Excessive trophoblast apoptosis and dysfunction are critical events in the pathogenesis of preeclampsia (PE). Soluble endoglin (sENG) is elevated in PE; however, its direct role in regulating trophoblast biology remains incompletely understood. In this study, we investigated whether sENG promotes trophoblast apoptosis and impairs trophoblast function, and further explored the underlying molecular mechanisms. Placenta-targeted sENG overexpression was induced in pregnant mice via an AAV6 vector driven by the FLT1 promoter, and PE-like phenotypes including hypertension and proteinuria were evaluated. Transforming growth factor-β (TGF-β)-Smad2/3 signaling, CD39 expression, extracellular ATP (eATP) levels, and P2X7 receptor (P2X7R) expression were analyzed in placental tissues and maternal serum. In vitro, HTR-8/SVneo trophoblasts were treated with recombinant sENG, and trophoblast biological functions and signaling protein expression were assessed by CCK-8, wound healing, Transwell, immunofluorescence, and western blotting. Rescue experiments were performed using exogenous TGF-β1 and a P2 × 7R inhibitor. Key signaling changes were further validated in placental tissues from patients with PE and healthy controls. In mice, sENG overexpression triggered hypertension, proteinuria, fetal growth restriction, and placental apoptosis, accompanied by inhibition of the TGF-β1-Smad2/3-CD39 pathway. In vitro, sENG induced dose-dependent apoptosis by suppressing Smad2/3 phosphorylation, downregulating CD39, and elevating eATP levels; these effects were reversed by exogenous TGF-β1. BzATP-mediated P2X7R activation contributed to trophoblast dysfunction and apoptosis. In human PE placentas, reduced phosphorylation of Smad2/3 and decreased CD39 expression, together with increased P2X7R expression, were associated with enhanced apoptotic signaling. sENG induces trophoblast apoptosis and dysfunction through the TGF-β-Smad2/3-CD39-eATP-P2 × 7R signaling axis, contributing to PE pathogenesis and representing a promising molecular candidate for future therapeutic research into PE.

IntroductionMaternal vascular dysfunction and impaired placental angiogenesis are key features of preeclampsia (PE). Umbilical cord blood-derived endothelial colony-forming cells (CB-ECFCs) contribute to fetal endothelial repair and vascular network formation, but their phenotype in PE remains incompletely understood.MethodsWe investigated whether the matricellular protein CCN1 is associated with CB-ECFC senescence and dysfunction. CB-ECFCs were isolated from pregnancies complicated by PE (n = 8) and healthy pregnancies (n = 9). Cellular senescence was assessed by SA-β-galactosidase staining, qPCR, and Western blotting of senescence-associated markers and senescence-associated secretory phenotype factors. Control-derived CB-ECFCs were exposed to recombinant CCN1, with or without the gamma-secretase inhibitor DAPT, and senescence-associated proteins, cleaved Notch1, the redox-responsive protein HO-1, the inflammatory factor TNF-α, and the DNA damage-response marker gamma-H2AX were examined.ResultsPE-derived CB-ECFCs showed reduced colony yield, increased senescence-associated markers, and higher secreted and cell-associated CCN1 expression. Recombinant CCN1 increased senescence-associated markers, cleaved Notch1, HO-1, TNF-α, and γ-H2AX and impaired CB-ECFC metabolic activity, migration, and tube formation. DAPT attenuated selected molecular changes and partially restored tube formation but not wound closure.DiscussionThese findings identify increased CCN1 as a feature of PE-derived CB-ECFCs and support an association between CCN1 exposure, Notch1 signaling, oxidative stress, and CB-ECFC senescence and dysfunction. The CCN1/Notch1 axis may represent a potential mechanism contributing to fetoplacental endothelial dysfunction in PE.

