| The gonadotropin-releasing hormone（Gn RH）/Gn RH receptor（Gn RHR）system played an important role in female reproductive tissues and participated in the process of the local invasion and embryo implantation of trophoblast cells in the form of autocrine/paracrine.In recent years,the synthetic analogues of Gn RH,including agonists（Gn RH-a）and antagonists（Gn RH-ant）,have been widely used in assisted reproductive technology.The advantages of Gn RH-ant included milder stimulation,shorter duration of treatment,and lower risk of ovarian hyperstimulation syndrome（OHSS）.However,many studies indicated that the clinical pregnancy rate of Gn RH-ant protocol was lower than that of Gn RH-a protocol,the possible reasons might be correlated with endometrial receptivity.Our clinical data also confirmed this viewpoint,which suggested that the negative effects of Gn RH-ant on clinical pregnancy outcomes arised primarily from its negative effects on endometrial receptivity,rather than embryo quality;However,the related molecular mechanisms are still not completely clear at present.Therefore,it is particularly important to further elucidate the molecular regulatory mechanisms of Gn RH-ant on endometrial receptivity.Previous studies have found that C-kit was expressed in oocytes,and the activation of this receptor was important to the normal development of follicles,embryo development and embryo implantation.Recent studies have reported the presence of C-kit expression in human and mouse endometrium and suggested that C-kit receptor might be closely related to the improvement of endometrium receptivity.However,the specific mechanisms of Gn RH-ant action was not completely understood,and whether the effects of Gn RH-ant on receptivity involved C-kit has not been reported.To this end,this study used human primary stromal cells to conduct in-depth research on the molecular mechanisms of Gn RH-ant on endometrial receptivity with Gn RH receptor activator and inhibitor,RT-PCR,Western Blot,immunofluorescence staining,cell migration and adhesion,Gn RHR gene knocked down and C-kit overexpressed and other experimental techniques.We found that patients treated with Gn RH-ant had a higher rate of high-quality embryos,while the embryo implantation rate,clinical pregnancy rate and live birth rate were significantly lower than those treated with Gn RH-a,indicating that Gn RH-ant protocol was likely to have a negative impacts on endometrial receptivity.In order to reveal its potential mechanisms,we conducted in vitro cell experiments and found that Gn RH-ant significantly down-regulated the expression of endometrial receptivity marker HOXA10 in decidualization.In addition,Gn RH-ant remarkably inhibited the migration of endometrial stromal cells and their adhesion to trophoblast cells.Subsequent Western Blot results showed that Gn RH-ant inhibited the expression and activation of C-kit in stromal cells,and the expression of MMP-9,a downstream protein closely related to cell migration,was also significantly decreased.To verify whether the inhibitory effect of Gn RH-ant was acted through C-kit,we subsequently added the C-kit inhibitor（imatinib）to decidual stromal cells and found that the expression level of MMP-9 and cell migration ability decreased significantly.Subsequent cell adhesion experiments showed that Gn RH-ant could obviously inhibit the adhesion between endometrial stromal cells and trophoblast cells.When imatinib was added to the stromal cells,the adhesion of the two cell groups was also significantly weakened.In order to verify the relationship between Gn RHR and C-kit,after Gn RHR knockdown by si RNA,compared with Gn RH-a group,the overall level and activation level of C-kit as well as the expression levels of HOXA10 and MMP-9 were still inhibited by Gn RH-ant treatment.At the same time,the migration ability and adhesion ability of Gn RH-ant group were significantly lower than those of control group.After overexpression of C-kit,the migration ability of endometrial stromal cells and the expression of MMP-9 were significantly increased,and the adhesion ability of deciduated stromal cells and trophoblast cells was significantly enhanced.This study preliminarily confirmed that,in decidual stage of endometrium,Gn RH-ant could down-regulate the expression level of C-kit and weaken its activation,which leaded to decrease the expression of downstream protein MMP-9,inhibition of cell migration ability as well as blockade of adhesion between stromal and trophoblast cells.Consequently,these could impair endometrial receptivity and embryo implantation.The results of this study reveales a molecular mechanism of Gn RH-ant effects on endometrial receptivity,which opens up new ideas for clinical optimization of IVF-ET treatment and has important theoretical significance and potential application value.Part I:The comparison of the clinical outcomes between Gn RH antagonist and agonist protocol in vitro fertilization-embryo transferObjective:To investigate the difference of clinical outcomes between two groups of patients treated with Gn RH antagonist and depot Gn RH agonist protocol during in vitro fertilization-embryo transfer cycles.Methods:The clinical data of 18026 infertile patients who underwent fresh IVF-ET cycles in Center for Assisted Reproduction of Jiangxi Maternal and Child Health Hospital from January 2014 to September 2021 were retrospectively analyzed.All patients were 20-34 years old and had regular menstruation,>1 year of infertility,and antral follicle count（AFC）of 5-20.There were 16750 patients in Gn RH-a group and 1276 patients in Gn RH-ant group.Potential confounding factors of