Study Of Platelet-rich Plasma In The Treatment Of Thin Endometrium

BackgroundAssisted reproductive technology has become an important technical method to solve the problem of infertility.The development of this thecnology has solved the problem of embryo quality,but the change of endometrial receptivity caused by thin endometrium is still one of the main factors of cycle cancellation and embryo implantation failure in assisted reproductive technology[1].?Chinese Expert Consensus on Diagnosis and Treatment of Abnormal Endometrium in Assisted Reproductive Technology?and Canadian Fertility and Andrology Society(CFAS)defined the thin endometrium as endometrium<7mm on ovulation day or on the day of injection of human chorionic gonadotropin(HCG)during fresh in vitro fertilization(IVF)cycle or the day of progesterone initiation during freezing-thawing embryo transfer(FET)cycle[2-3].However,the thin endometrium is not limited to the thinning of the endometrium,but also includes a variety of endometrial receptivity changes such as abnormal blood flow in the functional layer of the endometrium and changes in factors affecting endometrial receptivity.Clinically,the treatment of thin endometrium mainly includes hormone therapy,low-dose aspirin,minimally invasive endometrium stimulation,and improvement of endometrium blood perfusion,in order to improve endometrium thickness,blood flow and other receptivity effects.However,due to the inexact clinical effect of the above treatment methods and the large individual differences among patients,the treatment effect is not ideal,and it is urgent to explore new effective treatment methods.The stem cell,as a kind of undifferentiated cell population with self-renewal and multi-directional differentiation potential,has unique advantages in the field of regenerative medicine and has been widely studied in many medical fields.Among them,there have been reports on the application of stem cells in endometrial repair and successful pregnancy.Although there have been successful reports,the application of stem cells is limited by their limited source,severe trauma and immunogenicity[4].In recent years,with the continuous deepening of research in the field of stem cells,the research of endometrium mesenchymal stem cells(EnMSCs)has also made major breakthroughs.EnMSCs has high proliferative activity and can differentiate into bone,fat,muscle cells and chondrocytes,showing a strong multidirectional differentiation potential.Studies have shown that EnMSCs can be induced by estradiol to differentiate into endometrial epithelial cells in vitro[5].In a mouse model.EnMSCs can reconstruct endometrial tissue and increase endometrial thickness[6].Tan[7]and Zheng[8]also achieved good efficacy by transplanting menstrual blood-derived EnMSCs for the treatment of severe Ashman syndrome.The above research results suggest that EnMSCs play an important role in improving the growth and function of the endometrium.However,the long-distance homing and local colonization of vitro stem cell applications are very difficult,and the number of stem cells needed for clinical treatment is large.Besides,the addition of fetal bovine serum and allogeneic growth factors in the process of replication and amplification is also against the ethical scope.Can we find a safe and effective self-resource method to culture stem cells?More interestingly,hypothesizing that this autologous resource could awaken the function of the stem cells in vivo would yield a therapeutic effect that would be twice as effective with less effort.Platelet-rich plasma(PRP)is a kind of plasma with high concentration of platelets obtained by centrifuging venous blood.When PRP is activated,? granules are released to produce a variety of growth factors,which mainly include transforming growth factor(TGF),vascular endothelial growth factor(VEGF),platelet-derived growth factor(PDGF),epidermal growth factor(EGF)and insulin growth factor(IGF),etc.These growth factors can promote cell proliferation,effectively change the biological micro-environment of the injury site,promote cell migration and new matrix formation,thus promoting tissue repair and regeneration,which have a positive effect on wound healing after injury[9].In addition,PRP has its own unique advantages such as simple operation,safety and economy,and less damage,so it is widely used in orthopedics,stomatology,dermatology,cosmetic surgery,etc.[10-12].Based on the above basic theoretical research and related applications,the application of PRP is gradually introduced into the field of reproduction and treated as a new safe and effective autologous biomaterial for the treatment of thin endometrium.In view of the urgent need for treatment of thin endometrium,stem cells therapy is effective but limited in application and difficult to be popularized,and autologous PRP containing multiple growth factors can affect various cell functions,application safety and other excellent characteristics,we designed this project to study the roles of PRP in the treatment of thin endometrium.ObjectiveFirstly,this study evaluated the effects of PRP on the biological functions of proliferation,migration and adhesion of EnMSCs in vitro.Then,a mouse model of thin endometrium was established to explore the effect and potential mechanism of PRP in the treatment of thin endometrium by observing the changes of endometrial morphology,receptivity indexes and EnMSCs markers in vivo after PRP intrauterine perfusion in mice with thin endometrium.Finally,autologous PRP intrauterine perfusion was applied to patients with thin endometrium who are planning to undergo FET to observe the clinical effect of intrauterine perfusion PRP in the treatment of thin endometrium.We aim to find a new effective method to treat