Homozygous ACTL9 mutations cause irregular mitochondrial sheath arrangement and abnormal flagellum assembly in spermatozoa and male infertility.

PURPOSE: To identify novel variants in ACTL9 and new phenotypes responsible for male infertility. METHODS: Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing (WES). Computer-assisted sperm analysis (CASA) was used to test the motility of spermatozoa. The ultrastructure of flagella and the mitochondrial sheath were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunostaining was used to validate the localization and expression of ACTL9 and ACTL7A. An Actl9-mutated mouse model was used to validate the phenotypes by CASA and TEM. RESULTS: We identified novel homozygous variants in ACTL9 in two independent Chinese families. Spermatozoa with ACTL9 mutations showed decreased CASA parameters and a higher proportion of spermatozoa with abnormal morphology, exhibiting coiled flagella and a thickened midpiece. The spermatozoa were characterized by chaotic or irregular '9+2' structures and irregular mitochondrial sheath arrangements in the flagellum. Actl9 knock-in mice also showed abnormal CASA parameters and irregular '9+2' structures in flagella. CONCLUSIONS: Our study expands the mutation spectrum and phenotypic spectrum of ACTL9.

A single-cell transcriptome atlas of human euploid and aneuploid blastocysts.

Aneuploidy is frequently detected in early human embryos as a major cause of early pregnancy failure. However, how aneuploidy affects cellular function remains elusive. Here, we profiled the transcriptomes of 14,908 single cells from 203 human euploid and aneuploid blastocysts involving autosomal and sex chromosomes. Nearly all of the blastocysts contained four lineages. In aneuploid chromosomes, 19.5% ± 1.2% of the expressed genes showed a dosage effect, and 90 dosage-sensitive domains were identified. Aneuploidy leads to prevalent genome-wide transcriptome alterations. Common effects, including apoptosis, were identified, especially in monosomies, partially explaining the lower cell numbers in autosomal monosomies. We further identified lineage-specific effects causing unstable epiblast development in aneuploidies, which was accompanied by the downregulation of TGF-ß and FGF signaling, which resulted in insufficient trophectoderm maturation. Our work provides crucial insights into the molecular basis of human aneuploid blastocysts and may shed light on the cellular interaction during blastocyst development.

Human umbilical cord-derived mesenchymal stem cell transplantation improves the long COVID.

No effective treatments can ameliorate symptoms of long COVID patients. Our study assessed the safety and efficacy of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) in the treatment of long COVID patients. Ten long COVID patients were enrolled and received intravenous infusions of UC-MSCs on Days 0, 7, and 14. Adverse events and clinical symptoms were recorded, and chest-high-resolution CT (HRCT) images and laboratory parameters were analyzed. During UC-MSCs treatment and follow-up, we did not observe serious adverse events, the symptoms of long COVID patients were significantly relieved in a short time, especially sleep difficulty, depression or anxiety, memory issues, and so forth, and the lung lesions were also repaired. The routine laboratory parameters did not exhibit any significant abnormalities following UC-MSCs transplantation (UMSCT). The proportion of regulatory T cells gradually increased, but it was not statistically significant until 12 months. The proportion of naive B cells was elevated, while memory B cells, class-switched B-cells, and nonswitched B-cells decreased at 1 month after infusion. Additionally, we observed a transient elevation in circulating interleukin (IL)-6 after UMSCT, while tumor necrosis factor (TNF)-α, IL-17A, and IL-10 showed no significant changes. The levels of circulating immunoglobulin (Ig) M increased significantly at month 2, while IgA increased significantly at month 6. Furthermore, the SARS-CoV-2 IgG levels remained consistently high in all patients at Month 6, and there was no significant decrease during the subsequent 12-month follow-up. UMSCT was safe and tolerable in long COVID patients. It showed potential in alleviating long COVID symptoms and improving interstitial lung lesions.

