BACKGROUND:Spermiogenesis is a highly specialized differentiation process involving by extensive nuclear remodeling and cytoplasmic reorganization. Disruption of these events is a major cause of male infertility, however the genetic determinants underlying human spermiogenic failure remain incompletely understood. NUP210L, which encodes a testis-enriched nucleoporin, has recently been implicated in male infertility, although direct evidence in humans is limited and murine models do not fully recapitulate the human phenotype. OBJECTIVE:To investigate the genetic basis and spermiogenic consequences of a homozygous NUP210L variant in a patient with severe oligoasthenoteratozoospermia (OAT), and to evaluate its impact on fertilization. MATERIALS AND METHODS:A consanguineous family with an affected male proband was studied. Whole-exome sequencing followed by Sanger validation was performed to identify the causative variant. Comprehensive sperm analyses, including light and transmission electron microscopy, immunofluorescence, Western blotting, and chromomycin A3 staining, were conducted to assess ultrastructure, protein localization, and chromatin remodeling. Fertilization outcomes of intracytoplasmic sperm injection (ICSI) with or without artificial oocyte activation (AOA) were evaluated. RESULTS:A homozygous in-frame deletion in NUP210L (c.1501_1503del, p.Ser501del) was identified. Patient sperm exhibited marked spermiogenic defects, including impaired chromatin condensation, acrosomal abnormalities, mitochondrial sheath disorganization, and fibrous sheath dysplasia. Although NUP210L protein levels were preserved, immunofluorescence revealed abnormal accumulation and mislocalization, suggesting functional impairment rather than loss of expression. Chromomycin A3 staining indicated significant histone retention, consistent with defective nuclear remodeling. Notably, the sperm-specific oocyte activation factor PLCζ was undetectable, consistent with total fertilization failure (TFF) following conventional ICSI. In contrast, ICSI combined with AOA successfully restored fertilization and enabled blastocyst development. CONCLUSION:This study identifies a homozygous NUP210L variant associated with severe spermiogenic defects and impaired sperm function. The findings support a role for NUP210L in human spermiogenesis that is not fully recapitulated in murine models. The successful rescue of fertilization by AOA further suggests that NUP210L-related defects are associated with impaired oocyte activation, providing human-based evidence that nucleoporin dysfunction contributes to male infertility.

BackgroundThis study aims to investigate the epidemiological data and trends of male infertility aged 20-49 years in Asia from 1990 to 2021.MethodsData for this study was obtained from the Global Burden of Disease (GBD) 2021 public database. Trends in prevalence, years lived with disability (YLDs), age-standardized prevalence rate (ASPR), and age-standardized YLD rate (ASYR), along with their correlation with the sociodemographic index (SDI), were analyzed. A join-point regression model was developed to assess trends across different time segments. In addition, an autoregressive integrated moving average (ARIMA) model was employed for predictions extending to 2040.ResultsFrom 1990 to 2021, the burden of male infertility among individuals aged 20-49 years increased both globally and across all Asian regions. In East Asia, the burden was the highest in 1990 and had an initial rapid escalation through the early 1990s, then declined steadily since 1995. South and Southeast Asia experienced significantly accelerating upward trends, with South Asia saw the steepest increases. High-income Asia Pacific and Central Asia sustained relatively low disease burdens and stable trends throughout the study period.ConclusionThe disease burden in East Asia, South Asia, and Southeast Asia remains significant, with a notable upward trend observed in South Asia and Southeast Asia. Targeted interventions and policies are needed based on the epidemiological characteristics and changing trends of different Asian regions.

