GnRH antagonist weakens endometrial stromal cells growth ability by decreasing c-kit receptor expression

GnRH antagonist weakens endometrial stromal cells growth ability by decreasing c-kit receptor expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research GnRH antagonist weakens endometrial stromal cells growth ability by decreasing c-kit receptor expression Ding-Fei Xu, Pei-Pei Liu, Lu Fan, Qi Xie, Zhi-Qin Zhang, Jun Tan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-845752/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2022 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted 13 You are reading this latest preprint version Abstract Background Several surveys have reported that patients treated with gonadotropin-releasing hormone antagonist (GnRH-ant) protocol showed a significantly lower rate of implantation and clinical pregnancy compared to GnRH agonist (GnRH-a) protocol during in vitro fertilization-fresh embryo transfer. Subsequent studies imputed this poor outcome to the negative effects of GnRH-ant on endometrial receptive. However, the mechanisms were not fully understood. Methods The clinical data of 2815 patients undergoing fresh embryo transfer in our center were analyzed. Human endometrial stromal cells (ESCs) from healthy women undergoing elective pregnancy termination of a normal pregnancy at 8–10 weeks gestation were treated with GnRH-analogs or imatinib (c-kit receptor inhibitor). Results The clinical data showed that the endometrial thickness on HCG Day were significantly lower in GnRH-ant group. Although no difference of embryo quality in these two groups, GnRH-ant group showed remarkably decreased rate of HCG positive, embryo implantation and pregnancy. Moreover, GnRH-ant significantly reduced the proliferation and induced the apoptosis of ESCs. Furthermore, the expression and activation of c-kit receptor, which played pivotal roles during embryo implantation, were observably decreased by GnRH-ant. Inhibiting the activation of c-kit by imatinib remarkably suppressed the proliferation and promoted the apoptosis of ESCs. Additionally, the phosphorylation of AKT and expression of Cyclin D1, which were closely related with cellular growth, were distinctly lessened after treating with imatinib. Conclusions In summary, our study showed that GnRH-ant weakened the activization of c-kit receptor by decreasing its expression, causing the impaired growth ability of ESCs. Our findings provided a new insight into the effects of GnRH-ant on endometrium. Endocrinology & Metabolism GnRH-ant c-kit receptor ESCs growth ability Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Gonadotropin-releasing hormone antagonist (GnRH-ant), which could competitively bind with GnRH receptor (GnRHR) and reversibly inhibited hypothalamic-pituitary-gonadal axis, has been used in assisted reproduction for several decades from the first generation to the recent third generation [ 1 ]. Based on the superiority of convenience, flexibility and safety, GnRH-ant protocol has been one of the most commonly therapeutic schedules in in vitro fertilization-embryo transfer (IVF-ET) [ 2 ]. However, the unsatisfactory clinical outcomes of this protocol, compared to GnRH-a protocol, has restrained its promotion and application in clinic. Many clinical investigations have reported a lower fresh embryo implantation and pregnancy rates in GnRH-ant protocol than those in GnRH-a protocol [ 3 – 5 ]. Similarly, Bukulmez’s research also showed a lower HCG positive and clinical pregnancy rates in GnRH-ant protocol when compared to GnRH-a protocol under the no difference of embryo quality between these two protocols [ 6 ]. Additionally, these worse clinical outcomes from GnRH-ant protocol were only occurred in fresh embryo transfer but not in freezing embryo transfer [ 7 ], suggesting that the adverse effects of GnRH-ant on endometrium might be the key reason. In fact, many researches have demonstrated that the endometrial thickness could be an important factor which influenced the pregnancy after embryo transfer [ 8 – 11 ]. Patients with endometrial thickness less than 8 mm showed an obviously lower implantation and pregnancy rate [ 12 ]. Significantly, recent studies have proved that patients treated with GnRH-ant protocol showed a remarkably thinner endometrial thickness than patients treated with GnRH-a protocol [ 13 , 14 ]. These results implied that one of the adverse effects of GnRH-ant on endometrium was to weaken endometrial thickness. Nonetheless, the related molecular mechanism was still unclear. Previous studies have found the expression of c-kit receptor