OBJECTIVE:To test the hypothesis that women assigned to a natural ovulation regimen before frozen embryo transfer compared with a programmed regimen would have an increased chance of a healthy live birth and a reduced risk of pre-eclampsia or eclampsia. DESIGN:Multicentre, randomised, parallel group, assessor blinded clinical trial. SETTING:24 academic fertility centres in China. PARTICIPANTS:4376 ovulatory women (aged 20-40 years) planning to undergo a frozen single blastocyst transfer. INTERVENTIONS:Eligible participants were randomised (1:1) to receive a natural ovulation regimen or a programmed regimen of hormone replacement for endometrial preparation. Endometrial preparation and frozen embryo transfer timing were determined in the natural ovulation regimen group by monitoring natural follicle development and measuring serum levels of luteinising hormone, oestradiol, and progesterone. In the programmed regimen group, endometrial preparation was achieved by sequential administration of oestrogen and progesterone. MAIN OUTCOMES AND MEASURES:Primary outcomes were a healthy live birth and pre-eclampsia or eclampsia after a frozen embryo transfer. Secondary outcomes were cycle cancellation, biochemical pregnancy, clinical pregnancy, ongoing pregnancy, pregnancy loss, ectopic pregnancy, live birth, birth weight, and maternal, fetal, and neonatal complications. RESULTS:In the intention-to-treat analyses, 910 (41.6%) of 2185 patients in the natural ovulation regimen group and 890 (40.6%) of 2191 in the programmed regimen group achieved a healthy live birth (relative ratio 1.03 (95% confidence interval (CI) 0.96 to 1.10); P=0.49). The risk of pre-eclampsia was lower in the natural ovulation regimen group among patients who achieved clinical pregnancy than in the programmed regimen group (2.9% (38 of 1302) v 4.6% (61 of 1326); 0.63 (0.43 to 0.94); P=0.02). The incidences of early pregnancy loss (12.1% (158 of 1302) v 15.2% (201 of 1326); 0.80 (0.66 to 0.97)), placental accreta spectrum (1.8% (24 of 1302) v 3.6% (48 of 1326); 0.51 (0.31 to 0.83)), caesarean section (69.5% (776 of 1117) v 75.6% (831 of 1100); 0.92 (0.87 to 0.97)), and postpartum haemorrhage (2.0% (22 of 1117) v 6.1% (67 of 1100); 0.32 (0.20 to 0.52)) were lower in the natural ovulation regimen group. No differences between groups were observed for birth weight or neonatal complications. The rate of cycle cancellation was higher in the natural ovulation regimen (16.2% (354 of 2185) v 11.5% (251 of 2191), P<0.001). The prespecified per protocol and subgroup analyses yielded results consistent with the intention-to-treat analyses. CONCLUSIONS:In ovulatory women, a natural ovulation regimen for endometrial preparation was as effective as programmed regimen in terms of achieving a healthy live birth after frozen embryo transfer, but with a lower risk of maternal complications during pregnancy. TRIAL REGISTRATION:Chinese Clinical Trial Registry ChiCTR2200057990.

Adenomyosis (AM) is a common gynecological disorder in reproductive-age women, with impaired endometrial receptivity as a key contributor to infertility. However, a comprehensive single-cell atlas of the eutopic endometrium during the implantation window remains lacking, particularly regarding the roles of specific subpopulations in shaping the implantation microenvironment. This gap has limited mechanistic insights into AM-associated implantation failure. We performed 10x Genomics single-cell RNA sequencing of mid-secretory eutopic endometrium from three primary infertile AM patients and three parous controls, validated by qRT-PCR, immunohistochemistry, and immunofluorescence. AM-derived cells accounted for a higher proportion of epithelial, perivascular, and proliferative populations, whereas stromal and immune cell fractions remained largely unchanged. Notably, an expanded epithelial subpopulation characterized by the stem cell markers LGR5 and SOX9 exhibited excessive proliferative activity and strengthened tight junctions, collectively disrupting the establishment of a receptive epithelial phenotype. In the stroma, a DIO2+ decidual subpopulation with senescence-associated secretory phenotype (SASP) features was markedly reduced in AM, accompanied by downregulation of inflammatory mediators and key decidualization markers, indicative of impaired decidualization. Cell–cell communication analysis revealed marked remodeling of epithelial–stromal crosstalk in AM. Hyperactive epithelial PTN and loss of ncWNT could be linked to aberrant proliferative activity and polarity alterations, while reduced epithelial DKK1 and IGF2 signaling, together with stromal ncWNT loss, may partially explain deficient decidualization. In summary, our findings indicate that excessive proliferation and impaired differentiation of epithelial cells, along with insufficient inflammatory activation and decidualization defects in stromal cells, jointly underlie reduced endometrial receptivity in AM. Moreover, altered epithelial signaling may further compromise stromal decidualization.

