Feiyang Diao

STUDY QUESTION: Are there new genetic factors responsible for male infertility characterized by fertilization defects despite normal sperm motility? SUMMARY ANSWER: We identified the novel pathogenetic gene actin-related protein T3 (ACTRT3) as one of primary causes for male infertility characterized by fertilization defects. WHAT IS KNOWN ALREADY: To date, only a few genes have been reported to be responsible for fertilization defects in male infertility despite normal sperm motility. ACTRT3, as one of the actin-related proteins, is specifically expressed highly in murine testes and interacts with the protein Profilin3 to form a testis-specific complex during spermatogenesis. STUDY DESIGN, SIZE, DURATION: The ACTRT3 variants were identified by whole-exome sequencing in a cohort of 248 infertile couples characterized by fertilization defects (fertilization rate ≤ 30% in their IVF/ICSI attempts) despite normal sperm concentration (≥15 × 106/ml) and normal motility (progressive motility ≥32%; motility of PR (progressive)+NP (non-progressive) ≥40%). Thirty-six hours after cell transfection, immunoprecipitation was performed to detect the interaction between ACTRT3 and actin-like 7A (ACTL7A)/phospholipase C zeta 1 (PLCZ1) (n = 3 biological replicates). A knock-in (KI) mouse model was generated by CRISPR-Cas9. Sexually mature males (10 weeks old) were used for fertility testing (n = 3 mice per group, lasts 6–8 months), semen parameter analysis (n = 3 mice per group), western blot (n = 3 biological replicates), immunofluorescence (n = 3 biological replicates), IVF (n ≥ 3 biological replicates), ICSI (n = 3 biological replicates), scanning electron microscopy (SEM, n = 3 mice per group), transmission electron microscopy (TEM, n = 3 mice per group), Ca2+ oscillations (n ≥ 3 biological replicates), and other functional assays between 2022 and 2025. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ACTRT3 variants were identified by whole-exome sequencing and confirmed by Sanger sequencing. ACTRT3 KI mouse was constructed to discover the pathogenic mechanism. We first checked ultrastructure of the sperm through periodic acid-schiff staining, SEM, and TEM. Western blotting, immunofluorescence, and immunoprecipitation were further performed to clarify the molecular mechanism of the ACTRT3 variants. MAIN RESULTS AND THE ROLE OF CHANCE: We identified compound-heterozygous (NM_032487.5: c. C41G (p. Ser14Trp) and c. C687delA (p. Lys229fs)) and homozygous variants (NM_032487.5: c.712C>T (p. Gln238Ter)) in ACTRT3 in two infertile males, and functional studies in transgenic KI mouse models confirmed the pathogenicity of the Actrt3 variants. Defects in ACTRT3 caused decreased protein levels of ACTL7A and disrupted localization of PLCZ1 in sperm, which resulted in acrosome detachment, further impacted the binding between sperm and the zona pellucida of oocytes and caused fertilization failure. In addition, sperm with defective ACTRT3 cannot initiate calcium oscillations, which leads to embryo arrest. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: More cases are needed to confirm the genetic contribution of ACTRT3 variants to male infertility. In addition, it will be more persuasive and informative by adding the rescue experiments of artificial oocyte activation through SrCl2 which is used to active calcium ion oscillation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest the causal relationship between ACTRT3 variants and male infertility caused by fertilization defects, and partially revealing the pathological mechanisms of ACTRT3 variants. This provides new insights into the understanding of male infertility due to fertilization problems and provides references for future clinical treatment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (82325021, 32130029, and 82201767) and the Fundamental and Interdisciplinary Disciplines Breakthrough Plan of the Ministry of Education of China (No. JYB2025XDXM508). TRIAL REGISTRATION NUMBER: N/A

Purpose To study reproductive outcomes of transferring embryos with mosaic chromosomal aneuploidies unrelated to target loci in preimplantation genetic testing for structural rearrangement (PGT-SR). Methods A retrospective analysis of 1712 PGT-SR cycles (involving 1373 couples) from a single center was conducted. Comprehensive chromosomal analysis was performed on trophectoderm biopsy samples using whole-genome amplification combined with next-generation sequencing. Clinical outcomes after single embryo transfer, including clinical pregnancy rate, miscarriage rate, live birth rate, and ectopic pregnancy rate, were compared between mosaic embryo transfers and completely euploid embryo transfers. Results In PGT-SR cycles, euploid embryo transfers (1338 cycles) demonstrated a clinical pregnancy rate of 65.17%, live birth rate of 58.67%, miscarriage rate of 6.05%, and ectopic pregnancy rate of 0.45%, whereas mosaic embryo transfers (165 cycles) exhibited comparable outcomes with 58.18%, 54.55%, 3.64%, and 0%, respectively. The subgroup analysis by mosaic degree which revealed low-level mosaicism (20%–29% and 30%–49%) had outcomes similar to euploid embryos. High-level mosaicism (50%–69%) demonstrated significantly reduced clinical pregnancy (OR = 0.20, 95% CI 0.06–0.54) and live birth rates (OR = 0.20, 95% CI 0.06–0.56), though miscarriage remained comparable. No significant outcome variations were observed across chromosomal types. Conclusions In PGT-SR cycles, embryos with low-level mosaicism (20%–49%) unrelated to target loci demonstrate equivalent clinical pregnancy and live birth outcomes to completely euploid embryos. Although high-level mosaicism (50%–69%) is likely associated with reduced live birth rates, these embryos still retain potential for healthy live births.

