| BackgroundEmbryo implantation requires the endometrium to be in the secretory phase.From 5 to 7 days after ovulation,we called it "implantation window".Whether the embryo can be successfully implanted or not depends on the endometrium's receptivity to the embryo,that is,the endometrium's receptivity.Endometrial thickness is determine the important premise of receptive,a large number of experimental results showed that patients undergoing in vitro fertilization and embryo transfer treatment process,if the fresh embryo transfer in endometrial thickness was less than or equal to 7 mm,the clinical pregnancy rate decreased significantly,and higher rate of abortion during early pregnancy,and patients accept frozen embryos transplant in the process of recovery,endometrial thickness less than 8 mm patients,the clinical pregnancy rate was lower than that in group endometrial greater than or equal to 8 mm[1].The above experimental results showed that the endometrial thickness greatly affected the endometrial receptivity of the embryo and determined whether the embryo implantation could be successful.In the process of in vitro fertilization-embryo transfer,some patients transfer embryos into the female uterus,but the endometrium was too thin due to various causes,leading to the failure of embryo transplantation,or the endometrium thickness met the standard of embryo transfer,but the pregnancy had not been successful after multiple embryo transfer.Effect the receptivity of endometrium of etiology is very complex,uterine apoxesis repeatly,abortion,and endometrial tuberculosis,as well as acute or chronic endometritis were likely to lead to endometrial pathological change,the endometrium becomes weak,In the middle of the corpus luteum the thickness of endometrium is still less than 7 mm called thin endometrium.There are different ways to treat thin endometrium,and different patients have different therapeutic effects on different treatment methods.Many patients with stubborn thin endometrium do not work well with existing methods.Current treatment methods lack specificity According to the current situation,We regard patients less than age of 40,accumulative total at least 2 pieces of blastocyst transplant or 4 pieces of cleavage stage embryos still did not get clinical pregnancy patients,as repeated implantation failure patients,the leading cause of implantation failure in summary is divided into two aspects,the receptivity of endometrium and embryo factors,in addition to the embryo itself factors,abnormal intrauterine environment resulting in the decrease of the receptivity of endometrium is one of the important factors.Therefore,in the premise that the quality of embryos has been obtained and cannot be changed,how to enhance endometrial receptivity is a hot issue that reproductive physicians pay attention to patients with repeated implantation failure.Autologous platelet-rich plasma is mainly obtained by centrifugation.It is a product of highly concentrated platelets.The role of platelets is that after they are successfully activated,they will actively release various factors,such as tumor necrosis factor-?(TNF-?)and insulin-like growth factors.(IGF)and epidermal growth factor(EGF).They involved in tissue repair,including cell chemotaxis,angiogenesis,and accelerating the secretion of cell matrix.Due to the particularity of PRP to promote cell reproduction,it has been used in the treatment of bone tissue and chronic wound healing[4].Because PRP is derived from autologous blood,it avoids the risk of cross infection and is relatively safe to use.According to the physiological characteristics of PRP,we hypothesized that the role of PRP in promoting cell proliferation and repairment could be applied to the field of repair of female endometrium,so as to improve the clinical intrauterine environment of patients with endometrial receptivity abnormalities that lead to the failure of embryo transplantation,and to improve their clinical pregnancy rate.PRP were occasional reports on the application in the field of reproduction[5-7],but most of them were case reports,lack of large sample research,and lack of basic research on the effect of PRP on endometrial receptivity.ObjectiveThis study evaluated the effect of PRP on the proliferation of RL95-2 cells cultured in vitro,observing the optimal concentration of PRP to promote cell proliferation in vitro.Explored the feasibility of PRP on 3D culture of RL95-2 cells,To observe whether PRP can increase adhesion on RL95-2 cells on 3D cell growth scaffolds made by electrostatic spinning technologyand perfused.PRP was perfused into the uterine cavity of SD rats with thin endometrial model to observe whether PRP could change the endometrial thickness and receptivity of rats,and repair the functional layer of rats' endometrium after chemical damage by anhydrous ethanol.Observe the clinical repeated implantation failure and thin endometrial patients through the PRP cavity Changes in clinical pregnancy rate,embryonic abortion rate and embryo implantation rate after perfusion therapy.To investigate the feasibility of PRP in treating thin endometrium,improving endometrial receptivity and improving clinical pregnancy rate in patients with repeated implantation failure.MethodsTake 17.5 ml of fasting venous blood from 5 healthy volunteers,adding 2.5 ml of anticoagulantuse,two-step centrifugation to separate PRP,mix PRP with 10%CaC12 buffer to activate it.The activated PRP was co-cultured