Bi-allelic variants in DNHD1 cause flagellar axoneme defects and asthenoteratozoospermia in humans and mice.

Asthenoteratozoospermia, defined as reduced sperm motility and abnormal sperm morphology, is a disorder with considerable genetic heterogeneity. Although previous studies have identified several asthenoteratozoospermia-associated genes, the etiology remains unknown for the majority of affected men. Here, we performed whole-exome sequencing on 497 unrelated men with asthenoteratozoospermia and identified DNHD1 bi-allelic variants from eight families (1.6%). All detected variants were predicted to be deleterious via multiple bioinformatics tools. Hematoxylin and eosin (H&E) staining revealed that individuals with bi-allelic DNHD1 variants presented striking abnormalities of the flagella; transmission electron microscopy (TEM) further showed flagellar axoneme defects, including central pair microtubule (CP) deficiency and mitochondrial sheath (MS) malformations. In sperm from fertile men, DNHD1 was localized to the entire flagella of the normal sperm; however, it was nearly absent in the flagella of men with bi-allelic DNHD1 variants. Moreover, abundance of the CP markers SPAG6 and SPEF2 was significantly reduced in spermatozoa from men harboring bi-allelic DNHD1 variants. In addition, Dnhd1 knockout male mice (Dnhd1‒/‒) exhibited asthenoteratozoospermia and infertility, a finding consistent with the sperm phenotypes present in human subjects with DNHD1 variants. The female partners of four out of seven men who underwent intracytoplasmic sperm injection therapy subsequently became pregnant. In conclusion, our study showed that bi-allelic DNHD1 variants cause asthenoteratozoospermia, a finding that provides crucial insights into the biological underpinnings of this disorder and should assist with counseling of affected individuals.

Deleterious variants in X-linked RHOXF1 cause male infertility with oligo- and azoospermia.

Oligozoospermia and azoospermia are two common phenotypes of male infertility characterized by massive sperm defects owing to failure of spermatogenesis. The deleterious impact of candidate variants with male infertility is to be explored. In our study, we identified three hemizygous missense variants (c.388G>A: p.V130M, c.272C>T: p.A91V, and c.467C>T: p.A156V) and one hemizygous nonsense variant (c.478C>T: p.R160X) in the Rhox homeobox family member 1 gene (RHOXF1) in four unrelated cases from a cohort of 1201 infertile Chinese men with oligo- and azoospermia using whole-exome sequencing and Sanger sequencing. RHOXF1 was absent in the testicular biopsy of one patient (c.388G>A: p.V130M) whose histological analysis showed a phenotype of Sertoli cell-only syndrome. In vitro experiments indicated that RHOXF1 mutations significantly reduced the content of RHOXF1 protein in HEK293T cells. Specifically, the p.V130M, p.A156V, and p.R160X mutants of RHOXF1 also led to increased RHOXF1 accumulation in cytoplasmic particles. Luciferase assays revealed that p.V130M and p.R160X mutants may disrupt downstream spermatogenesis by perturbing the regulation of doublesex and mab-3 related transcription factor 1 (DMRT1) promoter activity. Furthermore, ICSI treatment could be beneficial in the context of oligozoospermia caused by RHOXF1 mutations. In conclusion, our findings collectively identified mutated RHOXF1 to be a disease-causing X-linked gene in human oligo- and azoospermia.

CFAP65 is required in the acrosome biogenesis and mitochondrial sheath assembly during spermiogenesis.

Asthenoteratospermia is a common cause of male infertility. Recent studies have revealed that CFAP65 mutations lead to severe asthenoteratospermia due to acrosome hypoplasia and flagellum malformations. However, the molecular mechanism underlying CFAP65-associated sperm malformation is largely unclear. Here, we initially examined the role of CFAP65 during spermiogenesis using Cfap65 knockout (Cfap65-/-) mice. The results showed that Cfap65-/- male mice exhibited severe asthenoteratospermia characterized by morphologically defective sperm heads and flagella. In Cfap65-/- mouse testes, hyper-constricted sperm heads were apparent in step 9 spermatids accompanied by abnormal manchette development, and acrosome biogenesis was abnormal in the maturation phase. Moreover, subsequent flagellar elongation was also severely affected and characterized by disrupted assembly of the mitochondrial sheath (MS) in Cfap65-/- male mice. Furthermore, the proteomic analysis revealed that the proteostatic system during acrosome formation, manchette organization and MS assembly was disrupted when CFAP65 was lost. Importantly, endogenous immunoprecipitation and immunostaining experiments revealed that CFAP65 may form a cytoplasmic protein network comprising MNS1, RSPH1, TPPP2, ZPBP1 and SPACA1. Overall, these findings provide insights into the complex molecular mechanisms of spermiogenesis by uncovering the essential roles of CFAP65 during sperm head shaping, acrosome biogenesis and MS assembly.