on animal embryo and endometrium [ 15 ], suggesting the crucial role of c-kit receptor in the process of animal embryo implantation. C-kit receptor belonged to a member of III type RTK family which could be stimulated by stem cell factor (SCF). Activated c-kit receptor was associated with multiple kinds of biological events, including proliferation, differentiation, migration and apoptosis, in many types of cells [ 16 , 17 ]. Our previous study has found the expression of c-kit receptor on human embryo and demonstrated that added exogenous SCF to activate c-kit receptor could significantly promote embryo development [ 18 ]. Recently, we surprisingly found the expression of c-kit receptor on human endometrial stromal cells (ESCs). Based on the regulatory function on the growth ability of many kinds of cells, we speculated that c-kit receptor might also influenced the growth ability of human ESCs. Furthermore, whether its regulatory effect is associated with GnRH-ant is still unknown. Therefore, the present study is to explore the effects of GnRH-ant and c-kit receptor on the growth of human ESCs, and to investigated the regulatory relationship between GnRH-ant and c-kit receptor. Methods Reagents The following reagents were used in this study: rabbit anti-GnRHR (Abcam, UK), mouse anti-c-kit (CST, USA), rabbit anti-c-kit (CST, USA), rabbit anti-phospho-c-kit (CST, USA), rabbit anti-AKT (CST, USA), rabbit anti-phospho-AKT (CST, USA), rabbit anti-Cyclin D1 (CST, USA), mouse anti-β-actin (Santa Cruz, USA), Donkey anti rabbit Alexa flour 488 (Thermo Fisher Scientific, USA), Donkey anti rabbit Alexa flour 594 (Thermo Fisher Scientific, USA), GnRH-a (Decapeptyl; Ferring), GnRH-ant (Cetrotide; Serono) and Imatinib (Biovision, USA). Patients The present study retrospectively analyzed the clinical data of 2815 patients undergoing fresh embryo transfer in Reproductive Medicine Center of Jiangxi Maternal and Child Health Hospital from Jan 2016 to Dec 2020. All patients were in good physical and mental condition. The inclusion criteria for all patients included age ≤ 37 years, body mass index (BMI) of 15–25 kg/m 2 , 1.1 < anti-Müllerian hormone (AMH) < 5.0, 5 ≤ antral follicle count (AFC) ≤ 20, and less than twice IVF-ET experiences. Women with a history of the following procedures or disorders were excluded: uterine malformation, ovarian surgery, radiotherapy or chemotherapy, premature ovarian failure, ovarian dysfunction, adenomyosis, polycystic ovarian syndrome, thyroid dysfunction, recurrent implantation failure (failed to achieve a pregnancy more than three times), submucosal fibroids, intrauterine adhesion, hydrosalpinx, and patients (women or men) with abnormal chromosomes. Study design and groups 2815 patients were divided into GnRH-ant group (563) and GnRH-a group (2252) according to the different protocols. In GnRH-ant group, recombinant human FSH (rhFSH, Merck-Serono, German) was used on day 2 or 3 of the menstrual cycle. The initial dosage (112.5–225 IU/day) was determined based on age, BMI, AFC, and AMH. The dose of rhFSH were adjusted according to ovarian response as monitored by ultrasonography and serum estradiol (E 2 ) levels. GnRH antagonist (Cetrorelix, Merck Serono, Switzerland) at a daily dose of 0.25 mg was started when the largest follicle exceeded 12 mm. Both GnRH antagonist and rhFSH were stopped and a single injection of 6000–8000 IU of hCG (Merck-Serono, German) was administered when the dominant follicle was ≥ 19 mm in diameter or at least 2 follicles were ≥ 18 mm in diameter. Oocyte retrieval was performed 36–40 hours later under transvaginal ultrasound guidance. In GnRH-a group, a standard full dose of gonadotropin-releasing hormone agonist (3.75 mg, GnRH agonist, Ipsen, France) was used in the second day of menstrual cycle for down regulation. Pituitary down regulation (Endometrial thickness ≤ 5 mm, serum FSH < 5 mIU/mL, LH < 5 mIU/mL, E 2 < 50 pg/mL) was confirmed with transvaginal ultrasound and endocrine examination after 28–30 days. The initial dosage (112.5–225 IU/day) was determined based on age, BMI, AFC, and AMH. The dose of rhFSH were adjusted according to ovarian response as monitored by ultrasonography and serum estradiol levels. The HCG trigger process was the same as described above. Embryo assessment The embryo assessment criteria were executed as the previously described [ 18 ]. A good quality embryo should consist of 7–9 blastomeres with a uniform size, and the fragment proportion should be less than 20% at day 3 for human 3PN embryos after fertilization. The good quality embryo rate refers to the number of good quality