Search

Abstract: Male infertility is a worldwide health issue, affecting 8%-12% of the global population. Oligoasthenoteratozoospermia (OAT) represents a severe type of male infertility, characterized by reduced sperm count and motility and an increased frequency of sperm with aberrant morphology. Using whole-exome sequencing, this study identified a novel missense mutation (c.848C>A, p.A283E) in the coiled-coil domain-containing 34 gene (CCDC34) in a consanguineous Pakistani family. This rare mutation was predicted to be deleterious and to affect the protein stability. Hematoxylin and eosin staining of spermatozoa from the patient with OAT revealed multiple morphological abnormalities of the flagella and transmission electron microscopy indicated axonemal ultrastructural defects with a lack of outer dynein arms. These findings indicated that CCDC34 plays a role in maintaining the axonemal ultrastructure and the assembly or stability of the outer dynein arms, thus expanding the phenotypic spectrum of CCDC34 missense mutations., (Copyright © 2024 Copyright: ©The Author(s)(2024).)

Aberrant sperm protamination is linked to sperm dysmorphology and nuclear chromatin condensation. Yet, its effects on sperm cytoplasmic maturation remain largely unexplored. The relationships of protamines, sperm morphology, DNA damage, and cytoplasmic remodeling were illustrated in this study to provide fresh perspectives on the mechanisms of male infertility. A total of 205 infertile males were allocated into 5 groups according to the percentage of spermatozoa exhibiting abnormal morphology within their samples. Sperm concentration, motility, abnormal sperm morphology, cytoplasmic droplets (CDs), and excess residual cytoplasm (ERC) were analyzed according to the World Health Organization manual (2010). Sperm nuclear vacuoles (NVs) were determined by propidium iodide (PI) staining. Sperm protamine expressions (P1 and P2) were detected by western blot. DNA damage was measured by acridine orange test (AOT) to calculate the proportion of sperm with single-strand DNA breaks (SSBs). Our data showed that sperm concentration and motility in infertile males significantly decreased with the severity of abnormal sperm morphology (both P < 0.01). P1 level, P1/P2 ratio, and SSB rate increased with the severity of sperm dysmorphology, whilst the P2 level decreased (all P < 0.01). NVs, CDs, and ERC were more common in males with sperm dysmorphology and positively correlated with the SSB rate (all P < 0.01). The relationships between the SSB rate and the P1/P2 ratio were also significant ( P < 0.01). Aberrant protamination may cause sperm dysmorphology and compromise male fertility by impairing sperm's nucleus and cytoplasm maturation, with the P1/P2 ratio potentially serving as a valuable indicator of sperm quality and male fertility., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Phospholipase C zeta (PLCζ) is a key sperm-borne oocyte-activating factor that triggers Ca 2+ oscillations and the subsequent block to polyspermy following gamete fusion. Mutations in PLCZ1 , the gene encoding PLCζ, cause male infertility and intracytoplasmic sperm injection (ICSI) fertilization failure; and PLCζ expression and localization patterns are significantly correlated with ICSI fertilization rate (FR). However, in conventional in vitro fertilization (cIVF), whether and how sperm PLCζ affects fertilization remain unclear. Herein, we identified one previously reported and two novel PLCZ1 mutations associated with polyspermy in vitro that are characterized by excessive sperm-zona binding and a delay in pronuclei (PN) formation. Immunofluorescence staining and oocyte activation testing revealed that virtually all spermatozoa from patients lacked functional PLCζ and were thus unable to evoke Ca 2+ oscillations. ICSI with an artificial oocyte activation treatment successfully rescued the polyspermic phenotype and resulted in a live birth. Furthermore, we analyzed PLCζ in an additional 58 males after cIVF treatment in the Reproductive and Genetic Hospital of CITIC-Xiangya (Changsha, China) between February 2019 and January 2022. We found that the proportion of spermatozoa that expressed PLCζ was positively correlated with both 2PN rate and total FR. The optimal cutoff value below which males were likely to experience low FR (total FR ≤30%) after cIVF was 56.7% for the proportion of spermatozoa expressing PLCζ. Our study expands the mutation and the phenotypic spectrum of PLCZ1 and further suggests that PLCζ constitutes a promising biomarker for identifying low FRs cases in cIVF due to sperm-related oocyte activation deficiency and that sperm PLCζ analysis may benefit the wider male population and not only men with ICSI failure., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Male germ cells employ specific metabolic pathways throughout their developmental stages. In a previous study, we discovered heightened expression of pyruvate kinase M (PKM), a pivotal glycolytic enzyme, in spermatogonia and spermatids. To gain deeper insights into PKM's roles in spermatogenesis, sperm function, and male fertility, we engineered a conditional-knockout mouse model ( Pkm -vKO mice) to selectively disrupt the Pkm gene within germ cells. Despite maintaining regular testicular histology and sperm morphology, the male Pkm -vKO mice were infertility, characterized by significant impairments in sperm motility and adenosine triphosphate (ATP) generation. In addition, Pkm -null spermatozoa exhibited similar deficits in protein tyrosine phosphorylation linked to capacitation, as well as compromised performance in in vitro fertilization experiments. To conclude, PKM's presence is not obligatory for the entirety of spermatogenesis in male germ cells; however, it emerges as a critical factor influencing sperm motility and overall male fertility., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Variations in the dynein axonemal heavy chain gene, dynein axonemal heavy chain 6 ( DNAH6 ), lead to multiple morphological abnormalities of the flagella. Recent studies have reported that these deficiencies may result in sperm head deformation. However, whether DNAH6 is also involved in human acrosome biogenesis remains unknown. The purpose of this study was to investigate DNAH6 gene variants and their potential functions in the formation of defective sperm heads and flagella. Whole-exome sequencing was performed on a cohort of 375 patients with asthenoteratozoospermia from the First Affiliated Hospital of Anhui Medical University (Hefei, China). Hematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy were performed to analyze the sperm morphology and ultrastructure. Immunofluorescence staining and Western blot analysis were conducted to examine the effects of genetic variants. We identified three novel deleterious variants in DNAH6 among three unrelated families. The absence of inner dynein arms and radial spokes was observed in the sperm of patients with DNAH6 variants. Additionally, deficiencies in the acrosome, abnormal chromatin compaction, and vacuole-containing sperm heads were observed in these patients with DNAH6 variants. The decreased levels of the component proteins in these defective structures were further confirmed in sperm from patients with DNAH6 variants using Western blot. After intracytoplasmic sperm injection (ICSI) treatment, the partner of one patient with a DNAH6 variant achieved successful pregnancy. Overall, novel variants in DNAH6 genes that contribute to defects in the sperm head and flagella were identified, and the findings indicated ICSI as an effective clinical treatment for such patients., (Copyright © 2023 Copyright: © The Author(s)(2023).)