STUDY QUESTION: What is the minimum effective dose of SHR7280, a novel oral GnRH antagonist, to prevent a premature LH surge in women undergoing controlled ovarian hyperstimulation for ART? SUMMARY ANSWER: SHR7280 at 200 mg once every 12 h (Q12h) was the minimal effective dose to suppress an LH surge and was associated with high-quality embryos, and a high clinical pregnancy rate. SHR7280 was well-tolerated and safe. WHAT IS KNOWN ALREADY: Currently, all approved GnRH antagonists for preventing a premature LH surge are injectable, peptide-based formulations, which can cause inconvenience and injection site reactions. There is a significant unmet need for an orally available GnRH antagonist to address this issue. SHR7280 has previously demonstrated effective suppression of LH levels in healthy volunteers in phase 1 trials. STUDY DESIGN, SIZE, DURATION: This multi-center, open-label, dosing-finding phase 2 trial was conducted between 26 January 2022 and 3 August 2023 in 85 infertile women. PARTICIPANTS/MATERIALS, SETTING, METHODS: The starting dose of SHR7280 was 300 mg Q12h, followed by dose exploration in two lower dose groups (200 mg Q12h and 200 mg Q24h) sequentially. Dose exploration was terminated if an LH surge occurred. SHR7280 was administrated orally starting on Day 5 of recombinant human FSH stimulation until hCG administration. The primary endpoint was the rate of premature LH surge inhibition during SHR7280 treatment. MAIN RESULTS AND THE ROLE OF CHANCE: Eighty-five patients received SHR7280: 300 mg Q12h, n = 40; 200 mg Q12h, n = 42; 200 mg Q24h, n = 3. The mean ( ± SD) duration of SHR7280 treatment was 5.7 ± 1.2, 5.7 ± 1.3, and 3.7 ± 0.6 days, respectively. The rate of LH surge inhibition was 99% (95% CI 94–100) in all patients, 100% (95% CI 91–100) in the 300 mg Q12h group, 100% (95% CI 92–100) in the 200 mg Q12h group, and 67% (95% CI 9–99) in the 200 mg Q24h group. Two hundred milligrams Q12h was established as the minimal effective dose. Embryological and pregnancy outcomes were comparable in the 300 and 200 mg Q12h groups. In the 200 mg Q12h group, the mean ( ± SD) number of oocytes retrieved per patient, two-pronucleate zygotes, and high-quality embryos was 10.7 ± 4.6, 6.9 ± 3.2, and 3.9 ± 2.6, respectively; among patients receiving fresh embryo transfer, 62% (95% CI 44–78) were tested positive for serum β-hCG and 53% (95% CI 35–70) were confirmed to be clinically pregnant per transfer. No spontaneous ovulation occurred on the day of oocyte retrieval. In all 85 patients, treatment-related adverse events were reported in 1 (1%; mild in severity) patient. LIMITATIONS, REASONS FOR CAUTION: The sample size was moderate and there was no standard peptide-based GnRH antagonist as a control group. Additionally, the performance of SHR7280 in specific populations, such as patients with diminished ovarian reserve and at high risk of OHSS, remains to be established. Further research is also needed to determine the recommended dose in patients with high BMI, intending for frozen embryo transfer, and using a GnRH agonist as trigger. Moreover, the study included only Chinese patients and allowed up to 2 embryos per transfer. The efficacy and safety of SHR7280 in other racial groups and regions require further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Findings from this phase 2 trial suggest that an oral GnRH antagonist could be an effective alternative for preventing a premature LH surge in ART. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Jiangsu Hengrui Pharmaceuticals. Z.L., Yu.S., C.S., Hong.C., and K.S. were employees of Jiangsu Hengrui Pharmaceuticals at the time of study. All other authors have no conflicts of interests to declare. TRIAL REGISTRATION NUMBER: NCT05082233.

