The Effect Of Dyslipidemia On Endometrial Receptivity And Mechanism Of CXCL8 In Embryo Implantation

Background: Obesity is not only related to the occurrence and development of dyslipidemia,hypertension and diabetes,but also closely related to the reproductive health of childbearing-aged women.Obese women have reduced fecundity,increased risk of infertility and recurrent spontaneous miscarriage.In the assisted reproductive technology(ART)scenario,some obese infertile women still face the risk of implantation failure even after the high-quality embryo transfer,indicating that their endometrial receptivity(ER)may be affected.Obesity is an abnormal accumulation of adipose tissue caused by lipid metabolism disorders.Dyslipidemia is one of the direct manifestations of lipid metabolism disorder,and highly related to the occurrence and development of obesity and cardiovascular diseases.Unlike obesity,which has been widely studied,few studies have explored the impacts and potential mechanisms of maternal blood lipid levels on pregnancy outcomes after ART attempts.Several studies revealed that dyslipidemia was related to adverse pregnancy outcomes.It needs further clarification whether it should be blamed for the poor oocyte-embryo quality or the disrupted ER.On the one hand,abnormal blood lipid levels are related to the embryo quality after IVF,although it is still debatable.On the other hand,there are evidence that fatty acids,lipid derivatives such as lysophosphatidic acid,prostaglandins,and endogenous cannabinoid systems play crucial roles in embryo implantation and decidualization.As such,this study hypothesizes that dyslipidemia may affect the pregnancy outcome of infertile women after ART attempts by interfering with ER.Relevant research had been conducted to explore the potential mechanisms.Objective:Part Ⅰ: To explore the impact of dyslipidemia,specific types of dysregulated blood lipids,and the degree of elevated lipid levels on the pregnancy outcomes of the first IVF/ICSI-ET cycle among infertile patients.Part II: To explore the effects of dyslipidemia on the endometrial window of implantation(WOI)in patients with recurrent implantation failure(RIF),and screen for potential ER associated key gene.Part III: To verify the m RNA and protein expression levels of the potential ER associated key gene CXCL8,and SOD2 which may interact with the CXCL8.To explore the possible mechanisms by which CXCL8 participates in the embryo implantation.To explore the possible mechanism by which dyslipidemia affects CXCL8 m RNA expression and protein secretion.Methods:Part Ⅰ: This part retrospectively analyzed the clinical data of 5030 infertile patients who underwent their first IVF/ICSI-ET cycle.Patients were divided based on the presence or absence of dyslipidemia,and baseline data and pregnancy outcomes were compared between the two groups.Propensity score matching(PSM)was performed to adjust for the potential confounders.Women with dyslipidemia were matched in a 1:1ratio to the control group participants.Baseline characteristics and pregnancy outcomes were compared between the two groups.Subgroup analysis based on the degree of elevated blood lipids was also conducted,and patients were divided into the borderline increase group,the increase group and the control group.The multivariable logistic regression model was adopted to demonstrate the association between dyslipidemia,specific types of dysregulated blood lipids,the degree of elevated lipid levels and live birth respectively.Each model was adjusted for age,BMI,main aetiology of infertility,ovarian stimulation protocols,endometrial thickness and number of transferred embryos.Part II: According to the inclusion and exclusion criteria,eligible RIF patients were recruited at the Reproductive Medicine Center of Xiangya Hospital,Central South University,from June 2020 to June 2022.Participants were assigned to the experimental group and the control group based on whether they were willing to perform rs ERT.Based on blood lipid levels,both groups were further divided into the dyslipidemia group and the non-dyslipidemia group.Endometrial samples were collected from the experimental group on P+5/LH+7/ovulation+5 days for rs ERT,and personalized embryo transfer(p ET)was guided by the result in the subsequent frozen-thawed embryo transfer(FET)cycles.The control group did not perform rs ERT and only underwent conventional embryo transfer.Pregnancy outcomes were compared among the four groups.By comparing the endometrial transcriptomic characteristics of patients with dyslipidemia who also had displaced WOI and patients had normal WOI and blood lipid levels,the ER associated key genes were identified.Part III: By adopting IHC,RT-q PCR,Western Blot and ELISA to verify the expressions of CXCL8 and SOD2 m RNA and protein in the two groups.The effects of CXCL8 on migration and invasion of HTR-8/SVneo cells were