm head/neck region. A δ(392-422)-specific inhibitory peptide against GABRD blocked the progesterone-induced acrosome reaction and the associated increase in intracellular Ca2+. Similarly, an inhibitory effect against both progesterone-induced Ca2+ influx and the acrosome reaction was observed with a P2X2 receptor antagonist. The lack of synergism between the GABRD and P2X2 inhibitors suggests that these two receptors are playing a role in the same pathway. Furthermore, a co-immunoprecipitation experiment demonstrated that GABRD could undergo protein-protein interactions with the Ca2+-conducting P2X2 receptor. This interaction between the receptors could be reduced following progesterone (10µM) inducement. Significantly reduced GABRD expression was observed in spermatozoa from infertile patients with reduced acrosome reaction capacity, suggesting that normal expression of GABRD is critical for the sperm acrosome reaction and thus male fertility. The results of the present study indicate that GABRD represents a novel progesterone receptor or modulator in spermatozoa that is responsible for the progesterone-induced Ca2+ influx required for the acrosome reaction through its interaction with the P2X2 receptor.

Efficient transfection of DNA into primarily cultured rat sertoli cells by electroporation.

The expression of exogenous DNA in Sertoli cells is essential for studying its functional genomics, pathway analysis, and medical applications. Electroporation is a valuable tool for nucleic acid delivery, even in primarily cultured cells, which are considered difficult to transfect. In this study, we developed an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation under different conditions. After transfection of plasmid into Sertoli cells, enhanced green fluorescent protein (EGFP) expression could be easily detected by fluorescent microscopy, and cell survival was evaluated by dye exclusion assay using Trypan blue. In terms of both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping the voltage constant (250 V), relatively high cell survival (76.5% ± 3.4%) and transfection efficiency (30.6% ± 5.6%) were observed with a pulse length of 20 µm. The number of pulses significantly affected cell survival and EGFP expression (P < 0.001). Cell survival clearly decreased following one to three pulses, from 83.9% ± 6.1% to 3.2% ± 1.1%, with EGFP expression increasing from 41.8% ± 9.4% to 66.7% ± 5.2%. The yield of positive cells increased with increasing concentration of plasmid DNA (range, 10-50 µg/ml), from 14.0% ± 2.8% to 35.0% ± 6.3%, but cell viability steadily decreased following 20 µg/ml plasmid DNA, from 73.1% ± 4.9% to 57.0% ± 6.6%. Compared with two popular cationic lipid transfection methods, the transfection efficiency of electroporation (21.5% ± 5.7%) was significantly higher than those of Lipofectamine 2000 (2.9% ± 1.0%) and Effectene (1.9% ± 0.8%) in this experiment (P < 0.001). We describe the process of optimizing electroporation conditions, and the successful electroporation of plasmid DNA into primarily cultured Sertoli cells. Our results indicate that the method of electroporation is more suitable than other approaches for the transfection of Sertoli cells.

Significant improvement of sperm DNA quality after microsurgical repair of varicocele.

Varicocele is currently the most common irregularity identified in males that is associated with impaired spermatogenesis. It primarily presents in the form of decreased sperm count and motility, abnormal morphology, and significantly increased sperm DNA fragmentation. Several studies have shown that surgical repair improves semen parameters and increases the odds of spontaneous pregnancy. However the exact effect of surgical repair treatment remains controversial. Therefore, the aim of our study was to evaluate the effectiveness of microsurgical repair by comparing common semen parameters and sperm DNA fragmentation index (DFI). We evaluated infertile men (n = 19) who underwent microsurgical subinguinal varicocelectomy for treatment of clinical varicocele before and 3 months after surgery. Normozoospermic men (n = 19) were considered as the normal control group. Semen parameters improved significantly after surgery when compared with that before surgery, but still significant differences with the normal control group were observed. In comparison, sperm DNA integrity improved significantly after surgery (percentage DFI decreased from 28.4 ± 15.6% before surgery to 22.4 ± 12.9%, at 3 months post surgery) to similar levels as the normal control group. These results suggest that microsurgical repair may be considered as a treatment option in infertile men with palpable varicocele.

Effect of varicocelectomy on testicular volume in children and adolescents: a meta-analysis.

OBJECTIVE: To evaluate the effect of surgical intervention on catch-up growth as determined by a decreased testicular volume discrepancy in children and adolescents with varicocele. METHODS: A systematic search was performed using MEDLINE and the PubMed database and cross-referenced as of October 28, 2011 using the terms "varicocele," "children," "adolescent," "surgery," and "testicular volume." All relevant studies were of the testicular volume discrepancy variance before and after surgical repair. The outcomes included the number of patients with initial testicular atrophy and those with catch-up growth after surgical repair. The database search, quality evaluation, and data extraction were independently performed by 2 reviewers. RESULTS: Of 75 studies, 14 were included for analysis and involved 1475 patients. The combined analysis showed that the testicular volume discrepancy was significantly reduced after surgery in the ≥10% group (P < .00001) and ≥20% group (P < .00001), respectively. No difference was found between the 2 groups (P = .70). Taken together, the number of patient with testicular volume disproportion in all pediatric and adolescent varicocele patients significantly decreased after surgery (P < .00001). The average proportion of catch-up growth was 76.4% (range 52.6%-93.8%). CONCLUSION: The meta-analysis suggested clear advantages of surgical intervention on reducing testicular hypotrophy when the discrepancy is ≥10% in children and adolescents with varicocele. Additional prospective and controlled studies are warranted to elucidate the treatment of children and adolescents with varicocele.