embryos divided by the total number of all embryos. Blastocyst formation was determined and graded by using the system of Gardner and Schoolcraft [ 19 ]. The blastulation rate refers to the number of blastocysts divided by the total number of all embryos. The good quality blastulation rate refers to the number of good quality blastocyst divided by the total number of all blastocysts. The assessment was made in a blinded manner by two embryologists. Primary human ESCs isolation, culture and treatment Primary human decidual ESCs were isolated from the decidual tissue of healthy multipara women (aged 25–32 years) undergoing elective surgical termination of a normal pregnancy at 8 to 10 weeks of gestation. The informed consents from all patients were obtained before the initiation of this study. According to the standard protocol [ 20 , 21 ], the human decidual tissue was minced and treated with type IV collagenase and DNase type I in a shaking water bath at 37℃ for 90 minutes. The cell digest was then passed through a 70 µm filter, both decidual stromal and epithelial cells were collected in tube. Then, decidual stromal cells were separated from epithelial cells with a 45 µm filter. The stromal cells were subsequently pelleted by centrifugation at 1000 rpm for 5 minutes. The cell pellets were washed once, resuspended, and cultured in Dulbecco modified Eagle medium containing 25 mM glucose, Lglutamine, antibiotics and supplemented with 10% fetal bovine serum at 37℃. ESCs was confirmed by detecting the expression of vimentin protein via immunohistochemical analysis (Supplementary Fig. 1). The cells were used after reaching 70–80% confluence. ESCs were treated for 120 hours with different concentrations of GnRH-a, GnRH-ant (10 − 8 , 10 − 5 , and 2x10 − 5 mol/L) or imatinib (0 µM, 4 µM, 8 µM, 16 µM, 24 µM, 32 µM). Cells were subsequently collected for cell proliferation assay, flow cytometry and western blot. Cell proliferation assay 5000 cells/well were planted into 96-well plates in quintuplicate wells. Normally, cells were placed in the culture medium with different concentrations of GnRH analogs for 24, 48, 72, 96 and 120 hours, a total of 10 µL CCK8 reagent (APExBio, USA) was added to each well. After incubating for 2 hours at 37°C, the absorbance at 450 nm per well was measured using microplate reader. Each group of experiment was repeated for three times. Flow cytometry The suspended cells were collected by centrifugation. Centrifuge 1000g, centrifugation time 5 minutes at 2–8℃. Cultured cells needed to be digested with EDTA-free trypsin, then terminated with serum-containing medium, centrifuged at 1000g for 5 minutes, supernatant removed, and washed with PBS resuspension. After centrifugal precipitation, the PBS was resuspended, transferred into the flow tube, washed once with PBS, centrifuged at 1000g for 5 minutes, and the supernatant was discarded. Cells were suspended with 400 UL × Annexin binding solution at a concentration of approximately 1×10 6 cells/ml. 5 UL Annexin V-FITC staining solution (BestBio, China) was added to the cell suspension, gently mixed and incubated for 15 minutes at 2–8℃ in the dark. After adding 10 UL PI staining solution, gently mixed and incubated for 15 minutes at 2–8℃ in the dark. Immediately detected by flow cytometry. Each group of experiment was repeated for three times Immunofluorescence staining The methods were described previously [ 22 ]. Cells were fixed with 4% paraformaldehyde at 4℃ for 10 minutes and washed with PBS three times. The cells were sealed with 2% triton-100 solution at room temperature for 30 minutes. The primary antibody diluted by blocking solution was added and incubated overnight at 4°C. The next day, cells were washed with PBS three times, 5 minutes each time. The fluorescent secondary antibodies were added and incubated for 1 hour at room temperature in the dark, then the cells were washed with PBS for 3 times, 5 minutes each time. Fluoroshield mounting medium (containing DAPI) with 1:10 dilution was added and stored at 4°C in the dark for fluorescence microscopy observation. Western blot Total proteins were extracted from cells using the RIPA lysis buffer containing protease inhibitors (Applygen, China) and phosphatase inhibitors (Sigma, USA). The protein concentrations were determined by NanoDrop 2000c spectrophotometer using BCA protein assay kit (Applygen, China). After loading equal amount of protein samples, SDS-PAGE (12% sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed. The proteins were then transferred to a PVDF membrane (Merck-Millipore, USA). After blocking with Tris