IntroductionProgestin can inhibit the pituitary luteinising hormone (LH) surge during ovarian stimulation for in vitro fertilisation (IVF) and studies show progestin-primed ovarian stimulation (PPOS) is effective in blocking the LH surge in IVF. More and more centres are using PPOS because this regimen appears simpler and cheaper. This study aims to compare the euploidy rate of blastocysts following the PPOS protocol and the gonadotropin-releasing hormone antagonist protocol in women undergoing preimplantation genetic testing for aneuploidy (PGT-A).Methods/analysisThis is a randomised trial. A total of 400 women undergoing PGT-A will be enrolled and randomised according to a computer-generated randomisation list to either (1) the antagonist group: an antagonist given once daily from day 6 of ovarian stimulation till the day of the ovulation trigger; or (2) the PPOS group: dydrogesterone from the first day of ovarian stimulation till the day of ovulation trigger. The primary outcome is the euploidy rate of blastocysts.Ethics/disseminationAn ethical approval was granted from the ethics committee of assisted reproductive medicine in Shanghai JiAi Genetics and IVF institute (JIAIE2020-03). A written informed consent will be obtained from each woman before any study procedure is performed, according to good clinical practice. The results of this randomised trial will be disseminated in a peer-reviewed journal.Trial registration numberNCT04414748.

BackgroundThe molecular mechanisms underlying window of implantation (WOI) displacement in patients with recurrent implantation failure (RIF) remain unclear. This study aims to explore the transcriptomic signatures of endometrium with normal and displaced WOIs and to identify the causes of endometrial receptivity (ER) abnormalities and WOI displacement in RIF patients.MethodsIn this study, 40 RIF patients were recruited and underwent personalized embryo transfer (pET) guided by the predicted results of endometrial receptivity diagnosis (ERD) model. Transcriptome analysis of endometrium from patients with clinical pregnancies after pET was performed to identify differentially expressed genes (DEGs) associated with WOI displacement. Gene expression data from HRT and natural cycle endometrium were compared to identify specific gene expression patterns of ER-related genes during WOI.ResultsThe ERD results indicated that 67.5% of RIF patients (27/40) were non-receptive in the conventional WOI (P+5) of the HRT cycle. The clinical pregnancy rate in RIF patients improved to 65% (26/40) after ERD-guided pET, indicating the effectiveness of transcriptome-based WOI prediction. Among the 26 patients with clinical pregnancy, the gene expression profiles of P+5 endometrium from advanced (n=6), normal (n=10) and delayed (n=10) WOI groups were significantly different from each other. Furthermore, 10 DEGs identified among P+5 endometrium of 3 groups were involved in immunomodulation, transmembrane transport and tissue regeneration, which could accurately classify the endometrium with different WOIs. Additionally, a large number of ER-related genes showed significant correlation and similar gene expression patterns in P+3, P+5, and P+7 endometrium from HRT cycles and LH+5, LH+7, and LH+9 endometrium from natural cycles.ConclusionOur study shows that ER-related genes share similar gene expression patterns during WOI in both natural and HRT cycles, and their aberrant expression is associated with WOI displacements. The improvement of pregnancy outcomes in RIF patients by adjusting ET timing according to ERD results demonstrates the importance of transcriptome-based endometrial receptivity assessment and the clinical efficiency of ERD model.

Introduction In recent years, the use of frozen embryo transfers (FET) has rapidly increased following the freeze-all strategy due to the advantages of increased maternal safety, improved pregnancy rates, lower ectopic pregnancy rates and better obstetric and neonatal outcomes. Currently, there is still no good scientific evidence to support when to perform FET following a stimulated in vitro fertilisation (IVF) cycle in the freeze-all strategy.Methods/analysis This will be a randomised controlled trial. A total of 828 women undergoing their first FET following their first stimulated IVF cycle in the freeze-all strategy will be enrolled and randomised into one of the following groups according to a computer-generated randomisation list: (1) the immediate group, in which FET will be performed in the first menstrual cycle following the stimulated IVF cycle; or (2) the delayed group, in which FET will be performed at least in the second menstrual cycle following the stimulated IVF cycle. The primary outcome will be live birth, which is defined as the delivery of any infants at ≥22 gestational weeks with heartbeat and breath.Ethics/dissemination Ethical approval was granted by the Ethics Committee of Assisted Reproductive Medicine at the Shanghai JiAi Genetics & IVF Institute (JIAI E2019-15). Written informed consent will be obtained from each woman before any study procedure is performed, according to good clinical practice. The results of this trial will be disseminated in a peer-reviewed journal.Trial registration number NCT04371783