determined by the wound healing test and the Transwell test.The effect of CXCL8 on adhesion of HTR-8/SVneo cells was determined by CXCL8 knockdown model in Ishikawa cells.The effects of lipid metabolism disorder on CXCL8 m RNA and protein expression were determined by palmitic acid(PA)intervention.Based on blood lipid levels and whether WOI was displaced or not,RIF patients who received rs ERT in Part II were divided into four groups:non-dyslipidemia and normal WOI,non-dyslipidemia and displaced WOI,dyslipidemia and normal WOI,dyslipidemia and displaced WOI.The endometrial CXCL8 and SOD2 m RNA expression were determined by RT-q PCR to preliminarily explore the relationship between the two genes.Results:Part Ⅰ:1.According to the presence or absence of dyslipidemia,1903 infertile patients were assigned to the dyslipidemia group and 3127 patients were assigned to the control group.There were significant differences between the two groups in terms of age,BMI,infertility duration,gravidity,parity,main aetiology of infertility,blood lipid levels,basal FSH,AFC,ovarian stimulation protocols,duration of Gn,total Gn dose,hormonal levels on h CG day,endometrial thickness,endometrial type,and fertilization method(P<0.05).Specifically,the endometrial thickness on h CG day in the dyslipidemia group was significantly lower than the control group,while the proportion of C-type endometrium was significantly increased in the dyslipidemia group(P<0.05).The number of oocytes retrieved and MII oocytes,MII oocyte rate,number of good-quality embryos,good-quality embryo rate,and number of transferred embryos did not exhibit significant between-group differences.Compared with the control group,dyslipidemia group had higher early and late miscarriage rates,lower term birth and live birth rates(P<0.05).Other pregnancy outcomes were comparable between the two groups(P>0.05).2.By adopting PSM,the two groups were matched for age,BMI,main aetiology of infertility,ovarian stimulation protocols,endometrial thickness and number of transferred embryos.There were 1686 patients in the dyslipidemia group and 1686 in the control group.Except for blood lipid levels,E2 level and endometrial type on h CG day,the two groups were comparable in terms of the baseline characteristics.Specifically,the proportion of C-type endometrium on h CG day in the dyslipidemia group was higher than that in the control group(P<0.05).Term birth and live birth rates in the dyslipidemia group were significantly lower than those in the control group(P<0.05).3.By adopting multivariate logistic regression analysis,the dyslipidemia group had a significantly lower live birth rate compared to the control group(adjusted OR 0.87,95% CI 0.77-0.98,P=0.026).Moreover,abnormal TC concentrations(≥5.20 mmol/)were negatively associated with the live birth rate(adjusted OR 0.86,95% CI 0.75-0.98,P=0.028).4.The subgroup analysis included 3127 infertile women without dyslipidemia,1214 women with borderline increased lipid concentrations and 567 infertile women with increased serum lipid concentrations.Compared with the control group,the increase group had lower term birth and live birth rates,and higher late miscarriage rate(P<0.05).The differences between the borderline increase group and the control group were not significant(P>0.05).5.By adopting multivariate logistic regression analysis,women with increased serum lipid concentrations were less likely to have a live birth compared with the control group(adjusted OR 0.78,95% CI 0.64-0.94;P=0.011).The borderline increase grcoup had less possibility of a live birth even though the difference was not significant(adjusted OR 0.93,95% CI 0.81-1.07;P = 0.300).Part II:1.There were 316 RIF patients who met the inclusion and exclusion criteria,including 159 in the experimental group and 157 in the control group.There were 64 patients with dyslipidemia in the study group,while61 people with dyslipidemia in the control group.In the experimental group,compared to the non-dyslipidemia counterparts,women with dyslipidemia had significantly higher displaced WOI rate(62.50% vs.34.74%,P=0.001).Compared to the control group,rs ERT-guided p ET significantly improved the positive β-h CG rate,intrauterine pregnancy rate and embryo implantation rate in the experimental group(P<0.05).2.By comparing the endometrial transcriptomic characteristics of patients with dyslipidemia who also had displaced WOI and patients had normal WOI and blood lipid levels,239 DEGs were identified,including78 up-regulated genes and 161 down-regulated genes.GO enrichment analysis showed that the four most significant biological processes for up-regulated DEGs were response to ethanol,mitotic cell cycle,negative regulation of ossification and regulation of mitotic nuclear division.Down-regulated DEGs mainly involved in aging,inflammatory reaction,inter-cellular signal transduction,immune response,positive