Two missense STK11 gene variations impaired LKB1/adenosine monophosphate-activated protein kinase signaling in Peutz-Jeghers syndrome.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is a rare hereditary neoplastic disorder mainly associated with serine/threonine kinase 11 (STK11/LKB1) gene mutations. Preimplantation genetic testing can protect a patient's offspring from mutated genes; however, some variations in this gene have been interpreted as variants of uncertain significance (VUS), which complicate reproductive decision-making in genetic counseling. AIM: To identify the pathogenicity of two missense variants and provide clinical guidance. METHODS: Whole exome gene sequencing and Sanger sequencing were performed on the peripheral blood of patients with PJS treated at the Reproductive and Genetic Hospital of Citic-Xiangya. Software was employed to predict the protein structure, conservation, and pathogenicity of the two missense variation sites in patients with PJS. Additionally, plasmids were constructed and transfected into HeLa cells to observe cell growth. The differences in signal pathway expression between the variant group and the wild-type group were compared using western blot and immunohistochemistry. Statistical analysis was performed using one-way analysis of variance. P < 0.05 was considered statistically significant. RESULTS: We identified two missense STK11 gene VUS [c.889A>G (p.Arg297Gly) and c.733C>T (p.Leu245Phe)] in 9 unrelated PJS families who were seeking reproductive assistance. The two missense VUS were located in the catalytic domain of serine/threonine kinase, which is a key structure of the liver kinase B1 (LKB1) protein. In vitro experiments showed that the phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) at Thr172 and LKB1 at Ser428 were significantly higher in transfected variation-type cells than in wild-type cells. In addition, the two missense STK11 variants promoted the proliferation of HeLa cells. Subsequent immunohistochemical analysis showed that phosphorylated-AMPK (Thr172) expression was significantly lower in gastric, colonic, and uterine polyps from PJS patients with missense variations than in non-PJS patients. Our findings indicate that these two missense STK11 variants are likely pathogenic and inactivate the STK11 gene, causing it to lose its function of regulating downstream phosphorylated-AMPK (Thr172), which may lead to the development of PJS. The identification of the pathogenic mutations in these two clinically characterized PJS patients has been helpful in guiding them toward the most appropriate mode of pregnancy assistance. CONCLUSION: These two missense variants can be interpreted as likely pathogenic variants that mediated the onset of PJS in the two patients. These findings not only offer insights for clinical decision-making, but also serve as a foundation for further research and reanalysis of missense VUS in rare diseases.

Immunological Indicators of Recurrent Pregnancy Loss: A Mendelian Randomization Study.

Recurrent pregnancy loss (RPL) is thought to be related to maternal-fetal immune tolerance disorders. Immune monitoring of RPL patients mainly involves two aspects: inflammatory factors and immune cells. However, most observational studies have reported controversial findings. This study aimed to confirm whether abnormal inflammatory factors and immune cells in peripheral blood may lead to RPL, and guide clinical immune monitoring. We demonstrated causality using two-sample Mendelian randomization. Sensitivity analysis, reverse Mendelian randomization and meta-analysis were used to enhance the effectiveness of the results. There was a causal relationship between the level of IL-12 (OR = 1.78, 95% CI = 1.25-2.55; P = 0.00149) and RPL for 41 inflammatory factors. We screened 5 groups of immune cell subtypes that were causally associated with RPL: switched memory B-cell absolute count (OR = 0.66, 95% CI = 0.49-0.87, P = 0.00406), IgD + CD24 + B-cell absolute count (OR = 0.69, 95% CI = 0.53-0.88, P = 0.00319), CD39 + resting CD4 regulatory T-cell %CD4 regulatory T-cell (OR = 0.86, 95% CI = 0.78-0.95, P = 0.00252), activated & resting CD4 regulatory T-cell %CD4 regulatory T-cell (OR = 0.89, 95% CI = 0.82-0.97, P = 0.00938) and CD45 RA + CD28-CD8 + T-cell %CD8 + T-cell (OR = 0.99, 95% CI = 0.98-1.00, P = 0.01231). In terms of inflammatory factors, a causal relationship between IL-12 and RPL in peripheral blood was confirmed. We also identified five immune cell phenotypes that play a protective role. This suggests that there may be protective B cells and CD8 + T-cell subsets in peripheral blood, and the protective effect of Tregs was proved again. Immune monitoring of peripheral blood in patients with RPL seems to be necessary and the foundation for precision medicine.

[Genetic analysis and assisted reproductive guidance for two infertile patients with rare small supernumerary marker chromosomes].