Context: Dyslipidemia adversely affects reproduction outcomes; however, its relation with repeated implantation failure (RIF) remains unclear. Objective: This study aims to analyze the impact of dyslipidemia on assisted reproductive technology (ART) outcomes, endometrial transcriptome, and microbiome of RIF women. Design: A retrospective real-world analysis and prospective study. Setting: University clinic. Patients: 6499 infertile women, including 5618 non-RIF and 881 RIF. Interventions: Dyslipidemia. Main Outcome Measures: Implantation rate (IR), clinical pregnancy rate (CPR), and live birth rate (LBR) were compared in RIF women with or without dyslipidemia. Results of endometrial studies, including RNA sequencing (RNA-seq)-based endometrial receptivity test (ERT), 16S rRNA-based microbiome study, next-generation sequencing (NGS)-based gene comparison, and bulk RNA-seq deconvolution analysis were analyzed. Results: The prevalence of dyslipidemia in RIF women was significantly higher. In dyslipidemia women, IR, CPR, and LBR were significantly lower, and the prevalence of nonreceptive ERT and the presence of endometrial pathogenic bacteria were higher than those of controls. After the personalized treatment, CPR 73.3% and LBR 60.0% were achieved in dyslipidemia women. NGS revealed that 176 differentially expressed genes in the endometrium of RIF women with dyslipidemia compared to those without, suggesting highly enriched in cholesterol and steroid biosynthesis and monocyte differentiation processes. An increased endometrial CD56dim natural killer cells and macrophage M1/M2 ratio with dysregulated immune factors were found by bulk RNA-seq deconvolution analysis. Conclusion: RIF women with dyslipidemia have significantly poorer ART outcomes. In women with dyslipidemia, immune homeostasis was breached in the luteal phase endometrium, implicating a possible immune mechanism for dyslipidemia-related implantation failure.

Y-chromosome AZFc deficiency, which includes the DAZ gene family, is one of the most common genetic causes of severe male infertility. Epidemiological and molecular evidence suggest a developmental link between abnormalities in germ cell development and testicular germ cell tumor (TGCT), but whether and how DAZ deficiency is associated with TGCT susceptibility remains unclear. Using a specific human embryonic stem cell (hESC) model, we constructed and characterized a DAZ-deleted cell line (SKLRMe001-A) and compared its efficiency of induction into human primordial germ cell-like cells (hPGCLCs) with that of the H1 control line. We assessed intersections between embryonic-like networks and HMGA1-P53-MAPK/PI3K pathways to explore the potential connection between DAZ deficiency and TGCT susceptibility. DAZ deficiency significantly reduced hPGCLC induction efficiency and downregulated early lineage markers such as SOX17, BLIMP1, and TFAP2C, accompanied by S-phase shortening, cell cycle dysregulation, and increased apoptosis. RNA sequencing and enrichment analyses indicated sustained upregulation of HMGA1 and pointed to dysregulated homeostasis of P53 stress responses and PI3K/AKT and MAPK signaling axes. These findings are consistent with features of TGCT, including maintenance of embryonic-like programs, activation of proliferative signals, and heightened DNA damage responses. Therefore, we propose that lineage differentiation defects and mismatches between stress and survival signaling caused by DAZ deficiency may produce in vivo a population of "undifferentiated and susceptible" cells that increase the risk of TGCT, while also explaining the developmental basis of male infertility. Using a human in vitro model, this study links DAZ deficiency to both infertility and TGCT susceptibility and identifies HMGA1 as a central transcriptional-signaling hub. This finding provides molecular clues for risk stratification and early surveillance in populations with DAZ deficiency and suggests that interventions targeting HMGA1 and the PI3K/AKT and MAPK pathways may simultaneously improve germline differentiation and reduce potential tumor risk.

Dcaf17, also known as DDB1- and CUL4-associated factor 17, is a member of the DCAF family and acts as the receptor for the CRL4 ubiquitin E3 ligase complex. Several previous studies have reported that mutations in Dcaf17 cause Woodhouse-Sakati Syndrome (WSS), which results in oligoasthenoteratozoospermia (OAT) and male infertility. As a model to explore the role of Dcaf17 in the male reproductive system, we created Dcaf17-deficient male golden hamsters using CRISPR-Cas9 technology, the results of which demonstrate that deletion of Dcaf17 led to abnormal spermatogenesis and infertility. To uncover the underlying molecular mechanisms involved, we conducted single-cell RNA sequencing (scRNA-seq) analysis to evaluate the effect of Dcaf17 deficiency on transcriptional levels in spermatogenic cells during various stages of spermatogenesis. These data emphasize the significant regulatory role played by Dcaf17 in early spermatogenic cells, with many biological processes being affected, including spermatogenesis, and protein degradation. Dysregulation of genes associated with these functions ultimately leads to abnormalities. In summary, our findings highlight the critical function of Dcaf17 in spermatogenesis and male fertility and clarify the specific stage at which Dcaf17 exerts its effects, while simultaneously providing a novel animal model for the study of Dcaf17.