Age-related diminished ovarian reserve (DOR) leads to declining fertility, miscarriage, and systemic health issues. Cell-free fat extract (CEFFE) has demonstrated therapeutic effects in various tissues. In our previous study, CEFFE successfully improved ovarian function in mice without adverse effects; however whether it can prevent DOR remains unclear. This study aimed to determine if early intervention with CEFFE can delay DOR and extend reproductive lifespan, exploring its potential as a novel clinical approach for women with DOR. The mice in the Prophylactic group received tail vein injections of 200 μL of CEFFE per mouse every 3 days for three months; the Control group were administered an equivalent volume of saline injections. Post-treatment outcomes assessed included body weight, ovarian weight, follicle count, embryo quality, production rates, and levels of serum hormones. Safety was evaluated via organ weights and hematoxylin and eosin staining in parents and offspring. The impact of CEFFE on cell proliferation, hormone synthesis, oxidative stress, and senescence was assessed via Cell counting Kit-8 assays, enzyme-linked immunosorbent assays, and reverse transcription quantitative real-time PCR, 1,1′,3,3′-tetraethyl-5,5′,6,6′-tetrachloroimidacarbocyanine iodide and Senescence-associated beta-galactosidase staining. Compared with the Control group, CEFFE treatment effectively preserved ovarian function in aging mice, improving ovarian weight, hormone levels, and follicular development, and reduced follicular atresia. CEFFE treatment enhanced embryo quality and development, induced a higher pregnancy rate, reduced the number of abnormal pregnancies, and increased the litter size and the number of live births. CEFFE demonstrated favorable safety in the Prophylactic group and their offspring, with no significant adverse effects on organ morphology or coefficients, except for increased liver weight in treated mice. Transcriptomic analysis suggested CEFFE influences cell proliferation, hormone response, and oxidative stress pathways in ovarian granulosa cells, which was verified in primary mouse granulosa cells. In vitro studies demonstrated that CEFFE pre-treatment alleviated the phenotype of Control mice by inhibiting oxidative stress, promoting proliferation, and enhancing hormone secretion. Early prophylactic administration of CEFFE in adult mice can prevent DOR and extend their reproductive lifespan by inhibiting granulosa cell senescence.

Reduced lymphoid enhancer-binding factor 1 (LEF1) expression in patients with adenomyosis during the mid-secretory phase leads to impaired endometrial receptivity, affecting embryo implantation. This study investigated the molecular mechanisms underlying reduced endometrial receptivity in 25 adenomyosis patients and 25 controls. Functional experiments were conducted using human endometrial stromal cells (HESCs) and TERT-immortalized HESCs(T-HESCs), with final validation performed using a mouse model. Western blot and quantitative real-time polymerase chain reaction (RT-qPCR) analyses revealed that patients with adenomyosis showed a marked decrease in LEF1 expression in the stromal cells of the endometrium during the mid-secretory phase. In vitro experiments demonstrated that LEF1 knockdown in stromal cells led to impaired decidualization. Transcriptome sequencing, dual-luciferase reporter assays, and chromatin immunoprecipitation (ChIP) experiments showed that LEF1 could bind to the promoter region of interleukin (IL)-11 and promote its transcription, and IL-11 expression was also found to be downregulated in adenomyosis patients. Overexpression of IL-11 rescued the impaired decidualization caused by decreased LEF1 expression. In the in vitro co-culture model, LEF1/IL-11 knockdown led to a reduction in embryo implantation area, which was partially restored upon IL-11 overexpression. In the adenomyosis mouse model, we observed a decrease in LEF1 expression and a reduction in implantation sites compared to control mice, accompanied by impaired decidualization and receptivity. Notably, supplementation with IL-11 restored the number of implantation sites. The decrease in fertility due to reduced endometrial receptivity in adenomyosis patients is a significant clinical issue in assisted reproductive technology. This research provides insights into one potential molecular mechanism underlying this decreased receptivity, with a specific focus on the reduced expression of LEF1 in the endometrial stromal cells during the mid-secretory phase in adenomyosis patients. Our findings offer new perspectives for clinical strategies to improve endometrial receptivity in patients with adenomyosis, potentially enhancing their chances of successful pregnancy.