buffered saline containing 0.05% Tween-20 (TBST) and 5% non-fat dry milk or 5% BSA for 1 hour, the membrane was incubated with corresponding antibodies at 4℃ overnight, washed in TBST, followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies for 1 hour. Visualization of the proteins was detected with ECL chemiluminescence. Beta-actin was used as a loading control. The intensity values were assessed and analyzed with Image J software. (n = 4 for per lane) Statistical analysis Statistical analyses were conducted by using SPSS 24.0 software (SASInstitute Inc.), and all data were expressed as means ± standard errors of the means (s.e.m.s) or percentage (%). Results among experimental groups were analyzed by student’s t-test or one-way ANOVA. For all tests, P-value < 0.05 was considered statistically significant. Results General characteristics No differences were found in general characteristics between these two groups (Table 1 ). Table 1 Comparison of the general information of the two groups of patients GnRH-ant (n = 563) GnRH-a (n = 2252) P-value Age (y) 30.99 ± 4.55 31.01 ± 4.2 > 0.05 BMI (Kg/m 2 ) 21.7 ± 5.35 21.67 ± 3.96 > 0.05 Duration of infertility (y) 4.22 ± 3.26 4.24 ± 3.1 > 0.05 Antral follicle count (n) 10.94 ± 3.96 10.95 ± 3.71 > 0.05 Endometrial thickness (mm) 7.03 ± 2.3 7.08 ± 2.19 > 0.05 Intrauterine adhesions rate (%) 5.51 5.24 > 0.05 Scar uterus rate (%) 10.83 10.83 > 0.05 AMH (ng/mL) 2.76 ± 1.04 2.77 ± 1.04 > 0.05 Basal FSH (mIU/mL) 6.19 ± 21.28 6.48 ± 15.01 > 0.05 Basal E2 (pg/mL) 66.86 ± 163.07 57.28 ± 238.25 > 0.05 Basal P (ng/mL) 1.2 ± 3.33 1.55 ± 4.26 > 0.05 Basal LH (mIU/mL) 4.44 ± 3.79 4.49 ± 3.45 > 0.05 Note: The data were expressed as mean ± SD. Abbreviations: BMI, Body Mass Index; AMH, anti-mullerian hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; P, progesterone; Comparison of the IVF outcomes between GnRH-ant and GnRH-a groups Firstly, we compared the effects of ovulation promotion between GnRH-ant and GnRH-a groups (Table 2 ). Among the two groups, the levels of E 2 and P on HCG day were similar ( P > 0.05 ), but the initial dose of Gn, duration of Gn used, total dose of Gn and endometrial thickness on HCG Day were significantly lower in GnRH-ant group than those in GnRH-a group ( P < 0.01 ), whereas the LH level on HCG Day was higher in GnRH-ant group ( P < 0.01 ). Then, we investigated the clinical outcomes from these two groups (Table 3 ). There were no differences in the number of oocytes retrieved, 2PN fertilization rate, 2PN cleavage rate, high quality embryo rate and high quality blastulation rate between these two groups ( P > 0.05 ). However, GnRH-ant group showed remarkably decreased rate of HCG positive, embryonical implantation and clinical pregnancy when compared to GnRH-a group ( P < 0.01 ), implying the worse clinical outcomes in GnRH-ant group. Table 2 Comparison of the effect of ovulation promotion between two groups of patients GnRH-ant (n = 563) GnRH-a (n = 2252) P-value Initial dose of Gn (IU) 212.92 ± 75.17 177.83 ± 69.62 < 0.01 Duration of Gn used (d) 9.11 ± 1.47 11.19 ± 1.92 < 0.01 Total dose of Gn (IU) 2132.1 ± 631.08 2391.2 ± 857.17 < 0.01 Endometrial thickness on HCG day (mm) 7.51 ± 2.27 9.09 ± 2.61 0.05 LH on HCG day (mIU/mL) 2.14 ± 1.51 1.06 ± 0.94 0.05 Note: The data were expressed as mean ± SD. Abbreviations: HCG, Human Chorionic Gonadotropin; LH, luteinizing hormone; E2, estradiol; P, progesterone; Table 3 Comparison of clinical outcomes between two groups of patients GnRH-ant (n = 563) GnRH-a (n = 2252) P- value Number of oocytes retrieved 11.26 ± 3.65 11.44 ± 3.58 > 0.05 2PN fertilization rate (%) 60.3 61.64 > 0.05 2PN cleavage rate (%) 95.75 95.82 > 0.05 High quality embryo at day 3 rate (%) 28.59 28.22 > 0.05 High quality blastulation rate (%) 36.2 34.01 > 0.05 Number of transfer embryos 1.91 ± 1.7 1.88 ± 1.83 > 0.05 HCG positive rate (%) 62.95 71.72 < 0.01 Implantation rate (%) 40 47.62 < 0.01 Clinical pregnancy rate (%) 55.41 64.04 < 0.01 GnRH-ant reduced the proliferation and induced the apoptosis of human isolated ESCs. CCK8 and Flow cytometry were used to investigated the effects of GnRH-ant on the growth ability of human isolated ESCs, which was confirmed by immunohistochemical analysis (Supplementary Fig. 1). Little differences were found in the concentrations of GnRH-ant and GnRH-a at 10 − 8 and 10 − 5 mol/L. When the concentration was elevated to 2x10 − 5 mol/L, GnRH-ant obviously reduced the proliferation and induced the apoptosis of ESCs (Fig. 1 ). GnRH-ant decreased the expression of c-kit receptor in human isolated ESCs. As shown in the results of immunofluorescence