We examined a cohort of 93 cystic fibrosis (CF) male patients who were pancreatic-sufficient (PS-CF; n=40) or pancreatic-insufficient (PI-CF; n = 53). Complex semen examination was performed, including standard semen analysis, quantitative karyological analysis (QKA) of immature germ cells (IGCs), transmission electronic microscopy (TEM), biochemical analysis, and sperm DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) assay. Azoospermia was diagnosed in 83 (89.2%) patients. The other 10 (10.8%) patients were found to be nonazoospermic and showed various spermatological diagnoses (asthenozoospermia, n = 2; asthenoteratozoospermia, n = 3; oligoasthenozoospermia, n = 1; oligoasthenoteratozoospermia, n = 3; and normozoospermia, n = 1) with no specific morphological abnormalities. Oligospermia was detected in 89.2% azoospermic and 30.0% nonazoospermic patients. Low seminal pH (<7.0) was found in 74 (89.2%) of 83 azoospermic patients. Moderate leukocytospermia (2.0 × 10 6 -2.2 × 10 6 ml -1 ) was revealed in 2.4% azoospermic and 40.0% nonazoospermic semen samples. The signs of partial meiotic arrest at prophase I were found in 4 of 6 nonazoospermic patients examined by QKA of IGCs. The content of fructose and citrate was low in oligospermic and normal in nonoligospermic semen samples. An increased percentage (>30%) of spermatozoa with noncondensed ("immature") chromatin was revealed in 2 of 6 nonazoospermic semen samples analyzed by TEM., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Several preventive measures, including vaccination, have been implemented owing to the severe global effect of coronavirus disease 2019 (COVID-19), but there is still limited evidence in the effect of this disease and vaccination against it on male fertility. Therefore, this study is to compare sperm parameters of infertile patients with or without COVID-19 infection and the effect of COVID-19 vaccine types on them. Semen samples of infertile patients were collected consecutively at Universitas Indonesia - Cipto Mangunkusumo Hospital (Jakarta, Indonesia). COVID-19 was diagnosed by rapid antigen or polymerase chain reaction (PCR) tests. Vaccination was performed with three types of vaccine, namely inactivated viral vaccine, messenger RNA (mRNA) vaccine, and viral vector vaccine. Spermatozoa were then analyzed on the World Health Organization recommendations, and DNA fragmentation was assayed with the sperm chromatin dispersion kit. The results showed that the COVID-19 group experienced a significant decrease in sperm concentration and progressive motility (both P < 0.05), but there was no significant change in morphology or sperm DNA fragmentation index (DFI; both P > 0.05). The viral vector vaccine caused a decrease in morphology as well as an increase in DFI compared with the control (both P < 0.05), meanwhile results for those who were vaccinated with the inactivated and mRNA types were not significant compared with the control (both P > 0.05). We conclude that COVID-19 has negative effects on sperm parametes and sperm DNA fragmentation, and we found that the viral vector vaccines affect sperm parameter values and DNA fragmentation negatively. Further studies with a larger population and longer follow-up are needed to confirm the results., (Copyright © 2023 Copyright: © The Author(s)(2023).)