Objective To test the hypothesis that a freeze-all strategy would increase the chance of live birth compared with fresh embryo transfer in women with low prognosis for in vitro fertilisation (IVF) treatment. Design Pragmatic, multicentre, randomised controlled trial. Setting Nine academic fertility centres in China. Participants 838 women with a low prognosis for IVF treatment defined by ≤9 oocytes retrieved or poor ovarian reserve (antral follicle count <5 or serum anti-Müllerian hormone level <8.6 pmol/L). Interventions Eligible participants were randomised (1:1) to undergo either frozen embryo transfer or fresh embryo transfer on the day of oocyte retrieval. Participants in the frozen embryo transfer group had all of their embryos cryopreserved and underwent frozen embryo transfer later. Participants in the fresh embryo transfer group underwent fresh embryo transfer after oocyte retrieval. Main outcome measures The primary outcome was live birth, defined as the delivery of neonates with a heartbeat and respiration at ≥28 weeks’ gestation. Secondary outcomes were clinical pregnancy, singleton or twin pregnancy, pregnancy loss, ectopic pregnancy, birth weight, maternal and neonatal complications, and cumulative live birth after embryo transfers within one year after randomisation. Results In an intention-to-treat analysis, the rate of live birth was lower in the frozen embryo transfer group than in the fresh embryo transfer group (32% (132 of 419) v 40% (168 of 419); relative ratio 0.79 (95% confidence interval 0.65 to 0.94); P=0.009). The frozen embryo group had a lower rate of clinical pregnancy than the fresh embryo group (39% (164 of 419) v 47% (197 of 419); 0.83 (0.71 to 0.97)). The cumulative live birth rate was lower in the frozen embryo transfer group compared with the fresh embryo transfer group (44% (185 of 419) v 51% (215 of 419), 0.86 (0.75 to 0.99)). No difference was observed in birth weight, incidence of obstetric complications, or risk of neonatal morbidities. Conclusions Fresh embryo transfer may be a better choice for women with low prognosis in terms of live birth rate compared with a freeze-all strategy. The treatment strategies that prevent fresh embryo transfers, such as accumulating embryos with back-to-back cycles or performing routine preimplantation genetic testing for aneuploidy, warrant further studies in women with a low prognosis. Trial registration Chinese Clinical Trial Registry ChiCTR2100050168.

STUDY QUESTION:How does allograft inflammatory factor-1 (AIF-1) affect endometrial receptivity in women with recurrent implantation failure (RIF)? SUMMARY ANSWER:Significant upregulation of AIF-1 in the endometrial stromal cells of women with RIF inhibits cell proliferation and decidualization via the p38 mitogen-activated protein kinase (MAPK) phosphorylation, thereby reducing endometrial receptivity. WHAT IS KNOWN ALREADY:RIF is a challenging clinical issue, with AIF-1, a cytokine-inducible protein linked to allograft rejection, potentially contributing to its pathogenesis. However, the precise mechanisms remain elusive. STUDY DESIGN, SIZE, DURATION:This study analyzed endometrial tissue samples from women diagnosed with RIF and a control group of fertile women from December 2018 to December 2023. Single-cell RNA sequencing (scRNA-seq) data from public datasets (GSE111976, GSE250130, GSE183837) were integrated to characterize AIF-1 expression patterns in endometrium. Isolated human endometrial stromal cells (HESCs) from the human endometrium and an endometrial stromal cell line were used for in vitro analysis, and an in vivo mouse model with AIF-1 overexpression in the uterus was employed to evaluate implantation outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS:Mid-secretory endometrial samples were collected from the 18 patients with RIF and 18 control patients; endometrial samples from another five different phases during the menstrual cycle were collected from 30 additional control patients. Quantitative PCR, western blot, immunohistochemical and immunofluorescence analyses, and RNA sequencing were conducted to determine the expression levels of AIF-1 and various markers. Cell proliferation, decidualization, and trophoblast outgrowth were measured. AIF-1 overexpression and gene silencing were achieved by plasmid injection and short hairpin RNA, respectively. For in vivo experiments, CD-1 mice with intrauterine injection of an AIF-1 plasmid were used. Phosphorylation of p38 was inhibited by PD169316. MAIN RESULTS AND THE ROLE OF CHANCE:Based on scRNA-seq analysis and our own endometrial tissue detection, AIF-1 was significantly increased in HESCs in patients with RIF compared with their control group during the mid-secretory phase. AIF-1 overexpression resulted in reduced cell proliferation, inadequate cell decidualization, and diminished embryo outgrowth in in vitro experiments, and it reduced the number of embryo implantation sites in CD-1 mice; these effects were mitigated by PD169316, an inhibitor of p38 MAPK. LIMITATIONS, REASONS FOR CAUTION:Although the study establishes a link between increased AIF-1 expression in endometrial stromal cells and reduced endometrial receptivity, the role of AIF-1 in endometrial macrophages during embryo implantation remains unclear. WIDER IMPLICATIONS OF THE FINDINGS:The findings suggest that targeting the AIF-1 and p38 MAPK pathway could serve as a promising therapeutic strategy to improve endometrial receptivity in RIF patients. STUDY FUNDING/COMPETING INTEREST(S):This study was supported by the National Key R&D Program of China (No. 2022YFC2703800) and the National Natural Science Foundation of China [Nos. 82271703; 82371704; 82071596; 82071712; 82101800; 81701513]. The authors declare that they have no conflict of interests with the contents of this manuscript. TRIAL REGISTRATION NUMBER:N/A.