Single-cell RNA sequencing technology in human spermatogenesis: Progresses and perspectives.

Spermatogenesis, a key part of the spermiation process, is regulated by a combination of key cells, such as primordial germ cells, spermatogonial stem cells, and somatic cells, such as Sertoli cells. Abnormal spermatogenesis can lead to azoospermia, testicular tumors, and other diseases related to male infertility. The application of single-cell RNA sequencing (scRNA-seq) technology in male reproduction is gradually increasing with its unique insight into deep mining and analysis. The data cover different periods of neonatal, prepubertal, pubertal, and adult stages. Different types of male infertility diseases including obstructive and non-obstructive azoospermia (NOA), Klinefelter Syndrome (KS), Sertoli Cell Only Syndrome (SCOS), and testicular tumors are also covered. We briefly review the principles and application of scRNA-seq and summarize the research results and application directions in spermatogenesis in different periods and pathological states. Moreover, we discuss the challenges of applying this technology in male reproduction and the prospects of combining it with other technologies.

Physiological and genetic convergence supports hypoxia resistance in high-altitude songbirds.

Skeletal muscle plays a central role in regulating glucose uptake and body metabolism; however, highland hypoxia is a severe challenge to aerobic metabolism in small endotherms. Therefore, understanding the physiological and genetic convergence of muscle hypoxia tolerance has a potential broad range of medical implications. Here we report and experimentally validate a common physiological mechanism across multiple high-altitude songbirds that improvement in insulin sensitivity contributes to glucose homeostasis, low oxygen consumption, and relative activity, and thus increases body weight. By contrast, low-altitude songbirds exhibit muscle loss, glucose intolerance, and increase energy expenditures under hypoxia. This adaptive mechanism is attributable to convergent missense mutations in the BNIP3L gene, and METTL8 gene that activates MEF2C expression in highlanders, which in turn increases hypoxia tolerance. Together, our findings from wild high-altitude songbirds suggest convergent physiological and genetic mechanisms of skeletal muscle in hypoxia resistance, which highlights the potentially medical implications of hypoxia-related metabolic diseases.

Bioinformatic Analysis of Key Biomarkers and Infiltrating Immune Cells in Nonalcoholic Fatty Liver Disease.

