Mingrong Lv

Background: Male infertility is a complex, multifactorial disorder that affects ∼7% of men worldwide. Variants in DNAH and CFAP genes are well‐established causes of asthenoteratozoospermia, but the contribution of TTC ‐family genes ( TTC12 , TTC21A , TTC29 ) to sperm dysfunction and ICSI outcome remains poorly documented. Objectives: To investigate the genetic and clinical implications of TTC gene variants in men with asthenoteratozoospermia and to evaluate the ICSI outcomes of affected individuals. Materials and Methods: We performed whole‐exome sequencing and bioinformatic filtering in 843 men with asthenoteratozoospermia. Candidate variants were validated by Sanger sequencing, and the effects on protein expression were assessed using western blotting and immunofluorescence. Sperm morphology and ultrastructure were examined by light and transmission electron microscopy. Clinical outcomes were evaluated following ICSI treatment, including a total of 18 TTC ‐variant patients and DNAH1 ‐variant men. Results: Thirteen TTC variants in 11 unrelated patients (1.3%) were identified in this cohort, including two homozygous TTC12 variants, six homozygous plus two compound‐heterozygous TTC21A variants, and one homozygous TTC29 variants. Variants induced markedly reduced TTC proteins expression and disorganized axonemes and peri‐axonemal structures. Clinical data from ICSI cycles demonstrated that fertilization could be achieved, importantly the first reported live birth for TTC21A variants, although blastocyst rates were lower than in DNAH1 ‐mutated controls. Conclusion: This study not only expanded the genotype–phenotype spectrum of variations in the TTC gene family, but also built a bridge between molecular genetics and clinical application, demonstrating the translational path from gene discovery to effective treatment.

The formation of N6-methyladenosine (m6A) is the most prevalent internal RNA modification in eukaryotic transcripts and plays an essential role in spermatogenesis. PRRC2A is an m6A reader that is indispensable for meiotic progression and is essential for maintaining spermatocyte identity during mouse spermatogenesis. However, its contribution to human spermatogenesis remains unknown. In this study, we investigated a man diagnosed with non-obstructive azoospermia (NOA) and two with severe oligozoospermia, all carrying biallelic PRRC2A variants. All patients exhibited impaired spermatogenesis. The patient with NOA showed marked arrest at meiotic metaphase I or delayed entry into metaphase. RNA-seq and m6A-seq data demonstrated that his PRRC2A deficiency was accompanied by altered chromatoid body–associated RNA regulatory architecture and affected histone-to-transition protein exchange during spermiogenesis. Despite the presence of PRM2-positive spermatids, testicular spermatozoa retrieved from the NOA patient failed to achieve fertilization via TESE–ICSI, possibly due to impaired DNA integrity arising from a perturbed histone-to-protamine transition. In the remaining cases, repeated ART attempts resulted in fertilization failure or early embryonic arrest. A successful pregnancy was ultimately achieved only through donor sperm IVF-ET. The concordance between molecular defects and consistently poor fertilization outcomes across independent individuals supports a pathogenic role for PRRC2A deficiency. We suggest that the disruption of an m6A reader compromises meiotic progression, as well as sperm functional competence and fertilization capacity, although the limited sample size precludes formal statistical inference. By linking epitranscriptomic dysregulation to adverse ART outcomes, this study expands the molecular basis of severe male factor infertility and has direct implications for genetic diagnosis, prognosis of fertilization potential, and reproductive counselling.

Severe asthenoteratozoospermia, a prevalent cause of male infertility, has increasingly been associated with ARMC2 variants that cause Multiple Morphological Abnormalities of the Sperm Flagella (MMAF). Although ARMC2 is also expressed in other ciliary structures, no studies have yet reported a link between ARMC2 gene variants and other symptoms of Primary Ciliary Dyskinesia (PCD). Here, we performed whole-exome sequencing (WES) on Chinese subjects with MMAF to identify potential genetic variants. Sanger sequencing was used to validate the candidate variants. Sperm morphology was assessed using modified hematoxylin and eosin (H&E) staining, and transmission electron microscopy (TEM) was performed to observe the ultrastructural defects of the sperm flagella. Western blot analysis and immunofluorescence (IF) of spermatozoa were performed to evaluate variations in structural protein. Additionally, intracytoplasmic sperm injection (ICSI) was applied for assisted fertilization. We identified two compound heterozygous ARMC2 variants and one homozygous variant (P1: c.1030_1042del, p.T344fs/c.1331G > A, p.R444H; P2:c.1264C > T, p.R422X) in two unrelated individuals. Notably, in addition to MMAF, individual P2 exhibited classic symptoms of PCD in the lungs, including recurrent airway infections, bronchitis, and rhinosinusitis. Morphological and ultrastructural analyses of the spermatozoa obtained from the two individuals revealed dramatic disorganization in axonemal and peri-axonemal structures, as well as the absence of the axonemal central pair complex (CPC). Immunoblotting and immunofluorescence assays revealed the reduced expression of ARMC2 and the abnormality of various axonal structural proteins. Further assisted reproduction outcomes showed that one of the individuals conceived successfully after Intracytoplasmic Sperm Injection (ICSI). Overall, this study significantly expanded the mutational phenotype of ARMC2, marking the first discovery of PCD-related pulmonary phenotypes outside of the reproductive system. This work establishes the association between ARMC2 and typical PCD and lays the groundwork for further investigation into the molecular mechanisms of ARMC2 in both flagellogenesis and ciliogenesis.

Bi-allelic variants in DNAH11 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on human sperm flagellar and their involvement in male infertility remain largely unknown. A collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11 variants. In this study, we identified seven novel variants in the DNAH11 gene in four asthenoteratozoospermia subjects. These variants led the absence of DNAH11 proteins and ultrastructure defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. The levels of ODA protein DNAI2 and axoneme related proteins were down regulated, instead of inner dynein arms (IDA) proteins DNAH1 and DNAH6. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11, resulting in the aberrant assembly of sperm flagella and contributing to asthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants.

