Study On Endometrial Receptivity Of GnRH-ant Protocol By Single Cell Transcriptome Sequencing

Objective:To explore the application of antagonist GnRH-ant protocol in in vitro fertilization（IVF）and clinical pregnancy outcome in normal ovarian response population,and to further explore the mechanism of affecting the endometrial receptivity of antagonist GnRH-ant protocol.Methods:1.The clinical data of patients with normal ovarian response to in vitro fertilization and embryo transfer in the Reproductive Center of 901 Hospital of Joint Logistics support Force of Chinese people’s Liberation Army from May 2020 to May2021 were analyzed retrospectively.According to the standard of anovulation,301patients with antagonist GnRH-ant protocol were enrolled in the group,and 248patients with long follicular phase GnRH-ant protocol in the same period were selected as the control group.The ovulation induction process and clinical outcome of the two GnRH-ant protocols were compared.Multivariate logistic regression analysis was used to further analyze the clinical outcomes of the two groups.After correcting the confounding factors such as age,years of infertility,scheme,AMH,Gn days,HCG day P level,HCG day endometrial thickness and the number of embryos transferred,the above factors were used as independent variables for multivariate logistic regression analysis to explore the influencing factors of clinical outcome.two.Furthermore,the single cell transcriptional group sequencing technique was used to detect the difference of endometrial cell composition between the antagonist GnRH-ant protocol and the control group during the implantation window,and to further detect the differential gene expression of endometrial cells.the biological processes and affected biological pathways of differential genes were analyzed in order to explore the mechanism of antagonist GnRH-ant protocols affecting endometrial receptivity.Results:1.There was no significant difference in age,body mass index,years of infertility,anti-Mullerian hormone and type of infertility between the antagonist group and the control group（P>0.05）.The Gn dose（1850.08±721.02 vs 2347.55±820.24 U/L）,Gn time（9.73±1.97vs10.96±2.60 days）,number of eggs obtained（11.71±3.90vs12.53±4.13）and HCG day endometrial thickness（10.55±2.44 vs11.12±2.33 mm）in the antagonist GnRH-ant protocol were significantly lower than those in the control group.The daily E₂ level of HCG（2824.50±2432.05 vs 2786.15±1308.94pg/m L）and the daily P level of HCG（1.23±0.47 vs 1.14±0.34U/L）had no significant difference compared with the control group.The clinical pregnancy rate（49.83%vs 61.69%）,embryo implantation rate（35.82%vs 43.57%）and live birth rate（41.20%vs 53.23%）in the antagonist GnRH-ant protocol were significantly lower than those in the control group（P<0.05）.The 2PN fertilization rate（52.75%vs 54.35%）,the number of transferable embryos（4.57±2.47vs4.15±2.66）,the number of high quality embryos（4.27±2.08vs4.50±3.65）,the rate of high quality embryos（69.07%vs 66.04%）and the number of transplanted embryos in the antagonist group were not significantly different from those in the control group（P>0.05）.There was no significant difference in abortion rate（14.00%vs 13.73%）and ectopic pregnancy rate（3.33%vs 1.96%）between the two groups and the control group（P>0.05）.The results of multivariate logistic regression analysis showed that the GnRH-ant protocol was an independent factor affecting the clinical pregnancy rate,embryo implantation rate and live birth rate in IVFfresh embryo transfer cycle.two.Both the GnRH-ant protocol and control groups had 10 cell clusters in the endometrial implantation window,with a significantly higher proportion of CD16NK cells in the T/NK cell cluster subgroup compared to the control group（9.45%vs2.41%）in the GnRH-ant protocol,while the proportion of CD56NK cells was significantly lower than the control group（19.05%vs 32.05%）.The GO enrichment analysis of endometrial stromal cells showed that the upregulated differentially expressed genes were mainly related to the composition of the extracellular matrix,and the differentially expressed genes enriched in this function were mainly COL4A1and COL4A2.The enrichment analysis of KEGG pathway in endometrial epithelial cells showed that the upregulated differentially expressed genes are mainly involved in the extracellular matrix receptor signaling pathway,and the main genes involved in this pathway include TGFBI.Conclusions:1.In the population with normal ovarian response,the clinical pregnancy rate,embryo implantation rate and live birth rate of fresh embryo transfer in antagonist group were lower than those in GnRH-a protocol.2.GnRH-ant protocol is an independent factor affecting the clinical outcome of IVF fresh embryo transfer cycle.3.GO enrichment analysis of differentially expressed genes showed that differentially expressed genes were enriched in biological processes such as extracellular collagen synthesis and extracellular matrix formation.KEGG pathway analysis showed that the extracellular matrix receptor pathway was enriched,which was related to endometrial receptivity.