OBJECTIVE: To carry out cytogenetic and molecular genetic analysis for two infertile patients carrying rare small supernumerary marker chromosomes (sSMC). METHODS: Two infertile patients who received reproductive and genetic counseling at CITIC Xiangya Reproductive and Genetic Hospital on October 31, 2018 and May 10, 2021, respectively were selected as the study subjects. The origin of sSMCs was determined by conventional G banding, fluorescence in situ hybridization (FISH) and copy number variation sequencing (CNV-seq). Microdissection combined with high-throughput whole genome sequencing (MicroSeq) was carried out to determine the fragment size and genomic information of their sSMCs. RESULTS: For patient 1, G-banded karyotyping and FISH revealed that he has a karyotype of mos47,XY,del(16)(p10p12),+mar[65]/46,XY,del(16)(p10p12)[6]/48,XY,del(16)(p10p12),+2mar[3].ish mar(Tel 16p-,Tel 16q-,CEP 16-,WCP 16+). CNV analysis has yielded a result of arr[GRCh37]16p12.1p11.2(24999364_33597595)×1[0.25]. MicroSeq revealed that his sSMC has contained the region of chromosome 16 between 24979733 and 34023115 (GRCh37). For patient 2, karyotyping and reverse FISH revealed that she has a karyotype of mos 47,XX,+mar[37]/46,XX[23].rev ish CEN5, and CNV analysis has yielded a result of seq[GRCh37]dup(5)(p12q11.2)chr5:g(45120001_56000000)dup[0.8]. MicroSeq results revealed that her sSMC has contained the region of chromosome 5 between 45132364 and 55967870(GRCh37). After genetic counseling, both couples had opted in vitro fertilization (IVF) treatment and preimplantation genetic testing (PGT). CONCLUSION: For individuals harboring sSMCs, it is vital to delineate the origin and structural characteristics of the sSMCs for their genetic counseling and reproductive guidance. Preimplantation genetic testing after microdissection combined with high-throughput whole genome sequencing (MicroSeq-PGT) can provide an alternative treatment for carrier couples with a high genetic risk.

SARS-CoV-2 infection is detrimental to pregnancy outcomes after embryo transfer in IVF/ICSI: a prospective cohort study.

BACKGROUND: To explore whether SARS-CoV-2 infection affects the pregnancy outcomes of assisted reproductive techniques (ART). METHODS: A prospective cohort study recruited patients for embryo transfer from December 01, 2022, to December 31, 2022. All patients were closely followed up for SARS-CoV-2 infection after embryo transfer. The SARS-CoV-2 "diagnosed group" was defined as RNA or antigen-positive. The SARS-CoV-2 "suspected infection group" was defined as having apparent SARS-CoV-2 symptoms without an RNA or antigen test, while the "uninfected group" was defined as having a negative SARS-CoV-2 RNA or antigen test and no SARS-CoV-2 symptoms. RESULTS: A total of 1330 patients participated in the study, 687 of whom were in the SARS-CoV-2 diagnosed group, 219 in the suspected infection group, and 424 in the uninfected group. There was no significant difference in basic characteristics among the three groups. The clinical pregnancy rate was 68% in the SARS-CoV-2 diagnosed group, 63% in the uninfected group, and 51% in the suspected infection group (P < 0.001). The ongoing pregnancy rate was 58% in the SARS-CoV-2 diagnosed group, 53% in the uninfected group, and 45% in the suspected infection group (P < 0.001). Upon analyzing the factors influencing clinical pregnancy, it was found that suspected infection (odds ratio [OR] 0.618, 95% CI 0.444-0.862, P = 0.005) and the short time (≤ 22 days) between embryo transfer and SARS-CoV-2 infection (OR 3.76, 95% CI 1.92-8.24, P < 0.001) were not conducive to clinical pregnancy. In addition, the concurrent presence of fever and dizziness/headache SARS-CoV-2 symptoms (OR 0.715, 95% CI 0.526-0.972, P = 0.032) decreased the clinical pregnancy rate. However, vaccination administered 2-3 times (OR 1.804, 95% CI 1.332-2.444, P < 0.001) was associated with an improvement in clinical pregnancy rate. CONCLUSIONS: This prospective cohort study shows that SARS-CoV-2 infection in a short period of time after embryo transfer is not conducive to clinical pregnancy. Reproductive physicians should advise patients to avoid SARS-CoV-2 infection shortly after embryo transfer. Meanwhile, women should be encouraged to vaccinate at least 2-3 times before embryo transfer or pregnancy.

A hemizygous loss-of-function variant in BCORL1 is associated with male infertility and oligoasthenoteratozoospermia.

