| Background With the incidence of infertility increasing,assisted reproductive technology(ART)rapid development,in vitro fertilization-embryo transfer(in vitro fertilization and embryo transfer,IVF-ET)and its derivative technology benifit many infertility patients.However,A large number of couples are still unable to undergo pregnancy after several IVF treatments,resulting in a serious decline in the quality of life,and every failure will bring a huge financial burden.RIF has become a hot topic and difficult to study in the field of assisted reproduction.The key factors in the success of the embryo implantation are the mother and the embryo.Therefore,how to improve the quality of the embryo,improve the endometrial receptivity,promote maternal dialogue and immune tolerance become the main research in the field of assisted reproductive treatment of RIF direction.Recently Yoshioka et al reported that the use of intraperitoneal perfusion through HCG culture PBMC and fresh PBMC suspension can significantly improve the clinical pregnancy rate and the implantation rate of patients with the repeated implantation failure(RIF).Human Peripheral blood mononuclear cells(PBMCs)mainly include T lymphocytes,B lymphocytes and monocytes.Cytokines secreted by PBMCs,such as IL-1?,IL-1? and TNF-?,plays an important role in embryonic implantation.The mechanism may be associated with a variety of inflammatory factors secreted by PBMC can induce endometrial differentiation and the opening of the bed opening and endometrial receptivity.Objective 1.To study the effect of autologous PBMC on the expression of endometrium receptivity-related makers LIF and integrin of RIF patients under in vitro.2.To study the effect of PBMC on the expression of LIF receptor on embryo co-cultured in vitro.3.To study the effects of autologous PBMC intrauterine administration on pregnancy outcome of patients with RIF receiving frozen/thawed embryo transfer.Material and Methods Part 1 Effect of autologous PBMC on the expression of endometrium receptivity-related makers LIF and integrin of RIF patients under in vitro.1.The endometrial tissue and PBMCs were obtained from RIF patients from May 2015 to May 2016 at the Second Affiliated Hospital of Zhengzhou University.The endometrial glandular epithelium and stromal cells were isolated.2.Endometrial cells were cultured with the progesterone-containing culture medium in vitro to simulated human endometrial environment,the day adding estrogen and progesterone culture medium was code Day0.3.Cultured to Day2,the experimental group(Group A)added 0.5ml PBMC suspension by a density of 3×106 / ml.Continue cultured in vitro to Day6.4.Exchange the culture medium every other day,and the supernatant were collected in Day1,3,5 to be measured.5.The concentration of LIF and integrin ?V?3 in culture medium was detected by enzyme-linked immunosorbent assay.Part 2 Effect of PBMC on expression of LIF receptor on embryo in co-culture Model of Embryo and Endometrium in Vitro 1.A total of 58 embryos were collected with signed consent by the couples.2.Endometrial cells were cultured with the progesterone-containing culture medium in vitro to simulated human endometrial environment,the day adding estrogen and progesterone culture medium was code Day0.3.Group A: when cultured to Day2,add 0.5ml PBMC suspension by a density of 3×106 / ml.Placed embryo in Day3,continue cultured in vitro to Day6.Collect the formed blastocysts to be detected? 4.Group B: when cultured to Day2,add equal volume of culture medium.Placed embryo in Day3,continue cultured in vitro to Day6.Collect the formed blastocysts to be detected? 5.Group C: A total of 10 embryos were detected directly.6.Immunofluorescence assay was used to detect the expression of LIFR on embryos and blastocysts Part 3 Effects of autologous PBMC intrauterine administration on outcomes of receiving frozen/thawed embryo transfer in patients with RIF 1.A total of 78 patients who received frozen/thawed embryo transfer between January 2015 to December 2016 in our clinic were enrolled in this study.All patients had experienced three or more failures of IVF-ET therapy without poor ovarian reserve(FSH <15 m IU/m L)and were less than 40 years of age.2.The patients were randomized into two groups: a PBMC treated group(n=56)receiving intrauterine administration of autologous PBMC before ET,while a control group(n=22)receiving intrauterine administration of a equal volume of culture medium.3.The frozen/thawed embryo transfer procedure was performed as previously described elsewhere,and the receptivity of the endometrium was prepared by spontaneous menstrual cycle(ovulation day coded as Day 0)or hormone replacement therapy(progesterone injection day coded as day 0).4.Embryo transfer was performed on the day 3 at the early cleavage stage.5.In the PBMC treated group,blood samples(4ml)were obtained from patients on day 2 and PBMC were isolated,then 200 ul of PBMC(3106/ml)suspension was gently administered to the cavity.6.The pregnancy test was performed 12 days after ET,and an ultrasound examination was performed 35 days after a positive pregnancy test to visualize the number and the location of gestational sacs and confirm fetal heart pulsations.Results Part 1 Effect of autologous PBMC on the expression of endometrium receptivity-related makers LIF and integrin of RIF patients under in vitro.1.There was no significant difference in LIF concentration between the two groups on the Day 3(P>0.05).Compared with the endometrial group,the concentration of LIF in the culture medium of the group added PBMC was significantly increased on the Day 5(P<0.05).2.There was no significant difference in Integrin ?V?3 concentration between the two groups on the Day 3(P>0.05).Compared with the endometrial group,the concentration of Integrin ?V?3 in the culture medium of the group added PBMC was significantly increased on the Day 5(P<0.05).Part 2 Effect of PBMC on expression of LIF receptor on embryo in co-culture Model of Embryo and Endometrium in Vitro 1.In this study,10 embryos of D3 were detected,and 10 blastocysts were formed in the control group.The experimental group formed 12 blastocysts.The blastocyst formation rates of the experimental group and the control group were 50.0% and 41.7% respectively,the difference was not statistically significant(P> 0.05).2.The difference of immunofluorescence OD value of the LIFR on the embryo between the three groups was significant(P<0.05).Compared two groups respectively,the OD value of the experimental group stronger than the control group,and the two blastocysts group all stronger than the cleavage stage embryo.Part 3 Effects of autologous PBMC intrauterine administration on outcomes of receiving frozen/thawed embryo transfer in patients with RIF 1.There was no significant difference in PBMC treatment group and control group between the mean age,infertility period,infertility type,the number of previous transplant cycles and the number of previous transplanted embryos(P> 0.05).2.The implantation rate and clinical pregnancy rate of PBMC treatment group were higher than those of control group(P <0.05).Conclusions 1.Autologous PBMC can increase the concentration of LIF and integrin ?V?3 in endometrial cells in vitro,and then up-regulate endometrial receptivity of patients with RIF.2.PBMC can promote the expression of LIF receptor on embryo under coculture of endometrium.3.Autologous PBMC intrauterine perfusion can improve the embryo implantation rate and clinical pregnancy rate of patients with RIF receiving frozen/freeing embryo transfer. |
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