Effect of surgical repair on testosterone production in infertile men with varicocele: a meta-analysis.

OBJECTIVES: To determine the effect of surgical varicocele repair in improving testicular Leydig cell function as shown by increased testosterone production. METHODS: Eligible studies were searched in Medline and the Pubmed database, and cross-referenced as of 31 May 2011 using the terms "varicocele,""testosterone" and "surgery." The database search, quality assessment and data extraction were independently carried out by two reviewers. Only studies including patients with testosterone evaluation before and after surgery were considered for the analysis. A systematic review and meta-analysis was carried out for continues variables using random effect models. RESULTS: Out of 125 studies, a total of nine were selected, including 814 patients. The combined analysis showed that mean serum testosterone levels after surgical treatment increased by 97.48 ng/dL (95% confidence interval 43.73-151.22, P=0.0004) compared with preoperative levels. CONCLUSIONS: Surgical treatment of varicocele significantly increases testosterone production and improves testicular Leydig cell function.

[Influencing factors of sperm membrane fluidity].

Sperm membrane fluidity is one of the causes of male infertility, and it is thought to be related with temperature, reactive oxygen species, oxygen free radicals, anti-sperm antibodies, stilbestrol, and fenvalerate. A deeper insight into the influencing factors of sperm membrane fluidity is of vital importance for in vitro sperm preservation, revival of frozen-thawed sperm, in vitro fertilization and management of male infertility.

Homeostasis model assessment-insulin resistance (HOMA-IR), a key role for assessing the ovulation function in polycystic ovary syndrome (PCOS) patients with insulin resistance.

According to our research, we evaluated that for the ovulation function in polycystic ovary syndrome (PCOS) with IR, Homeostasis model assessment-insulin resistance (HOMA-IR) is a clinic , simple and practical and sensitive Index for assessing the ovualtion failure. Meanwhile, after anti-IR treatment, HOMA-IR is also a reliable and simple for accessing the recoverying ovulation function.

[Xianling Gubao Capsule improves semen quality].

OBJECTIVE: To observe the effect of the compound Chinese traditional drug Xianling Gubao Capsule on the semen quality of infertile males. METHODS: We treated 66 infertile men with Xianling Gubao Capsule for 24 months, and analyzed the semen quality and sperm morphology before and after the treatment. RESULTS: Two months after the medication, sperm concentration was increased by a small margin, but no statistically significant changes were observed in sperm vitality and motility (P > 0.05), the rate of morphologically normal sperm was significantly raised from 25.8% before treatment to 57.6% (P < 0.05) in those with the normal rate > or = 15%, but decreased from 53.0% to 25.8% (P < 0.05) in those with the normal rate < 9%. Among the 7 cases of oligospermia, the rate of morphologically normal sperm was elevated to an average of 10.9% after the 4-month medication, significantly different from the baseline rate of 5.8% (P < 0.05). Five spontaneous pregnancies and 1 successful IVF-ICSI were achieved during the treatment. CONCLUSION: Xianling Gubao Capsule can improve semen quality and significantly increase the percentage of morphologically normal sperm.

[Influence of cryoprotectant with glycerol and freezing-thawing procedure on the motility of human sperm].

OBJECTIVE: To investigate the effects of cryoprotectant with glycerol and the freezing-thawing procedure on the motility of METHODS: The motion characteristics of human sperm from 18 selected specimens were assessed by computer-assisted human sperm. sperm analysis before and after adding hyper-osmolarity cryoprotectant with glycerol and the freeze-thaw procedure, and the data were evaluated in pairs. RESULTS: The adding of cryoprotectant caused an increase in the proportion of rapid linear motile sperms (P < 0.05) and sperm velocities, including VCL, VSL and VAP (P < 0.005). But no changes were observed in the proportion of progressively motile sperms and sperm motility. Compared with the data from pre-frozen samples, velocities of post-thawed sperms and the percentage of motile sperms in each grade significantly declined (P < or = 0.01), so did ALH, while WOB, LIN and STR remained unchanged (P > 0.05). Significant differences in ALH, WOB, LIN and STR were observed only in comparison between the post-thawed and pre-treated samples. CONCLUSION: The number of Grade a sperms and sperm velocity increased after adding hyper-osmolarity cryoprotectant with glycerol. The sperm motile potential was impaired and even entirely destroyed in some cases by cryodamage. The influences on the sperm motion were differently induced in freezing-thawing procedures.

[Semen quality and sperm morphologic study of infertile men with varicocele].

OBJECTIVE: To examine the semen quality and the sperm morphology in infertile men with varicocele. METHODS: Semen from 98 infertile men with varicocele were studied and those of 130 normal semen donors were taken as the control. Semen analysis was performed based on the methods described in the WHO manual and sperm morphology was evaluated by WHO criteria. RESULTS: A significantly reduced percentage of normal morphologic sperm and of forward progression were found in patients with varicocele comparing with those of the control (P <0.001). The head defects were observed as the predominant type of sperm malformation. CONCLUSION: The varicocele increases malformed sperm in ejaculates, which may result from impaired male fertility by varicocele. Sperm morphologic assessment with WHO criteria provides a sensitive and practical measurement of sperm damage in infertile men with varicocele.