Oligoasthenoteratozoospermia (OAT) is a common type of male infertility; however, its genetic causes remain largely unknown. Some of the genetic determinants of OAT are gene defects affecting spermatogenesis. BCORL1 (BCL6 corepressor like 1) is a transcriptional corepressor that exhibits the OAT phenotype in a knockout mouse model. A hemizygous missense variant of BCORL1 (c.2615T > G:p.Val872Gly) was reported in an infertile male patient with non-obstructive azoospermia (NOA). Nevertheless, the correlation between BCORL1 variants and OAT in humans remains unknown. In this study, we used whole-exome sequencing to identify a novel hemizygous nonsense variant of BCORL1 (c.1564G > T:p.Glu522*) in a male patient with OAT from a Han Chinese family. Functional analysis showed that the variant produced a truncated protein with altered cellular localization and a dysfunctional interaction with SKP1 (S-phase kinase-associated protein 1). Further population screening identified four BCORL1 missense variants in subjects with both OAT (1 of 325, 0.31%) and NOA (4 of 355, 1.13%), but no pathogenic BCORL1 variants among 362 fertile subjects. In conclusion, our findings indicate that BCORL1 is a potential candidate gene in the pathogenesis of OAT and NOA, expanded its disease spectrum and suggested that BCORL1 may play a role in spermatogenesis by interacting with SKP1.

Bi-allelic variants in DNAH3 cause male infertility with asthenoteratozoospermia in humans and mice.

STUDY QUESTION: Are there other pathogenic genes for asthenoteratozoospermia (AT)? SUMMARY ANSWER: DNAH3 is a novel candidate gene for AT in humans and mice. WHAT IS KNOWN ALREADY: AT is a major cause of male infertility. Several genes underlying AT have been reported; however, the genetic aetiology remains unknown in a majority of affected men. STUDY DESIGN SIZE DURATION: A total of 432 patients with AT were recruited in this study. DNAH3 mutations were identified by whole-exome sequencing (WES). Dnah3 knockout mice were generated using the genome editing tool. The morphology and motility of sperm from Dnah3 knockout mice were investigated. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS SETTING METHODS: WES was performed on 432 infertile patients with AT. In addition, two lines of Dnah3 knockout mice were generated. Haematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunostaining, and computer-aided sperm analysis (CASA) were performed to investigate the morphology and motility of the spermatozoa. ICSI was used to overcome the infertility of one patient and of the Dnah3 knockout mice. MAIN RESULTS AND THE ROLE OF CHANCE: DNAH3 biallelic variants were identified in three patients from three unrelated families. H&E staining revealed various morphological abnormalities in the flagella of sperm from the patients, and TEM and immunostaining further showed the loss of the central pair of microtubules, a dislocated mitochondrial sheath and fibrous sheath, as well as a partial absence of the inner dynein arms. In addition, the two Dnah3 knockout mouse lines demonstrated AT. One patient and the Dnah3 knockout mice showed good treatment outcomes after ICSI. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This is a preliminary report suggesting that defects in DNAH3 can lead to asthenoteratozoospermia in humans and mice. The pathogenic mechanism needs to be further examined in a future study. WIDER IMPLICATIONS OF THE FINDINGS: Our findings show that DNAH3 is a novel candidate gene for AT in humans and mice and provide crucial insights into the biological underpinnings of this disorder. The findings may also be beneficial for counselling affected individuals. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from National Natural Science Foundation of China (82201773, 82101961, 82171608, 32322017, 82071697, and 81971447), National Key Research and Development Program of China (2022YFC2702604), Scientific Research Foundation of the Health Committee of Hunan Province (B202301039323, B202301039518), Hunan Provincial Natural Science Foundation (2023JJ30716), the Medical Innovation Project of Fujian Province (2020-CXB-051), the Science and Technology Project of Fujian Province (2023D017), China Postdoctoral Science Foundation (2022M711119), and Guilin technology project for people's benefit (20180106-4-7). The authors declare no competing interests.

A case report of a normal fertile woman with 46,XX/46,XY somatic chimerism reveals a critical role for germ cells in sex determination.