Objective:To evaluate clinical effectiveness of endometrial preparation of letrozole stimulation protocol for frozen-thawed embryo transfer (FET) in patients with endometriosis.Methods:In this prospective, controlled randomized trail, a total of 143 endometriosis women who undergwent FET in Shanghai Ji Ai Genetics and IVF Institute from January 2019 to December 2020 were randomly assigned to two groups according to computer digital: 69 patients received letrozole stimulation protocol (letrozole group) and 74 patients received hormone replacement therapy with gonadotropin-releasing hormone agonist (GnRH-a-HRT group). Clinical data between two groups were compared and the primary outcome was clinical pregnancy rate.Results:There were no significant differences in the number of embryos transferred, the proportion of blastocysts transferred, high-quality embryo rate and duration of endometrial preparation (all P>0.05). Peak estradiol levels [(417.8±102.7) ng/L], endometrial thickness on transfer day [(10.2±1.6) mm] and drug cost [(3 259.2±134.3) yuan] in GnRH-a-HRT group were significantly higher than those in letrozole group [(188.5±44.8) ng/L, P<0.001; (8.9±1.4) mm, P=0.007; (356.9±79.9) yuan, P<0.001]. The numbers of ultrasound scan (3.8±0.9) and venous samplings (2.8±0.9) in letrozole group were more than those in GnRH-a-HRT group (2.4±0.4, P=0.041; 1.4±0.4, P=0.022). The implantation rate, the clinical pregnancy rate, the twin pregnancy rate and the ectopic pregnancy rate were similar in both groups (all P>0.05). No significant difference was found in the miscarriage rate although miscarriage rate in GnRH-a-HRT group seemed higher than that of letrozole group (all P>0.05). Conclusion:Endometrial preparation of letrozole stimulation protocol for FET in patients with endometriosis showed similar pregnancy outcomes when compared with GnRH-a-HRT protocol. The drug cost is more economical in letrozole stimulation protocol.

Background:Today, approximately 10% of participants in assisted reproductive technology (ART) are defined as having recurrent implantation failure (RIF). Recent studies show that endometrial receptivity array can improve pregnancy and implantation rates by nearly 20% in women with RIF. However, these studies are limited, with little published data in the Chinese population. Recently, we have established a transcriptome-based endometrial receptivity assessment (Tb-ERA) method of predicting the endometrial window of implantation (WOI) using transcriptome-profiling data of different phases of the menstrual cycle from healthy fertile Chinese women by RNA-Seq. It is meaningful to conduct a randomized controlled trial (RCT) to assess the clinical efficiency of Tb-ERA in Chinese patients with RIF.Methods:In this RCT, a total of 200 RIF patients will be recruited and randomized into 2 groups. Patients in the Tb-ERA group will undergo a Tb-ERA test, after which embryo transfer time will be adjusted according to Tb-ERA results and embryo transfer will be performed again in the next cycle. Patients in the control group will not receive any interventions until the next transfer cycle. We will perform statistical analysis on both groups at the primary endpoint (clinical-pregnancy rate) and at secondary endpoints (rate of WOI displacement, embryo implantation, biochemical pregnancy, early abortion, and ectopic pregnancy). Implications: This study aims to evaluate the effectiveness of our Tb-ERA test in Chinese RIF patients and to determine that whether Tb-ERA could improve the clinical-pregnancy rate in these RIF patients.Trial registration:NCT04497558, registered August 4, 2020.

Objective: To define the transcriptomic signature with respect to human endometrial receptivity in Chinese women by next-generation sequencing and to develop a more refined and customized bioinformatic predictive method for endometrial dating in Chinese women. Design: Randomized. Setting: A tertiary hospital-based reproductive medicine center. Patient(s): Ninety healthy, fertile Chinese women. Intervention(s): Human endometrial biopsies. Main Outcome Measure(s): Gene expression of endometrial biopsies. Result(s): Ninety endometrial samples from healthy Chinese women during their menstrual cycles-including prereceptive (luteinizing hormone [LH] + 3 days/LH + 5 days), receptive (LH + 7 days), and post-receptive (LH + 9 days) phases-were subjected to transcriptomic analysis using messenger RNA (mRNA)-enriched RNA-Seq. Feature genes were obtained and used to train the predictor for endometrial dating, with 63 samples for the training set and 27 samples for the validation set. Differentially expressed genes (DEGs) were identified by comparing samples from different phases of the menstrual cycle. Based on the transcriptomic feature genes, we constructed a bioinformatic predictor for endometrial dating. The accuracy on assessment of the endometrium on days LH + 3, LH + 5, LH + 7, and LH + 9 was 100% in the training set and 85.19% in the validation set. Conclusion(s): Our transcriptomic profiling method can be used to monitor the window of implantation with regard to the endometrium in the Chinese population. This method potentially provides an evaluation of endometrial status, and can be used to predict a personal window of implantation by reproductive medicine clinicians. ((C) 2020 by American Society for Reproductive Medicine.)