Background: Dystrophinopathies are the most common X-linked inherited muscle diseases, and the disease-causing gene is DMD. Exonic duplications are a common type of pathogenic variants in the DMD gene, however, 5’ end exonic duplications containing exon 1 are less common. When assessing the pathogenicity of exonic duplications in the DMD gene, consideration must be given to their impact on the reading frame. Traditional molecular methods, such as multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS), are commonly used in clinics. However, they cannot discriminate the precise physical locations of breakpoints and structural features of genomic rearrangement. Long-read sequencing (LRS) can effectively overcome this limitation. Results: We used LRS technology to perform whole genome sequencing on three families and analyze the structural variations of the DMD gene, which involves the duplications of exon 1 and/or exon 2. Two distinct variant types encompassing exon 1 in the DMD Dp427m isoform and/or Dp427c isoform are identified, which have been infrequently reported previously. In pedigree 1, the male individuals harboring duplication variant of consecutive exons 1–2 in the DMD canonical transcript (Dp427m) and exon 1 in the Dp427c transcript are normal, indicating the variant is likely benign. In pedigree 3, the patient carries complex SVs involving exon 1 of the DMD Dp427c transcript showing an obvious phenotype. The locations of the breakpoints and the characteristics of structural variants (SVs) are identified by LRS, enabling the classification of the variants' pathogenicity. Conclusions: Our research sheds light on the complexity of DMD variants encompassing Dp427c/Dp427m promoter regions and emphasizes the importance of cautious interpretation when assessing the pathogenicity of DMD 5' end exonic duplications, particularly in carrier screening scenarios without an affected proband.

Preimplantation embryo arrest (PREMBA) is a common cause of female infertility and recurrent failure of assisted reproductive technology. However, the genetic basis of PREMBA is largely unrevealed. Here, using whole-exome sequencing data from 606 women experiencing PREMBA compared with 2,813 controls, we performed a population and gene–based burden test and identified a candidate gene, karyopherin subunit α7 (KPNA7). In vitro studies showed that identified sequence variants reduced KPNA7 protein levels, impaired KPNA7 capacity for binding to its substrate ribosomal L1 domain-containing protein 1 (RSL1D1), and affected KPNA7 nuclear transport activity. Comparison between humans and mice suggested that mouse KPNA2, rather than mouse KPNA7, acts as an essential karyopherin in embryonic development. Kpna2^–/– female mice showed embryo arrest due to zygotic genome activation defects, recapitulating the phenotype of human PREMBA. In addition, female mice with an oocyte-specific knockout of Rsl1d1 recapitulated the phenotype of Kpna2^–/– mice, demonstrating the vital role of substrate RSL1D1. Finally, complementary RNA (cRNA) microinjection of human KPNA7, but not mouse Kpna7, was able to rescue the embryo arrest phenotype in Kpna2^–/– mice, suggesting mouse KPNA2 might be a homologue of human KPNA7. Our findings uncovered a mechanistic understanding for the pathogenesis of PREMBA, which acts by impairing nuclear protein transport, and provide a diagnostic marker for PREMBA patients.

Purpose To investigate the genetic causes of polyspermy and total fertilization failure (TFF) in two independent male patients suffering from male infertility. Methods Immunofluorescence (IF) staining was used to detect the localization of the PLCζ protein in sperm and the maternal pronucleus in the zygote. Genomic DNA samples were extracted from the peripheral blood of patients and their families. The ExAC database was used to identify the frequency of corresponding mutations. The PLCZ1 mutations were validated by Sanger sequencing. The pathogenicity of the identified mutations and their possible effects on the protein were assessed using in silico tools and molecular modeling. Results We identified a reported homozygous mutation c.588C > A (p.Cys196Ter) and a compound heterozygous mutation c.2 T > C(p.Met1Thr)/c.590G > A (p.Arg197His) with one novel mutation in PLCZ1. The IF results showed that these multipronuclear zygotes formed as a result of polyspermy. In silico analysis predicted that the mutations result in disease-causing proteins. IF staining revealed that PLCζ is abnormally localized in the sperm samples from the two affected patients. Assisted oocyte activation (AOA) successfully rescued polyspermy and TFF and achieved pregnancy in two patients with the PLCZ1 mutation. Conclusion We identified a homozygous mutation in PLCZ1 (c.588C > A [p.Cys196Ter]) in a male patient with polyspermy after in vitro fertilization (IVF) as well as a compound heterozygous mutation c.2 T > C(p.Met1Thr)/c.590G > A (p.Arg197His) with one novel mutation in a male patient with fertilization failure after intracytoplasmic sperm injection (ICSI), and we provide evidence that the homozygous mutation can cause polyspermy and the compound heterozygous mutation can cause fertilization failure.