Individuals with 46,XX/XY chimerism can display a wide range of characteristics, varying from hermaphroditism to complete male or female, and can display sex chromosome chimerism in multiple tissues, including the gonads. The gonadal tissues of females contain both granulosa and germ cells. However, the specific sex chromosome composition of the granulosa and germ cells in 46,XX/XY chimeric female is currently unknown. Here, we reported a 30-year-old woman with secondary infertility who displayed a 46,XX/46,XY chimerism in the peripheral blood. FISH testing revealed varying degrees of XX/XY chimerism in multiple tissues of the female patient. Subsequently, the patient underwent preimplantation genetic testing (PGT) treatment, and 26 oocytes were retrieved. From the twenty-four biopsied mature oocytes, a total of 23 first polar bodies (PBs) and 10 second PBs were obtained. These PBs and two immature metaphase I (MI) oocytes only displayed X chromosome signals with no presence of the Y, suggesting that all oocytes in this chimeric female were of XX germ cell origin. On the other hand, granulosa cells obtained from individual follicles exhibited varied proportions of XX/XY cell types, and six follicles possessed 100% XX or XY granulosa cells. A total of 24 oocytes were successfully fertilized, and 12 developed into blastocysts, where 5 being XY and 5 were XX. Two blastocysts were transferred with one originating from an oocyte aspirated from a follicle containing 100% XY granulosa cells. This resulted in a twin pregnancy. Subsequent prenatal diagnosis confirmed normal male and female karyotypes. Ultimately, healthy boy-girl twins were delivered at full term. In summary, this 46,XX/XY chimerism with XX germ cells presented complete female, suggesting that germ cells may exert a significant influence on the sexual determination of an individual, which provide valuable insights into the intricate processes associated with sexual development and reproduction.

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.

Extended application of PGT-M strategies for small pathogenic CNVs.

PURPOSE: The preimplantation genetic testing for aneuploidy (PGT-A) platform is not currently available for small copy-number variants (CNVs), especially those < 1 Mb. Through strategies used in PGT for monogenic disease (PGT-M), this study intended to perform PGT for families with small pathogenic CNVs. METHODS: Couples who carried small pathogenic CNVs and underwent PGT at the Reproductive and Genetic Hospital of CITIC-Xiangya (Hunan, China) between November 2019 and April 2023 were included in this study. Haplotype analysis was performed through two platforms (targeted sequencing and whole-genome arrays) to identify the unaffected embryos, which were subjected to transplantation. Prenatal diagnosis using amniotic fluid was performed during 18-20 weeks of pregnancy. RESULTS: PGT was successfully performed for 20 small CNVs (15 microdeletions and 5 microduplications) in 20 families. These CNVs distributed on chromosomes 1, 2, 6, 7, 13, 15, 16, and X with sizes ranging from 57 to 2120 kb. Three haplotyping-based PGT-M strategies were applied. A total of 89 embryos were identified in 25 PGT cycles for the 20 families. The diagnostic yield was 98.9% (88/89). Nineteen transfers were performed for 17 women, resulting in a 78.9% (15/19) clinical pregnancy rate after each transplantation. Of the nine women who had healthy babies, eight accepted prenatal diagnosis and the results showed no related pathogenic CNVs. CONCLUSION: Our results show that the extended haplotyping-based PGT-M strategy application for small pathogenic CNVs compensated for the insufficient resolution of PGT-A. These three PGT-M strategies could be applied to couples with small pathogenic CNVs.

Mosaic variegated aneuploidy syndrome with tetraploid, and predisposition to male infertility triggered by mutant CEP192.

In this study, we report on mosaic variegated aneuploidy (MVA) syndrome with tetraploidy and predisposition to infertility in a family. Sequencing analysis identified that the CEP192 biallelic variants (c.1912C>T, p.His638Tyr and c.5750A>G, p.Asn1917Ser) segregated with microcephaly, short stature, limb-extremity dysplasia, and reduced testicular size, while CEP192 monoallelic variants segregated with infertility and/or reduced testicular size in the family. In 1,264 unrelated patients, variant screening for CEP192 identified a same variant (c.5750A>G, p.Asn1917Ser) and other variants significantly associated with infertility. Two lines of Cep192 mice model that are equivalent to human variants were generated. Embryos with Cep192 biallelic variants arrested at E7 because of cell apoptosis mediated by MVA/tetraploidy cell acumination. Mice with heterozygous variants replicated the predisposition to male infertility. Mouse primary embryonic fibroblasts with Cep192 biallelic variants cultured in vitro showed abnormal morphology, mitotic arresting, and disruption of spindle formation. In patient epithelial cells with biallelic variants cultured in vitro, the number of cells arrested during the prophase increased because of the failure of spindle formation. Accordingly, we present mutant CEP192, which is a link for the MVA syndrome with tetraploidy and the predisposition to male infertility.