Purpose This study aims to investigate the epidemiological data and trends of male infertility aged 20 to 49 years in Asia from 1990 to 2021. Methods Data for this study was obtained from the GBD 2021 public database. Trends in prevalence, disability-adjusted life years (DALYs), age-standardized prevalence rate (ASPR), and age-standardized DALY rate (ASDR), along with their correlation with the Socio-Demographic Index, were analyzed. A join-point regression model was developed to assess trends across different time segments. Additionally, an autoregressive integrated moving average (ARIMA) model was employed for predictions extending to 2040. Results The burden of male infertility among individuals aged 20 to 49 years significantly increased globally and in East Asia, South Asia, and Southeast Asia from 1990 to 2021, with the South Asia region exhibiting the largest increase and the fastest growth rate. East Asia exhibited the highest disease burden, whereas Central Asia and the high-income Asia Pacific region had the lowest. Predictions for 2040 indicate that this upward trend will continue both globally and within Asian regions. Conclusion The disease burden in East Asia, South Asia, and Southeast Asia remains significant, with a notable upward trend observed in South Asia and Southeast Asia. Targeted interventions and policies are needed based on the epidemiological characteristics and changing trends of different Asian regions.

Spermatozoa experience a long and tough transit in male and female genital tracts before successful fertilization. Glycosylation helps spermatogenesis, epididymal maturation, passing through cervical mucus, avoiding killing of the female immunologic system, and shaking hands between sperm and egg. Changes in glycosylations along the transit ensure that the right things happen at the right time and place on spermatozoa. Aberrant glycosylations on spermatozoa will negatively affect their fertility. Thus, we developed a lectin array method to examine the glycocalyx of spermatozoa, which will help observe glycosylations occurring on spermatozoa in a normal or abnormal conditions, such as spermatozoa with DEF126 mutation and poor freezability. Intriguingly, binding levels of ABA (Agaricus bisporus agglutinin), a lectin marking the inner layer of the glycocalyx, were changed in these subfertile spermatozoa, which indicates that the integrity of glycocalyx is critical for sperm fertility. In this chapter, we reviewed the impacts of glycosylations on sperm fertility, the lectin array method, and its potential application for sperm function assessment.

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic biological research, drug development, and screening. This paper presents a simple, rapid, and highly efficient in vitro model for embryo implantation. In this protocol, we first introduce mouse blastocyst acquisition and human endometrial adenocarcinoma cells (Ishikawa) preparation for implantation, followed by the co-culture method for mouse embryos and Ishikawa cells. Finally, we conducted a study to assess the impact of varying concentrations of 17-β-estradiol (E2) and progesterone (P4) on embryo adhesion rates based on this model. Our findings revealed that high concentrations of E2 significantly reduced embryo adhesion, whereas the addition of progesterone could restore the adhesion rate. This model offers a simple and fast platform for evaluating and screening molecules involved in the adhesion process, such as cytokines, drugs, and transcription factors controlling implantation and endometrial receptivity.

Background Actin-like 7 A (ACTL7A) is essential for acrosome formation, fertilization and early embryo development. ACTL7A variants cause acrosome detachment responsible for male infertility and early embryonic arrest. In this study, we aim to explore the additional functions of ACTL7A beyond the process of acrosome biogenesis and investigate the possible underlying mechanisms. Methods Nuclear morphology analysis was used to observe the sperm head shape of ACTL7A -mutated patients. Actl7a knock-out (KO) mouse model was generated. Immunofluorescence and transmission electron microscopy (TEM) were performed to analyze the structure of spermatids during spermiogenesis. Tandem mass tags labeling quantitative proteomics strategy was employed to explore the underlying molecular mechanisms. The expression levels of key proteins in the pathway were analyzed by western blotting. Intracytoplasmic sperm injection (ICSI)-artificial oocyte activation (AOA) technology was utilized to overcome fertilization failure in male mice with a complete knockout of Actl7a . Results The new phenotype of small head sperm associated with loss of ACTL7A in patients was discovered, and further confirmed in Actl7a -KO mice. Immunofluorescence and TEM analyses revealed that the deletion of ACTL7A damaged the formation of acrosome-acroplaxome-manchette complex, leading to abnormalities in the shaping of sperm heads. Moreover, a proteomic analysis of testes from WT and Actl7a -KO mice revealed that differentially expressed genes were notably enriched in PI3K/AKT/mTOR signaling pathway which is strongly associated with autophagy. Inhibition of autophagy via PI3K/AKT/mTOR signaling pathway activation leading to PDLIM1 accumulation might elucidate the hindered development of manchette in Actl7a -KO mice. Remarkably, AOA successfully overcame fertilization failure and allowed for the successful production of healthy offspring from the Actl7a complete knockout male mice. Conclusions Loss of ACTL7A causes small head sperm as a result of defective acrosome-acroplaxome-manchette complex via autophagy inhibition. ICSI-AOA is an effective technique to rescue male infertility resulting from ACTL7A deletion. These findings provide essential evidence for the diagnosis and treatment of patients suffering from infertility.

