The Research On LPAR3for Human Endometrial Receptivity

Implantation in humans is an intricate process that involves complex interactions between the embryo and the uterus during the initial period of gestation. There are three important conditions for successful implantation:good embryo quality, high endometrial receptivity and a synchronized dialogue between maternal and embryonic tissues. The endometrium is not receptive to embryo implantation except for during a strict frame of time called the'implantation window'. The uterine receptivity is regulated by several kinds of molecular signals, such as specific growth factors, cytokines, lipid mediators, adhesion molecules, and transcription factors. They are further regulated by female sex steroids, prostaglandins (PGs) and peptide hormones. The expression level of any factors is abnormal will contribute to change the endometrial receptivity and lead to reduce the pregnancy rate.Lysophosphatidic acid (LPA) is a growth factor that triggers many cellular responses. These responses include cell survive, proliferation, differentiation, inhibition of gapjunction communication and action cytoskeletal rearrangements. LPA also plays an important role on reproduction, including stimulation of oocyte maturation, the preimplantation development of two-or four-cell embryos to the blastocyst stage and embryo transport in the oviduct. It could be produced through the hydrolysis of phospholipids by extracellular lysophospholi-pase D or by activating platelets, erythrocytes, postmitotic neurons, ovarian and cervical cancer cells, adipocytes and mast cells. LPA functions through its G protein-couple receptors, LPA1-4. Mice kicking out LPA1and LPA2reproduce normally, but LPA3-null mice showed reduced litter size and altered embryo spacing, embryo crowding, and delayed implantation. It provided additional evidence for its role in murine implantation. Expression of LPA3in the mouse uterus is strictly regulated during early pregnancy. Within the mouse uterus, LPA3mRNA increased on2.5days post coitus (2.5d.p. c), peaking around3.5d. p.c, peri-implantation period, then returned to the basal levels on4.5d. p. c. through the end of pregnancy. In the generation domain, there is lack of information about the function of LPA3on endometrial receptivity until now. Recent research showed that LPA3expression is low during secretory phase of endometrium in those patients with repeated implantation failure and endometriosis.Controlled ovarian hyperstimulation (COH) can get many germ cell by using large amounts promoting ovulation drug. Long protocol controlled ovarian hyperstimulation is the most commonly used protocol that can get good quality eggs, high fertilization rate and high qualified embryo rate, but the implantation rate is still low. GnRHa long time superovulation program put a negative effect in the balance of internal hormone which decreasing the endometrial receptiblity. So to find the reason of decreasing in endometrial receptiblity and find an efficient means to solve the problem is with good clinical significance. So far, there is no reported about the expression of LPA3with Long protocol controlled ovarian hyperstimulation in human endometrium during periimplantation periode.Polycystic ovary syndrome (PCOS) patients with endocrine disorder caused by abnormal secretion of estrogen and progesterone and rising of luteinizing hormone (LH), which reduces the receptivity of the endometrium of PCOS patients. Endometrial receptivity in patients with PCOS is mainly concentrated in the steroid hormone receptors, integrins, matrix metalloproteinases, and homeobox factor. LPA3play an important role in the reproductive process. In addition, the clinical part of the study, to improve PCOS endometrial receptivity, artificial frozen-thawed embryo transfer cycles with pituitary suppression was used. The purpose is to improve the receptivity of the endometrium of women with PCOS.In this study, by using RT-PCR, Western blot and immunohis-tochemistry study the expression of LPA3with normal menstrual cycle of women, Long protocol controlled ovarian hyperstimulation in human endometrium during periimplantation periode and menstrual cycle of PCOS. This study is to reveal that the important role of LPA3in the process of establishing endometrial receptivity. Divided into four parts, the three parts of the experimental part, the last part of the clinical part, the experimental methods and results are as follows:Part1:Cyclic regulation of LPA3in human endometriumObject:The aims of the present study were to characterize LPA3mRNA and protein in human endometrium during the normal human menstrual cycle.Methods:Forty-three normally cycling volunteers without reproductive disorders were randomized to undergo endometrial sampling on a specific cycle day. Samples were assessed for relative LPA3mRNA expression using real-time PCR and for LPA3protein using immunohistochemistry and Western blot.Results:ⅰ. LPA3mRNA and protein can be detected in endometrial tissues throughout the human menstrual cycle.ⅱ. The expression of LPA3mRNA and protein significantly increased during the early and late secretory phase compared with other menstrual phases. Its expression dropped rapidly in the mid-secretory and remained low in the whole proliferative phase.iii. In proliferative samples, LPA3protein was detected and localized primarily to the cytoplasm of the luminal and glandular epithelial cells. Early secretory immunostaining for LPA3protein was markedly elevated in both the epithelial and stromal cells. Samples from the midsecretory phase (the implantation window) showed a markedly reduced intensity of LPA3staining in glandular epithelial cells and stromal cells. Late secretory immunostaining for LPA3protein expression was elevated in the nuclei of stromal cells. Thus, the intensity of immunohistochemistry staining for LPA3generally parallels the mRNA and Western blot changes.