| ObjectiveRepeated Implantation Failure(RIF)is generally determined when accumulatively transferred several high-quality embryos fail to successfully achieve clinical pregnancy following multiple treatment cycles of in vitro fertilization or intracytoplasmic sperm injection(IVF/ICSI).At present,it is one of the most urgent problems which should be solved in the field of assisted reproduction,which causes extreme physical and mental suffering to patients and their families.The pathogenesis of RIF is complicated.In recent years,with the continuous rise of reproductive immunology,immunological factors at the maternal-fetal interface have become a research focus,especially the role of peripheral blood immune cells in maternal-fetal communication and maternal remodeling.Among existing research,most believes that the frequency of T lymphocytes,NK cells and other related immunocompetent cells expression greatly affects the stability of the immune system at the maternal-fetal interface during the period of embryo implantation.However,the expression and mechanism of related cytokines in RIF have not been clarified clearly.In this study,we studied the expression of IL-2,IL-4,IL-6,IL-10,IL-17A,IFN-y,TNF-α,TNF-γ,GM-CSF,G-CSF,Th1/Th2 cell balance and NK cell in peripheral blood of RIF patients and those with successful clinical pregnancy after first transplantation to explore the influence and the regulatory mechanism of immunological factors in peripheral blood on embryo implantation.Then it may act as the biological markers of RIF to provide accurate basis for clinical diagnosis and treatment.Methods1.From March 2020 to December 2020,41 cases with repeated implantation failure were collected from the Reproductive Center of Qilu Hospital of Shandong University as RIF group,29 cases with a successful pregnancy in the first IVF/ICSI-ET cycle were collected as the control group.Of the 41 patients in the RIF group,18 cases obtained clinical pregnancy in this cycle,of which 10 cases obtained complete follow-up data after pregnancy;and 14 cases of the 29 patients in the control group obtained complete follow-up data after pregnancy.2.Both groups of patients received freeze-thaw embryo transfer(FET)in this treatment cycle.Patients in the RIF group were given relevant individualized treatment in this cycle,including the appropriate application of low-dose aspirin,prednisone,low molecular weight heparin and immunosuppressants(cyclosporine,hydroxychloroquine,and so on).3.Peripheral blood was drawn from the two groups of patients in the middle of the luteal phase of the cycle before transplantation and the time when a live intrauterine pregnancy was diagnosed by transvaginal ultrasound.4.Flow high-throughput multi-factor detection technology was used to detect IL-2,IL-4,IL-6,IL-10,IL-17A,IFN-γ,TNF-α,TNF-β,GM-CSF and G-CSF in peripheral blood.While the percentage of NK cells in peripheral blood was detected by flow cytometry.5.To study the differences and changes of multiple cytokine levels,Th1/Th2 balance and NK cell expression in peripheral blood before and after pregnancy in the both groups and also analyze the relationship between those immunological factors and related clinical indicators.Results1.There was no significant difference in the type of infertility,number of abortions,BMI,AMH,basal endocrine and thyroid function between the two groups(P>0.05),The age and infertility years of the RIF group were significantly higher,while the thickness of the endometrium was significantly lower(P<0.05).2.Compared with the control group,the patients in the RIF group had lower MII cleavage rate and the total dosage of Gn were also lower,both of which have significant difference(P<0.05).While there were no significant differences between RIF group and control group in terms of fertilization method,duration of Gn,number of oocytes retrieved,number of MII cleavage,number of 2PN,2PN rate,number of transferable embryos,number of high-quality embryos and high-quality embryo rate(P>0.05).3.Compared with the control group,the level of IL-6 in the middle luteal phase of the RIF group was significantly higher,while IL-10 and G-CSF were lower(P<0.05).In addition,the levels of IL-2,IL-4,IL-17A,IFN-γ and GM-CSF in RIF group were higher than those of the control group,the levels of TNF-α and TNF-β were lower,but the differences were not statistically significant(P>0.05).4.Compared with the control group,the ratios of IL-2/IL-10 and IFN-γ/IL-10 in the middle luteal phase of the RIF group were higher,both of which have significant difference(P<0.05).In addition,IL-2/IL-4,TNF-α/IL-10,TNF-β/IL-4 and TNF-β/IL-10 were higher,IFN-y/IL-4 and TNF-α/IL-4 were lower,but the differences were not statistically significant(P>0.05).5.In the middle luteal phase,there was no significant difference in NK cells between the RIF group and the control group(P>0.05).6.In the RIF group,the serum IL-4 level in the middle luteal phase was positively correlated with the basal FSH level(r=0.3385,P=0.0304),the TNF-β level was negatively correlated with the thickness of the endometrium(r=-0.3232,P=0.0419),and the percentage of NK cells was positively correlated with age(r=0.3455,P=0.0269);while in the control group,IL-4 was negatively correlated with age(r=-0.4744,P=0.0093),the level of IL-6 was negatively correlated with the years of infertility(r=-0.3 731,P=0.0462),and the percentage of NK cells was positively correlated with the TSH level(r=0.4274,P=0.0208).In addition,other cytokines and NK cells had no obvious correlation with each clinical index.7.In the RIF group,the levels of serum IL-10 and TNF-α after pregnancy were significantly higher,the ratio of IL-2/IL-10 was significantly lower than that before pregnancy(P<0.05).While there was no significant difference regarding the expression of NK cells in peripheral blood before and after pregnancy(P>0.05).8.In the control group,before and after pregnancy,the level of serum IL-6 after pregnancy was significantly higher,the ratio of TNF-α/IL-4 was significantly lower than that before pregnancy(P<0.05).While there was no significant difference regarding the expression of NK cells in peripheral blood before and after pregnancy(P>0.05).9.After pregnancy,the serum G-CSF level and TNF-α/IL-4 of the RIF group were higher,while the expression of NK cells was lower,both of which have significant differences(P<0.05).10.There was no significant difference in the expression of β-HCG between the two groups,which was measured at 10-12 days after transplantation(P>0.05).Conclusions1.During the peri-implantation period,the patients of the RIF group have higher serum IL-6 levels and lower IL-10 and G-CSF levels,showing that excessive of IL-6 or deficient in IL-10 and G-CSF can adversely affect embryo implantation,which may be an immunological factor that leads to repeated implantation failures.2.During the peri-implantation period,the overall trend in the peripheral blood of patients with RIF is that the level of Th1 type cytokines with pro-inflammatory activity increases,while the level of Th2 type anti-inflammatory cytokines decreases,which means that the ratio of Th1/Th2 increases.Therefore,we can speculate that Th1/Th2 immunity imbalance may be one of the immunological mechanisms leading to the failure of embryo implantation.3.Most of the cytokines in the peripheral blood of RIF patients increase after pregnancy,but Thl/Th2 balance gradually shifts to Th2 type,which may be beneficial to embryo implantation and the maintenance of normal pregnancy.4.The level of IL-6 and NK cells in RIF patients decreases significantly after pregnancy,which may be related to the application of individualized treatment to the patients with RIF in this cycle,or may be related to the limitation of sample size,and further research is still needed.5.Serum Thl/Th2 and associated cytokine levels are involved in the pathogenesis of RIF.Monitoring the changes of Th1/Th2 ratio and associated cytokines in peripheral blood of RIF patients is expected to be an effective predictor of pregnancy outcome after IVF/ICSI-ET,which may become a new target for RIF immunotherapy. |
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