| Background of the study In vitro fertilization-embryo transfer(IVF-ET)technology has been developed for more than 40 years so far,and with the improvement and continuous advancement of the technology,pregnancy outcomes including clinical pregnancy rate and live birth rate of women undergoing IVF-ET have significantly improved.The success of IVF-ET depends on two basic elements involved in implantation: a good quality embryo and a good endometrium.With advances in embryo laboratory technology,quality embryos can be screened prior to implantation and thus the embryo issue has been largely resolved.In contrast,the problem of reduced endometrial tolerance which tends to be more common and more difficult to diagnose and treat.Due to changes in lifestyle and other factors,mechanical,pharmacological,microbiological and other causes of endometrial injury are becoming more common in clinical practice.Endometrial injury can directly lead to thinning of the endometrium,with or without or fibrosis,resulting in decreased endometrial tolerance,failure of embryo implantation and consequent infertility.Thin endometrium due to injury has been one of the most challenging problems in the field of assisted reproduction.The endometrium is the tissue that covers the inner layer of the uterus and is the tissue that the embryo directly contacts during implantation,and it is the main factor that affects embryo implantation and pregnancy outcome.The endometrium is composed of two layers,the functional layer closer to the uterine cavity and the basal layer below the functional layer.The basal layer is the source of endometrial regeneration and produces a new functional layer each month.During each menstrual cycle,the superficial functional layer of the endometrium sheds and regrows from the basal layer below,while the basal layer does not shed.This physiological shedding and regeneration is needed to accommodate embryo implantation and does not cause adverse consequences such as trauma,inflammation or infection.However,when the endometrium is severely damaged,such as repeated or invasive curettage,endometritis,uterine ischemia,and other pathological conditions,the basal layer of the uterus is damaged and the ability of the cells of the basal layer to add value to generate a functional layer is impaired,which is most commonly and directly manifested by the thinning of the endometrium.At the same time,the damaged endometrium may undergo pathological repair in the process of completing self-repair,producing fibrosis of varying degrees,which in severe cases may even lead to occlusion of the uterine cavity due to uterine adhesions.The risk of thin endometrium is not only the cosmetic thinning,but also the reduction of endometrial tolerance,including the reduction of blood flow to the functional layer of the endometrium and the alteration of various cytokines that affect endometrial tolerance.Women with a thin endometrium have impaired fertility,as evidenced by reduced pregnancy rates,poor pregnancy outcomes and recurrent miscarriages.Therefore,after endometrial damage occurs,improvement of thin endometrium and prevention of fibrosis become the key to treatment.The main treatment options for thin endometrium in current clinical practice include hormonal therapy such as prolonging or increasing the dose of estrogen,improving endometrial blood flow such as using sildenafil and low-dose aspirin in order to increase endometrial thickness and improve endometrial blood flow.However,the efficacy of the above treatments is not certain.Some studies have also reported that bone marrow-derived mesenchymal stem cells(BMDSCs)can improve the regeneration of damaged endometrium in mouse models.However,there are still some unresolved questions regarding the safety and availability of BMDSCs,including immunogenic reactions and effects on endometrial proliferative diseases such as endometriosis and infections.Therefore the search for new therapeutic approaches has become an urgent issue.Platelet-rich plasma is plasma containing a high concentration of platelets obtained by centrifugation of one’s own blood.Platelets contain three types of particles,of which the a-particles contain a variety of growth factors and cytokines such as PDGF(platelet-derived growth factor),as well as cytokines PF4(platelet factor 4)and CD40-L,among others.The physiological function of platelets in the body is to prevent blood loss at the site of vascular injury,thus playing an important role in hemostasis.The physiological process begins with platelet adhesion and aggregation,followed by the formation of a procoagulant surface that leads to thrombin production and fibrin formation.Platelets also promote repair at the site of injury,release substances that promote tissue repair,promote cell proliferation,migration,and influence the reactivity of blood vessels and other blood cells in angiogenesis and inflammation.At the same time,these growth factors and cytokines are essential for the leukocytes to the location of injury,and for the induction and regulation of the proliferation and migration of other cell types involved in tissue repair.Based on the property of platelet-rich plasma to promote tissue repair,and endometrial injury is macroscopically a tissue injury,then