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BACKGROUND: Homeodomain (HD) proteins contain an evolutionarily conserved helix-turn-helix (HTH) DNA-binding motif and act as transcription factors to control gene expression. A previous study showed that the HD gene Homez is highly enriched in adult testes. However, the role of HOMEZ in spermatogenesis and male fertility remains unknown. METHODS: Using CRISPR/Cas9 technology, Homez mutant mice were generated and performed histological, immunofluorescence, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot and mating assays to analyze the phenotype of Homez mutants. RESULTS: Molecular phylogenetic analyses indicated that the HOMEZ is evolutionarily conserved among mammalian species. qRT-PCR and Western blot analyses showed that Homez is highly expressed in the testis, with a relatively increased expression trend during spermatogenesis. Homez mutant males were viable and showed no differences in body and testis weight compared to their wild-type. In addition, mating between Homez mutant males and wild-type females produced normal litter sizes. Moreover, histopathology detected complete spermatogenesis in the seminiferous tubules and mature spermatozoa in the epididymides from Homez knockout males. Furthermore, significantly increased transcription of three Zhx genes were found in Homez mutatnt testes compared with wild-type testes. CONCLUSIONS: Homez knockout mice are fertile and are not essential for germ cell development. These findings could prevent unnecessary duplicative work by other groups.

Human oocyte maturation arrest represents one of the severe conditions for female patients with primary infertility. However, the genetic factors underlying this human disease remain largely unknown. The spindle assembly checkpoint (SAC) is an intricate surveillance mechanism that ensures accurate segregation of chromosomes throughout cell cycles. Once the kinetochores of chromosomes are correctly attached to bipolar spindles and the SAC is satisfied, the MAD2L1BP, best known asp31comet, binds MAD2 and recruits the AAA+-ATPase TRIP13 to disassemble the mitotic checkpoint complex (MCC), leading to the cell cycle progression. In this study, by whole-exome sequencing (WES), we identified homozygous and compound heterozygousMAD2L1BPvariants in three families with female patients diagnosed with primary infertility owing to oocyte metaphase I (MI) arrest. Functional studies revealed that the protein variants resulting from the C-terminal truncation of MAD2L1BP lost their binding ability to MAD2. cRNA microinjection of full-length or truncated MAD2L1BP uncovered their discordant roles in driving the extrusion of polar body 1 (PB1) in mouse oocytes. Furthermore, the patient’s oocytes carrying the mutatedMAD2L1BPvariants resumed polar body extrusion (PBE) when rescued by microinjection of full-lengthMAD2L1BPcRNAs. Together, our studies identified and characterized novel biallelic variants inMAD2L1BPresponsible for human oocyte maturation arrest at MI, and thus prompted new therapeutic avenues for curing female primary infertility.

In mammals, the production of mature oocytes necessitates rigorous regulation of the discontinuous meiotic cell-cycle progression at both the transcriptional and post- transcriptional levels; however, the factors underlying this sophisticated but explicit process during oocyte development remain largely unclear. Here we characterized the function of N-acetyltransferase 10 (Nat10), which was previously recognized as a “writer” for N4-acetylcytidine (ac4C) deposited on RNA molecules. We generated two germline-specific Nat10 knockout mouse models in the embryonic gonad and in postnatal growing oocytes, and another two tamoxifen-inducible Nat10 deletion models. We provided genetic evidence showing that Nat10 is essential for oocyte meiotic prophase I progression, oocyte growth and maturation in mice. Intriguingly, we discovered that Nat10 is required for sculpting the maternal transcriptome through timely degradation of polyA tail mRNAs during the Maternal-to-Zygotic transition (MZT). Importantly, we developed a novel method that outperformed the conventional methods for examining the polyA tail length (PAT), termed Hairpin Adaptor-polyA tail length (HA-PAT), in terms of the cost, sensitivity and efficiency. In summary, these findings altogether provide solid genetic evidence that unveils the indispensable role of maternal Nat10 in oocyte development, and lay a solid foundation for future mechanistic studies of varied domains of NAT10.