Setd2 is the only enzyme that catalyzes histone H3 lysine 36 trimethylation (H3K36me3) on virtually all actively transcribed protein-coding genes, and this mechanism is evolutionarily conserved from yeast to human. Despite this widespread and conserved activity, Setd2 and H3K36me3 are dispensable for normal growth of yeast but are absolutely required for mammalian embryogenesis, such as oocyte maturation and embryonic vasculogenesis in mice, raising a question of how the functional requirements of Setd2 in specific developmental stages have emerged through evolution. Here, we explored this issue by studying the essentiality and function of Setd2 in zebrafish. Surprisingly, the setd2-null zebrafish are viable and fertile. They show Mendelian birth ratio and normal embryogenesis without vascular defect as seen in mice; however, they have a small body size phenotype attributed to insufficient energy metabolism and protein synthesis, which is reversable in a nutrition-dependent manner. Unlike the sterile Setd2-null mice, the setd2-null zebrafish can produce functional sperms and oocytes. Nonetheless, related to the requirement of maternal Setd2 for oocyte maturation in mice, the second generation of setd2-null zebrafish that carry no maternal setd2 show decreased survival rate and a developmental delay at maternal-to-zygotic transition. Taken together, these results indicate that, while the phenotypes of the setd2-null zebrafish and mice are apparently different, they are matched in parallel as the underlying mechanisms are evolutionarily conserved. Thus, the differential requirements of Setd2 may reflect distinct viability thresholds that associate with intrinsic and/or extrinsic stresses experienced by the organism through development, and these epigenetic regulatory mechanisms may serve as a reserved source supporting the evolution of life from simplicity to complexity.

Preimplantation embryos in vivo are exposed to various growth factors in the female reproductive tract that are absent in in vitro embryo culture media. Cell-free fat extract exerts antioxidant, anti-ageing, and ovarian function-promoting effects. However, its effects on embryo quality are yet to be investigated. We assessed the effect of cell-free fat extract supplementation on embryo culture using a naturally ageing mouse model. We assessed the model’s efficacy in influencing embryo development and pregnancy rates in older women with in vitro fertilization failure. In addition, we performed immunofluorescence staining, multiplex immunoassay, whole-genome amplification and DNA sequencing, time-lapse embryo monitoring, and in vitro experiments. Cell-free fat extract-supplemented media has a suitable osmolarity and pH and contains high levels of bioactive growth factors. Cell-free fat extract promoted embryo development and implantation in aged mice, probably by increasing embryo growth rate, inhibiting cell apoptosis, and promoting blastocyst adhesion. Clinical results showed that the cell-free fat extract group had significantly higher rates of the day 3 available and high-quality embryos than the control group, and the rate of usable embryos tended to be higher in the cell-free fat extract group. Furthermore, implantation and clinical pregnancy rates improved in the cell-free fat extract group than in the control group. Our study implies that cell-free fat extract supplementation can promote embryo development and clinical outcomes and may serve as a rescue strategy for older women with in vitro fertilization failure.