Phospholipase Cζ (PLCζ) is composed of XY domain, EF-hand domains and a C2 domain, which specifically hydrolyzes phosphatidylinositol (4,5)-bisphosphate (PIP 2) in the oocyte cytoplasm but not on the plasma membrane through the classical PIP 2→(1,4,5)-inositol trisphosphate (IP 3)+diacylglycerol (DAG) signal transduction pathway, which causes oscillation of calcium ion in oocytes, and plays an irreplaceable role in activating oocytes and initiating gamete development. Clinical data have shown that the reduced or absent expression level of PLCζ can lead to the inability of sperm activating oocytes and are closely related to various sperm abnormalities, ultimately leading to male subfertility and even infertility. In summary, this article reviews the structure and biological functions of PLCζ, the clinical diseases caused by abnormal PLCζ and their following therapeutic progresses.

Obtaining high-quality embryos is one of the key factors to improve the clinical pregnancy rate of assisted reproductive technologies (ART). So far, the clinical evaluation of embryo quality depends on embryo morphology. However, the clinical pregnancy rate is still low. Therefore, new indicators are needed to further improve the evaluation of embryo quality. Several studies have shown that the decrease of sperm-specific protein actin-like 7A (ACTL7A) leaded to low fertilization rate, poor embryo development, and even infertility. The aim of this study was to study whether the different expression levels of ACTL7A on sperm can be used as a biomarker for predicting embryo quality. In this study, excluding the factors of severe female infertility, a total of 281 sperm samples were collected to compare the ACTL7A expression levels of sperms with high and low effective embryo rates and analyze the correlation between protein levels and in-vitro fertilization (IVF) laboratory outcomes. Our results indicated that the ACTL7A levels were significantly reduced in sperm samples presenting poor embryo quality. Furthermore, the protein levels showed a significant correlation with fertilization outcomes of ART. ACTL7A has the potential to be a biomarker for predicting success rate of fertilization and effective embryo and the possibility of embryo arrest. In conclusion, sperm-specific protein ACTL7A has a strong correlation with IVF laboratory outcomes and plays important roles in fertilization and embryo development.

Androgen insensitivity syndrome (AIS) is an X-linked recessive genetic disease characterized by disorders of sex development, commonly caused by mutations of the androgen receptor (AR) gene. Herein, we identified a novel hemizygous mutation (c.2118T > A, p. Asn706Lys) of AR resulting in complete androgen insensitivity syndrome (CAIS) in twins. This missense mutation contributed to significantly decreased mRNA transcription and protein expression. In addition, structure model analysis showed that Asn706Lys resulted in loss of hydrogen bond with Asp891 and reduced protein stability. Furthermore, the mutant AR failed to bind to ligand due to the loss of hydrogen bond with dihydrotestosterone (DHT). This disrupted the translocation of AR protein from cytoplasm to nucleus after hormone stimulation. Our findings firstly demonstrated the novel mutation of c.2118T > A in AR directly caused CAIS. This contributed to expanding the AR mutational spectrum and revealed the pathogenic mechanism of AIS, as well as facilitating precise diagnosis and genetic counseling.