As a specific type of asthenoteratozoospermia, multiple morphological abnormalities of the sperm flagella (MMAF) is characterized by composite abnormalities, including absent, short, coiled, angulation, and irregular-caliber flagella. Mutations in cilia- and flagella-associated protein 43 ( CFAP43 ) are one of the main causative factors of MMAF established to date. To identify whether there are other CFAP43 mutations related to MMAF and to determine the clinical outcomes of assisted reproductive technology for patients with MMAF harboring different mutations, we recruited and screened 30 MMAF-affected Chinese men using a 22-gene next-generation sequencing panel. After systematic analysis, seven mutations in CFAP43 , including five novel mutations and two previously reported mutations, were identified from four families and related to MMAF in an autosomal recessive pattern. Papanicolaou staining, immunofluorescence, and electronic microscopy further clarified the semen characteristics and abnormal sperm morphologies, including disorganized axonemal and peri-axonemal structures, of the CFAP43 -deficient men. The female partners of two patients were pregnant after undergoing assisted reproductive technology through intracytoplasmic sperm injection, and one of them successfully gave birth to a healthy boy. This study significantly expands the mutant spectrum of CFAP43 , and together with the available information regarding male infertility and MMAF, provides new information for the genetic diagnosis and counseling of MMAF in the future., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Approximately 31% of patients with 22q11.2 deletion syndrome (22q11.2DS) have genitourinary system disorders and 6% of them have undescended testes. Haploinsufficiency of genes on chromosome 22q11.2 might contribute to the risk of 22q11.2DS. In this study, we used mice with single-allele deletion in mitochondrial ribosomal protein L40 ( Mrpl40 +/- ) as models to investigate the function of Mrpl40 in testes and spermatozoa development. The penetrance of cryptorchidism in Mrpl40 +/- mice was found to be higher than that in wild-type (WT) counterparts. Although the weight of testes was not significantly different between the WT and Mrpl40 +/- mice, the structure of seminiferous tubules and mitochondrial morphology was altered in the Mrpl40 +/- mice. Moreover, the concentration and motility of spermatozoa were significantly decreased in the Mrpl40 +/- mice. In addition, data-independent acquisition mass spectrometry indicated that the expression of genes associated with male infertility was altered in Mrpl40 +/- testes. Our study demonstrated the important role of Mrpl40 in testicular structure and spermatozoa motility and count. These findings suggest that Mrpl40 is potentially a novel therapeutic target for cryptorchidism and decreased motility and count of spermatozoa., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Male infertility is a major reproductive disorder, which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality. To date, five male patients with biallelic loss-of-function (LOF) variants of PARN-like ribonuclease domain-containing exonuclease 1 ( PNLDC1 ) have been reported to experience infertility with nonobstructive azoospermia. The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia (OAT) in a patient from a Chinese Han family. Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant (NM&#95;173516.2, c.142C>T, p.Gln48Ter) in PNLDC1 . Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype, including microcephaly, head tapering, and globozoospermia. Consistently, peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome. Furthermore, the disomy rate of chromosomes in the patient's spermatozoa was significantly increased compared with that of a fertile control sample. We reported an LOF variant of the PNLDC1 gene responsible for OAT., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Male infertility caused by idiopathic oligoasthenospermia (OAT) is known as idiopathic male infertility. Glutathione S-transferase (GST) and fluoride may play important roles in idiopathic male infertility, but their effects are still unknown. Our study examined the relationship between GST polymorphisms and fluoride-induced toxicity in idiopathic male infertility and determined the underlying mechanism. Sperm, blood, and urine samples were collected from 560 males. Fluoride levels were measured by a highly selective electrode method, and GST genotypes were identified using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Semen parameters, DNA fragmentation index (DFI), mitochondrial membrane potential (MMP), and oxidative stress (OS) biomarkers were statistically assessed at the P < 0.05 level. Compared with healthy fertile group, semen parameters, fluoride levels, OS biomarkers, sex hormone levels, and MMP and DFI levels were lower in the idiopathic male infertility group. For glutathione S-transferase M1 (GSTM1[-]) and glutathione S-transferase T1 (GSTT1[-]) or glutathione S-transferase P1 (GSTP1) mutant genotypes, levels of semen fluoride, OS, MMP, and DFI were considerably higher, and the mean levels of sperm parameters and testosterone were statistically significant in GSTM1(+), GSTT1(+), and GSTP1 wild-type genotypes. Both semen and blood fluoride levels were associated with oxidative stress in idiopathic male infertility patients. Elevated fluoride in semen with the genotypes listed above was linked to reproductive quality in idiopathic male infertility patients. In conclusion, GST polymorphisms and fluorine may have an indicative relationship between reproductive quality and sex hormone levels, and OS participates in the development of idiopathic male infertility., Competing Interests: None

Spermatogenesis is regulated by several Y chromosome-specific genes located in a specific region of the long arm of the Y chromosome, the azoospermia factor region (AZF). AZF microdeletions are the main structural chromosomal abnormalities that cause male infertility. Assisted reproductive technology (ART) has been used to overcome natural fertilization barriers, allowing infertile couples to have children. However, these techniques increase the risk of vertical transmission of genetic defects. Despite widespread awareness of AZF microdeletions, the occurrence of de novo deletions and overexpression, as well as the expansion of AZF microdeletion vertical transmission, remains unknown. This review summarizes the mechanism of AZF microdeletion and the function of the candidate genes in the AZF region and their corresponding clinical phenotypes. Moreover, vertical transmission cases of AZF microdeletions, the impact of vertical inheritance on male fertility, and the prospective direction of research in this field are also outlined., Competing Interests: None

Congenital bilateral absence of the vas deferens (CBAVD) is observed in 1%-2% of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. CFTR is one of the most well-known genes related to male fertility. The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia (NOA). CFTR mutations are highly polymorphic and have established ethnic specificity. Compared with F508Del in Caucasians, the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis. However, whether p.G970D participates in male infertility remains unknown. Herein, a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA. Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation (4.1%, 5/122), excluding polymorphic sites. Furthermore, we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients. The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells. In spermatocyte GC-2(spd)ts (GC2) Cftr p.G965D cells, RNA splicing variants were detected and CFTR expression decreased, which may contribute to the phenotypes associated with impaired spermatogenesis. Thus, this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells., Competing Interests: None

Competing Interests: None

This study aimed to identify genetic causes responsible for multiple morphological abnormalities of the sperm flagella (MMAF) in the Han Chinese population. Three primary infertile males with completely immobile sperm and MMAF were enrolled. Whole-exome sequencing and Sanger sequencing were performed to identify disease-causing genes. Subsequently, morphological and ultrastructural analyses of sperm flagella were investigated. The probable impact of genetic variants on protein function was analyzed by online bioinformatic tools and immunofluorescence assay. Three patients with dynein axonemal heavy chain 1 (DNAH1) gene compound heterozygous variations were identified. DNAH1 c.7435C>T, p.R2479X and c.10757T>C, p.F3586S were identified in the patient from Family 1, c.11726&#95;11727delCT, p.P3909fs and c.12154delC, p.L4052fs were found in the patient from Family 2, and c.10627-3C>G and c.11726&#95;11727delCT, p.P3909fs existed in the patient from Family 3. Four of these variations have not been reported, and all the mutations showed pathogenicity by functional effect predictions. The absence of the center pair and disorganization of the fibrous sheath were present in sperm flagella at the ultrastructural level. Moreover, the expression of DNAH1 was absent in spermatozoa from the participants, validating the pathogenicity of the variants. All three couples have undergone intracytoplasmic sperm injection (ICSI), and two couples of them became pregnant after the treatment. In conclusion, the newly identified DNAH1 mutations can expand the mutational and phenotypic spectrum of MMAF genes and provide a theoretical basis for genetic diagnosis in MMAF patients. It is recommended to conduct genetic screening in male infertility patients with MMAF and provide rational genetic counseling, and ICSI might be an optimal strategy to help with fertilization and conception for patients with DNAH1 mutations.