Embryo implantation requires precise endometrial receptivity, which is critically dependent on progesterone-induced stromal decidualization during the mid-secretory phase. In recurrent implantation failure (RIF), this process is impaired, yet the underlying mechanisms remain poorly understood. Here, by integrating bulk and single-cell transcriptomic data, we found that secretoglobin, family 2A, member 1 (SCGB2A1) was dynamically expressed in the human endometrium, with peak expression in the stromal compartment during the mid-secretory phase. This expression pattern was validated in samples from fertile control women, whereas in individuals with RIF, SCGB2A1 was significantly downregulated in both the endometrial stroma and uterine fluid during this critical window for decidualization. Functionally, SCGB2A1 was transcriptionally regulated by the progesterone signaling axis, and its knockdown in endometrial stromal cells impaired decidual marker expression and cytoskeletal remodeling. Mechanistically, we reported for the first time that SCGB2A1 physically interacted with protein kinase B (AKT), as demonstrated by RNA-seq, proteomic analyses, and co-immunoprecipitation assays. Loss of SCGB2A1 disrupts this interaction, resulting in aberrant AKT activation, increased AKT/forkhead box O1 (FOXO1) phosphorylation, and impaired FOXO1 nuclear translocation, ultimately suppressing decidual marker expression. Notably, treatment with an AKT inhibitor partially rescued the decidualization defects caused by SCGB2A1 deficiency. These findings reveal a previously unrecognized molecular defect in RIF, implying that SCGB2A1 may contribute to progesterone-mediated decidualization and could serve as a potential marker for assessing endometrial receptivity.

Endometrial hyperplasia (EH), a precursor lesion to endometrial carcinoma, poses a significant challenge to women's health. However, current EH treatment involves a critical bottleneck because the rigid structures of conventional levonorgestrel (LNG)-releasing intrauterine system leads to poor adaptability. Here, we designed a novel self-expanding injectable intrauterine scaffold by first constructing a three-dimensional porous biomimetic extracellular matrix network via electrospinning. The elastic network was subsequently optimized through the thermal crosslinking-condensation reaction of polylactic acid and gelatin. By regulating the proportion of short nanofibers, we refined the microporous scaffold structure, enabling rapid self-expansion upon exposure to uterine fluid after injection and dynamic fit to the uterine cavity. The resulting scaffold efficiently loaded LNG through physical binding and exhibited excellent biocompatibility. In particular, it exerted therapeutic effects through a tripartite synergistic mechanism: suppressing excessive endometrial cell proliferation, promoting apoptosis in abnormal endometrial cells, and inhibiting pathological angiogenesis. In EH rat models, the scaffold effectively remodeled uterus morphology-reducing uterine wet weight from 0.91 ± 0.21 to 0.59 ± 0.11 g and restoring endometrial thickness from 558.2 ± 58.04 to 463.50 ± 61.01 μm, approaching the normal thickness of 420.4 ± 40.04 μm. It also significantly improved pathological features, with glandular density decreased from 22.97 ± 4.43 to 14.22 ± 4.27 per high power field (HP), close to the normal count of 14.28 ± 2.52 per HP. Our intelligent scaffold, with its injectability, self-expanding adaptability, and biomimetic therapeutic functions, may resolve the clinical limitations of conventional rigid intrauterine materials, enabling effective EH treatment.

Background: Among the POSEIDON criteria, group 3 and group 4 have an expected low prognosis. For those patients with inadequate ovary reserve, embryo accumulated from consecutive oocyte retrieval cycles for multiple frozen-thawed embryo transfers (FET) has become more common. It is necessary to inform them of the pregnancy outcomes after single or multiple FET cycles before the treatment. However few studies about cumulative live birth rate (CLBR) for those with low prognosis have been reported. Methods: This retrospective study included 4712 patients undergoing frozen embryo transfer cycles from July 2015 to August 2020. Patients were stratified as POSEIDON group 3, group 4, control 1 group (< 35 years) and control 2 group (≥35 years). The primary outcome is CLBRs up to six FET cycles and the secondary outcomes were LBRs per transfer cycle. Optimistic approach was used for the analysis of CLBRs and the depiction of cumulative incidence curves. Results: Under optimistic model analyses, control 1 group exhibited the highest CLBR (93.98%, 95%CI 91.63%-95.67%) within 6 FET cycles, followed by the CLBR from women in POSEIDON group 3（92.51%，95%CI 77.1-97.55）was slightly lower than that in control 1 group. The CLBR of POSEIDON group 4（55% ,95%CI 39.34%-70.66%）was the lowest and significantly lower than that of control 2 group（88.7%, 95%CI 80.68%-96.72%）. Further, patients in POSEIDON group 4 reached a CLBR plateau after 5 FET cycles. Conclusions: The patients of POSEIDON group 3 may not be considered as traditional “low prognosis” in clinical practice as extending the number of FET cycles up to 6 can archive considerably CLBR as control women. While for the POSEIDON group 4, a simple repeat of the FET cycle is not recommended after four failed FET cycles, some strategies such as PGT-A may be beneficial.