Context: The liver is both the largest metabolic and the largest immune organ and is closely related to the mechanisms of disease development. Clarifying the immune environment of the NAFLD liver to determine its interactions with biomarkers would be beneficial in exploring the mechanisms of disease development. Objective: The study aimed to identify biomarkers and immune cells associated with nonalcoholic fatty liver disease (NAFLD) and to analyze the correlation between key genes and immune cells in NAFLD, to improve the understanding of the mechanisms underlying NAFLD and provide potential therapeutic targets. Design: The research team performed a genetic study. Setting: The study took place at Qingdao, Shandong Province, China. Outcome Measures: The research team: (1) obtained the NAFLD-related datasets GSE63067, GSE48452, and GSE89632 from the Gene Expression Omnibus (GEO) database; (2) analyzed immune-cell infiltrates using single-sample gene set enrichment analysis (ssGSEA) to determine the hub immune cells; (3) selected the differentially expressed genes (DEGs) between the NAFLD and normal samples and screened them to identify the hub genes; (4) evaluated the efficiency of the hub genes using receiver operating characteristic (ROC) curves; and (5) analyzed the correlations between hub genes and immune cells. Results: The research team: (1) found 28 differential immune cells; (2) identified monocytes as the hub immune cells; (3) identified 55 DEGs; (4) comparing the top 10 genes, identified five hub genes: S100 calcium binding proteins A12 (S100A12), S100A9, S100A8, selectin L (SELL), and sex hormone binding globulin (SHBG); (5) for all five, the area under the ROC curve (AUC) was greater than 0.6-training set: AUCSA00A12 = 0.699, AUCSELL = 0.743, AUCS100A9 = 0.735, AUCSHBG = 0.752, and AUCS100A8 = 0.703; and validation set: AUCSA00A12 = 0.852, AUCSELL = 0.905, AUCS100A9 = 0.819, AUCSHBG = 0.830, and AUCS100A8 = 0.822; (6) negatively correlated SHBG with immune cells (P > .05, r=-0.09); and (7) positively correlated S100A12, S100A9, S100A8, and SELL with immune cells-rS100A8 = 0.40, rS100A9 = 0.50, rS100A12 = 0.38, and rSELL = 0.42, respectively. Conclusions: Based on bioinformatic analyses, the progression of NAFLD may involve monocytes through promotion of liver inflammation. The hub genes S100A12, S100A9, S100A8, SELL, and SHBG are potential biomarkers that may be useful as diagnostic tools or therapeutic targets for NAFLD.

Trace the profile and function of circular RNAs in Sertoli cell only syndrome.

Studies increasingly show the involvement of circular RNAs (circRNAs) in several diseases. This study aims to explore the circRNA expression pattern in the testicular tissues of patients with Sertoli only cell syndrome (SCOS) and their potential functions. High throughput circRNA microarray analysis indicated that 399 circRNAs were upregulated and 1195 were down-regulated (fold change >2, P < 0.05) in SCOS relative to obstructive azoospermia (OA). The hsa_circRNA_101222, hsa_circRNA_001387, hsa_circRNA_001153, hsa_circRNA_101373 and hsa_circRNA_103864 were validated by qRT-PCR. Furthermore, the hosting genes of the differentially expressed circRNAs (DEcircRNAs) were enriched in biological processes related to cell cycle and intercellular communication. Also, the overlapping genes between the hosting genes of SCOS-related DEcircRNAs and those highly expressed in Sertoli cells of non-obstructive azoospermia (NOA) were enriched in immune cell development and cell communication. Taken together, aberrantly expressed circRNAs likely mediate SCOS development by regulating the function of Sertoli cells and the spermatogenic microenvironment.

A multi-faceted comparative perspective on elevational beta-diversity: the patterns and their causes.

The observed patterns and underlying mechanisms of elevational beta-diversity have been explored intensively, but multi-dimensional comparative studies remain scarce. Herein, across distinct beta-diversity components, dimensions and species groups, we designed a multi-faceted comparative framework aiming to reveal the general rules in the observed patterns and underlying causes of elevational beta-diversity. We have found that: first, the turnover process dominated altitudinal patterns of species beta-diversity (ßsim > ßsne), whereas the nestedness process appeared relatively more important for elevational trait dissimilarity (ßfuncsim < ßfuncsne); second, the taxonomic turnover was relative higher than its phylogenetic and functional analogues (ßsim > ßphylosim/ßfuncsim), conversely, nestedness-resultant trait dissimilarity tended to be higher than the taxonomic and phylogenetic measures (ßfuncsne > ßsne/ßphylosne); and third, as elevational distance increased, the contradicting dynamics of environmental filtering and limiting similarity have jointly led the elevational patterns of beta-diversity, especially at taxonomic dimension. Based on these findings, we infer that the species turnover among phylogenetic relatives sharing similar functional attributes appears to be the main cause of shaping the altitudinal patterns of multi-dimensional beta-diversity. Owing to the methodological limitation in the randomization approach, currently, it remains extremely challenging to distinguish the influence of the neutral process from the offset between opposing niche-based processes. Despite the complexities and uncertainties during species assembling, with a multi-dimensional comparative perspective, this work offers us several important commonalities of elevational beta-diversity dynamics.

Risks associated with cryopreserved semen in a human sperm bank during and after the COVID-19 pandemic.