Defects in phospholipase C zeta cause polyspermy and low fertilization after conventional IVF: not just ICSI failure.

ABSTRACT: Phospholipase C zeta (PLCζ) is a key sperm-borne oocyte-activating factor that triggers Ca2+ 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 Ca2+ 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.

Transcriptomic profiles reveal the characteristics of oocytes and cumulus cells at GV, MI, and MII in follicles before ovulation.

BACKGROUND: The oocyte and its surrounding cumulus cells (CCs) exist as an inseparable entity. The maturation of the oocyte relies on communication between the oocyte and the surrounding CCs. However, oocyte evaluation is primarily based on morphological parameters currently, which offer limited insight into the quality and competence of the oocyte. Here, we conducted transcriptomic profiling of oocytes and their CCs from 47 patients undergoing preimplantation genetic testing for aneuploidy (PGT-A). We aimed to investigate the molecular events occurring between oocytes and CCs at different stages of oocyte maturation (germinal vesicle [GV], metaphase I [MI], and metaphase II [MII]). Our goal is to provide new insights into in vitro oocyte maturation (IVM). RESULTS: Our findings indicate that oocyte maturation is a complex and dynamic process and that MI oocytes can be further classified into two distinct subtypes: GV-like-MI oocytes and MII-like-MI oocytes. Human oocytes and cumulus cells at three different stages of maturation were analyzed using RNA-seq, which revealed unique transcriptional machinery, stage-specific genes and pathways, and transcription factor networks that displayed developmental stage-specific expression patterns. We have also identified that both lipid and cholesterol metabolism in cumulus cells is active during the late stage of oocyte maturation. Lipids may serve as a more efficient energy source for oocytes and even embryogenesis. CONCLUSIONS: Overall, our study provides a relatively comprehensive overview of the transcriptional characteristics and potential interactions between human oocytes and cumulus cells at various stages of maturation before ovulation. This study may offer novel perspectives on IVM and provide a reliable reference data set for understanding the transcriptional regulation of follicular maturation.

Novel biallelic ASTL variants are associated with polyspermy and female infertility: A successful live birth following ICSI treatment.

Fertilization of the egg by the sperm is the first vital stage of embryogenesis. In mammals, only one sperm is incorporated into the oocyte. Polyspermy is a key anomaly of fertilization that is generally lethal to the embryo. To date, only a few causative genes for polyspermy have been reported. In a recent study, a homozygous variant in astacin-like metalloendopeptidase (ASTL), which encodes the ovastacin enzyme that cleaves ZP2 to prevent polyspermy, was found to be associated with female infertility characterized by polyspermy in vitro. Herein, we identified two ASTL variants in a Chinese woman likely responsible for her primary infertility and polyspermy in in vitro fertilization. Both variants were located within the key catalytic domain and predicted to alter hydrogen bonds, potentially impairing protein stability. Moreover, expression and immunoblot analyses in CHO-K1 cells indicated abnormal ovastacin zymogen activation or decreased enzyme stability. Intracytoplasmic sperm injection treatment successfully bypassed the defect in polyspermy blocking and resulted in a live birth. Our study associates ASTL variants with human infertility and further supports the contribution of this gene to blocking polyspermy in humans. Our findings expand the spectrum of ASTL mutations and should facilitate the diagnosis of oocyte-borne polyspermy.

Navigation of Knowledge: the Impact of COVID-19 on Pregnancy-a Bibliometric Analysis.