with RL95-2 cells,the proliferation curve of RL95-2 cells in the PRP group at different concentrations was measured by the MTT method according to the different concentrations of PRP and FBS in the culture medium,The prepared PRP was placed in a 96-well plate and divided into two groups,A and B,according to whether it was activated or not,the growth factors VEGF,TNF-?,IGF,and PRP released at different time periods in vitro were measured by the ELISA method.The concentrations of PRP secreted by growth factor in different time periods were observed.PRP and 3D cultured RL95-2 cells were co-cultured for 7 days.After paraffin-embedded sections,HE staining was performed to observe whether PRP can promote the activity of RL95-2 cells in the 3D culture scaffold.Use the activated PRP as the 3D scaffold.RL95-2 was planted on PRP as seed cells,and the survival of cells on the PRP scaffold was observed.Immunohistochemical staining was performed on cells cultured in 3D to observe whether RL95-2 cells could normally express progesterone receptor under 3D culture conditions.Chemical damage of rat endometrium was caused by anhydrous ethanol intrauterine perfusion to obtain rat thin endometrial animal model.After successful modeling,PRP intrauterine perfusion was performed on the rats in the treatment group,and the repair effect of PRP on the endometrium of rats after intrauterine perfusion was observed by HE staining.By observing the endometrial vimentin,vascular endothelial growth factor and integrin in rats,it was confirmed whether PRP can improve the endometrial receptivity of rats after intrauterine perfusion.Observe patients with thin endometrium and repeated implantation failure patients by uterine cavity perfusion PRP 2-3 times uterine endometrial thickness changes before and after the infusion.For patients with thin endometrium,hysteroscopy was performed 3-5 days after menstruation,and micro-stimulation was performed on the wall of the uterus respectively with micro-scissorswhen.When lining thickness reach standard after transplantation,then frozen embryo transplantation,recovery in patients with treatment group of related experimental data statistics,including pregnancy rate,miscarriage rate and birth.Result(1)Co-cultivation of PRP and RL95-2 cells can promote the proliferation of RL95-2 cells.The growth curve of RL95-2 cells shows that the growth rate is the fastest in the culture medium with 1%PRP concentration combined with serum concentration 2%and 10%.And cell proliferation was obvious after 72 hours of culture.The proliferation activity of RL95-2 cells co-cultured with PRP in serum-free medium increased significantly,and with the increase of PRP concentration,the proliferation activity increased,The proliferative activity of RL95-2 cells at low concentrations(1%and 2%)was better than that at high concentrations(5%and 10%).(2)PRP can continuously secrete VEGF,TNF-?,PDGF,EGF and other growth factors after activation in vitro,and the growth factor concentration continues to increase with the increase of days.(3)PRP can promote the adhesion of RL95-2 cells in the 3D cell culture scaffold;using PRP as the 3D scaffold,culturing RL95-2 cells,and paraffin-embedded sections,the growth of RL95-2 on the surface of the scaffold can be seen.The scaffold has small voids and no cell growth inside.(4)Rat intrauterine perfusion with PRP can increase the thickness of thin endometrium in rats,and the protein levels of vimentin,integrin ?3,and vascular endothelial growth factor in the PRP intrauterine perfusion group are higher than those in the thin endometrial group.(5)Through intrauterine perfusion of PRP in patients with repeated implantation failure,the condition of this group of patients can be alleviated,and the receptivity of the endometrium will be significantly enhanced,clinical pregnancy rate was not significant compared with the normal control group.The uterine cavity of patients with thin uterus is perfused through PRP uterine cavity,and the condition of the patient's endometrium was also improved,and its thickness was increase accordingly,which reduces the cancellation rate of embryo transfer cycles.In conclusion(1)PRP culture in vitro can promote the proliferation of RL95-2 cells,and the combination of low-concentration PRP and low-concentration fetal bovine serum can promote the proliferation of RL95-2 cells,which is higher than that of the culture medium simply adding serum.(2)PRP can promote the adhesion and proliferation of RL95-2 cells on the 3D scaffold,and it has the possibility of preparing 3D cell culture models,providing possible carrier options for intrauterine transplantation of endometrial epithelial cells or stem cells.(3)PRP intrauterine perfusion of rat endometrium can protect rat endometrium from chemical damage caused by anhydrous ethanol,and improve the tolerance of rat thin endometrium.(4)The clinical pregnancy rate,abortion rate and birth rate were similar to those of the control group after PRP intrauterine perfusion in patients with repeated implantation failure.The uterine cavity of patients with thin endometrial perfusion with PRP,the thickness of the endometrium of the patients in the treatment group increased,which reduced the cancellation rate of frozen embryo resuscitation cycles,and achieved a certain clinical pregnancy rate,and did not increase the birth abnormalities and maternal complications of offspring Risk of disease. |
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