Effects Of Progesterone Levels In The Late Follicular Phase On The Receptivity Of Endometrium In Patients With Moderate Ovarian Responses

Objective: To retrospectively analyse the effects of high progesterone on outcomes of in vitro fertilization-embryo transfer in patients with different ovarian responses in the controlled ovarian hyperstimulation(COH).We explore the changes of endometrial histology, morphology and the expression of integrin ?v?3, LIF, IL-1 under high levels of progesterone in late follicular phase in patients with moderate ovarian responses.Using RNA-seq analysis, we have investigated endometrial biopsies obtained from the patients with moderate ovarian.The expression of selected genes was further validated by quantitative RT-PCR. By using stringent bioinformatic analysis of the date, we explore the possible mechanisms that might be critical for the establishment of the window of implantation in humans.In order to take appropriate measures to avoid elevated levels of progesterone based on ovarian responses,or we must provide a suitable strategy for patients after elevation of progesterone level.Methods: 1.Effects of high progesterone in late follicular phase on outcomes of IVF-ET in patients with different ovarian responses during COH.The date of 3841 cycles undergoing IVF-ET in our reproductive Center between January 2003 and December 2013 were retrospectively analyzed.According to the number of oocyte retrieved, this study was divided into the high ovarian response group(oocyte retrieval?20, 842 cycles), the moderate ovarian response group(5<oocyte retrieval<20, 2008 cycles), and the low ovarian response group(oocyte retrieval ?5, 991cycles).The P levels are divided into the following 8 intervals:<1.00, 1.00�1.25, 1.25�1.5, 1.5�1.75, 1.75�2.00, 2.00�2.25, 2.25�2.5, and >2.5 ng/ml in the patients.Clinical pregnancy rates were calculated for all P intervals.The cut-off values of serum P were determined based on odds ratio and 95% confidence interval obtained by comparison of clinical pregnancy rates between each P interval and the lowest P interval. According to the cut-off value, the clinical pregnancy rate, implantation rate and various clinical parameters were compared between the two groups in different ovarian responses. The factors of elevated progesterone level in patients with different ovarian responses were analyzed by logistic regression analysis.2.The effects of high progesterone in late follicular phase on endometrial histology, pinopodes and the expression of integrin ?v?3, LIF, IL-1 in patients with moderate ovarian responses during COH.This study collects endometrium of 29 patients with moderate ovarian responses who were under long agonist program treatments in IVF-ET cycles with cancelling of implantation and signed the informed consent during April 2014 to Dcember 2015. According to the progesterone level on the day of HCG administration, eighteen patients have high levels of serum progesterone and eleven patients have normal levels of serum progesterone.Endometrial samples were obtained from these patients in the 5 days after oocyte retrieval. Each endometrial tissue was divided into three pieces: one piece was put into the pre-configured 2% glutaraldehyde PBS fixative after rinsed with sterilesaline three times, one piece was placed into a 10% neutral formalin solution after rinsed with sterilesaline, the other one was immersed quickly into liquid nitrogen.Using HE staining to observe the endometrial histology change between the two groups. The change of endometrium pinocytosis sudden was observed by scanning electron microscope. The expression of integrin ?v?3, LIF and IL-1 were detected by immunohistochemical method and western blot method.3.Transcriptomic changes during the high progesterone in the late follicular in the endometrium of the patients with moderate ovarian responses detected by RNA-seq.This study collects endometrium of 12 patients with moderate ovarian responses who were under long agonist program treatments in IVF-ET cycles with cancelling of implantation and signed the informed consent during April 2014 to Dcember 2015.According to the progesterone level on the day of HCG administration, six patients have high levels of serum progesterone and six patients have normal levels of serum progesterone. Endometrial samples were obtained from these patients in the 5 days after oocyte retrieval. Each sample was distributed into two after rinsed with RNase-free water and then immersed quickly into liquid nitrogen.By using RNA-seq analysis, we have investigated the gene expression profiles.The expression of the differentially expressed genes was further validated by quantitative RT-PCR. And we analyed the function of these differential genes.Results:1. Adverse effect of high progesterone in late follicular phase on outcomes of IVF-ET in patients with different ovarian responses during COH.With increase in ovarian response, the cut-off values of serum P on the day of human chorionic gonadotrophin(h CG) rose, and respectively were 2.5 ng/ml in the high ovarian response group, 2.25 ng/ml in the moderate ovarian response group, and 1.5 ng/ml in the low ovarian response group.With the rise of progesterone levels, the clinical pregnancy rate and ovarian response group embryo grow rate were significantly decreased(P < 0.05), but the fertilization rate, cleavage rate, embryo rate and high quality embryo rate has no significant difference(P > 0.05). The number of oocyte retrieval and Gn dose were all positively correlated with an increased P level by logistic regression analysis(P < 0.05).2. The adverse effects of high progesterone in late follicular phase on endometrial histology, pinopodes and the expression of integrin ?v?3, LIF, IL-1 in patients with moderate ovarian responses during COH.The proportion in the mid-secretory phase in the high progesterone group were higher than the group with normal progesterone(P < 0.05).The pinopodes were wrinkled in the high progesterone group.And the pinopodes were developing and mature in the normal progesterone group.The expression of endometrial integrin ?v?3(0.212�0.05 vs 0.47�0.032), LIF(0.137�0.027 vs 0.531�0.056), IL-1(0.264�0.018 vs 0.515�0.023)in the high progesterone group were lower than the normal group(P < 0.05).3. The endometrial transcriptomes may be changed under the high progesterone in the late follicular phase of patients with moderate ovarian responses by the RNA-seq.There were 31 significant difference genes between high progesterone and the normal group, 15 of which were up regulated and 16 of which down regulated. And then the differentially expressed genes were verified by RT-PCR. SLC7A4 DDX5 IFI6, HLA- DRB4, HLA- DRB5, SCGB1D2 were consistent with the results of RNA-seq analysis.Premature progesterone in late follicular phase rise may alter the endometrial gene expression and affect the endometrial receptivity.Conclusions:1.This suggests that an increased P level on the day of HCG may fail to affect the quality of embryos and oocytes, but decreases embryo implantation rate and clinical pregnancy rate probably by changing the endometrial receptivity. With increase in ovarian response, the cut-off values of serum P on the day of HCG rose. The number of oocyte retrieval and Gn dose were all positively correlated with an increased P level.2. In these patients with moderate ovarian responses, the endometrial histology, morphology and molecular expression were changed.The elevated progesterone may lead to the early maturation of the endometrium during implantation phase, leading to a failure of the embryo-endometrium dialogue.3. The endometrial transcriptomes during implantation phase may be changed undering the high progesterone in the latefollicular phase by the RNA-seq and the expression of the differentially expressed genes was validated by quantitative RT-PCR.This may provide the corresponding gene targets for exploring the molecular mechanisms.