During recent decades, the association between mutations in ubiquitin-specific protease 26 (USP26) and male infertility remains doubtful. We conducted this meta-analysis to evaluate the association between mutations in USP26 and male infertility according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines. It was registered in the International Prospective Register of Systematic Reviews (PROSPERO; CRD42021225251). PubMed, Web of Science, and Scopus were systematically searched for comparative clinical studies, which were written in English and provided eligible data. Studies were included when they compared USP26 mutations in azoospermic, oligozoospermic, and asthenozoospermic patients with controls with normal sperm parameter values or whose partners had experienced spontaneous pregnancy. Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated with random effect models. Overall, twelve studies with 3927 infertility patients and 4648 healthy controls were included. The association between overall USP26 mutations and infertility was not significant (OR = 1.60, 95% CI: 0.51-5.01). For specific mutations, the pooled ORs were 1.65 (95% CI: 1.02-2.69) for cluster mutation (including 370-371insACA, 494T>C, and 1423C>T), 1.80 (95% CI: 0.35-9.15) for c.576G>A, 1.43 (95% CI: 0.79-2.56) for c.1090C>T, and 3.59 (95% CI: 2.30-5.59) for c.1737G>A. Our results suggest that several mutations (cluster mutation, c.1737G>A) may play roles in male infertility, while others (c.576G>A and c.1090C>T) do not show notable associations with male infertility. More high-quality clinical researches are needed for validation., Competing Interests: None

To explore the relationship between genetic polymorphisms of metabolic enzymes such as CYP1A1, CYP2D6, GSTM1, GSTT1, and GSTP1 and idiopathic male infertility. By observing the efficacy of antioxidants in the treatment of idiopathic male infertility, the effect of metabolic enzyme gene polymorphisms on antioxidant therapy in patients with idiopathic male infertility was prospectively studied. This case-control study included 310 men with idiopathic infertility and 170 healthy controls. The cytochrome P450 1A1 (CYP1A1), cytochrome P450 2D6 (CYP2D6), glutathione S-transferase M1 (GSTM1), glutathione S-transferase T1 (GSTT1), and glutathione S-transferase P1 (GSTP1) genotypes in peripheral blood samples were analyzed by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). The idiopathic male infertility group was treated with vitamin C, vitamin E, and coenzyme Q10 for 3 months and followed up for 6 months. GSTM1(-), GSTT1(-), and GSTM1/T1(-/-) in the idiopathic male infertility groups were more common than those in the control group. The sperm concentration, motility, viability, mitochondrial membrane potential (MMP), and seminal plasma total antioxidant capacity (T-AOC) level in patients with GSTM1(-), GSTT1(-), and GSTM1/T1(-/-) were lower than those in wild-type carriers, and the sperm DNA fragmentation index (DFI), 8-hydroxy-2'-deoxyguanosine (8-OH-dG), and malondialdehyde (MDA) and nitric oxide (NO) levels were higher. Therefore, oxidative damage may play an important role in the occurrence and development of idiopathic male infertility, but antioxidant therapy is not effective in male infertility patients with GSTM1 and GSTT1 gene deletions., Competing Interests: None

Large numbers of microbes can be present in seminal fluid, and there are differences in the semen microbiota between normal and abnormal semen samples. To evaluate the semen microbiota in patients with leukocytospermia, 87 seminal fluid samples, including 33 samples with a normal seminal leukocyte count and 54 samples with leukocytospermia, were obtained for a cross-sectional analysis. Twenty samples with a normal seminal leukocyte count had normal sperm parameters (Control group), and 13 samples with a normal seminal leukocyte count were from asthenozoospermia patients (Ast group). However, 32 samples with leukocytospermia were from asthenozoospermia patients (LA group), and only 22 samples with leukocytospermia had normal sperm parameters (Leu group). The 16S ribosomal RNA (rRNA) gene sequencing method was used to sequence the microbiota in the seminal fluid, and multiple bioinformatics methods were utilized to analyze the data. Finally, the results showed that the worse sperm parameters were observed in the leukocytospermia-related groups. Semen microbiota analysis found that there was increased alpha diversity in the leukocytospermia-related groups. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the primary phyla in the seminal fluid. Two microbiota profiles, namely, Lactobacillus-enriched and Streptococcus-enriched groups, were identified in this study. The majority of the samples in the groups with a normal seminal leukocyte count could be categorized as Lactobacillus-enriched, whereas the majority of the leukocytospermia samples could be categorized as Streptococcus-enriched. Our study indicated that males with leukocytospermia have worse sperm parameters and a different semen microbiota composition compared to males with a normal seminal leukocyte count., Competing Interests: None

