| Objectives To analyze the prevalence and influencing factors of recurrent implantation failure(RIF)in patients receiving assisted reproductive technology therapy by crosssectional study;On this basis,single cell sequencing,flow cytometry,in situ hybridization,immunofluorescence,3D imaging and bioinformatics were used to study the features of mouse uterine pericytes,and its regulation of the functions of uterus in different periods such as early uterine development,early pregnancy and peri-implantation,to explore the possible mechanisms of the uterine pericytes participation in RIF,including the distribution,markers,gene expression,signal pathways and immune response to inflammation of uterine pericytes;To find the specific molecular markers of uterine pericytes in early pregnancy.The results will lay a foundation for further revealing the etiology and mechanisms of RIF,and maybe provide new bases for discovering the diagnosis and treatment methods of RIF.Methods 1 The clinical data of 3318 cycles received ART in reproductive medicine department of a maternal and child health hospital from January 2018 to December 2020 were analyzed using a cross-sectional study method,and the prevalence of RIF was calculated.The clinical data of different pregnancy outcomes in patients with RIF and nonRIF cycles and ≥3 cycles were compared and analyzed.2 Whole tissue immunostaining,tissue clearing and two-photon 3D imaging were used to observe the dynamic distribution of uterine pericytes in newborn mice during the early stage of uterine development,and verified the expression of pericytes markers.Single-cell isolation by 10 X Genomics platform,single-cell sequencing of pericytes,the heterogeneity of uterine pericytes was predicted to be different from that of other tissue.The results were verified by immunofluorescence staining.3 A total of 16 adult CD1 female WT mice were selected and divided into 5 groups according to different treatments,including 4 of D4＿WT group,4 of D4＿LPS3h group(LPS was injected at D4 7:00 a.m.and sacrificed at D4 10:00 a.m.),and 4 of D4＿LPS24h group(LPS was injected at D3 10:00 a.m.andsacrificed at D4 10:00 a.m.),4 of D5＿IS group(implantation site uterine stromal cells from D5 wild-type mice),and 4 of D5＿NIS group(non-implantation site uterine stromal cells from the same mouse in D5＿IS group).The uterine pericytes of different treatment groups were obtained by fluorescence activated cell sorting(FACS)and RNA-seq was performed.Through analysis of differential genes such as GO,KEGG and GSEA,the possible functions of peri-implantation uterine cells were predicted.4 Some of the previously reported marker genes of pericytes in different tissues were selected for in situ hybridization(ISH)experiment verification,in order to explore the specific marker molecules of uterine pericytes except NG2.Results 1 There were 3184 cycles in the non-RIF group and 134 cycles in the RIF group,and the prevalence of RIF was 4.04%.The increase of maternal age in different cycles was a risk factor for RIF,and its OR(95% CI)was 1.127(1.081-1.175),P<0.001.Higher AMH value and total egg harvest were protective factors for RIF,and their OR(95% CI)were0.845(0.739-0.965)and 0.888(0.827-0.953),P values were 0.013 and 0.001,respectively.The increase of maternal age in different cycles was a risk factor for clinical pregnancy in patients with ≥3 cycles,and the OR(95% CI)was 1.098(1.015-1.188),P=0.019.Higher endometrial thickness was a protective factor for clinical pregnancy in patients with ≥3 cycles,and its OR(95% CI)was 0.786(0.665～0.929),P=0.005.2 Pericytes existed in the early stage of uterine development.NG2 positive pericytes signal appeared around CD31 positive endothelial cells from the first day of postnatal day(PND1).NG2 canmark pericytes in early pregnancy.During D1 to D8 of early pregnancy,there were some pericytes expressing ERα and PR,and this phenomenon was not obvious in other tissues.3The uterine pericytes obtained by FACS were specific,and the transcripts of uterine pericytes were significantly different in diverse conditions.The difference between the different treatment groups is small,the sample parallelism is good.Compared with the D4＿WT group,the upregulation genes of uterine pericytes in the D4＿LPS3h and D4＿LPS24h groups mainly enriched in up-regulated inflammatory pathways,such as natural immune regulation,positive regulation of defense response,cytokines signaling,positive regulation of cytokines production,response to LPS,leukocyte migration,cytokine receptors interaction signaling,chemokine signaling and other functions or pathways were significantly enriched.The down-regulated genes were mainly concentrated in the basal function of pericytes under normal physiological conditions,such as extracellular matrix composition,collagen fiber composition,muscle migration,muscle contraction,etc.Compared with D4＿WT group,up-regulated genes in D5＿IS group were mainly enriched in proliferation related functions and signaling pathways.Ed U staining results further confirmed this conclusion,the proportion of Ed U and NG2 double positive cells in D4＿WT group was 4.32%,while the proportion was as high as 29.98% in D5＿IS group.In addition,compared with D5＿NIS group,thedown-regulatedgenesin D5＿IS group was enriched in functions and signaling pathways that maintain vascular permeability.Such as vascular development regulation,positive regulation of cell adhesion,cell junction,endothelial cell migration,vascular permeability regulation and so on.This suggests that pericytes at D5 implantation site may be involved in the upregulation of vascular permeability.4 ISH showed that although all of these pericyte markers could label uterine pericytes in early pregnancy,some of them had non-specific signals in the muscular layer or stroma,such as Cox4i2,Kcnj8,and Foxs1.In contrast,Rgs5 was not expressed in the muscular layer,and its expression pattern in the uterus during early pregnancy was similar to that of NG2.Conclusions 1 The prevalence of RIF in the survey area was 4.04%.The increase of maternal age,lower AMH value and less total number of eggs harvested in different cycles were the influence factors of RIF.The increase of maternal age and lower endometrial thickness in different cycles were the influence factors of clinical pregnancy in patients with≥3 cycles.2 Uterine pericytes are formed in the early stage of uterine development.Compared with pericytes in other tissues,uterine pericytes have some heterogeneity.NG2 can label uterine pericytes in early uterine development and early pregnancy.The expression of estrogen and progesterone receptors in pericytes during early pregnancy suggests that pericytes may regulate endometrial thickness and uterine vascular permeability through expression of estrogen and progesterone receptors,thus contributing to the occurrence of normal implantation and recurrent implantation failure.3 The uterine pericytes at D5 implantation site not only have high proliferation ability,but also have the function of regulating vascular permeability.In addition,uterine pericytes in early pregnancy can also respond to LPS stimulation and participate in inflammatory response.Peri-implantation functions of pericytes may play an essential role in normal implantation and recurrent implantation failure.4 In early pregnancy,in addition to NG2,Rgs5 is also an important marker gene of uterine pericytes.Moreover,Tbx2 may be a specific transcriptional regulation factor in uterine pericytes.Figure 31;Table 24;Reference 354... |
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