The cell division cycle 20 (CDC20) protein is a co-activator of anaphase-promoting complex/cyclosome (APC/C), required for mitotic exit and also meiotic exit, containing seven WD40 repeats in the C-terminus responsible for protein-protein interactions. Recently, a previous study has shown that biallelic mutations in CDC20 are causative for female infertility with abnormalities in oocyte maturation and embryonic development. This study is to further identify new mutations of CDC20 and the prevalence of variants in our cohort. A cohort of 50 primary infertile females with oocyte maturation abnormality and early embryonic arrest were recruited. Genomic DNA was isolated from peripheral blood samples. Mutation screening of all the coding regions of CDC20 was performed by Sanger sequencing. The pathogenicity of the identified variants on the CDC20 protein was accessed in silico. Two CDC20 variants, a nonsense mutation p.R262* and a missense mutation p.A211T, identified in one female of 50 unrelated affected individuals, accounting for a relative small proportion of this cohort (2%). In silico analysis revealed that the p.R262* would cause no production of protein or a truncated protein lacking five WD40 repeats in the C-terminus; and that p.A211T may interfere with the formation of a deep hydrophobic pocket and thus disturb the binding of CDC20 protein to the substrates of APC/C. This study identified two novel mutations in CDC20, further expanding the mutation spectrum of this gene. Our findings further confirm that biallelic mutations in CDC20 occur in a proportion of infertile females with oocyte maturation abnormality and early embryonic arrest.

Currently, the cryopreservation of human spermatozoa must overcome the adverse effects of excessive oxidation. In this study, we aimed to evaluate the effect of supplementation of cryopreservation medium with cyanidin-3-Ο-glucoside (C3G) on sperm quality. Semen samples were obtained from men with normozoospermia according to WHO criteria (n = 39). The sperm parameter values were compared after cryopreservation in medium supplemented with and without C3G.Compared with the control group (without additive), low doses (50 μM and 100 μM) of C3G improved sperm viability and motility and decreased the reactive oxygen species (ROS) of spermatozoa, while high doses (200 μM) of C3G did not obviously enhance sperm quality. The amount of DNA fragmentation index (DFI) and high DNA stainability (HDS) after freezing were higher in the control group than in the C3G supplementation groups. Low-concentration C3G supplementation (50 μM) was negatively correlated with sperm ROS levels (r = -0.2, p = 0.03). Collectively, our findings suggest that C3G could be an efficient semen cryoprotectant that ameliorates oxidative stress in human sperm during cryopreservation.

CD49a

PURPOSE: This study aims to identify the genetic causes of 12 women with primary infertility characterized by primarily oocyte maturation abnormality and consequent early embryonic arrest. METHODS: Genomic DNA was isolated from peripheral blood samples. Whole-exome sequencing was performed on the probands, and the identified variants were confirmed by Sanger sequencing. The pathogenicity of the identified variants on the protein was accessed in silico. And we used qRT-PCR to detect the possible effects of the novel mutation on the mRNA level of NLRP5. RESULTS: A novel homozygous frameshift variant (p.V429Efs*30) in NLRP5 and compound heterozygous variants with a novel frameshift variant (p.A297Efs*20) and a recurrent variant (c. 223-14_223-2delCCCTCCTGTTCCA) in PATL2 were identified in two unrelated affected individuals. qRT-PCR showed an obvious decrease of the mutant NLRP5 mRNA. In addition, the truncated proteins of NLRP5 and PATL2 were predicted to be non-functional due to the deletion of the most or the whole region of the critical functional domain(s) respectively. CONCLUSIONS: This study identified novel mutations in NLRP5 and PATL2, further expanding the mutational and phenotypic spectrum of both genes. This is the first report of the NLRP5 mutations that associates with oocyte maturation abnormality in humans. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-022-02412-4.