Background The gonadotrophin-releasing hormone antagonist (GnRH-ant) protocol in controlled ovarian stimulation (COS) has distinct advantages and become widespread. However, the dose-dependent disturbance of GnRH-ant on endometrial immune factors may negatively impact endometrial receptivity, potentially contributing to lower clinical pregnancy rates in fresh embryo transfer cycles, thereby diminishing the benefits of this protocol. Identifying strategies to mitigate these adverse effects on the endometrium is crucial for improving pregnancy outcomes in fresh embryo transfer cycles following this COS protocol. Prednisone, a primary immunosuppressive agent, has been proposed as a means to counteract the negative effects of GnRH-ant on the endometrium. This study designed a multi-center randomized clinical trial to compare the efficacy of combining prednisone with a fixed full-dose GnRH-ant protocol versus a flexible half-dose GnRH-ant protocol and a conventional fixed full-dose GnRH-ant protocol. Methods and Findings We conducted a randomized, controlled, open-label clinical trial across three reproductive centers in China from April 2019 to November 2022, with follow-up completed in August 2023. Of the 5,042 identified patients aged 20 to 35 years undergoing in vitro fertilization and embryo transfer (IVF-ET) with the GnRH-ant protocol, 2,052 patients without contraindications for fresh embryo transfer were enrolled. Participants were randomly assigned to three groups: Group A received a fixed full-dose GnRH-ant combined with prednisone 10 mg per day from the start day of stimulation until 11 to 14 days after embryo transfer; Group B received a flexible half-dose GnRH-ant; and Group C received a fixed full-dose GnRH-ant. The primary outcome, clinical pregnancy rate (CPR), was defined as the ultrasound confirmation of an intrauterine gestation sac 30 to 35 days after embryo transfer, divided by the number of cases that underwent transfer. Of the enrolled patients, 1,512 (73.7%) underwent embryo transfer. Group A demonstrated a significantly higher CPR (63.1%) compared to Group B (54.7%; rate difference (RD) 8.4%, 95% confidence interval (CI) 2.4%–14.5%, P = 0.007) and Group C (46.4%; RD 16.7%, 95% CI 10.7%–22.7%, P < 0.001). The cancellation rate of fresh embryo transfer in Group A (18.7%) was similar to that in Group C (19.9%), but significantly lower than in Group B (24.1%). No significant differences in embryo laboratory results or in adverse events were observed among the groups. Conclusions In patients undergoing IVF with the GnRH-ant protocol, the addition of low dose prednisone significantly improved the CPR without increasing adverse effects. These findings suggest an optimal strategy to enhance the success of the antagonist protocol in IVF-ET. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial ChiCTR1900021024 ### Clinical Protocols ### Funding Statement Yes ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ruijin Hospital Ethics Committee, Shanghai JiaoTong University School of Medicine I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All relevant data are within the manuscript and its Supporting Information files.

Abnormal interaction between granulosa cells and oocytes causes disordered development of ovarian follicles. However, the interactions between oocytes and cumulus granulosa cells (CGs), oocytes and mural granulosa cells (MGs), and CGs and MGs remain to be fully explored. Using single-cell RNA-sequencing (scRNA-seq), we determined the transcriptional profiles of oocytes, CGs and MGs in antral follicles. Analysis of scRNA-seq data revealed that CGs may regulate follicular development through the BMP15-KITL-KIT-PI3K-ARF6 pathway with elevated expression of luteinizing hormone receptor (LHR). Because internalization of the LHR is regulated by Arf6, we constructed LHRN316S mice by CRISPR/Cas9 to further explore mechanisms of follicular development and novel treatment strategies for female infertility. Ovaries of LHRN316S mice exhibited reduced numbers of corpora lutea and ovulation. The LHRN316S mice had a reduced rate of oocyte maturation in vitro and decreased serum progesterone levels. Mating LHRN316S female mice with ICR wild type male mice revealed that the infertility rate of LHRN316S mice was 21.4% (3/14). Litter sizes from LHRN316S mice were smaller than those from control wild type female mice. The oocytes from LHRN316S mice had an increased rate of maturation in vitro after progesterone administration in vitro. Furthermore, progesterone treated LHRN316S mice produced offspring numbers per litter equivalent to WT mice. These findings provide key insights into cellular interactions in ovarian follicles and provide important clues for infertility treatment.