thin endometrium.Methods1.The venous blood of 5 healthy volunteers was collected,separated by two-step centrifugation to make and activate PRP.The expression patterns of EnMSCs surface markers CD14,CD19,CD34,CD45,CD73,CD90,CD105 and HLA-DR were detected by flow cytometry.EnMSCs also were cultured in adipogenic,osteogenic and chondrogenic differentiation medium,and the cell morphology,lipid droplets,mineralized nodules and chondrogenic formation were observed to identify EnMSCs.Then EnMSCs were cultured in medium with different concentrations of PRP,and the proliferation curves of each group at 24h,48h,72h and 96h were monitored by CCK-8 method.Transwell method and scratch test were used to detect the effects of different concentrations of PRP on the migration and adhesion of EnMSCs.2.95%ethanol was perfused into the uterine cavity to make the thin endometrial mouse model,after which PRP was perfused into the uterine cavity.The changes of endometrium thickness,area and microvessels were observed by HE staining.Endometrial function and receptivity indexes were detected by immuno-histochemistry,ELISA and Western-blot,including Cytokeratin,Vimentin,VEGF,Leukemia inhibitory factor(LIF)and interleukin-6(IL-6).The surface markers of EnMSCs,CD90 and CD 105,were detected by immunofluorescence double staining method to observe the changes of EnMSCs in vivo.All above experiments were to investigate the effect and mechanism of intrauterine perfusion PRP in repairing thin endometrium.3.Venous blood was collected from 20 thin endometrial infertility patients undergoing FET treatment,PRP was separated and prepared by two-step centrifugal method,then PRP was infused intrauterine aseptic in the same cycle,and continued artificial cycle replacement therapy.When the endometrium reached the thickness of embryo transfer(?7mm),progesterone was added to transform the endometrium and FET was performed.Provide appropriate luteal phase support and measure serum?-HCG levels.Calculate and analyze the endometrial thickness,morphology and blood flow,and follow up with positive P-HCG and clinical pregnancy.Results(1)Different concentrations of PRP were added to the culture medium to culture EnMSCs in vitro.It was found that different concentrations of PRP had different effects on the proliferation of EnMSCs.The proliferation activity of EnMSCs in the 2%PRP group was the largest.The proliferation activity of EnMSCs in 1%PRP and 4%PRP groups was slightly lower than that in 2%PRP group.The proliferation activity of EnMSCs in 5%PRP and 0.5%PRP groups was the weakest(p<0.01).(2)PRP can promote the migration of EnMSCs.Among them,the migration rate of EnMSCs cultured in 2%PRP medium was the highest.EnMSCs in 1%PRP group and 4%PRP group followed.The migration rate of EnMSCs was the lowest in 5%PRP group and 0.5%PRP group.(3)Different concentrations of PRP also have different effects on the adhesion ability of EnMSCs:the adhesion ability of EnMSCs cultured in 2%PRP medium was the strongest(p<0.01),and there was no significant difference in the adhesion ability of other groups(p>0.05).(4)By making the thin endometrial mouse model and performing uterine-cavity perfusion PRP treatment,we found that the uterine cavity perfusion PRP can improve the endometrial receptivity of thin endometrial mice,including the improvement of endometrial morphology(endometrial thickness,area and the increase of microvessels)and the improvement of the receptivity indexes of endometrium(CK9,VIM,VEGF,LIF).In addition,PRP can also inhibit the release of the inflammatory factor IL-6.Immunofluorescence was used to trace the expression of EnMSCs in the endometrium of mice,and we found that EnMSCs increased after intrauterine infusion of PRP in vivo.(5)By observing the clinical effect of autologous PRP intrauterine perfusion therapy for thin endometrial patients undergoing FET,we found that in 20 patients,the mean endometrial thickness was 5.55±0.71mm before PRP treatment and 7.82±1.04mm after PRP treatment(p<0.0001).The endometrial morphology and sub-endometrial blood flow signal were also significantly improved.After intrauterine perfusion of PRP,the endometrium of all patients reached the thickness of transplantation,among which 12 patients achieved clinical pregnancy.The pregnancy rate was 60%,the implantation rate was 39.4%,and the early abortion rate was 16.7%.Conclusion1.In this study,different concentrations of PRP were used to culture EnMSCs in vitro,and it was found that within the range of 2%PRP concentration,the ability of proliferation,migration and adhesion of EnMSCs increased with the increase of PRP concentration in a dose-dependent manner.However,the above effects showed a decreasing phenomenon after the treatment of higher concentrations of PRP,suggesting that different concentrations of PRP had different effects on EnMSCs.It is the first to verify the effects of PRP on the proliferation,migration and adhesion of EnMSCs and the relationship with its concentration.2.PRP can replace FBS and single growth factor for the culture of EnMSCs,and provide amplification support for clinical application of EnMSCs in the treatment of thin endometrium and other diseases.3.Intrauterine infusion of PRP into thin endometrium mice can promote the repair of damaged endometrium proliferation,improve endometrium receptivity,and reduce endometrium inflammation.This effect may be related to the effect of PRP on the biological function of EnMSCs in vivo.4.Intrauterine infusion of PRP in infertile women with thin endometrial could increase endometrial thickness,improve blood flow,avoid cycle cancellations,and facilitate embryo implantation to obtain clinical pregnancy,and also did not increase the risk of maternal complications and abnormalities of offspring.