BackgroundHepatitis B virus (HBV) infection poses a substantial threat to human health, impacting not only infected individuals but also potentially exerting adverse effects on the health of their offspring. The underlying mechanisms driving this phenomenon remain elusive. This study aims to shed light on this issue by examining alterations in paternally imprinted genes within sperm.MethodsA cohort of 35 individuals with normal semen analysis, comprising 17 hepatitis B surface antigen (HBsAg)-positive and 18 negative individuals, was recruited. Based on the previous research and the Online Mendelian Inheritance in Man database (OMIM, https://www.omim.org/), targeted promoter methylation sequencing was employed to investigate 28 paternally imprinted genes associated with various diseases.ResultsBioinformatic analyses revealed 42 differentially methylated sites across 29 CpG islands within 19 genes and four differentially methylated CpG islands within four genes. At the gene level, an increase in methylation of DNMT1 and a decrease in methylation of CUL7, PRKAG2, and TP53 were observed. DNA methylation haplotype analysis identified 51 differentially methylated haplotypes within 36 CpG islands across 22 genes.ConclusionsThis is the first study to explore the effects of HBV infection on sperm DNA methylation and the potential underlying mechanisms of intergenerational influence of paternal HBV infection.

Purpose To identify the genetic cause of a cryptorchidism patient carrying a non-canonical splicing variant highlighted by SPCards platform in RXFP2 and to provide a comprehensive overview of RXFP2 variants with cryptorchidism correlation. Methods We identified a homozygous non-canonical splicing variant by whole-exome sequencing and Sanger sequencing in a case with cryptorchidism and non-obstructive azoospermia (NOA). As the pathogenicity of this non-canonical splicing variant remained unclear, we initially utilized the SPCards platform to predict its pathogenicity. Subsequently, we employed a minigene splicing assay to further evaluate the influence of the identified splicing variant. Microdissection testicular sperm extraction (micro-TESE) combined with intracytoplasmic sperm injection (ICSI) was performed. PubMed and Human Genome Variant Database (HGMD) were queried to search for RXFP2 variants. Results We identified a homozygous non-canonical splicing variant (NM_130806: c.1376-12A > G) in RXFP2, and confirmed this variant caused aberrant splicing of exons 15 and 16 of the RXFP2 gene: 11 bases were added in front of exon 16, leading to an abnormal transcript initiation and a frameshift. Fortunately, the patient successfully obtained his biological offspring through micro-TESE combined with ICSI. Four cryptorchidism-associated variants in RXFP2 from 90 patients with cryptorchidism were identified through a literature search in PubMed and HGMD, with different inheritance patterns. Conclusion This is the first cryptorchidism case carrying a novel causative non-canonical splicing RXFP2 variant. The combined approach of micro-TESE and ICSI contributed to an optimal pregnancy outcome. Our literature review demonstrated that RXFP2 variants caused cryptorchidism in a recessive inheritance pattern, rather than a dominant pattern.

Background Bi-allelic variants in DNAH11 and DNAH5 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on sperm flagellar and their involvement in male infertility remain elusive. Methods A collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11/DNAH5 variants. Results The study identified seven novel variants in the DNAH11 gene in four subjects and two novel variants in the DNAH5 gene in one individual with oligoasthenoteratozoospermia. These variants led to structural defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. However, the patient carrying DNAH5 variants did not achieve a favorable pregnancy outcome. Conclusions The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11/DNAH5, resulting in the aberrant assembly of sperm flagella and contributing to oligoasthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants.

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.

Introduction: Infertility is a major disease affecting human life and health, among which male factors account for about half. Asthenoteratozoospermia accounts for the majority of male infertility. High-throughput sequencing techniques have identified numerous variants in genes responsible for asthenoteratozoospermia; however, its etiology still needs to be studied.Method: In this study, we performed whole-exome sequencing on samples from 375 patients with asthenoteratozoospermia and identified two HYDIN compound heterozygous variants, a primary ciliary dyskinesia (PCD)-associated gene, in two unrelated subjects. H&E staining, SEM were employed to analyze the varies on sperm of patients, further, TEM was employed to determine the ultrastructure defects. And westernblot and immunostaining were chose to evaluate the variation of structural protein. ICSI was applied to assist the mutational patient to achieve offspring.Result: We identified two HYDIN compound heterozygous variants. Patient AY078 had novel compound heterozygous splice variants (c.5969-2A>G, c.6316+1G>A), altering the consensus splice acceptor site of HYDIN. He was diagnosed with male infertility and PCD, presenting with decreased sperm progressive motility and morphological abnormalities, and bronchial dilatation in the inferior lobe. Compared to the fertile control, HYDIN levels, acrosome and centrosome markers (ACTL7A, ACROSIN, PLCζ1, and Centrin1), and flagella components (TOMM20, SEPT4, SPEF2, SPAG6, and RSPHs) were significantly reduced in HYDIN-deficient patients. Using intracytoplasmic sperm injection (ICSI), the patient successfully achieved clinical pregnancy. AY079 had deleterious compound heterozygous missense variants, c.9507C>G (p. Asn3169Lys) and c.14081G>A (p. Arg4694His), presenting with infertility; however, semen samples and PCD examination were unavailable.Discussion: Our findings provide the first evidence that the loss of HYDIN function causes asthenoteratozoospermia presenting with various defects in the flagella structure and the disassembly of the acrosome and neck. Additionally, ICSI could rescue this failure of insemination caused by immobile and malformed sperm induced by HYDIN deficiency.