Poor endometrial receptivity results in embryo implantation failure. Acquisition of endometrial receptivity involves substantial structural alterations in the cytoskeleton and plasma membrane of epithelial cells, which facilitate embryo adhesion. However, the underlying molecular mechanism remains largely unknown. In this study, we identified that α-actinin-1 (ACTN1) was significantly downregulated in the mid-secretory phase of the endometrium compared with other phases; however, ACTN1 significantly increased in women with recurrent implantation failure (RIF). In Ishikawa and human endometrial epithelial cells (HEECs), ACTN1 overexpression significantly decreased NEBL levels, enhanced F-actin fiber levels, and caused a notable impairment in blastocyst adhesion, which mimicked the process of embryo adhesion. However, NEBL overexpression notably restored adhesion. Moreover, NEBL expression was reduced in patients with RIF compared with that in controls. Finally, our data showed that ACTN1 upregulation impaired endometrial receptivity in women with RIF, possibly by regulating NEBL expression and subsequent cell-adhesion capability.

Asthenoteratospermia is a significant cause of male infertility. FAM71D (Family with sequence similarity 71, member D), as a novel protein exclusively expressed in the testis, has been found to be associated with sperm motility. However, the association of FAM71D mutation with male infertility has yet to be examined. Here, we conducted whole-exome sequencing and identified a homozygous missense mutation c.440G > A (p. Arg147Gln) of FAM71D in an asthenoteratospermia-affected man from a consanguineous family. The FAM71D variant is extremely rare in human population genome databases and predicted to be deleterious by multiple bioinformatics tools. Semen analysis indicated decreased sperm motility and obvious morphological abnormalities in sperm cells from the FAM71D-deficient man. Immunofluorescence assays revealed that the identified FAM71D mutation had an important influence on the assembly of sperm structure-related proteins. Furthermore, intra-cytoplasmic sperm injection (ICSI) treatment performed on the infertile man with FAM71D variant achieved a satisfactory outcome. Overall, our study identified FAM71D as a novel causative gene for male infertility with asthenoteratospermia, for which ICSI treatment may be suggested to acquire good prognosis. All these findings will provide effective guidance for genetic counselling and assisted reproduction treatments of asthenoteratospermia-affected subjects.

Abstract Background Significant lower genital tract (LGT) dysbiosis and an associated lower rate of clinical pregnancy after in vitro fertilization–frozen embryo transfer (IVF-FET) among polycystic ovary syndrome (PCOS) patients have been previously reported by our group. We aimed to assess whether transvaginal Lactobacillus supplementation can reverse LGT dysbiosis and further improve perinatal outcomes in PCOS patients after IVF-FET. Methods/design This is a protocol for a multicenter, open-label, randomized controlled trial in China. Women diagnosed with PCOS who are undergoing IVF-FET treatment will be recruited. Allocation to the intervention/control arms at a ratio of 1:1 will be executed by an electronic randomization system. Participants in the intervention arm will receive the live Lactobacillus capsule vaginally for 10 consecutive days before embryo transfer, while those in the control arm will receive standard individualized care. The primary outcomes will be the clinical pregnancy rate, implantation rate, and live birth rate. 16S rRNA sequencing and liquid chromatography–mass spectrometry will be conducted to evaluate the LGT microbiome and systemic metabonomics before and after the intervention. A sample of 260 participants will provide 95% power to detect a 20% increase in the rate of clinical pregnancy (α = 0.025, one-tailed test, 15% dropout rate). A total of 300 participants will be recruited. Discussion This is the first large and multicenter randomized controlled trial aimed at assessing the efficacy of transvaginal Lactobacillus supplementation on restoring the LGT microbiome and improving perinatal outcomes in PCOS patients after IVF-FET. This pragmatic trial is promising for increasing the rates of clinical pregnancy and live birth in PCOS patients after IVF-FET. Ethics and dissemination Ethical review approval was obtained from the Medical Research Ethics Committees of the International Peace Maternity and Child Health Hospital of Shanghai Jiao Tong University (15 October 2020, GKLW 2020-29). To maximize dissemination, these findings will be reported in open access publications in journals with high impact, and oral and poster conference presentations will be performed. Trial registration ChiCTR ChiCTR2000036460. Registered on 13 September 2020, https://www.chictr.org.cn/showproj.html?proj=59549 .