RESEARCH QUESTION: What are the risks associated with cryopreserved semen collected during and after the coronavirus disease 2019 (COVID-19) pandemic wave in Wuhan, China? DESIGN: Retrospective cohort study involving young adult men who were qualified sperm donors at the Hunan Province Human Sperm Bank (China) during the pandemic wave (1 January 2020 to 30 January 2020) and after the wave and return to work (7 April 2020 to 30 May 30 2020). One hundred paired semen and blood specimens from 100 donors were included. One-step single-tube nested quantitative real-time polymerase chain reaction (OSN-qRT-PCR) was used to detect SARS-CoV-2. Moreover, to control the unacceptable risk of false-negative results, a second round of screening was performed with pooled RNA from negative semen samples using crystal digital PCR (cd-PCR). RESULTS: For individual blood and semen samples, the target genes, namely the nucleocapsid protein (N) and open reading frame (ORF-1ab) genes, tested negative in all of the 100 paired samples. Further, as per cd-PCR results, there were >20,000 droplets per well in the RNA for each combined sample and no positive droplets were present for either of the aforementioned target genes. A total of 100 paired semen and blood samples from these two groups tested negative for SARS-CoV-2. CONCLUSIONS: Cryopreserved semen at the Hunan Province Human Sperm Bank during and after the COVID-19 pandemic wave was free of SARS-CoV-2 and was judged safe for external use in the future.

Intra-Cytosplamic sperm injection outcomes with fresh and cryopreserved human epidydimal sperm from patients with obstructive azoospermia.

Techniques for the cryopreservation of epididymal sperm was are widely used in clinical practice. However, given the unique characteristics of sperm from patients with obstructive azoospermia, epididymal sperm cryopreservation is more difficult because of low count and weak motility; therefore, conventional methods of sperm cryopreservation may not result in the best outcomes. We used the micro-straw method to store small quantities of sperm obtained from patients with severe oligozoospermia or azoospermia and achieved successful deliveries in the previous study. This retrospective study of ICSI cycles included the first ICSI cycles of fresh or frozen/thawed epididymal sperm that were performed in patients suffering from obstructive azoospermia who were admitted to the CITIC-Xiangya Hospital of Reproduction and Genetics of China from June 1, 2015 to June 31, 2019. A total of 2441 patients with obstructive azoospermia were divided according to the use of fresh (n = 2342) or frozen/thawed (n = 99) epididymal sperm. The results showed that the fertilisation rate was higher with fresh epididymal sperm than that with frozen/thawed epididymal sperm (85.14% vs. 79.26%, respectively; p = 0.000). However, the rates of embryo cleavage, high-quality embryos, clinical pregnancy, miscarriage, singletons and birth defect were similar between fresh and frozen/thawed epididymal sperm (98.28% vs. 99.13%, 60.34% vs. 57.29%, 67.90% vs. 70.51%, 8.12% vs. 10.91%, 57.76% vs. 49.09%, 1.59% vs. 1.45%respectively; p = 0.088, 0.109, 0.628, 0.462,0.203 and 0.686). In addition, the short-term cryostorage of small quantities of epididymal sperm did not affect clinical outcomes. The results indicated that in cases of obstructive azoospermia, cryostorage of small quantities epididymal sperm is a reliable option.

ß-estradiol promotes the growth of primary human fetal spermatogonial stem cells via the induction of stem cell factor in Sertoli cells.

BACKGROUND: Mammalian spermatogenesis is responsible for male fertility and is supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). Sertoli cells provide a supportive microenvironment for SSCs, in part by the production of stem cell factor (SCF), which is a potent regulator of spermatogonia proliferation and survival. METHODS: We investigated the novel role of ß-estradiol in modulating the proliferation and apoptosis of fetal SSCs via the regulation of SCF secretion in Sertoli cells isolated from human fetal testes. The proliferation of SSCs in the co-culture system was determined by colony formation and BrdU incorporation assays. TUNEL assay was used to measure SSC apoptosis in co-culture in response to treatment with control, ß-estradiol, or the combination of ß-estradiol and the estrogen receptor inhibitor ICI 182780. RESULTS: In the system with purified human fetal Sertoli cells (MIS+/c-Kit-/AP-), ß-estradiol upregulated the production of SCF in a dose- and time-dependent manner. In the co-culture system of primary human fetal SSCs (c-Kit+/SSEA-4+/Oct-4+/AP+) and Sertoli cells (MIS+), ß-estradiol markedly increased the proliferation of SSCs. Moreover, SSC apoptosis was significantly inhibited by ß-estradiol and was completely reversed by the combination of ß-estradiol and ICI 182780. CONCLUSION: Here we report, for the first time, that ß-estradiol can induce the increase of SCF expression in human fetal Sertoli cells and regulates the growth and survival of human fetal SSCs. These novel findings provide new perspectives on the current understanding of the role of estrogen in human spermatogenesis.