OBJECTIVE:We aim to clarify the comparative clinical efficacy of ovulation induction and artificial cycle (AC) protocols for frozen-thawed embryo transfer (FET) in reproductive-aged women with polycystic ovary syndrome (PCOS), given the conflicting findings and ongoing controversy surrounding this issue in existing studies. METHODS:This was a retrospective cohort study conducted at a public fertility center in China, which included a total of 1331 women with PCOS who underwent FET between January 2021 and December 2024; the study compared the efficacy of two endometrial preparation interventions during FET, ovulation induction cycles (n = 543) and AC (n = 788). The primary outcome measure was live birth rate, while the secondary outcome measures were categorized into three types: clinical outcomes, maternal outcomes and perinatal outcomes. RESULTS:Endometrial thickness on the day of progesterone administration was significantly greater in the ovulation induction group (10.3 (9.0-12.2) vs. 9.2 (8.0-10.8), p < 0.001) than in the AC group. Following adjustment for potential confounding factors, the ovulation induction cycle group exhibited a significantly higher clinical pregnancy rate (59.5% vs. 51.5%, aOR = 1.3, 95%CI: 1.1-1.7) and live birth rate (48.8% vs. 39.7%, aOR = 1.4, 95%CI: 1.1-1.7) relative to the artificial cycle group. Perinatal outcomes were comparable between AC and ovulation induction. CONCLUSIONS:In conclusion, ovulation induction significantly improves live birth rates over artificial cycles in PCOS women undergoing FET, with comparable perinatal safety. Despite the limitations of retrospective, single-center data, this study provides real-world evidence to guide endometrial preparation. Prospective randomized trials are warranted to confirm these findings.

Purpose To compare differences in euploidy rates for blastocysts in preimplantation genetic testing for aneuploidy (PGT-A) cycles after gonadotropin-releasing hormone agonist (GnRH-a) long and short protocols, GnRH-antagonist (GnRH-ant) protocol, progestin-primed ovarian stimulation and mild stimulation protocols, and other ovary stimulation protocols. Methods This was a retrospective cohort study from the Assisted Reproductive Medicine Department of Shanghai First Maternity and Infant Hospital. A total of 1657 PGT-A cycles with intracytoplasmic sperm injection after different controlled ovary hyperstimulation protocols were analyzed, and a total of 3154 embryos were biopsied. Differences in euploidy rate per embryo biopsied, embryo euploidy rate per oocyte retrieved and cycle cancellation rate were compared. Results For the PGT-A cycles, the euploidy rate per embryo biopsied was lower in the GnRH-ant protocol than in the GnRH-a long protocol (53.26% vs. 58.68%, respectively). Multiple linear regression showed that the GnRH-ant protocol was associated with a lower euploidy rate per embryo biopsied (β = −0.079, P = 0.011). The euploidy rate per embryo biopsied was not affected by total gonadotropin dosage, duration of stimulation and number of oocytes retrieved. The embryo euploidy rate per oocyte retrieved was similar in all protocols, and was negatively correlated with the total number of oocytes retrieved (β = −0.003, P = 0.003). Conclusion Compared with the GnRH-a long protocol, the GnRH-ant protocol was associated with a lower euploidy rate per embryo biopsied. The total gonadotropin dosage, duration of stimulation and number of oocytes retrieved did not appear to significantly influence euploidy rates.

BackgroundOligoasthenoteratozoospermia (OAT) is a common cause of male infertility, of which the causes remain largely unknown. Recently, BCORL1 was identified as a contributor to male infertility from non-obstructive azoospermia (NOA) to OAT. ObjectivesTo identify novel and hotspot variants in BCORL1 from infertile men with OAT and reveal their outcomes of assisted reproductive treatments (ARTs). Materials and methodsForty-six infertile men characterized by OAT were recruited from 2017 to 2022. Variants in OAT patients were identified by whole-exome sequencing (WES) and verified by Sanger sequencing. Papanicolaou staining was used for sperm morphology analysis. Pathogenicity of BCORL1 variants were analyzed by bioinformatics analysis, and further confirmed in vitro by using recombinant plasmids and cells. Meanwhile, ARTs were performed on these patients to investigate the appropriate clinical treatment strategy. ResultsWe identified a novel hemizygous missense variant (NM_021946: c.G4171A; p.G1391R) and a recurrent variant (NM_021946: c.T2615G; p.V872G) in BCORL1 from four OAT patients. Notably, routine semen assessment and Papanicolaou staining revealed a special OAT phenotype of patients with BCORL1 variants, whose rare mature sperm characterized by acephalic and abnormal acrosome. Pathogenicity analysis showed the interaction between BCORL1 with histone deacetylases (HDACs) were disrupted after variance, accompanied with epigenetic alterations and finally the orderly transcriptions of spermatogenetic genes were interfering. Besides, clinical record presented the poor outcomes of ARTs in these patients with BCORL1 variants. Discussion and conclusionsOur findings further expand the variant spectrum of BCORL1 related to OAT, and provide new evidences that BCORL1 acts as an important transcriptional regulator, participating in epigenetic regulation and directing the expression of key genes throughout spermatogenesis. The outcomes of ARTs will facilitate the genetic counseling and clinical treatment of infertile men with BCORL1 variants in the future.