Human autologous sperm freezing involves ejaculated sperm, and testicular or epididymal puncture sperm freezing, and autologous sperm freezing is widely used in assisted reproductive technology. In previous studies, researchers have tried to cryopreserve sperm from mammals (rats, dogs, etc.) using a −80°C freezer and have achieved success. It is common to use liquid nitrogen vapor rapid freezing to cryopreserve human autologous sperm. However, the operation of this cooling method is complicated, and the temperature drop is unstable. In this study, we compared the quality of human ejaculation and testicular sperm after liquid nitrogen vapor rapid freezing and −80°C freezing for the first time. By analyzing sperm quality parameters of 93 ejaculated sperm and 10 testicular sperm after liquid nitrogen vapor rapid freezing and −80°C freezing, we found reactive oxygen species (ROS) of sperm of the −80°C freezer was significantly lower than liquid nitrogen vapor rapid freezing. Regression analysis showed that progressive motility, ROS, and DNA fragmentation index (DFI) in post-thaw spermatozoa were correlated with sperm progressive motility, ROS, and DFI before freezing. For the freezing method, the −80°C freezer was positively correlated with the sperm progressive motility. Among the factors of freezing time, long-term freezing was negatively correlated with sperm progressive motility and ROS. Although freezing directly at −80°C freezer had a slower temperature drop than liquid nitrogen vapor rapid freezing over the same period, the curves of the temperature drop were similar, and slight differences in the freezing point were observed. Furthermore, there were no statistically significant differences between the two methods for freezing testicular sperm. The method of direct −80°C freezing could be considered a simplified alternative to vapor freezing for short-term human sperm storage. It could be used for cryopreservation of autologous sperm (especially testicular sperm) by in vitro fertilization centers.Clinical Trial Registration: (website), identifier (ChiCTR2100050190).

In vitro fertilization-embryo transfer (IVF-ET) technology make it possible for infertile couples to conceive a baby successfully. Nevertheless, IVF-ET does not guarantee success. Frozen embryo transfer (FET) is an important supplement to IVF-ET. Many factors are correlated with the outcome of FET which is unpredictable. Machine learning is a field of study that predict various outcomes by defining data attributes and using relevant data and calculation algorithms. Machine learning algorithm has been widely used in clinical research. The present study focuses on making predictions of early pregnancy outcomes in FET through clinical characters, including age, body mass index (BMI), endometrial thickness (EMT) on the day of progesterone treatment, good-quality embryo rate (GQR), and type of infertility (primary or secondary), serum estradiol level (E2) on the day of embryo transfer, and serum progesterone level (P) on the day of embryo transfer. We applied four representative machine learning algorithms, including logistic regression (LR), conditional inference tree, random forest (RF) and support vector machine (SVM) to build prediction models and identify the predictive factors. We found no significant difference among the models in the sensitivity, specificity, positive predictive rate, negative predictive rate or accuracy in predicting the pregnancy outcome of FET. For example, the positive/negative predictive rate of the SVM (gamma = 1, cost = 100, 10-fold cross validation) is 0.56 and 0.55. This approach could provide a reference for couples considering FET. The prediction accuracy of the present study is limited, which suggests that there may be some other more effective predictors to be developed in future work.

Declined quality and quantity of sperm is currently the major cause of patients suffering from infertility. Male germ cell development is spatiotemporally regulated throughout the whole developmental process. While it has been known that exogenous factors, such as environmental exposure, diet and lifestyle, et al, play causative roles in male infertility, recent progress has revealed abundant genetic mutations tightly associated with defective male germline development. In mammals, male germ cells undergo dramatic morphological change (i.e., nuclear condensation) and chromatin remodeling during post-meiotic haploid germline development, a process termed spermiogenesis; However, the molecular machinery players and functional mechanisms have yet to be identified. To date, accumulated evidence suggests that disruption in any step of haploid germline development is likely manifested as fertility issues with low sperm count, poor sperm motility, aberrant sperm morphology or combined. With the continually declined cost of next-generation sequencing and recent progress of CRISPR/Cas9 technology, growing studies have revealed a vast number of disease-causing genetic variants associated with spermiogenic defects in both mice and humans, along with mechanistic insights partially attained and validated through genetically engineered mouse models (GEMMs). In this review, we mainly summarize genes that are functional at post-meiotic stage. Identification and characterization of deleterious genetic variants should aid in our understanding of germline development, and thereby further improve the diagnosis and treatment of male infertility.