the two groups were controlled by propensity score matching method,and the matching ratio was 1:4.After matching,950 cases were in Gn RH-ant group and 3800 cases were in Gn RH-a group.Results:After propensity score matching（PSM）,there was no statistical difference in the basal levels between two regimens in the HCG-day P level and normal fertilization rate.In the Gn RH-ant group,the initial Gn dose（211.45±69.24 vs.174.11±70.87 IU,p<0.001）,the HCG-day LH（1.80 vs.0.90 m IU/ml,p<0.001）,and high-quality embryo rate（29.33%vs.27.89%,p=0.029）were higher than the Gn RH-a group.While the total Gn dose（1969.99±640.97 vs.2346.24±9742.98 IU,p<0.001）,duration of Gn stimulation（8.93±1.72 vs.11.22±2.02 days,p<0.001）,endometrial thickness on the HCG day（10.4±2.38 vs.11.13±2.54 mm,p<0.001）,E2 on the HCG day（1718 vs.1872.5 pg/ml,p<0.001）,fresh cycle transfer rate（38.21%vs.80.58%,p<0.001）,the incidence of moderate to severe OHSS（0.21%vs.1.29%,p=0.04）,and the number of oocytes（9 vs.11,p<0.001）were lower in the Gn RH-ant group.There were 363 transfer cycles in the Gn RH-ant group and 3062 transfer cycles in the Gn RH-a group.Compared with the Gn RH-a group,the embryo implantation rate（40.22%vs.51.92%,p<0.001）,positive HCG rate（63.64%vs.73.51%,p<0.001）,clinical pregnancy rate（55.37%vs.66.53%,p<0.001）,and live birth rate（47.66%vs.58.1%,p<0.001）were significantly lower in the Gn RH-ant group.Conclusions:Comparing the Gn RH-a protocol and Gn RH-ant protocol commonly used in IVF-ET treatment in our center,we found that although the rate of high-quality embryos in patients with Gn RH-ant protocol was significantly higher than that in patients with Gn RH-a protocol,the HCG day endometrial thickness,embryo implantation rate,clinical pregnancy rate and live birth rate were significantly lower in the Gn RH-ant protocol,implying that Gn RH-ant might have a negative effect on endometrial receptivity.Part II: The molecular mechanism of Gn RH antagonist affecting decidual stromal cell migration and adhesionObjective: To investigate the molecular mechanism of Gn RH antagonist on migration and adhesion of decidual stromal cells.Methods: Human endometrial cells were collected for primary culture,and treated with relevant receptor antagonists and activators.RT-PCR,Western Blot,immunofluorescence staining,cell migration and adhesion were experimentally employed to elucidate the molecular mechanism.Results:（1）We isolated human endometrial stromal cells and used immunofluorescence and Western blot to confirm that Gn RH receptor and C-kit were expressed abundantly in human endometrial stromal cells,suggesting that Gn RH receptor and C-kit might play a potential role in regulating the biological functions of human endometrial stromal cells.（2）The expression of endometrial receptivity marker HOXA10 was significantly reduced in the stromal cells after treating with Gn RH-ant,suggesting that Gn RH-ant negatively regulated endometrial receptivity.（3）The migration ability of endometrial stromal cells was significantly inhibited after Gn RH-ant treatment.Meanwhile,compared with Gn RH-a group,the expression of MMP-9 protein,which was closely related to cell migration ability,was significantly decreased in Gn RH-ant group.（4）After establishing the co-culture system of human stromal cells and JAR trophoblast spheroids,simultaneously treated with Gn RH-a or Gn RH-ant,the cell adhesion ability of Gn RH-ant group was significantly decreased（vs Vehicle control）.（5）The expression of C-kit protein in Gn RH-ant group was significantly reduced,and the activation degree of this receptor was also significantly decreased.However,there was no difference in receptor ligand SCF expression.（6）After treatment with C-kit inhibitor imatinib for 24 h,the migration ability of decidual stromal cells was obviously reduced,and the expression of MMP-9 was also significantly reduced.（7）In the co-culture system of human stromal cells and JAR trophoblast spheroids,C-kit inhibitor imatinib was simultaneously treated for 4 h,24 h,48 h and 72 h,respectively.The results showed that the adhesion between decidual stromal cells and trophoblast cells continued to decrease with time after imatinib treatment.（8）After Gn RHR knockdown,the administration of Gn RH-ant treatment still suppressed the expression of C-kit（overall and activation level）,HOXA10 and MMP-9 expression levels（vs the Gn RH-a group）,the migration ability of the Gn RH-ant group was still lower in comparison with the Gn RH-a group.In co-culture with JAR trophoblast spheroids,the adhesion percentage at 2 h was lower in both Gn RHR-silenced groups（the Gn RH-ant group and the Gn RH-a group）vs the NC group,while this percentage was lower in the Gn RH-ant group than Gn RH-a group,and this trend was more pronounced at 72 h.（9）After overexpression of C-kit,the migration ability of endometrial stromal cells was significantly increased,the expression of MMP-9 was increased,and the adhesion between stromal cells and trophoblast spheroids was enhanced.Conclusion:Gn RH-ant might affect endometrial receptivity by weakening the function of ckit receptor through the following mechanism: Gn RH-ant antagonized Gn RHR receptor,subsequently reduced the expression of C-kit and its activation,as well as the expression of MMP-9,which in turn impaired the migration ability of endometrial stromal cells,hindered the adhesion process between embryos and endometrial mesenchymal cells,and finally resulting in embryo implantation failure.In addition,Gn RH-ant might also negavitely regulate the endometrial receptivity through nonGn RHR-dependent signals. |
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