STUDY QUESTION:Is there any difference in the ongoing pregnancy rate after immediate versus delayed frozen embryo transfer (FET) following a stimulated IVF cycle? SUMMARY ANSWER:Immediate FET following a stimulated IVF cycle produced significantly higher ongoing pregnancy and live birth rate than did delayed FET. WHAT IS KNOWN ALREADY:Embryo cryopreservation is an increasingly important part of IVF, but there is still no good evidence to advise when to perform FET following a stimulated IVF cycle. All published studies are retrospective, and the findings are contradictory. STUDY DESIGN, SIZE, DURATION:This was a randomised controlled non-inferiority trial of 724 infertile women carried out in two fertility centres in China between 9 August 2017 and 5 December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS:Infertile women having their first FET cycle after a stimulated IVF cycle were randomly assigned to either (1) the immediate group in which FET was performed in the first menstrual cycle following the stimulated IVF cycle (n = 362) or (2) the delayed group in which FET was performed in the second or later menstrual cycle following the stimulated IVF cycle (n = 362). All FET cycles were performed in hormone replacement cycles. The randomisation sequence was generated using an online randomisation program with block sizes of four. The primary outcome was the ongoing pregnancy rate, defined as a viable pregnancy beyond 12 weeks of gestation. The non-inferiority margin was -10%. Analysis was performed by both per-protocol and intention-to-treat approaches. MAIN RESULTS AND THE ROLE OF CHANCE:Women in the immediate group were slightly younger than those in the delayed group (30.0 (27.7-33.5) versus 31.0 (28.5-34.2), respectively, P = 0.006), but the proportion of women ≤35 years was comparable between the two groups (308/362, 85.1% in the immediate group versus 303/362, 83.7% in the delayed group). The ongoing pregnancy rate was 49.6% (171/345) in the immediate group and 41.5% (142/342) in the delayed group (odds ratios 0.72, 95% CI 0.53-0.98, P = 0.034). The live birth rate was 47.2% (163/345) in the immediate group and 37.7% (129/342) in the delayed group (odds ratios 0.68, 95% CI 0.50-0.92, P = 0.012). The miscarriage rate was 13.2% (26 of 197 women) in the immediate group and 24.2% (43 of 178 women) in the delayed group (odds ratios 2.10; 95% CI 1.23-3.58, P = 0.006). The multivariable logistic regression, which adjusted for potential confounding factors including maternal age, number of oocytes retrieved, embryo stage at transfer, number of transferred embryos/blastocysts, reasons for FET, ovarian stimulation protocol and trigger type, demonstrated that the ongoing pregnancy rate was still higher in the immediate group. LIMITATIONS, REASON FOR CAUTION:Despite randomisation, the two groups still differed slightly in the age of the women at IVF. The study was powered to consider the ongoing pregnancy rate, but the live birth rate may be of greater clinical interest. Conclusions relating to the observed differences between the treatment groups in terms of live birth rate should, therefore, be made with caution. WIDER IMPLICATIONS OF THE FINDINGS:Immediate FET following a stimulated IVF cycle had a significantly higher ongoing pregnancy and live birth rate than delayed FET. The findings of this study support immediate FET after a stimulated IVF cycle. STUDY FUNDING/COMPETING INTEREST(S):No external funding was used and no competing interests were declared. TRIAL REGISTRATION NUMBER:ClinicalTials.gov identifier: NCT03201783. TRIAL REGISTRATION DATE:28 June 2017. DATE OF FIRST PATIENT’S ENROLMENT:9 August 2017.

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.