Acephalic spermatozoa syndrome (ASS) is one of the most severe spermatogenic failures of all infertility in men. The cognition of ASS has experienced a tortuous process. Over the past years, with the in-depth understanding of spermatogenesis and the emergence of new genetic research technologies, the unraveling of the genetic causes of spermatogenic failure has become highly active. From these advances, we established a genetic background and made significant progress in the discovery of the genetic causes of ASS. It is important to identify pathogenic genes and mutations in ASS to determine the biological reasons for the occurrence of the disease as well as provide genetic diagnosis and treatment strategies for patients with this syndrome. In this review, we enumerate various technological developments, which have made a positive contribution to the discovery of candidate genes for ASS from the past to the present. Simultaneously, we summarize the known genetic etiology of this phenotype and the clinical outcomes of treatments in the present. Furthermore, we propose perspectives for further study and application of genetic diagnosis and assisted reproductive treatment in the future., Competing Interests: None

Thanks to tremendous advances in sequencing technologies and in particular to whole exome sequencing (WES), many genes have now been linked to severe sperm defects. A precise genetic diagnosis is obtained for a minority of patients and only for the most severe defects like azoospermia or macrozoospermia which is very often due to defects in the aurora kinase C (AURKC gene. Here, we studied a subject with a severe oligozoospermia and a phenotypic diagnosis of macrozoospermia. AURKC analysis did not reveal any deleterious variant. WES was then initiated which permitted to identify a homozygous loss of function variant in the zinc finger MYND-type containing 15 (ZMYND15 gene. ZMYND15 has been described to serve as a switch for haploid gene expression, and mice devoid of ZMYND15 were shown to be sterile due to nonobstructive azoospermia (NOA). In man, ZMYND15 has been associated with NOA and severe oligozoospermia. We confirm here that the presence of a bi-allelic ZMYND15 variant induces a severe oligozoospermia. In addition, we show that severe oligozoospermia can be associated macrozoospermia, and that a phenotypic misdiagnosis is possible, potentially delaying the genetic diagnosis. In conclusion, genetic defects in ZMYND15 can induce complete NOA or severe oligozoospermia associated with a very severe teratozoospermia. In our experience, severe oligozoospermia is often associated with severe teratozoospermia and can sometimes be misinterpreted as macrozoospermia or globozoospermia. In these instances, specific AURKC or dpy-19 like 2 (DPY19L2) diagnosis is usually negative and we recommend the direct use of a pan-genomic techniques such as WES., Competing Interests: None

Apparently balanced chromosomal structural rearrangements are known to cause male infertility and account for approximately 1% of azoospermia or severe oligospermia. However, the underlying mechanisms of pathogenesis and etiologies are still largely unknown. Herein, we investigated apparently balanced interchromosomal structural rearrangements in six cases with azoospermia/severe oligospermia to comprehensively identify and delineate cryptic structural rearrangements and the related copy number variants. In addition, high read-depth genome sequencing (GS) (30-fold) was performed to investigate point mutations causative of male infertility. Mate-pair GS (4-fold) revealed additional structural rearrangements and/or copy number changes in 5 of 6 cases and detected a total of 48 rearrangements. Overall, the breakpoints caused truncations of 30 RefSeq genes, five of which were associated with spermatogenesis. Furthermore, the breakpoints disrupted 43 topological-associated domains. Direct disruptions or potential dysregulations of genes, which play potential roles in male germ cell development, apoptosis, and spermatogenesis, were found in all cases (n = 6). In addition, high read-depth GS detected dual molecular findings in case MI6, involving a complex rearrangement and two point mutations in the gene DNAH1. Overall, our study provided the molecular characteristics of apparently balanced interchromosomal structural rearrangements in patients with male infertility. We demonstrated the complexity of chromosomal structural rearrangements, potential gene disruptions/dysregulation and single-gene mutations could be the contributing mechanisms underlie male infertility., Competing Interests: None

Nonobstructive azoospermia (NOA) is a common cause of infertility and is defined as the complete absence of sperm in ejaculation due to defective spermatogenesis. The aim of this study was to identify the genetic etiology of NOA in an infertile male from a Chinese consanguineous family. A homozygous missense variant of the membrane-bound O-acyltransferase domain-containing 1 (MBOAT1) gene (c.770C>T, p.Thr257Met) was found by whole-exome sequencing (WES). Bioinformatic analysis also showed that this variant was a pathogenic variant and that the amino acid residue in MBOAT1 was highly conserved in mammals. Quantitative polymerase chain reaction (Q-PCR) analysis showed that the mRNA level of MBOAT1 in the patient was 22.0% lower than that in his father. Furthermore, we screened variants of MBOAT1 in a broader population and found an additional homozygous variant of the MBOAT1 gene in 123 infertile men. Our data identified homozygous variants of the MBOAT1 gene associated with male infertility. This study will provide new insights for researchers to understand the molecular mechanisms of male infertility and will help clinicians make accurate diagnoses., Competing Interests: None

Acephalic spermatozoa syndrome is a rare type of teratozoospermia that severely impairs the reproductive ability of male patients, and genetic defects have been recognized as the main cause of acephalic spermatozoa syndrome. Spermatogenesis and centriole-associated 1 like (SPATC1L) is indispensable for maintaining the integrity of sperm head-to-tail connections in mice, but its roles in human sperm and early embryonic development remain largely unknown. Herein, we conducted whole-exome sequencing (WES) of 22 infertile men with acephalic spermatozoa syndrome. An in silico analysis of the candidate variants was conducted, and WES data analysis was performed using another cohort consisting of 34 patients with acephalic spermatozoa syndrome and 25 control subjects with proven fertility. We identified biallelic mutations in SPATC1L (c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X) from a patient whose sperm displayed complete acephalia. Both SPATC1L variants are rare and deleterious. SPATC1L is mainly expressed at the head-tail junction of elongating spermatids. Plasmids containing pathogenic variants decreased the level of SPATC1L in vitro. Moreover, none of the patient's four attempts at intracytoplasmic sperm injection (ICSI) resulted in a transplantable embryo, which suggests that SPATC1L defects might affect early embryonic development. In conclusion, this study provides the first identification of SPATC1L as a novel gene for human acephalic spermatozoa syndrome. Furthermore, WES might be applied for patients with acephalic spermatozoa syndrome who exhibit reiterative ICSI failures., Competing Interests: None