Sperm donor lifestyle survey: modifiable risk factors for potential sperm donors.

OBJECTIVES: To examine the association between modifiable lifestyle factors and the main semen parameter values, the number of qualified sperm donors, and to provide some sensible guidance for sperm donors. METHODS: Healthy men screened as potential sperm donors were recruited in the Hunan Province Human Sperm Bank of China from March 2019 to December 2019. Participants were invited to complete interviewer-assisted questionnaires on eleven items of information. Univariate and multivariate analyses were conducted to analyze which lifestyle factors collected by the questionnaire had an impact on the eligibility and main semen parameters of sperm donors. RESULTS: The eligibility of men as sperm donors was strongly influenced by the duration of abstinence (P = 0.002). The rate of eligibility sperm donors increased significantly with the number of days of abstinence. In addition, semen volume increased with abstinence time (P = 0.000). Exercise frequency (P = 0.025) and abstinence time (P = 0.000) were positively correlated with sperm concentration, and masturbation frequency was negatively correlated with sperm concentration (P = 0.013). Progressive sperm motility was significantly affected by abstinence time (P = 0.000) and bedtime (P = 0.047). CONCLUSIONS: Abstinence time was highly associated with semen parameters and donor qualification. Increase the abstinence time before donation may be meaningful in improving the proportion of eligible sperm donors.

Biallelic mutations in CFAP65 lead to severe asthenoteratospermia due to acrosome hypoplasia and flagellum malformations.

BACKGROUND: The genetic causes for most male infertility due to severe asthenozoospermia remain unclear. OBJECTIVE: Our objective was to identify unknown genetic factors in 47 patients with severe asthenozoospermia from 45 unrelated Chinese families. METHODS: We performed whole exome sequencing of 47 individuals with severe asthenozoospermia from 45 unrelated families. Mutation screening was performed in a control cohort of 637 individuals, including 219 with oligoasthenospermia, 195 with non-obstructive azoospermia and 223 fertile controls. Ultrastructural and immunostaining analyses of patients' spermatozoa were performed to characterise the effect of variants. RESULTS: One homozygous non-sense mutation (NM_194302, c.G5341T:p.E1781X), two compound heterozygous mutations (c.C2284T:p.R762X and c.1751delC:p.P584fs) and two compound heterozygous mutations (c.5714_5721del:p.L1905fs and c.C3021A:p.N1007K) were identified in CFAP65 of three individuals with completely immotile spermatozoa, respectively. No biallelic deleterious variants of CFAP65 were detected in the control cohort of 637 individuals. Ultrastructural and immunostaining analyses of spermatozoa from two patients showed highly aberrant sperm morphology with severe defects such as acrosome hypoplasia, disruption of the mitochondrial sheath and absence of the central pair complex. CONCLUSION: To the best of our knowledge, we are the first to report that CFAP65 mutations may cause spermatozoa to be completely immotile.

IQ motif containing D (IQCD), a new acrosomal protein involved in the acrosome reaction and fertilisation.