Since the outbreak of COVID-19, countries around the world have faced huge economic and social burdens. SARS-COV-2 may exist in nature for a long time due to the diversity of its different variants. Pregnant women and newborns as vulnerable groups will suffer serious health threats. Bibliometrics as a method of summarizing publications can be used to extract important achievements and hot topics in this field. We search the target publications from the Web of Science Core collection database, and then use Microsoft Office Excel, CiteSpace, R, Scimago, and VOSviewer for visual analysis. Finally, we included 1709 publications from 2998 institutions in 104 countries. The number of publications has exploded since the COVID-19 pandemic in 2019. Among them, the USA, China, Britain, and Italy have higher quantity and quality. We identified important journals, authors, keywords, and references in this field. Anxiety, stress, risk of pregnancy complications, and vaccine safety and acceptance have received extensive attention from scholars during the COVID-19 pandemic and will continue to be urgent issues to be addressed in the future. Most of the current studies fall into the category of case reports and clinical data analysis. COVID-19 has been linked to serious pregnancy complications and mental illness, and vaccination during pregnancy is recommended to protect both mother and fetus. Further large-scale cohort studies and discovery of molecular mechanisms are needed in this field.

The effect of carrier characteristics and female age on preimplantation genetic testing results of blastocysts from Robertsonian translocation carriers.

PURPOSE: To analyze factors affecting segregation and ploidy results from Robertsonian carriers, and determine chromosomes involved impact chromosome stability during meiosis and mitosis. METHODS: This retrospective study include 928 oocyte retrieval cycles from 763 couples with Robertsonian translocations undergoing preimplantation genetic testing for structural rearrangements (PGT-SR) using next-generation sequencing (NGS) between December 2012 and June 2020.The segregation patterns of the trivalent of 3423 blastocysts were analyzed according to the carrier's sex and age. A total of 1492 couples who received preimplantation genetic testing for aneuploidy (PGT-A) were included as the control group and matched according to maternal age and testing time stage. RESULTS: A total of 1728 (50.5%) normal/balanced embryos were identified from 3423 embryos diagnosed. The rate of alternate segregation in male Robertsonian translocation carriers was significantly higher than that in female carriers (82.3% vs. 60.0%, P < 0.001). However, the segregation ratio exhibited no difference between young and older carriers. Further, increasing maternal age decreased the proportion of transferable embryo cycle in both female and male carriers. And the ratio of chromosome mosaic from the Robertsonian translocation carrier group was significantly higher than that in the PGT-A control group (1.2% vs. 0.5%, P < 0.01). CONCLUSIONS: The meiotic segregation modes were affected by the carrier sex and were independent of the carrier's age. Advanced maternal age decreased the probability of obtaining a normal/balanced embryo. In additional, the Robertsonian translocation chromosome could increase the possibility of chromosome mosaicism during mitosis in blastocysts.

Novel homozygous variants in TTC12 cause male infertility with asthenoteratozoospermia owing to dynein arm complex and mitochondrial sheath defects in flagella.

Introduction: Tracing the genetic causes for male infertility due to asthenoteratozoospermia has revealed at least 40 causative genes, which provides valuable reference for the genetic testing of asthenoteratozoospermia in clinical practice. To identify deleterious variants in the human tetratricopeptide repeat domain 12 (TTC12) gene in a large cohort of infertile Chinese males with asthenoteratozoospermia. Methods: A total of 314 unrelated asthenoteratozoospermia-affected men were recruited for whole exome sequencing. The effects of the identified variants were evaluated by in silico analysis, and confirmed by in vitro experiments. Intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of assisted reproduction technique therapy. Results and Discussion: Novel homozygous TTC12 variants (c.1467_1467delG (p.Asp490Thrfs*14), c.1139_1139delA (p.His380Profs*4), and c.1117G>A (p.Gly373Arg)) were identified in three (0.96%) of the 314 cases. Three mutants were indicated to be damaging using in silico prediction tools, and were further confirmed by in vitro functional analysis. Hematoxylin and eosin staining and ultrastructural observation of the spermatozoa revealed multiple morphological abnormalities of flagella, with the absence of outer and inner dynein arms. Notably, significant mitochondrial sheath malformations were also observed in the sperm flagella. Immunostaining assays indicated that TTC12 is present throughout the flagella, and was strongly concentrated in the mid-piece in control spermatozoa. However, spermatozoa from TTC12-mutated individuals exhibited almost no staining intensity of TTC12 and outer and inner dynein arms components. The three men accepted ICSI treatment using their ejaculated spermatozoa, and two female partners successfully delivered healthy babies. Our findings provide direct genetic evidence that homozygous variants in TTC12 cause male infertility with asthenoteratozoospermia by causing dynein arm complex defects and mitochondrial sheath malformations in the flagellar. We also demonstrated that TTC12 deficiency-mediated infertility could be overcome by ICSI technology.