ⅳ. LPA3is in the low expression in the proliferative phase because of secretion of estrogen; In the late proliferative phase, follicular granulose cells begin to secrete progesterone, progensterone increased gradually after ovulation, LPA3increased during early secretory phase. In the mid-secretory phase E levels increased, LPA3decreased expression in uterine endometrial. In the late secretory phase decreased E level, LPA3in uterine endometrium on expression began to rise.Conclusion:ⅰ. LPA3during the human menstrual cycle of endometrium on the cyclic change, LPA3expression peaked in human peri-implantation. LPA3may participate in the establishment of endometrial receptivity, can be used as a predictor of endometrial receptivityⅱ. LPA3is regulated by progesterone and LH, estrogen by positive or negative feedback. Part2The study on the effect of long protocol controlled ovarian hyperstimulation by GnRHa on expression of peri-implantation window LPA3Object:To study on the effect of long protocol controlled ovarian hyperstimulation by GnRHa/rFSH/hCG on the endometrial LPA3expression.Methods:The people were divided into NC group and COH group. LPA3mRNA and protein expression were determined by RT-RCR and Western-blot form pd3to pd6in the two groups.Results:ⅰ. The peak expression was on the pd4in NC group, the surge was on pd3in COH group.ⅱ. The expression of LPA3significantly lower in COH group compared with NC group.ⅲ. COH group serum E2and P levels in the corresponding time points were higher than the NC control group, the E2/P ratio is also higher than the NC control group.Conclusion:ⅰ. Controlled ovarian hyperstimulation by GnRHa/rFSH/hCG may transform the level of LPA3, lead to disorder of implantation window,declin the endometrial receptivity.ⅱ. May be due to the super physiological doses of E2and P levels, as well as uncoordinated E2/P ratio caused the LPA3expression abnormalities duing COH. We may adjust the E2/P ratios, the expression of LPA3return to normal conditions, improve the IVF pregnancy rate. Part3Expression of LPA3in the endometrium of patients with PCOS during menstrual cycleObject:The aims of the present study were to characterize LPA3mRNA and protein in endometrium during the menstrual cycle of patients with PCOS.Methods:Samples were assessed for relative LPA3mRNA expression using real-time PCR and for LPA3protein using Western blot.Results:ⅰ. PCOS group, patients with menstrual cycle days to39.75days, significantly more than the number of days30.5days of the menstrual cycle of the NC control group (P<0.01). In the proliferative phase in the PCOS group, serum LH levels as8.61miu/ml higher than the NC group, proliferation of serum LH levels3.10miu/ml There were significant differences (P<0.01). Early in the secretory phase in patients with PCOS group serum P level of1.64ng/ml,6.36ng/ml, significantly lower than the NC group P level of4.46ng/ml,14.2ng/ml There were significant differences (P<0.05).ⅱ. Low expression of LPA3mRNA and protein in the proliferative phase in PCOS group, but it was higher in PCOS group than in NC control group in the proliferative phase. In the early secretory phase LPA3mRNA and protein expression was significantly increased and reached its peak, the sharp decline in the expression of mRNA and protein in the middle secretory phase. Expression of LPA3in the early secretory phase endometrium in PCOS group was significantly lower than NC control group.Conclusion:ⅰ. Because of LPA3abnormal expression in the proliferative phase,excessive proliferation s with endometrial with PCOS patients.ⅱ. PCOS group in the early secretory phase has low expression of LPA3, which may interfere with the establishment of endometrial receptivity, and reduced endometrial receptivityⅲ. PCOS group in the proliferative phase of serum levels of LH high expression and low P expression in secretory phase endometrium on state may relate to abnormal expression of LPA3. Part4Comparison of clinical outcomes of the two hormone replacement treatment for frozen-thawed embryo transfer cycle in patients with polycystic ovary syndromeObject:This study was designed to compare the clinical outcomes of the two hormone replacement treatment for frozen-thawed embryo transfer cycle in patients with polycystic ovary syndrome.Methods:Retrospective chart review of126patients with PCOS was done,2clinical protocols for preparing endometrium were carried out, including hormone replacement group(HRT, n=89) and artificial frozen-thawed embryo transfer cycles with pituitary suppression(GnRHa, n=37).Results:No significant differences were found in patient's age(29.53±3.39vs29.24±2.85years), body mass index (23.07±3.51vs22.20±2.96)and duration of infertility(3.14±1.89vs3.86±1.36 years)(P>0.05), also no significant differences were found in early abortion(16.3%vsl4.8%) and ectopic pregnancy risk(4.1%vs3.7%)(P>0.05). However, compared HRT group, GnRHa group achieved significantly higher clinical pregnancy (55.06%vs72.9%, P<0.05) and implantation (38.7%vs53.6%, P<0.05).Conclusion:It is an effective method for patients with PCOS by using hormone replacement with pituitary suppression for artificial frozen-thawed embryo transfer cycle.