whether platelet-rich plasma can be applied to tissue repair of endometrium,whether it can be applied to treat thin endometrium after endometrial injury,and whether it can promote endometrial development and embryo implantation,we conducted this project to initially explore the role of PRP in treating thin endometrium and prevention of fibrosis and improvement of pregnancy outcome in patients with thin endometrium.Objectives of the studyHuman endometrial cancer cells(HEC-151 cells)have the same biological properties as endometrial epithelial cells;therefore,HEC-151 cells were used instead of endometrial epithelial cells in this study.Firstly,we investigated the effect of PRP on the biological functions of HEC-151 cells including proliferation and migration;in animal experiments,we made a mouse model of thin endometrium after injury by injecting 95% ethanol into the uterine cavity,and divided the mice with thin endometrium and normal mice into three groups:(1)normal group,(2)experimental group: treated with PRP uterine perfusion after injury,and(3)control group.No PRP uterine perfusion treatment was given after injury.The expression of endometrial morphology,tolerance and fibrosis-related factors after intrauterine perfusion of PRP in mice with endometrial injury was analyzed by hematoxylin and eosin staining,protein blot analysis and reverse transcription-polymerase chain reaction.We also analyzed the number of embryo implantation sites and pregnancy outcomes in mice.Initially,we investigated the effect and potential mechanism of PRP in treating endometrial injury and improving pregnancy outcome.After that,we retrospectively analyzed the efficacy of PRP intrauterine perfusion in 75 patients with thin endometrium in a clinical setting and observed the effects of PRP intrauterine perfusion on endometrial thickness,blood flow,and pregnancy outcome in 86 patients diagnosed with thin endometrium during the same period as a control,with the aim of establishing a new method for the treatment of thin endometrium in a clinical setting.We conclude this study with a review and Meta-analysis of published studies on the use of PRP cavity infusion for the treatment of thin endometrium,in order to provide a more comprehensive analysis of the efficacy of PRP cavity infusion in thin endometrium.Study methods HEC-151 cells were cultured with gradient concentrations of PRP(20%,10%,5%,2.5%),and the proliferation curves were plotted using the CCK-8 method to detect the value-added of each group of cells at 24 h,48h,72 h and 96h;the effect of different concentrations of PRP on the migration ability of HEC-151 cells was examined using the Transwell method and scratch assay.The effect of different concentrations of PRP on the migration ability of HEC-151 cells was examined by Transwell and scratch assay.Finally,the effect of PRP on the cell cycle of HEC-151 cells was detected by flow cytometry,and the mechanism of the pro-proliferative effect of PRP was initially explored.Three experiments were conducted,95% ethanol was injected into the uterine cavity to make a thin endometrial mouse model after injury,and the changes of endometrial thickness and microvasculature were observed by HE staining after PRP treatment was given to the uterine cavity in experiment 1;endometrial function and tolerance markers were detected by immunohistochemistry,immunoblotting and reverse transcription PCR: cytokeratin(CK19),vimentin(Vimentin),VEGF,integrinβ3,and fibrosis marker expression;Experiment 2 investigated implantation outcomes[number of implantation sites(IS),embryo development,weight of IS] at mid-gestation to assess the effect of PRP treatment on day 12 of pregnancy growth and development.Experiment 3 assessed the pregnancy outcome of mice(time to conception,live birth rate,number of litters born.Retrospective analysis of 161 patients who underwent freeze-thaw embryo transfer in the current cycle after embryo transfer was cancelled due to endometrial thickness <7 mm on the previous transfer date.The two groups were divided into 86 patients in the hormone replacement group(control group)and 75 patients in the hormone replacement combined with PRP uterine perfusion group(observation group),and the endometrial thickening,subendometrial blood flow and clinical outcomes of the two groups were examined and analyzed before and after treatment.Results(1)HEC-151 cells were cultured in vitro by adding different concentrations of PRP to the culture medium PRP,and it was found that different final concentrations of PRP had different effects on the proliferation of HEC-151 cells,and the proliferation activity of HEC-151 cells in the 2% PRP group was the highest;the proliferation activity of HEC-151 cells in the 1% PRP and 4% PRP groups was slightly lower than that in the 2% PRP group;the proliferation activity of HEC-151 cells in the 5% PRP and 0.5% PRP groups was slightly lower than that in the 2% PRP group.group and 0.5% PRP group had the weakest proliferative activity of HEC-151cells(P<0.01).(2)PRP could promote the migration of HEC-151 cells.Among them,the migration rate of HEC-151 cells cultured in 2% PRP culture medium was the largest;HEC-151 cells in the 1 % PRP group and 4% PRP group were the second largest;HEC-151 cells in the 5% PRP group and 0.5% PRP group had the smallest migration rate.