Bone marrow-derived cells engraft to the uterine endometrium and contribute to endometriosis. This study sought to further confirm that estrogen can promote the migration of bone marrow mesenchymal stem cells (BMSCs) and to investigate the function of estrogen on the secretion of chemokines during BMSC migration. BMSCs were treated with or without 17β-estradiol, cultured with or without endometrial stromal cells (ESCs), or pretreated with or without AMD 3100 (an antagonist of the SDF-1α receptor) before co-culture. A migration assay was used to investigate the changes in the migration of BMSCs. The secretion of chemokines in the co-culture medium was detected by chemokine analysis, and the mRNA expression of SDF-1α in cells was tested using quantitative real-time PCR. The results revealed that the migration of BMSCs was promoted by ESC, and the migration ability of BMSCs was enhanced after treatment with 17β-estradiol (p < 0.05). Through chemokine analysis, we further showed that 17β-estradiol promoted the secretion of chemokines especially for SDF-1α (p < 0.05). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these chemokines were mainly linked to the cytokine signaling pathway and interaction with cytokines receptors. Furthermore, the expression of SDF-1α mRNA was significantly increased in the 17β-estradiol treatment group (p < 0.001), and the migration of BMSCs was blocked by the use of our SDF-1α antagonist (p < 0.01). Our results indicate that 17β-estradiol could promote the chemotaxis and migration of BMSCs by up-regulating the secretion of chemokines, especially SDF-1α. Our study provides additional evidence to support and supplement the stem cell theory of endometriosis.

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.

Objective: To investigate the clinical outcome of five different controlled ovarian hyperstimulation (COH) protocols including long,short,antagonist,clomiphene (CC) protocols and natural cycle for the women with advanced age undergoing IVF-ET.Methods: The laboratory and clinical outcomes data of 5 031 cycles including women aged ≥35 years undergone IVF-ET or ICSI with five different COH protocols in our center from 2014 to 2015 were retrospectively analyzed. The patients were divided into three groups according to different ages: 35-39 years,40-42 years,>42 years. The gonadotropin (Gn) dosage,duration,number of oocytes retrieved,MⅡoocytes,number of available embryos,normal fertilization rate,available embryo rate,cycle cancellation rate,clinical pregnant rate in fresh and frozen cycle,early miscarriage rate were compared among the groups.Results: There was no significant difference in the basic condition in the patients of different age with various kinds of COH protocol (P>0.05),expect that the basal FSH and anti-Mullerian hormone (AMH) levels in the natural cycle group was significantly lower than those in the other groups (P<0.05). Among the three different age group,Gn duration was the longest in long protocol group,and Gn dosage was the least in CC protocol (P<0.05). Meanwhile,the cycle cancellation rate in natural cycle was significantly higher than that in other groups (P<0.05). The clinical pregnant rate in short protocol and natural cycle (33.70% and 27.45%) was significantly lower than the other groups in 35-39 years group (P<0.05). The clinical pregnant rate in antagonist protocol and the natural cycle (18.95% and 13.33%) was significantly lower than that of the other groups in 40-42 years group (P<0.05). In the more than 42 years group,there was no significant difference in clinical pregnant rate among five protocols (P>0.05). There was no difference in early abortion rate among the different groups (P>0.05). Conclusions: Clomiphene protocol is worth to recommend for the advanced patients due to its lower dosage and less duration of Gn,and similar clinical pregnancy rate and early abortion rate as long protocol.

Objective:To compare the clinical effect between gonadotrophin-releasing hormone (GnRH) antagonist protocol and GnRH agonist long protocol in patients obtaining fresh embryo transfer.Methods:Clinical data of patients in the fresh embryo transfer cycle were analyzed retrospectively.Main clinical outcomes including superovulation and pregnant rates were compared.Results:A total of 706 cases were selected, 637 patients received GnRH agonist long protocol, and 69 patients received antagonist protocol.Compared with agonist group, the patients in antagonist group had significantly shorter duration of gonadotropin ([10.55±1.38] d vs [11.20±1.84] d), fewer doses ([1 699.50±611.25] U vs [2 196.75±765.75] U, P<0.05), more oocytes (12.11±5.20 vs 9.69±3.86, P<0.05), and more MⅡoocytes (10.15±4.65 vs 8.26±3.50, P<0.05).However, there was no significant difference in thickness of endometrium, fertilization rate, implantation rate, and pregnant rate between the two groups.Conclusions:Compared with the long GnRH agonist protocol, the GnRH antagonist protocol can bring out a substantial increase in the number of good quality embryos among the patients receiving fresh embryo transfer, but shows no advantage in pregnant rate.

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.

Background Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.).