The human endometrium is a highly regenerative tissue that is believed to have two main types of stem cells: endometrial mesenchymal/stromal stem cells (eMSCs) and endometrial epithelial stem cells (eESCs). So far, eMSCs have been extensively studied, whereas the studies of eESCs are constrained by the inability to culture and expand them in vitro. The aim of this study is to establish an efficient method for the production of eESCs from human endometrium for potential clinical application in intrauterine adhesion (IUA). Here we developed a culture condition with a combination of some small molecules for in vitro culturing and expansion of human SSEA-1+ cells. The SSEA-1+ cells exhibited stem/progenitor cell activity in vitro, including clonogenicity and differentiation capacity into endometrial epithelial cell-like cells. In addition, the SSEA-1+ cells, embedded in extracellular matrix, swiftly self-organized into organoid structures with long-term expansion capacity and histological phenotype of the human endometrial epithelium. Specifically, we found that the SSEA-1+ cells showed stronger therapeutic potential than eMSCs for IUA in vitro. In a rat model of IUA, in situ injection of the SSEA-1+ cells-laden chitosan could efficiently reduce fibrosis and facilitate endometrial regeneration. Our work demonstrates an approach for isolation and expansion of human eESCs in vitro, and an appropriate marker, SSEA-1, to identify eESCs. Furthermore, the SSEA-1+ cells-laden chitosan might provide a novel cell-based approach for IUA treatment. These findings will advance the understanding of pathophysiology during endometrial restoration which may ultimately lead to more rational clinical practice.

Early embryonic arrest is a challenge for in vitro fertilization (IVF). No genetic factors were previously revealed in the sperm-derived arrest of embryonic development. Here, we reported two infertile brothers presenting normal in conventional semen analysis, but both couples had no embryos for transfer after several IVF and intracytoplasmic sperm injection (ICSI). Whole-exome sequencing identified a homozygous missense mutation of ACTL7A in both brothers. This mutation is deleterious and causes sperm acrosomal ultrastructural defects. The Actl7a knock-in mouse model was generated, and male mutated mice showed sperm acrosomal defects, which were completely consistent with the observations in patients. Furthermore, the sperm from ACTL7A/Actl7a-mutated men and mice showed reduced expression and abnormal localization of PLCζ as a potential cause of embryonic arrest and failure of fertilization. Artificial oocyte activation could successfully overcome the Actl7a-mutated sperm-derived infertility, which is meaningful in the future practice of IVF/ICSI for the ACTL7A-associated male infertility.

Metabolic profile of follicular fluid (FF) has been investigated to look for biomarkers for oocyte quality. Resolvin E1 (RvE1), a potent pro-resolving mediator, was reported to have protective action in cell function. The study aimed to examine the predictive value of RvE1 for oocyte quality and to explore the cellular mechanism of RvE1 in improving oocyte competence. Metabolic profiles of 80 FF samples showed a higher level of RvE1 in group A (blastocysts scored ≥ B3BC and B3CB according to Gardner's blastocyst scoring system, N = 36) than that of group B (blastocysts scored < B3BC and B3CB, N = 44, P = 0.0018). The receiver operating characteristic (ROC) curve analysis showed that RvE1 level in FF below 8.96 pg/ml (AUC:0.75; 95%CI: 0.64–0.86; P = 0.00012) could predict poor oocyte quality with specificity of 97.22%, suggesting RvE1 as a potential biomarker to exclude inferior oocytes. Besides, the level of RvE1 was found to be significantly lower in FF than in serum (57.49 to 17.62 pg/ml; P=.0037) and was gradually accumulated in the culture medium of cumulus cells (CCs) during cell culture, which indicated that RvE1 came from both blood exudates and local secretion. The in vitro experiment revealed thecellular mechanism of RvE1 in improvingoocyte qualityby decreasing the cumulus cellapoptotic rate and increasing cell viability and proliferation. It is the first time thatthe role of RvE1 in reproduction is explored. In conclusion, RvE1 is valuable as a potential exclusive biomarker for oocyte selection andplays a role in improving oocyte quality.

Semen cryopreservation has been widely applied in assisted reproductive technologies and sperm bank, but it causes considerable impairments on sperm quality. It is necessary to find an evaluation indicator for determining the sperm-freezing tolerance.