Background Sperm cryopreservation is commonly used to preserve fertility but often decreases sperm quality by inducing oxidative stress. Pinus massoniana bark extract (Pinus massoniana, PMBE) exhibits strong antioxidant activity and has been used in traditional Chinese medicine. Objectives The objective was to determine whether adding PMBE to cryoprotectant could improve human sperm quality after cryo-resuscitation and to investigate the potential regulatory mechanisms. Methods PMBE was used as a cryoprotectant supplement. Hydrogen peroxide was applied to generate oxidative stress. The changes in sperm quality upon cryo-resuscitation and hydrogen peroxide treatment were measured by computer-aided sperm analysis and fluorescein isothiocyanate-labeled peanut (Pisum sativum) agglutinin staining. Oxidative stress was evaluated by dichlorofluorescein diacetate staining and the malondialdehyde content. The antioxidant capacity was determined by the glutathione peroxidase activity and the reduced glutathione content. Mass spectrometry was utilized to screen factors potentially regulated by PMBE, and the screened factors were evaluated by Western blot. Mouse in vitro fertilization was used to assess the effectiveness and safety of PMBE on the fertilization outcome of damaged spermatozoa. Results PMBE addition to cryoprotectant could effectively promote post-thaw vitality and acrosome reaction rates, reduce intracellular reactive oxygen species, and preserve antioxidant capacity and mitochondrial function in the presence of cryo-resuscitation and hydrogen peroxide treatment. Decreased in the expression of critical proteins such as inositol-3-phosphate synthase 1 (ISYNA1) and AKT1 upon resuscitation was rescued by PMBE supplementation, further reducing the cytoplasmic Nrf2 protein level and markedly increasing the nuclear level. PMBE dramatically enhanced the mRNA and protein expression of superoxide dismutase (SOD2). PMBE effectively alleviated the hydrogen peroxide-induced decline in the two-cell embryo rates. Conclusion Supplementing cryoprotectant with PMBE improves human sperm quality after cryo-resuscitation by reducing oxidative stress and stabilizing the functional proteins ISYNA1 and AKT1. PMBE addition improves the fertilization outcome of oxidative stress-damaged spermatozoa.

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.

Background Alginate matrix 3-dimensional culture offers the opportunity for the development and maturation of human secondary follicles in vitro. However, alginate may not be the most suitable culture system for human primordial/primary follicles in vitro. Thus, the innovation of alginate matrix 3-dimensional culture systems for human primordial/primary follicles could hold promise as an ideal approach to restoring fertility. Methods We extracted primordial/primary follicles from ovarian tissues collected from patients with non-ovarian benign gynecological conditions. Fibroblasts were isolated from dermal tissue from 1 male patient who had undergone posthectomy. The isolated human follicles were randomly divided into 2 groups and encapsulated within fibroblast-alginate-hydrogels or alginate hydrogels. The survival and growth of human primordial/primary follicles were measured after 21 days of in vitro culture. Results The dermal fibroblasts in alginate hydrogel microcapsules were round in shape, and were distributed as uniform clouds on the surface and gaps of the alginate. After 21 days of culture, the survival rate of follicles in the fibroblast-alginate group was higher than that of the alginate group (P<0.05). The diameter of follicles in the fibroblast-alginate group and the alginate group after 21 days of culture was 152.80±13.64 and 129.14±9.95 μm, respectively (P<0.05). After 21-day culture, the mean cpm (log-converted) for 3H-thymidine incorporated by granulosa cells in the fibroblast-alginate and alginate groups was 6.87±0.24 and 4.63±0.38, respectively (P<0.05). After 21 days of culture, the messenger RNA expression levels of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) were significantly higher in oocytes in fibroblast-alginate hydrogels than in those in alginate hydrogels (P<0.05). Conclusions Human fibroblasts are beneficial to the development of human follicles in 3-dimensional culture alginate gel systems over a long period of time. More studies are required to investigate the molecular biological mechanisms of human fibroblasts that promote follicle growth in vitro.