The acrosome is single, large, dense-core secretory granule overlying the nucleus of most mammalian spermatozoa. Its exocytosis, the acrosome reaction, is a crucial event during fertilisation. In this study we identified a new acrosome-associated gene, namely IQ motif containing D (IQCD), expressed nearly in multiple tissues with highest expression levels in the testis. In mouse testis, Iqcd transcript accumulated from Postnatal Day (PND) 1 to adulthood. However, expression of IQCD protein at the testicular development stage started primarily from PND 18 and increased in an age-dependent manner until plateauing in adulthood. IQCD was primarily accumulated in the acrosome area of round and elongating spermatids within seminiferous tubules of the testes during the late stage of spermiogenesis; this immunolocalisation pattern is similar in mice and humans. IQCD levels in spermatozoa were significantly lower in IVF patients with total fertilisation failure or a low fertilisation rate than in healthy men. Anti-IQCD antibody significantly inhibited the acrosome reaction and slightly reduced protein tyrosine phosphorylation levels in human spermatozoa, but specifically blocked murine IVF. IQCD interacted with mammalian homolog of C. elegans uncoordinated gene 13 (Munc13) in spermatozoa and may participate in acrosome exocytosis. In conclusion, this study identified a new acrosomal protein, namely IQCD, which is involved in fertilisation and the acrosome reaction.

Corrigendum to: IQ motif containing D (IQCD), a new acrosomal protein involved in the acrosome reaction and fertilisation.

The acrosome is single, large, dense-core secretory granule overlying the nucleus of most mammalian spermatozoa. Its exocytosis, the acrosome reaction, is a crucial event during fertilisation. In this study we identified a new acrosome-associated gene, namely IQ motif containing D (IQCD), expressed nearly in multiple tissues with highest expression levels in the testis. In mouse testis, Iqcd transcript accumulated from Postnatal Day (PND) 1 to adulthood. However, expression of IQCD protein at the testicular development stage started primarily from PND 18 and increased in an age-dependent manner until plateauing in adulthood. IQCD was primarily accumulated in the acrosome area of round and elongating spermatids within seminiferous tubules of the testes during the late stage of spermiogenesis; this immunolocalisation pattern is similar in mice and humans. IQCD levels in spermatozoa were significantly lower in IVF patients with total fertilisation failure or a low fertilisation rate than in healthy men. Anti-IQCD antibody significantly inhibited the acrosome reaction and slightly reduced protein tyrosine phosphorylation levels in human spermatozoa, but specifically blocked murine IVF. IQCD interacted with mammalian homolog of C. elegans uncoordinated gene 13 (Munc13) in spermatozoa and may participate in acrosome exocytosis. In conclusion, this study identified a new acrosomal protein, namely IQCD, which is involved in fertilisation and the acrosome reaction.

DMC1 mutation that causes human non-obstructive azoospermia and premature ovarian insufficiency identified by whole-exome sequencing.

BACKGROUND: The genetic causes of the majority of male and female infertility caused by human non-obstructive azoospermia (NOA) and premature ovarian insufficiency (POI) with meiotic arrest are unknown. OBJECTIVE: To identify the genetic cause of NOA and POI in two affected members from a consanguineous Chinese family. METHODS: We performed whole-exome sequencing of DNA from both affected patients. The identified candidate causative gene was further verified by Sanger sequencing for pedigree analysis in this family. In silico analysis was performed to functionally characterise the mutation, and histological analysis was performed using the biopsied testicle sample from the male patient with NOA. RESULTS: We identified a novel homozygous missense mutation (NM_007068.3: c.106G>A, p.Asp36Asn) in DMC1, which cosegregated with NOA and POI phenotypes in this family. The identified missense mutation resulted in the substitution of a conserved aspartic residue with asparaginate in the modified H3TH motif of DMC1. This substitution results in protein misfolding. Histological analysis demonstrated a lack of spermatozoa in the male patient's seminiferous tubules. Immunohistochemistry using a testis biopsy sample from the male patient showed that spermatogenesis was blocked at the zygotene stage during meiotic prophase I. CONCLUSIONS: To the best of our knowledge, this is the first report identifying DMC1 as the causative gene for human NOA and POI. Furthermore, our pedigree analysis shows an autosomal recessive mode of inheritance for NOA and POI caused by DMC1 in this family.

Disordered APC/C-mediated cell cycle progression and IGF1/PI3K/AKT signalling are the potential basis of Sertoli cell-only syndrome.