Although the thin endometrium (TE) has been widely recognized as a critical factor in implantation failure, the contribution of miRNA–mRNA regulatory network to the development of disease etiology remains to be further elucidated. This study performed an integrative analysis of the miRNA–mRNA expression profiles in the thin and adjacent normal endometrium of eight patients with intrauterine adhesion to construct the transcriptomic regulatory networks. A total of 1,093 differentially expressed genes (DEGs) and 72 differentially expressed miRNAs (DEMs) were identified in the thin adhesive endometrium of the TE group compared with the control adjacent normal endometrial cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the DEGs and the target genes of DEM were significantly enriched in angiogenesis, cell growth regulation, and Wnt signaling pathway. Multiple hub genes (CAV1, MET, MAL2, has-mir-138, ARHGAP6, CLIC4, RRAS, AGFG1, has-mir-200, and has-mir-429) were identified by constructing the miRNA–mRNA regulatory networks. Furthermore, a miRNA–mRNA pathway function analysis was conducted, and the hub genes were enriched in the FoxO signaling pathway, cell growth regulation, inflammatory response regulation, and regulation of autophagy pathways. Our study is the first to perform integrated mRNA-seq and miRNA-seq analyses in the thin adhesive endometrium and the control adjacent normal endometrial cells. This study provides new insights into the molecular mechanisms underlying the formation of thin endometrium.

The cell division cycle 20 (CDC20) protein is a co-activator of anaphase-promoting complex/cyclosome (APC/C), required for mitotic exit and also meiotic exit, containing seven WD40 repeats in the C-terminus responsible for protein-protein interactions. Recently, a previous study has shown that biallelic mutations in CDC20 are causative for female infertility with abnormalities in oocyte maturation and embryonic development. This study is to further identify new mutations of CDC20 and the prevalence of variants in our cohort. A cohort of 50 primary infertile females with oocyte maturation abnormality and early embryonic arrest were recruited. Genomic DNA was isolated from peripheral blood samples. Mutation screening of all the coding regions of CDC20 was performed by Sanger sequencing. The pathogenicity of the identified variants on the CDC20 protein was accessed in silico. Two CDC20 variants, a nonsense mutation p.R262* and a missense mutation p.A211T, identified in one female of 50 unrelated affected individuals, accounting for a relative small proportion of this cohort (2%). In silico analysis revealed that the p.R262* would cause no production of protein or a truncated protein lacking five WD40 repeats in the C-terminus; and that p.A211T may interfere with the formation of a deep hydrophobic pocket and thus disturb the binding of CDC20 protein to the substrates of APC/C. This study identified two novel mutations in CDC20, further expanding the mutation spectrum of this gene. Our findings further confirm that biallelic mutations in CDC20 occur in a proportion of infertile females with oocyte maturation abnormality and early embryonic arrest.

Alcohol consumption has commonly been associated with semen parameters. However, the association between alcohol intake and semen parameters in primary and secondary infertile men remains unclear. In this study, 776 infertile men from China were grouped according to alcohol intake: abstainers, moderate drinkers (

Problem Immune checkpoint molecules are receptors that can transmit inhibitory signals into cells to negatively modulate the immune response. However, their roles in NK cells during normal pregnancy remain poorly understood. Method of Study Peripheral blood samples were collected from women during the first, second and third trimesters of pregnancy. Peripheral blood NK (pNK) cells and T cells were analyzed for the expression of the immune checkpoint molecules TIGIT and PD-1 by flow cytometry. In addition, the ability of pNK cells to secret functional molecules was also evaluated. Results The expression of TIGIT on pNK cells increased gradually throughout pregnancy, whereas that of PD-1 showed the opposite pattern. However, on T cells, the expression of both TIGIT and PD-1 peaked during early pregnancy, and then declined gradually thereafter. Moreover, the expressions of granzyme B, IFN-γ and CD107a by pNK cells also decreased over the course of pregnancy. Compared with TIGIT- NK cells, TIGIT + NK cells possessed reduced expression of functional molecules. Conclusions As pregnancy progressed, the levels of immune checkpoint molecules expressed on pNK cells and T cells changed and the two molecules showed different trends. Furthermore, the secretion of functional molecules from pNK cells was negatively correlated with the trend of TIGIT expression, indicating TIGIT may play an important role in modulating the functions of pNK cells during pregnancy. Further study of TIGIT expression on pNK cells may enhance our understanding of its role in maintaining maternal-fetal tolerance and provide a useful marker for predicting instability during pregnancy.