Ectonucleotide pyrophosphatase-phosphodiesterase 3 (ENPP3), a protein detected in the human uterus, has been found to play an important role in the development and invasion of tumours. It was recently discovered that ENPP3 was upregulated during the window of implantation in the human endometrium but its functional relevance remains elusive. The objective was to determine ENPP3 expression in human endometrium and its roles in endometrial receptivity and embryo implantation. ENPP3 expression was analysed using immunohistochemistry and western blot assay. The effects of ENPP3 on embryo implantation were evaluated using a BeWo cell (a human choriocarcinoma cell line) spheroid attachment assay and BeWo cells were dual cultured with Ishikawa cells transfected with lentiviral vectors (LV5-NC or LV5-ENPP3) to mimic embryo implantation in a Transwell model. The effects of endometrial ENPP3 on factors related to endometrial receptivity were also determined. The results showed that ENPP3 was expressed in human endometrial epithelial cells and its expression levels changed during the menstrual cycle, peaking in the mid-secretory phase, corresponding to the time of embryo implantation. The overexpression of endometrial ENPP3 not only increased the embryo implantation rate but also had positive effects on the expression of factors related to endometrial receptivity in human endometrial cells. The results indicate that ENPP3 levels undergo cyclic changes in the endometrium and affect embryo adhesion and invasion via altering the expression of implantation factors in the human endometrium. Therefore, ENPP3 may play an important role in embryo implantation and may be a unique biomarker of endometrial receptivity.

Background: To examine the effects of dehydroepiandrosterone (DHEA) on in vitro fertilization (IVF) intracytoplasmic sperm injection (ICSI) and the levels of follicular fluid (FF) markers, namely, anti-Müllerian hormone (AMH), insulin-like growth factor (IGF)-1, bone morphogenetic protein (BMP)-15, and growth differentiation factor (GDF)-9, in patients with diminished ovarian reserves (DORs). Methods: 116 patients with DOR were randomized into two groups, DHEA group and control group. Each group contained 58 patients. The DHEA group received 75 mg/d of DHEA for 12 weeks prior to the start of IVF treatment, while the control group entered IVF treatment directly. All patients were treated with the same ovarian stimulation protocol. The primary outcome was high-quality embryo yield. Other IVF parameters, such as the clinical pregnancy rate, embryo survival rate, and intact blastomere rate, were compared between the two groups. FF samples from patients of both groups were collected to measure the levels of AMH, IGF-1, DHEA-sulfate, BMP-15, and GDF-9. Blood was also collected on day 3 of the menstrual cycle to define the baseline hormonal profile and to examine ovarian reserve markers. Results: The high-quality embryo yield was higher in DHEA group than that in control group (P = 0.033). AMH and IGF-1 concentrations in FF were significantly higher in DHEA group than that in the control group (2.83 ± 1.14 ng/L vs. 1.37 ± 0.55 ng/L, P = 0.000; 94.02 ± 38.28 ng/L vs. 74.03 ± 25.46 ng/L, P = 0.004, respectively). The BMP-15 level was also higher in DHEA group (relative expression were 1.80 ± 0.41) than that in control group (relative expression were 0.79 ± 0.16, P < 0.0001); however, there was no difference in GDF-9 expression between the two groups (relative expression were 1.29 ± 0.54 and 1.16 ± 0.50 respectively, P > 0.05) and in the clinical pregnancy rate between the two groups (13.79% vs. 7.27%, respectively, P > 0.05). Conclusions: In women with DOR undergoing IVF treatment, pretreatment with DHEA may increase the number of high-quality embryos, which may be due to increased levels of AMH, IGF-1, and BMP-15 in the FF. Trial Registration: NCT02866253.

Tamoxifen is administered for estrogen receptor positive (ER + ) breast cancers, but it can induce uterine endometrial cancer and non-alcoholic fatty liver disease (NAFLD). Importantly, ten years of tamoxifen treatment has greater protective effect against ER + breast cancer than five years of such treatment. Tamoxifen was also approved by the FDA as a chemopreventive agent for those deemed at high risk for the development of breast cancer. The side effects are of substantial concern because of these extended methods of tamoxifen administration. In this study, we found that anordrin, marketed as an antifertility medicine in China, inhibited tamoxifen-induced endometrial epithelial cell mitosis and NAFLD in mouse uterus and liver as an anti-estrogenic and estrogenic agent, respectively. Additionally, compared with tamoxifen, anordiol, the active metabolite of anordrin, weakly bound to the ligand binding domain of ER-α. Anordrin did not regulate the classic estrogen nuclear pathway; thus, it did not affect the anti-tumor activity of tamoxifen in nude mice. Taken together, these data suggested that anordrin could eliminate the side effects of tamoxifen without affecting its anti-tumor activity.