Semen analysis has long been used to evaluate male fertility. Recently, several sperm function tests have been developed. Of those, the sperm DNA fragmentation index (DFI), which describes the status of the sperm DNA, is thought to be a suitable parameter for evaluating male fertility. However, there have been no large-scale studies on the sperm DFI of Japanese men. Therefore, we investigated the feasibility of using an in-house flow cytometry-based sperm DFI analysis based on the sperm DNA fragmentation test of sperm chromatin structure assay (SCSA) to assess male fertility in Japan. This study enrolled 743 infertile and 20 fertile Japanese men. To evaluate reproducibility, inter- and intraobserver precision was analyzed. A receiver operating characteristic curve analysis was used to set a cutoff value for the sperm DFI to identify men who could father children by timed intercourse or intrauterine insemination. The variability of the sperm DFI among fertile volunteers was determined. The relationship between semen parameters and the sperm DFI was assessed by Spearman's rho test. A precision analysis revealed good reproducibility of the sperm DFI. The cutoff value of sperm DNA fragmentation in infertile men was 24.0%. Semen volume had no relationship with the sperm DFI. Sperm concentration, sperm motility, total motile sperm count, and percentage of normal-shaped sperm were significantly and negatively correlated with the sperm DFI. The median sperm DFI was smaller in fertile volunteers (7.7%) than that in infertile men (19.4%). Sperm DNA fragmentation analysis can be used to assess sperm functions that cannot be evaluated by ordinary semen analysis., Competing Interests: None

Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells (SSCs). However, such therapy for infertility has not been experimentally investigated yet. In this study, a mouse model with an X-linked testis-expressed 11 (TEX11) mutation (Tex11 PM/Y ) identified in azoospermia patients exhibited meiotic arrest due to aberrant chromosome segregation. Tex11 PM/Y SSCs could be isolated and expanded in vitro normally, and the mutation was corrected by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated endonuclease 9 (Cas9), leading to the generation of repaired SSC lines. Whole-genome sequencing demonstrated that the mutation rate in repaired SSCs is comparable with that of autonomous mutation in untreated Tex11 PM/Y SSCs, and no predicted off-target sites are modified. Repaired SSCs could restore spermatogenesis in infertile males and give rise to fertile offspring at a high efficiency. In summary, our study establishes a paradigm for the treatment of male azoospermia by combining in vitro expansion of SSCs and gene therapy., Competing Interests: None

Infertility affects about 15% of the world's population. In 40%-50% of infertile couples, a male factor underlies the problem, but in about 50% of these cases, the etiology of male infertility remains unexplained. Some clinical data show that lifestyle interventions may contribute to male reproductive health. Cessation of unhealthy habits is suggested for preserving male fertility; there is growing evidence that most preexisting comorbidities, such as obesity and metabolic syndrome, are highly likely to have an impact on male fertility. The analysis of genetic polymorphisms implicated in metabolic activity represents one of the most exciting areas in the study of genetic causes of male infertility. Although these polymorphisms are not directly connected with male infertility, they may have a role in specific conditions associated with it, that is, metabolic disorders and oxidative stress pathway genes that are potentially associated with an increased risk of male infertility due to DNA and cell membrane damage. Some studies have examined the impact of individual genetic differences and gene-diet interactions on male infertility, but their results have not been synthesized. We review the current research to identify genetic variants that could be tested to improve the chances of conceiving spontaneously through personalized diet and/or oral vitamin and mineral supplementation, by examining the science of genetic modifiers of dietary factors that affect nutritional status and male fertility., Competing Interests: None

Chromosomal abnormalities and Y chromosome microdeletions are considered to be the two more common genetic causes of spermatogenic failure. However, the relationship between chromosomal aberrations and Y chromosome microdeletions is still unclear. This study was to investigate the incidence and characteristics of chromosomal aberrations and Y chromosome microdeletions in infertile men, and to explore whether there was a correlation between the two genetic defects of spermatogenic failure. A 7-year retrospective study was conducted on 5465 infertile men with nonobstructive azoospermia or oligozoospermia. Karyotype analysis of peripheral blood lymphocytes was performed by standard G-banding techniques. Y chromosome microdeletions were screened by multiplex PCR amplification with six specific sequence-tagged site (STS) markers. Among the 5465 infertile men analyzed, 371 (6.8%) had Y chromosome microdeletions and the prevalence of microdeletions in azoospermia was 10.5% (259/2474) and in severe oligozoospermia was 6.3% (107/1705). A total of 4003 (73.2%) infertile men underwent karyotyping; 370 (9.2%) had chromosomal abnormalities and 222 (5.5%) had chromosomal polymorphisms. Karyotype analysis was performed on 272 (73.3%) patients with Y chromosome microdeletions and 77 (28.3%) had chromosomal aberrations, all of which involved sex chromosomes but not autosomes. There was a significant difference in the frequency of chromosomal abnormalities between men with and without Y chromosome microdeletions (P< 0.05)., Competing Interests: None