This study aims to compare the prevalence of sexually transmitted infections (STIs) with semen quality in men from couples with primary and secondary infertility. Semen samples were collected from 133 men who requested fertility evaluation. Seminal tract infection with Ureaplasma spp. (UU), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and herpes simplex virus-2 (HSV-2) was assessed by PCR-based diagnostic assays. Among all patients, the prevalence of STIs was higher in men from couples with primary infertility than that in men from couples with secondary infertility (39.7% vs 21.7%, P = 0.03). The prevalence of UU was 28.8% and 13.3% in men from couples with primary and secondary infertility, respectively. Men from couples with primary infertility were more likely to be positive for UU than men from couples with secondary infertility (P = 0.04). Regarding the UU subtype, the prevalence of Ureaplasma urealyticum (Uuu) and Ureaplasma parvum (Uup; including Uup1, Uup3, Uup6, and Uup14) did not differ between the two groups. No associations between the prevalence rates of MH, MG, and CT were found in men from either infertility group. A lower sperm concentration was associated with STI pathogen positivity in men with primary infertility according to the crude model (P = 0.04). The crude and adjusted models showed that semen volume (both P = 0.03) and semen leukocyte count (both P = 0.02) were independently associated with secondary infertility. These findings suggest the importance of classifying the type of infertility during routine diagnosis of seminal tract infections.

Among causes of infertility, teratozoospermia is characterised by a percentage of morphologically abnormal spermatozoa4%. Macrozoospermia, one form of monomorphic teratozoospermia, is observed in1% of cases of male infertility and is described as approximately 100% large-headed and/or multitailed spermatozoa. This study reports that an infertile man with large-head spermatozoa presenting compound heterozygosity aurora kinase C (AURKC) mutations (c.382CT, c.572CT) by whole-exome sequencing. Consequently, both two novel AURKC mutations had high probability of damage-causing and conserved across species and extremely low allele frequency in the population. Flow cytometry analysis revealed a high ratio of sperm DNA fragmentation. Two intracytoplasmic sperm injection (ICSI) procedures were attempted for the patient, but all were unsuccessful. These results indicate that sequence analysis should be performed for the variants of AURKC in Chinese patients with macrozoospermia.

Background Empty follicle syndrome (EFS) is a rare but severe condition in which no oocyte is recovered in female patients undergoing in vitro fertilization (IVF) after sufficient ovarian response to hormonal trigger. Accumulating evidence highlights the genetic basis of EFS occurrence. Methods In this study, we report a patient with primary infertility showing the characteristics of EFS from a consanguineous family. Under the treatment of assisted reproductive technique (ART), no oocyte was retrieved following the aspiration of mature follicles. Through whole‐exome sequencing (WES), we discovered a novel recessively transmitted mutation in ZP1 (c.769 C>T, p. Q257*). Results In vitro Co‐immunoprecipitation assays showed that mutant ZP1 protein failed to interact with either ZP2 or ZP3, which explains the degenerated oocytes in the patient with EFS. Conclusion Together, our data further expand the spectrum of ZP1 mutations that are associated with human EFS and thus provide novel insight into the diagnosis of EFS patients., ZP1 mutation causes empty follicle syndrome.