The cause of Sertoli cell-only syndrome (SCOS), a condition in which only Sertoli cells line the seminiferous tubules in the testis, is unknown. Three microarray data sets were downloaded from public databases and were used to compare SCOS and control group. A total of 291 genes differentially expressed (Log2 |FC| ≥ 1 and adjusted p value < 0.05) in SCOS patients. Further 238 genes were significantly downregulated, and 53 genes were significantly upregulated. To identify the hub genes in the differentially expressed genes, we constructed a protein-protein interaction network, and CCNB1, CCNA2, AURKA, KIF11, CCNB2, CDC6, PRC1, NCAPG, KIF2C and PLK4 were screened from the network for the downregulated genes. Since the upregulated genes could not form a network, we concentrated on the genes with a higher fold change, and CPA3, NFIB, LONRF2, LYVE1, ATP8B4, IGF1, ITPR1 and PLAT were identified as the top 50% fold change genes in any of the three microarray data sets. Among downregulated hub genes, CDC6, CCNA2, CCNB1 and CCNB2 were involved in APC/C-mediated cell cycle progression. Among key upregulated genes, IGF1 was involved in the PI3K/AKT pathway, while the other genes have not been reported in Sertoli or Leydig cells. In conclusion, SCOS appears to be caused by disordered APC/C-mediated cell cycle progression and PI3K/AKT signalling.

Human sperm acrosome function assays are predictive of fertilization rate in vitro: a retrospective cohort study and meta-analysis.

OBJECTIVE: To determine whether acrosome function scoring-including acrosomal enzyme (AE) levels and acrosome reaction (AR) results-can predict fertilization rate in vitro. METHODS: We examined the predictive value of acrosomal enzymes (AE) determined by spectrophotometry/N-α-benzoyl-DL-arginine-p-nitroanilide for fertilization rate (FR) in vitro in a retrospective cohort study of 737 infertile couples undergoing IVF therapy. Additionally, a meta-analysis was done for prospective cohort or case-control studies; the following summary measures were reported to expand upon the findings: pooled spearman correlation coefficient (Rs), standardized mean difference (SMD), sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic score (DS), diagnostic odds ratio (DOR), and area under the summary receiver operating characteristic curve (AUC). RESULTS: Lower AE levels determined by spectrophotometry with a cut-off value of <25µIU/106 spermatozoa were predictive of total fertilization failure (TFF) with moderate SEN (88.23%) and low SPE (16.50%). On meta-analysis, a total of 44 unique articles were selected, but given the multiple techniques described there was a total of 67 total datasets extracted from these 44 articles, comprising 5356 infertile couples undergoing IVF therapy. The AE levels or induced AR% was positively correlated with FR (Rs = 0.38, SMD = 0.79; Rs = 0.40, SMD = 0.86, respectively). Lower AE levels or induced AR% was predictive of lower fertilization rate with moderate accuracy (AUC = 0.78, AUC = 0.84, respectively); this was accompanied by low SEN/moderate SPE (0.57/0.85), moderate SEN/moderate SPE (0.79/0.87), respectively. For AE assay, the diagnostic performance in Asia (Rs = 0.24, SMD = 0.50) was inferior to that in North America (Rs = 0.54, SMD = 0.81) and Europe (Rs = 0.46, SMD = 0.92). Cryopreserved spermatozoa (SMD = 0.20, P = 0.204) were inferior to fresh spermatozoa (SMD = 0.89, P <  0.001). Sperm preparation yielded inferior results as compared to no preparation; spermatozoa after swim up were weak relevant (Rs = 0.27, P = 0.044); and there was no correlation for spermatozoa after a discontinuous gradient (SMD = 1.07, P >  0.05). Lower AE levels determined by fluorometry or substrate assay were used for predicting lower FR with low sensitivity and high specificity; the spectrophotometry assay had an uncertain predictive value. For induced AR assay, the diagnostic performance in the other areas was inferior to that in Africa (Rs = 0.65, SMD = 1.86). No preparation or double preparation yielded inferior results as compared to one preparation (Rs = 0.41); discontinuous gradient (Rs = 0.17, SMD = 0.47) was inferior to swim up (Rs =0.65, SMD = 1.51). Nonphysiological triggers (SMD = 0.81) did not differ from physiological triggers (SMD = 0.95) in general; ZP (Rs = 0.63) or mannose (Rs = 0.59) was superior to other physiological or nonphysiological triggers; and there was no correlation for human follicle fluid, progesterone, cyclic adenosine 3'-5'-phosphate analogue and phorbol ester-BSA-GlcNAc Neoglycoproteins with N-acetylglucosamine residues. Lower induced AR% determined by indirect immunofluorescence, direct immunofluorescence with lection, or triple stain was used for predicting lower FR, with moderate sensitivity/high specificity, moderate sensitivity/high specificity, or high sensitivity/low specificity. CONCLUSIONS: Although the correlation between acrosome function scoring and FR was significant, the assays were neither highly sensitive nor specific. Additionally, the diagnostic performance showed regional effects as well as an effect of the sperm preparation or assay method. More studies of multicenter, large-scale, careful design and synthesizing multiple sperm functional assays and oocyte quality assays are still needed in clinical settings to better predict fertilization outcome in IVF.