Sperm DNA fragmentation (SDF) has been linked with male infertility, and previous studies suggest that SDF can have negative influence on pregnancy outcomes with assisted reproduction. We performed a retrospective review of consecutive couples with a high SDF level that had intracytoplasmic sperm injection (ICSI) using testicular sperm (T-ICSI). We compared the T-ICSI outcomes to that of two control groups: 87 couples with failed first ICSI cycle and who had a second ICSI cycle using ejaculated sperm (Ej-ICSI), and 48 consecutive couples with high sperm chromatin structure assay (SCSA)-defined SDF (>15%) that underwent an ICSI cycle using ejaculated sperm after one or more failed ICSI cycles (Ej-ICSI-high SDF). The mean number of oocytes that were retrieved and the total number of embryos were not different among the three groups. The mean number of transferred embryos in the T-ICSI group was higher than the Ej-ICSI group but not significantly different than the Ej-ICSI-high SDF group (1.4, 1.2, and 1.3, respectively, P < 0.05). Clinical pregnancy rate in the T-ICSI group was not significantly different than the Ej-ICSI and Ej-ICSI-high SDF groups (48.6%, 48.2%, and 38.7%, respectively, P > 0.05). No significant difference was found in live birth rate when comparing T-ICSI to Ej-ICSI and Ej-ICSI-high SDF groups. The results suggest that pregnancy outcomes and live birth rates with T-ICSI are not significantly superior to Ej-ICSI in patients with an elevated SCSA-defined sperm DNA fragmentation and prior ICSI failure(s)., Competing Interests: None

Mice deficient in the transcription factor pleomorphic adenoma gene 1 (PLAG1) exhibit reproductive issues that are characterized, in part, by decreased progressive sperm motility in the male. However, the underlying cause of this impairment is unknown. As epididymal transit is critical for sperm maturation and motility, the morphology of the epididymis of Plag1-deficient mice was investigated and the spatial expression patterns of PLAG1 protein and mRNA were identified. Using X-gal staining and in situ hybridization, PLAG1 was shown to be widely expressed in both the epithelium and stroma in all regions of the mouse epididymis. Interestingly, the X-gal staining pattern was markedly different in the cauda, where it could be suggestive of PLAG1 secretion into the epididymal lumen. At all ages investigated, the morphology of epididymides from Plag1 knockout (KO) mice was aberrant; the tubule failed to elongate and coil, particularly in the corpus and cauda, and the cauda was malformed, lacking its usual bulbous shape. Moreover, the epididymides from Plag1 KO mice were significantly reduced in size relative to body weight. In 20% of Plag1-deficient mice, the left testicle and epididymis were lacking. The impaired morphogenesis of the epididymal tubule is likely to be a major contributing factor to the fertility problems observed in male Plag1-deficient mice. These results also establish PLAG1 as an important regulator of male reproduction, not only through its involvement in testicular sperm production, but also via its role in the development and function of the epididymis., Competing Interests: None

The azoospermia factor (AZF) region is important for spermatogenesis, and deletions within these regions are a common cause of oligozoospermia and azoospermia. Although several studies have reported this cause, the present research, to the best of our knowledge, is the first large-scale study assessing this factor in Japan. In this study, 1030 male patients with infertility who were examined for Y chromosome microdeletion using the polymerase chain reaction-reverse sequence-specific oligonucleotide (PCR-rSSO) method, a newly developed method for Y chromosome microdeletion screening, were included. The study enrolled 250 patients with severe oligospermia and 717 patients with azoospermia. Among the 1030 patients, 4, 4, 10, and 52 had AZFa, AZFb, AZFb+c, and AZFc deletions, respectively. The sperm recovery rate (SRR) of microdissection testicular sperm extraction in patients with AZFc deletions was significantly higher than that in those without AZF deletions (60.0% vs 28.7%, P = 0.04). In patients with gr/gr deletion, SRR was 18.7%, which was lower than that in those without gr/gr deletion, but was not statistically significant. In conclusion, our study showed that the frequency of Y chromosome microdeletion in male patients in Japan was similar to that reported in patients from other countries, and SRR was higher in patients with AZFc deletion., Competing Interests: None

The apoptosis that occurs in the immature testis under physiological conditions is necessary for male germ cell development, whereas improper activation of apoptosis can impair spermatogenesis and cause defects in reproduction. We previously demonstrated that in mice, the makorin-2 (Mkrn 2) gene is expressed exclusively in the testis and its deletion leads to male infertility. To understand the potential molecular mechanism, in this study, we found that levels of apoptosis in the testis were abnormally high in the absence of Mkrn 2. To identify specific gene(s) involved, we performed digital gene expression profiling (DGE) and pathway analysis via gene set enrichment analysis (GSEA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and we found that MKRN2 inhibits p53 apoptosis effector related to PMP22 (PERP) expression and that levels of the protein in sperm samples have an inverse correlation with infertility levels. GSEA additionally indicated that PERP is a negative regulator of spermatogenesis and that its ectopic expression induces male infertility. Further, Gene Expression Omnibus (GEO) dataset analysis showed that p53, upstream of PERP, was upregulated in oligoasthenoteratozoospermia (OAT). These observations suggest that Mkrn 2 is crucial for protecting germ cells from excessive apoptosis and implicate Mkrn 2-based suppression of the p53/PERP signaling pathway in spermatogenesis and male fertility., Competing Interests: None

The syndrome of multiple morphological abnormalities of the sperm flagella (MMAF) is a specific kind of asthenoteratozoospermia with a mosaic of flagellar morphological abnormalities (absent, short, bent, coiled, and irregular flagella). MMAF was proposed in 2014 and has attracted increasing attention; however, it has not been clearly understood. In this review, we elucidate the definition of MMAF from a systematical view, the difference between MMAF and other conditions with asthenoteratozoospermia or asthenozoospermia (such as primary mitochondrial sheath defects and primary ciliary dyskinesia), the knowledge regarding its etiological mechanism and related genetic findings, and the clinical significance of MMAF for intracytoplasmic sperm injection and genetic counseling. This review provides the basic knowledge for MMAF and puts forward some suggestions for further investigations., Competing Interests: None

Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein involved in both genomic and nongenomic estrogen signal transduction pathways. To date, the role of PELP1 protein has yet to be characterized in human sperm and has not been associated with sperm parameters. To confirm the presence of PELP1 in human sperm, fresh semen samples were obtained from 178 donors. The study was designed to establish both mRNA and protein presence, and protein cellular localization. Additionally, the number of PELP1-positive spermatozoa was analyzed in men with normal and abnormal semen parameters. Sperm parameters were assessed according to the World Health Organization (WHO) 2010 standards. The presence of PELP1 in spermatozoa was investigated using four precise, independent techniques. The qualitative presence of transcripts and protein was assessed using reverse transcription-polymerase chain reaction (RT-PCR) and western blot protocols, respectively. The cellular localization of PELP1 was investigated by immunocytochemistry. Quantitative analysis of PELP1-positive cells was done by flow cytometry. PELP1 mRNA and protein was confirmed in spermatozoa. Immunocytochemical analysis identified the presence of PELP1 in the midpieces of human sperm irrespective of sperm parameters. Becton Dickinson fluorescence-activated cell sorting (FACSCalibur™) analysis revealed a significantly lower number of PELP1-positive cells in males with normal semen parameters versus abnormal samples (42.78% ± 11.77% vs 61.05% ± 21.70%, respectively; P = 0.014). The assessment of PELP1 may be a time-saving method used to obtain information about sperm quality. The results of our study suggest that PEPL1 may be utilized as an indicator of sperm quality; thereby, PELP1 may be an additional biomarker useful in the evaluation of male infertility., Competing Interests: All authors declared no competing interests

In about half the cases of involuntary childlessness, a male infertility factor is involved. The PIWI-LIKE genes, a subclade of the Argonaute protein family, are involved in RNA silencing and transposon control in the germline. Knockout of murine Piwi-like 1 and 2 homologs results in complete infertility in males. The aim of this study was to analyze whether the mRNA expression of human PIWI-LIKE 1-4 genes is altered in ejaculated spermatozoa of men with impaired sperm characteristics. Ninety male participants were included in the study, among which 47 were with normozoospermia, 36 with impaired semen characteristics according to the World Health Organization (WHO) manual, 5 th edition, and 7 with azoospermia serving as negative control for the PIWI-LIKE 1-4 mRNA expression in somatic cells in the ejaculate. PIWI-LIKE 1-4 mRNA expression in the ejaculated spermatozoa of the participants was measured by quantitative real-time PCR. In nonazoospermic men, PIWI-LIKE 1-4 mRNA was measurable in ejaculated spermatozoa in different proportions. PIWI-LIKE 1 (100.0%) and PIWI-LIKE 2 (49.4%) were more frequently expressed than PIWI-LIKE 3 (9.6%) and PIWI-LIKE 4 (15.7%). Furthermore, a decreased PIWI-LIKE 2 mRNA expression showed a significant correlation with a decreased sperm count (P = 0.022) and an increased PIWI-LIKE 1 mRNA expression with a decreased progressive motility (P = 0.048). PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression exhibited a significant association with impaired sperm characteristics and may be a useful candidate for the evaluation of the impact of PIWI-LIKE 1-4 mRNA expression on male infertility.

Primary ciliary dyskinesia (PCD) is an autosomal-recessive disorder resulting from the loss of normal ciliary function. Symptoms include neonatal respiratory distress, chronic sinusitis, bronchiectasis, situs inversus, and infertility. However, only 15 PCD-associated genes have been identified to cause male infertility to date. Owing to the genetic heterogeneity of PCD, comprehensive molecular genetic testing is not considered the standard of care. Here, we provide an update of the progress on the identification of genetic factors related to PCD associated with male infertility, summarizing the underlying molecular mechanisms, and discuss the clinical implications of these findings. Further research in this field will impact the diagnostic strategy for male infertility, enabling clinicians to provide patients with informed genetic counseling, and help to adopt the best course of treatment for developing directly targeted personalized medicine.

Male infertility is a multifactorial syndrome encompassing a wide variety of disorders. In recent years, several genome-wide single-nucleotide polymorphism (SNP) association studies (GWAS) have been performed on azoospermia and/or oligozoospermia in different populations including two GWAS on nonobstructive azoospermia in China; however, the association of SNPs with idiopathic male infertility, especially asthenozoospermia and oligozoospermia, and their correlation with semen parameters are still not clear. To investigate genetic variants associated with idiopathic male infertility (asthenozoospermia, oligozoospermia, and oligoasthenozoospermia) in Chinese Han people, 20 candidate SNPs were selected from GWAS results and genotyped using the Sequenom MassARRAY assay. A total of 136 subfertile men and 456 healthy fertile men were recruited. rs6476866 in SLC1A1 (P = 1.919E-4, OR = 0.5905, 95% CI: 0.447-0.78) and rs10129954 in DPF3 (P = 0.0023, OR = 2.199, 95% CI: 1.311-3.689) were strongly associated with idiopathic male infertility. In addition, positive associations were observed between asthenozoospermia and rs215702 in LSM5 (P = 0.0016, OR = 1.479, 95% CI: 1.075-2.033) and between oligoasthenozoospermia and rs2477686 in PEX10 (P = 0.0011, OR = 2.935, 95% CI: 1.492-5.775). In addition, six SNPs (rs215702 in LSM5, rs6476866 in SLC1A1, rs10129954 in DPF3, rs1801133 in MTHFR, rs2477686 in PEX10, and rs10841496 in PED3A) were significantly correlated with semen quality alterations. Our results suggest that idiopathic male infertility in different ethnic groups may share the same mechanism or pathway. Cohort expansion and further mechanistic studies on the role of genetic factors that influence spermatogenesis and sperm progressive motility are suggested.