Abundant decidual natural killer (dNK) cells at the maternal-fetal interface are important during early pregnancy. However, functional subsets of dNK cells remain poorly understood. We describe a CD49a+PBX homeobox 1 (PBX1)+ dNK cell subset that promotes fetal development in humans and mice. The expression of PBX1 in dNK cells is up-regulated via the activated AKT1 pathway through the interaction of major histocompatibility complex G with the immunoglobulin-like transcript 2 receptor. PBX1 drives pleiotrophin and osteoglycin transcription in dNK cells, further promoting fetal development. Decreased PBX1 expression or the PBX1G21S mutant correlated with fetal growth restriction and pregnancy failure in patients with unexplained recurrent spontaneous abortion (URSA). Inactivation of Pbx1 in mouse dNK cells impairs fetal development by decreasing growth-promoting factors from CD49a+PBX1+ dNK cells. Impairment of PBX1 in dNK cells has positive correlation with URSA pathogenesis and may provide a potential marker for this condition.

Summary Natural killer (NK) cells are present in large populations at the maternal-fetal interface during early pregnancy. However, the role of NK cells in fetal growth is unclear. Here, we have identified a CD49a + Eomes + subset of NK cells that secreted growth-promoting factors (GPFs), including pleiotrophin and osteoglycin, in both humans and mice. The crosstalk between HLA-G and ILT2 served as a stimulus for GPF-secreting function of this NK cell subset. Decreases in this GPF-secreting NK cell subset impaired fetal development, resulting in fetal growth restriction. The transcription factor Nfil3, but not T-bet, affected the function and the number of this decidual NK cell subset. Adoptive transfer of induced CD49a + Eomes + NK cells reversed impaired fetal growth and rebuilt an appropriate local microenvironment. These findings reveal properties of NK cells in promoting fetal growth. In addition, this research proposes approaches for therapeutic administration of NK cells in order to reverse restricted nourishments within the uterine microenvironment during early pregnancy.

Objective: We tested the feasibility of the in vitro culture of fetal nucleated erythroblasts from maternal blood for noninvasive prenatal screening as a substitute for culturing fetal nucleated erythroblasts from fetal villi. Method: Nucleated blood cells separated via Percoll from 52 samples of fetal villi and maternal peripheral blood were cultured with or without magnetic-activated cell sorting glycophorin A (MACS-GPA+), and detected by an anti-hemoglobin-epsilon (FITC) antibody. Gender of the epsilon-positive cells were identified by FISH and further confirmed by PCR of the villi karyotype. Developmental stages of nucleated erythroblasts from villi and blood with MACS-GPA+ were analyzed by Wright-Giemsa staining. Results: In the maternal blood, epsilon-positive cells were found in 4 and 24 cultured samples with and without MACS-GPA+, respectively. Also, Y-signals were visualized in 3 out of 4 and in 15 out of 24 cases in the epsilon-positive cells. Although epsilon-positive cells were found in all villus samples irrespective of MACS-GPA+ sorting, Y-signals were visualized in 31 out of 52 cases. Proerythroblasts and basophilic erythroblasts occupied 7 and 1% in fetal and maternal samples (with MACS-GPA+), respectively. Conclusions: The in vitro culturing of fetal nucleated erythroblasts from maternal blood is not feasible with the current techniques for prenatal diagnosis, because the fetal nucleated erythroblast is not well developed in vitro. This may be attributed to the low proportion of these erythroblasts at an early stage in the fetal circulation and the low permeability of these cells to the maternal blood.

Natural killer (NK) cells accumulate at the maternal–fetal interface in large numbers, but their exact roles in successful pregnancy remain poorly defined. Here, we provide evidence that T H 17 cells and local inflammation can occur at the maternal–fetal interface during natural allogenic pregnancies. We found that decidual NK cells promote immune tolerance and successful pregnancy by dampening inflammatory T H 17 cells via IFN-γ secreted by the CD56 bright CD27 + NK subset. This NK-cell–mediated regulatory response is lost in patients who experience recurrent spontaneous abortions, which results in a prominent T H 17 response and extensive local inflammation. This local inflammatory response further affects the regulatory function of NK cells, leading to the eventual loss of maternal-fetal tolerance. Thus, our data identify NK cells as key regulatory cells at the maternal–fetal interface by suppressing T H 17-mediated local inflammation.