Background: To study the effects of cryopreservation on human sperm glycocalyx. Methods: The lectin binding profilings of sperm after freeze-thaw were compared by lectin microarray. Results: CryoSpermTM and direct fumigation were confirmed to be the optimized cryoprotectant and method by comparing the sperm recovery rate. In 91 lectins, 33 lectins were significantly changed after sperm cryopreservation. Among them, 9 lectins greatly decreased and 24 lectins mainly increased. The binding signals of MAA, PSA, ABA, and AIA were verified by FACS, and the results were consistent with that of lectin microarray. Conclusions: Sperm glycocalyx had significant changes after cryopreservation. The sialic acid, playing an important role in protecting sperm, was greatly lost, which exposed the inner carbohydrates. Thus, the glycocalyx damage due to the cryopreservation might be one of the reasons for low sperm fertility.

Clinical ovulation induction induces blood estrogen (E2) in excess of physiological levels, which can hinder uterine receptivity. In contrast, progesterone produces the opposite clinical effect, suggesting that it might be capable of recovering the lost receptivity resulting from exposure to high estrogen levels. Integrins are the most widely used biological markers for monitoring uterine conditions. We studied progesterone-induced changes in integrin β expression patterns as biomarkers for changes in uterine receptivity in response to increased estrogen levels.

Coating on the sperm surface, glycocalyx, plays a key role in sperm motility, maturation and fertilization. A comprehensive profile of sperm surface glycans will greatly facilitate both basic researches and clinical studies. Because of the capability of recognizing different glycan moieties, lectins are widely used in glycobiology. However, lacking high-throughput technology, limited lectins have been reported for analyzing the glycan of human sperm. In this study, we employed a lectin microarray for profiling the surface glycans of human sperm, on which 54 out of 91 lectins showed positive binding. Based on this technique, we compared lectin binding profiling of sperm with homozygous DEFB126 mutation (del/del) with that of wild type (wt/wt). DEFB126 was reported to contribute to the sialylation on sperm surface and its homozygous mutation was related to male subfertility. Six lectins (Jacalin/AIA, GHA, ACL, MPL, VVL and ABA) were found to develop lower binding affinity to sperm with del/del. Further validation showed that these lectins, especially ABA and MPL, can be potential biomarkers for clinical diagnosis of subfertility due to the mutation of DEFB126. Our research provides insight into the detection of some unexplained male subfertility and the lectin microarray is generally applicable for infertility/subfertility sperm biomarker discovery.

Glycosylation is one of the most abundant and functionally important protein post-translational modifications. As such, technology for efficient glycosylation analysis is in high demand. Lectin microarrays are a powerful tool for such investigations and have been successfully applied for a variety of glycobiological studies. However, most of the current lectin microarrays are primarily constructed from plant lectins, which are not well suited for studies of human glycosylation because of the extreme complexity of human glycans. Herein, we constructed a human lectin microarray with 60 human lectin and lectin-like proteins. All of the lectins and lectin-like proteins were purified from yeast, and most showed binding to human glycans. To demonstrate the applicability of the human lectin microarray, human sperm were probed on the microarray and strong bindings were observed for several lectins, including galectin-1, 7, 8, GalNAc-T6, and ERGIC-53 (LMAN1). These bindings were validated by flow cytometry and fluorescence immunostaining. Further, mass spectrometry analysis showed that galectin-1 binds several membrane-associated proteins including heat shock protein 90. Finally, functional assays showed that binding of galectin-8 could significantly enhance the acrosome reaction within human sperms. To our knowledge, this is the first construction of a human lectin microarray, and we anticipate it will find wide use for a range of human or mammalian studies, alone or in combination with plant lectin microarrays.