[Expression of IQCG in the human testis and its correlation with asthenospermia].

OBJECTIVE: To investigate the expression and location of IQ motif-containing G (IQCG) in the human testis, compare its expression in normal-motility sperm with that in the sperm of asthenospermia patients, and explore its possible mechanisms and its correlation with fertility. METHODS: The expression of the IQCG gene in the human testis was detected by RT-PCR and its location in the testis and sperm was determined by immunohistochemistry and immunofluorescence staining. Semen samples were collected from normal males, patients with asthenospermia, and fertile men that succeeded in artificial insemination with donor's sperm (AID), followed by analysis of the IQCG protein expression in different groups of samples by Western blot. RESULTS: Immunohistochemistry showed that IQCG was extensively expressed in the human testis, in the spermatocytes and spermatids, specifically in the sperm tail, weakly expressed or absent in the spermatogonial stem cells, and strongly expressed in the spermatogonial cells. The expression of IQCG was significantly lower in the asthenospermia patients than in the normal males (P= 0.041). Western blot manifested that IQCG was expressed in the semen of all the three groups of subjects, with statistically significant differences between the normal men and severe asthenospermia patients (P = 0.032) as well as between the fertile males and the severe asthenospermia group (P = 0.027) . CONCLUSIONS: IQCG may act on human sperm motility and its abnormal expression possibly reduces sperm motility and fertility. An insight into its action mechanisms may shed some new light on the etiology and treatment of asthenospermia.

[In vitro culture medium for sparse spermatozoa improves human sperm motility].

OBJECTIVE: To investigate whether in vitro culture medium (IVCM) for sparse spermatozoa can improve human sperm motility for the purpose of helping clinicians, laboratorians and patients choose a better strategy of assisted reproduction. METHODS: Semen samples were obtained from 178 males for routine semen examination from March to August 2016, including 151 cases of asthenozoospermia and 27 cases of normal sperm motility. A total of 200 µl was collected from each sample and divided into two equal portions and equal volumes of IVCM (experimental group) and F10 (1×) (control group) were added to the two portions, respectively, followed by 30-minute incubation at 37℃ in an incubator with 5% CO2. Sperm concentration, motility and viability and the percentages of progressively motile, non-progressively motile and immotile sperm were recorded before and after incubation. RESULTS: After activated with IVCM, neither the samples with asthenozoospermia nor those with normal sperm motility showed any statistically significant difference in sperm viability from the baseline or the control group (P>0.05). The rates of progressively and non-progressively motile sperm from the asthenozoospermia males were increased by 14.02% and 4.86% respectively, while that of immotile sperm decreased by 19.01% in the experimental group (P >0.01), and similar results were observed in the semen samples from the men with normal sperm motility. The percentage of reduced immotile viable sperm was positively correlated with that of immotile viable sperm in both the asthenozoospermia patients (r = 0.260, P <0.01) and the men with normal sperm motility (r = 0.679, P <0.01). CONCLUSIONS: IVCM can increase sperm motility without affecting sperm viability in men with either asthenozoospermia or normal sperm motility. The larger the proportion of immotile viable sperm, the higher the percentages of progressively and non-progressively motile sperm in the semen after IVCM activation, and this correlation is more significant in men with normal sperm motility than in asthenozoospermia patients.