PURPOSE:Tubulin beta eight class VIII (TUBB8) is essential for oogenesis, fertilization, and pre-implantation embryo development in human. Although TUBB8 mutations were recently discovered in meiosis-arrested oocytes of infertile females, there is no effective therapy for this gene mutation caused infertility. Our study aims to further reveal the infertility-causing gene mutations in the patient's family and to explore whether the infertility could be rescued by optimizing the conditions of embryo culture and finally achieve the purpose of making the patient pregnant. METHODS:Whole-exome sequence analysis and Sanger sequencing were performed on patients' family members to screen and identify candidate mutant genes. Construction of plasmids, in vitro transcription, microinjection of disease-causing gene cRNA, and immunofluorescence staining were used to recapitulate the infertility phenotype observed in patients and to understand the pathogenic principles. Simultaneously, overexpression of mutant and wild-type cRNA of the candidate gene in mouse oocytes at either germinal vesicle (GV) or metaphase II (MII) stage was performed in the rescue experiment. RESULTS:We first identified a novel heritable TUBB8 mutation (c.1041C>A: p.N347K) in the coding region which specifically affects the first mitosis and causes the developmental arrest of early embryos in a three-generation family. We further demonstrated that TUBB8 mutation could lead to abnormal spindle assemble. And moreover, additional expression of wild-type TUBB8 cRNA in the mouse oocytes in which the mutant TUBB8 were expressed can successfully rescue the developmental defects of resulting embryo and produce full-term offspring. CONCLUSIONS:Our study not only defines a novel mutation of TUBB8 causing the early cleavage arrest of embryos, but also provides an important basis for treating such female infertility in the future.

Poor oocyte quality is associated with early embryo developmental arrest and infertility. Maternal gene plays crucial roles in the regulation of oocyte maturation, and its mutation is a common cause of female infertility. However, how to improve oocyte quality and develop effective therapy for maternal gene mutation remains elusive. Here, we use Zar1 as an example to assess the feasibility of genome transfer to cure maternal gene mutation–caused female infertility. We first discover that cytoplasmic deficiency primarily leads to Zar1-null embryo developmental arrest by disturbing maternal transcript degradation and minor zygotic genome activation (ZGA) during the maternal-zygotic transition. We next perform genome transfer at the oocyte (spindle transfer or polar body transfer) and zygote (early pronuclear transfer or late pronuclear transfer) stages to validate the feasibility of preventing Zar1 mutation–caused infertility. We finally demonstrate that genome transfer either at the oocyte or at the early pronuclear stage can support normal preimplantation embryo development and produce live offspring. Moreover, those pups grow to adulthood and show normal fertility. Therefore, our findings provide an effective basis of therapies for the treatment of female infertility caused by maternal gene mutation.

Objective: To develop a unique approach using polarization microscopy (PM) to determine whether the presence of a spindle can be used as an indicator associated with fertilization failure 5 hours after short-term insemination.Design: Observational study.Setting: Assisted reproduction center.Patient(s): Eighty-five patients undergoing short-term insemination.Intervention(s): Oocytes imaged via PM at 4, 5, and 6 hours after standard insemination.Main Outcome Measure(s): Spindle visualization and fertilization rate, with rescue intracytoplasmic sperm injection (ICSI) results determined by rates of normal fertilization, abnormal fertilization, and good-quality embryo formation.Result(s): After standard insemination, comparisons of spindle visualization at three time points indicated that the predictive accuracy rates were 84.30% at 5 hours, 86.80% at 6 hours, and 62.20% at 4 hours, with the rates at 5 and 6 hours statistically significantly higher than at 4 hours. A spindle was present in 242 of the 788 metaphase-II oocytes 5 hours after insemination, and there were 204 failed fertilizations on day 1. The positive predictive value was 0.84. After rescue ICSI, the abnormal fertilization rate of the polar body group (assessed using the polar body visualization method) was statistically significantly higher than that of the PM group (assessed using the spindle visualization method) and the regular ICSI group (9.37%, 5.88%, and 4.87%, respectively).Conclusion(s): The presence of a spindle 5 hours after insemination in in vitro fertilization is an accurate indicator of unfertilized oocytes. Spindle imaging combined with rescue measures effectively prevents fertilization failure and decreases the polyspermy rate. (C)2017 by American Society for Reproductive Medicine.