regulation of cell migration and adhesion,decidualization,and regulation of vascular permeability in acute inflammatory reactions.KEGG enrichment analysis showed that the main pathways for up-regulated DEGs were neuroactive ligand-receptor interaction,thyroid hormone synthesis,insulin secretion,progesterone-mediated oocyte maturation,glucagon signaling pathway and insulin resistance.The main pathways for down-regulated DEGs were complement and coagulation cascades,viral protein interaction with cytokine and cytokine receptor,cytokine-cytokine receptor interaction,drug metabolism and pathogenic Escherichia coli infection.3.Using protein-protein interaction analysis,CXCL8 might be one of the key genes related to ER.There were 23 genes that may interact with the CXCL8,namely LBP,ATP12 A,TNFSF10,FLT3,TNFAIP6,LIF,CHI3L1,CCL21,ARG2,IGFBP1,MMP10,DEFB1,CD55,MUC5 B,TYMP,IL19,GPT,F2,CXCL14,SOD2,THBD,CD274 and EDN1.Part III:1.RIF patients with dyslipidemia and displaced WOI had significantly lower m RNA and protein expression levels of CXCL8 and SOD2(P<0.05),when compared to their counterparts with normal blood lipid levels and WOI.2.The wound healing assay showed after 8 and 16 hour-exposure to CXCL8(1-6ng/ml),the wound healing area significantly increased compared to the control group;while the wound healing area decreased when the concentration further increased to 10ng/ml(P<0.05).3.The Transwell assay showed that 1-6ng/ml CXCL8 significantly increased the migration and invasion ability of HTR-8/SVneo cells;while the ability began to decrease when the concentration further increased to10ng/ml(P<0.05)。4.The CXCL8 knockdown model of Ishikawa cells was successfully established.Compared to the NC si RNA group,the CXCL8 m RNA expression decreased 50% and CXCL8 protein secretion was significantly lower in the supernatant of CXCL8 si RNA group(P<0.05).The adhesion rate of HTR-8/SVneo cell spheres in the CXCL8 si RNA group was significantly lower than that in the NC si RNA group and the WT group(P<0.05).5.After 24 hour-exposure to the low dose of PA(80μmol/l),CXCL8 m RNA expression level was significantly lower than the control reagent group(P<0.05).SOD2 m RNA and supernatant CXCL8 secretion levels were comparable among the three groups.After 48 hour-exposure to the low dose of PA(80μmol/l),CXCL8 m RNA expression was lower than the control reagent group(P<0.05).The SOD2 m RNA level of 80μmol/l PA and control reagent group were significantly lower than the blank group(P<0.05).The supernatant CXCL8 secretion level in the PA group was significantly lower than the blank group(P<0.05).6.RIF patients with displaced WOI had significantly lower CXCL8 mRNA expression compared to their normal WOI counterparts,no matter whether they were dyslipidemic or not(P<0.05).The highest CXCL8 m RNA expression level was observed in patients had normal WOI and blood lipid levels,and the lowest level was seen in patients with dyslipidemia and displaced WOI.Moreover,patients with normal blood lipid level and displaced WOI had lower levels of CXCL8 m RNA expression than patients who were dyslipidemic but with normal WOI(P<0.05).The relative expression trends of SOD2 m RNA were similar to the CXCL8.The SOD2 m RNA expression was highest in the non-dyslipidemia and normal WOI group,while the dyslipidemia and displaced WOI group had the lowest level.Moreover,the SOD2 m RNA expression in the dyslipidemia and displaced WOI group was lower than the non-dyslipidemia and normal WOI group,and non-dyslipidemia and displaced WOI group(P<0.05).Conclusions:Part Ⅰ:1.Infertile women with dyslipidemia had a lower live birth rate following the first complete IVF/ICSI cycle compared with the control group,which might be ascribed to their compromised ER.2.Before ART’s attempts,dyslipidemia,especially serum TC level≥5.20 mmol/l,and increased degrees of serum lipid concentrations(TG,TC or LDL-C)were negatively associated with the live birth rate.Part II:1.Among RIF patients,participants with dyslipidemia had a higher displaced WOI rate than those with normal blood lipids.2.By adopting bioinformatics analysis,CXCL8 may be one of the key genes affecting embryo implantation under lipid metabolism disorders.Part III:1.CXCL8 could improve the migration and invasion ability of HTR-8/SVneo cells,and promoted the adhesion between HTR-8/SVneo and Ishikawa cells,suggesting that CXCL8 may be one of the most crucial chemokines participating in embryo implantation.2.The high-fat environment to some extent inhibits the m RNA expression of CXCL8 and SOD2,and CXCL8 protein secretion in Ishikawa cells.Taken the endometrial expression of CXCL8 and SOD2 m RNA among the four groups into consideration,we hypothesized that the high-fat environment may influence the CXCL8 m RNA expression by affecting the anti-oxidant effect represented by SOD2 in the endometrium.However,the specific mechanism needs to be further clarified.