BackgroundThe basal follicle stimulating hormone (FSH)/luteinizing hormone (LH) ratio is a useful predictor of ovarian response. In this study, we investigated whether the FSH/LH ratios during the entire controlled ovarian stimulation (COS) can be used as effective predictors of outcomes in women undergoing in vitro fertilization (IVF) treatment using the gonadotropin releasing hormone antagonist (GnRH-ant) protocol.MethodsA total of 1,681 women undergoing their first GnRH-ant protocol were enrolled in this retrospective cohort study. A Poisson regression model was used to analyze the association between the FSH/LH ratios during COS and embryological outcomes. Receiver operating characteristic analysis was performed to determine the optimal cutoff values for poor responders (≤ 5 oocytes) or poor reproductive potential (≤ 3 available embryos). A nomogram model was constructed to provide a tool for predicting the cycle outcomes of individual IVF treatments.ResultsThe FSH/LH ratios (at the basal day, stimulation day 6 (SD6) and trigger day) were significantly correlated with the embryological outcomes. The basal FSH/LH ratio was the most reliable predictor of poor responders with a cutoff value of 1.875 (area under the curve (AUC) = 72.3%, P < 0.05), or of poor reproductive potential with a cutoff value of 2.515 (AUC = 66.3%, P < 0.05). The SD6 FSH/LH ratio predicted poor reproductive potential with a cutoff value of 4.14 (AUC = 63.8%, P < 0.05). The trigger day FSH/LH ratio predicted poor responders with a cutoff value of 9.665 (AUC = 63.1%, P < 0.05). The basal FSH/LH ratio, combined with the SD6 and trigger day FSH/LH ratios, slightly increased these AUC values and improved the prediction sensitivity. The nomogram provides a reliable model with which to assess the risk of poor response or poor reproductive potential directly based on the combined indicators.ConclusionsFSH/LH ratios are useful predictors of poor ovarian response or reproductive potential throughout the entire COS with the GnRH antagonist protocol. Our findings also provide insights into the potential for LH supplementation and regimen adjustment during COS to achieve improved outcomes.

The damaged endometrium and the formation of fibrosis are key barriers to pregnancy and further lead to infertility. However, how to promote endometrium repair is always a challenge. Here, a bioactive injectable and self-healing hydrogel is developed by physically combination of thiolated polyethylene (PEG), Cu2+ and cell-free fat extract (CEFFE, CF) for endometrial regeneration and fertility. By inheriting the advantages of various active proteins contained in CEFFE, it could induce the overall repair of endometrial microenvironment for intrauterine adhesion (IUA). In vitro, CF@Cu-PEG reduces endometrial cell apoptosis by more than 50%, and increases angiogenesis by 92.8%. In the IUA mouse, injection of CF@Cu-PEG significantly reduces the rate of uterine hydrometra and prevents the formation of endometrial fibrosis. Remarkably, CF@Cu-PEG contributes to the repair of endometrial microstructure, especially increases the number of endometrial pinopodes, significantly improves endometrial receptivity, and increases the pregnancy rate of IUA mice from 7.14% to 66.67%. In summary, through the physically combination of CEFFE and Cu-PEG, the construction of loaded bioactive injectable hydrogel not only inhibits the IUA, but also induces the self-repair of endometrial cells in situ and improves fertility, providing a new strategy for IUA repair in clinical application.

Research question: What is the underlying mechanism of IVF and embryo transfer (IVF-ET) failure in patients with elevated peripheral blood natural killer cell (pNK) counts? Design: Patients undergoing IVF-ET cycles for tubal obstruction or pelvic adhesion (n = 486) were assigned to three groups: high (CD56(+)CD16(+)pNK >30% [n = 49]); medium (15< CD56(+)CD16(+)pNK <= 30% [n = 211]); and normal pNK groups (5 <= CD56(+)CD16(+)pNK <= 15% [n = 226]). Their general condition, previous pregnancy history and IVF outcomes were compared. Uterine fluid and endometrial tissue from patients in the high and normal pNK groups were collected during the mid-secretory phase and studied to elucidate the molecular mechanism underlying impaired endometrial receptivity. Results: The highest incidence of IVF-ET cycles (P < 0.0001) and biochemical pregnancy losses (P < 0.0001), and lowest implantation and clinical pregnancy rates (both P < 0.0001), were observed in patients with pNK over 30%. No significant difference was found in the number of previous miscarriages and rate of spontaneous miscarriage in IVF outcomes. Lower Septin11 (SEPT11) expression in the uterine fluid and endometrial epithelial cells (EEC), and higher endometrial IFN-gamma, was observed in patients with high pNK. Ishikawa cell and human endometrial epithelial cell (HEEC) adhesion was inhibited after SEPT11 knock-down. Elevated IFN-gamma decreased the SEPT11 protein levels in Ishikawa cells and HEECs. Conclusions: CD56(+)CD16(+)pNK above 30% may be a threshold for adverse IVF-ET outcomes. Low SEPT11 expression in EEC inhibits cell adhesion, which may cause impaired endometrial receptivity in patients with elevated pNK. The level of SEPT11 in mid-secretory uterine fluid could serve as a non-invasive marker to assess endometrial receptivity in these patients.