OBJECTIVE:To investigate the influence of Ureaplasma urealyticum (Uu) infection in the male reproductive tract on the outcomes of IVF and the clinical significance of preoperative Uu test by analyzing the correlation between the results of Uu culture before IVF-ET and the outcomes of IVF-ET.METHODS:Among 1,059 couples undergoing IVF-ET, we selected 973 after excluding genetic factors and divided them into a Uu negative and a Uu positive group according to the results of culture of Uu in the semen of the males. We compared the rates of IVF fertilization, oocyte cleavage, clinical pregnancy and abortion between the two groups, and analyzed the influence of Uu infection on IVF outcomes.RESULTS:Among the 973 selected subjects, 836 were Uu negative (group A) and 137 Uu positive (group B), and of the latter, 130 were restored to Uu negative after treatment (group B1) and the other 7 remained unchanged (group B2). No significant differences were found between groups A and B in the rates of IVF fertilization (81.6% vs 79.8%, P = 0.13), abnormal fertilization (11.8% vs 12.4%, P = 0.58) and oocyte cleavage (92.0% vs 92.1%, P = 0.94), nor between groups A and B2 (81.6% vs 89.8%, P = 0.10; 11.8% vs 13.2%, P = 0.75; 92.0% vs 92.5%, P = 0.10). Totally, 747 of the patients underwent embryo transfer, including 643 in group A and 104 in group B. There were no significant differences between groups A and B in the rates of clinical pregnancy (38.6% vs 34.7%, P = 0.44) and abortion (16.5% vs 22.2%, P = 0.39), nor between groups A and B2 (38.6% vs 33.3%, P = 0.79; 16.5% vs 0, P = 0.53).CONCLUSION:Uu infection in the male reproductive tract does not significantly affect the rates of IVF fertilization, oocyte cleavage, clinical pregnancy and abortion. However, more investigations with larger sample sizes of the cases restored from Uu positive to Uu negative are needed to lend further support to our findings.

目的:了解大连市西岗区孕妇缺铁性贫血的患病情况及相关因素,提出相应干预措施,降低孕产妇、围产儿死亡率及降低出生缺陷的发生率。方法:对5年中建立"孕产妇保健手册"的西岗区孕妇进行贫血调查,采静脉血检测血色素,并由专人负责登记、统计及采取相应干预措施。结果:共筛查孕妇6 858例,发生高危贫血515例,其发生率为7.5%;生理性贫血890例,发生率为12.98%;对早期、中期妊娠贫血进行铁剂治疗227例,用药2个月治愈221例,治愈率达97.36%;生理性贫血890例,其中补充铁剂一个疗程628例,未用药262例,一个月复查血色素发生高危贫血前者2例,后者182例,发生率分别为0.32%、69.47%。结论:发现孕妇贫血发生率较高,并随孕周增加发生率增高;以轻度贫血为主;治疗效果与贫血的程度相关;早期发现、早期治疗、定期复查及预防性补充铁剂,可降低孕妇贫血的发生率,提高其治愈率。

Objective: To evaluate the efficacy of intracytoplasmic sperm injection (ICSI) for rescuing of conventional in vitro fertilization (IVF) failure. Methods: Retrospective clinical study was involved in 11 women with non-male factor infertility undergoing conventional IVF in our center, 18 hours after conventional insemination failure, the oocytes were reinseminated by ICSI (late ICSI). Clinical pregnancy were identified 21 days after embryo transfer. Results 135 oocytes were got from 11 women (MII 107; MI 23; GV 5) and 2.8% (5/107) fertilized from conventional IVF. Late Intracytoplasmic sperm injection of 103 unfertilized oocytes resulted in 82 successful fertilization occurred in 105 (79.61%) of the survival oocytes and 61 cleave in 82 (74.39%). 25 embryos were transferred (include 3 embryos from conventional IVF) and no viable pregnancy resulted. Conclusion: Late ICSI, for a relatively successful means of rescuing conventional IVF cycles in which fertilization fails completely can increases the rate of fertilization and cleave but can't increase the rate of pregnancy. The further study will be focused on the relationship between low fertilization rate in conventional IVF cycle and low sperm motility.