(3)By making a mouse model of thin endometrium treated with intrauterine perfusion of PRP,it was found by experiment 1 that: intrauterine perfusion of PRP improved endometrial thickness and tolerance in mice with thin endometrium,including endometrial morphological indexes(increase in endometrial thickness,area and microvessels)and endometrial tolerance indexes(CK9,VIM,VEGF,integrin β3);in addition,PRP also prevents fibrosis(α-SMA,TGF-β).Specifically,H&E and MT staining confirmed a significant reduction in fibrosis and an increase in endometrial thickness in the PRP-treated group compared to the injured group.The endometrial area was 212.83 ± 15.84 μm 2 and 262.34 ± 12.33 μm 2 in the ethanol and PRP groups,respectively(p = 0.065).Immunohistochemistry and protein immunoblotting experiments revealed that the expression of CK9,VIM,VEGF,and integrin β3 was significantly enhanced in the PRP-treated group compared with the damaged group.In reverse transcription PCR analysis,CK9,VIM,VEGF,integrin β3 expression was significantly enhanced and α-SMA,TGF-β expression was significantly decreased in the PRP-treated group compared to the injury group.In Experiment 2,implantation sites and embryos were investigated to assess the effect of PRP treatment on the growth and development of mice at day 12 of pregnancy.Implantation sites in the normal group of mice showed normal gross morphology,while a smaller number of implantation sites in the untreated uterine horns of mice with thin endometrium after injury showed abnormalities.The number of implantation sites was significantly higher in the PRP-treated horns than in the untreated horns of thin endometrial mice(4.67 ± 1.01 vs.2.1 ± 0.75,P<0.01),but no delay in embryonic development was observed.No significant differences were observed between the horns of PRP-treated and untreated thin endometrial mice with respect to the weight distribution of implantation sites collected on day 12 of gestation(112 ± 31.0 vs.102 ± 30.4 mg).These results suggest that PRP treatment improves embryo implantation capacity,which is reflected in the greater number of implantation sites in thin endometrial mice treated with PRP uterine perfusion.In Experiment 3,it took an average of 2.2 ± 1.17 days to conceive in the normal group of mice and 21.5 ± 1.64 days in the thin endometrium mice,while only 6.5 ± 1.05 days in the PRP-treated thin endometrium mice(P<0.01).These results suggest that embryo implantation capacity is reduced by endometrial damage,but can be restored by PRP treatment,although not to the pre-damage level.All mice in the normal group gave birth to live pups,but the thin endometrium mice did not give birth to any live pups,and most of the PRP-treated thin endometrium mice gave birth to healthy pups(83.3%)(P<0.01).(4)The endometrial thickness increased in both groups after treatment,and the difference between before and after was statistically significant(P < 0.05);the increase in endometrial thickness in the observation group before and after treatment was higher than that in the control group,and the difference was statistically significant(P<0.05).The difference was statistically significant(P<0.05).The B +C blood flow type in the observation group on the luteal support day increased significantly compared with the B + C blood flow type in the control group;the B + C blood flow type in the observation group on the luteal support day increased significantly compared with the B + C blood flow type on the PRP infusion day.PI and RI were lower in the observation group on the luteal support day compared with the control group,and the difference was statistically significant(P<0.05);PI and RI were lower in the observation group on the luteal support day compared with the PRP perfusion day,and the difference was statistically significant P<0.05).Conclusion(1)AT the present study,HEC-151 cells were cultured in vitro with different concentrations of PRP,and it was found that the proliferation and migration ability of the cells increased with the increase of PRP concentration in a dose-dependent manner at 2% PRP concentration,while the above effects showed a decrease after using higher PRP concentration,suggesting that the effects of different concentrations of PRP on HEC-151 cells were different.The effect of PRP on the proliferation and migration of HEC-151 cells and the relationship between its concentration were investigated for the first time.(2)PRP can replace FBS and single growth factor to culture HEC-151 cells.(3)In animal experiments,PRP intrauterine perfusion treatment of thin endometrium mice promoted proliferation and repair of damaged endometrium,improved endometrial tolerance,increased endometrial blood flow and reduced endometrial fibrosis.This effect may be related to the fact that PRP affects the biological function of HEC-151 cells.It increases the number of implantation sites in the damaged uterus and improves the live birth rate in mice with thin endometrium.(4)In clinical practice,PRP intrauterine perfusion increased the thickness of thin endometrium,improved endometrial blood flow and increased embryo implantation and clinical pregnancy rates. |
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