staining, GnRH receptor and c-kit receptor were co-expressed on human isolated ESCs, indicating a possible potential regulatory relationship between these two receptors (Fig. 2 A). After treating human isolated ESCs with the concentration of GnRH-ant and GnRH-a at 2x10 − 5 mol/L, we found significantly reduced expression of c-kit receptor and phosphorylation c-kit receptor in GnRH-ant group (Fig. 2 B), suggesting c-kit signaling was attenuated by GnRH-ant. C-kit receptor regulated the growth ability of human isolated ESCs through AKT signaling pathway. Cultured human isolated ESCs were treated with different concentrations of imatinib (0 µM, 4 µM, 8 µM, 16 µM, 24 µM, 32 µM) to confirm the ideal dosage required to suppress the c-kit receptor activation. The results showed that with the increase concentration of imatinib, the reduction of proliferation and induction of apoptosis in ESCs were increasingly notable (Fig. 3 ). Due to most of ESCs were dead at 32 µM, we selected 24 µM concentration as the ideal dosage to treat ESCs. The results showed that after inhibiting the c-kit receptor activation by 24 µM imatinib in ESCs, the phosphorylation of AKT and the expression of cyclin D1 were significantly decreased (Fig. 4 ). Discussion In the normal menstrual cycle, endometrium would go through three phases: menstrual period, proliferative period and decidual period. During the decidual phase, the endometrium transformed into a receptive tissue that was suitable for embryo implantation [ 23 , 24 ]. In this process, ESCs were the main cell type providing nutrition for the implanting embryos [ 25 ]. Therefore, the abundant numbers of ESCs would directly decide the success of embryo implantation. In the present study, although there were no differences in embryo quality between GnRH-ant and GnRH-a group, GnRH-ant group showed unsatisfied clinical outcomes including lower rates of HCG positive, embryo implantation and clinical pregnancy. It was worth noting that a thinner endometrial thickness was observed in GnRH-ant group, suggesting that the appropriate endometrial thickness might be conducive to embryo implantation. Similar with our findings, many recent studies also found that the thinner endometrium was evidently related to the worse clinical outcomes in fresh and frozen-thaw embryo transfer [ 26 – 29 ]. These results reminded that one of the reasons resulting in worse clinical outcomes in GnRH-ant group might be the adverse effect of GnRH-ant on growth ability of human ESCs. Accordingly, we isolated human ESCs from human endometrial tissues and treated cells with different concentrations of GnRH-analog to investigated the effects of GnRH-ant on the growth ability of ESCs. As we expected that GnRH-ant significantly depressed the proliferation and promoted the apoptosis of ESCs, confirming the adverse influences of GnRH-ant on ESCs growth. However, how GnRH-ant negatively regulates the growth of ESCs is still unclear. Previous studies have reported the positive effects of c-kit receptor on growth ability of multiple kinds of cells [ 30 – 33 ]. Furthermore, recent studies have found the expression of c-kit receptor on animal embryo and endometrium, and proposed its important role in the process of embryo implantation [ 15 ]. Surprisingly, we found the expression of c-kit receptor on human ESCs, indicating that c-kit receptor might also regulate the growth ability of human ESCs. Whereafter, we treated human ESCs with imatinib (c-kit receptor inhibitor) and found that after inhibiting the activation of c-kit receptor by imatinib, ESCs showed remarkably decreased proliferation capacity and increased apoptosis level. Moreover, the phosphorylation of AKT and the expression of cyclin D1, which were closely associated with cell growth, were significantly weakened. These findings supported that activating c-kit receptor could accelerate the growth ability of human ESCs. Based on the above findings, it is reasonable to speculate that whether the adverse effects of GnRH-ant on human ESCs growth ability is correlated with the inactivation of c-kit receptor? To further verify our hypothesis, we subsequently treated ESCs with GnRH-ant at 2x10 − 5 mol/L for 120 hours. The results showed that GnRH-ant significantly suppressed the expression of c-kit receptor followed by the attenuated phosphorylation of c-kit receptor, implying that GnRH-ant could weaken the growth ability of human ESCs by impairing the activation level of c-kit receptor through suppressing c-kit receptor expression. Notably, recent study directed by Chen et al have demonstrated that GnRH-ant could disrupt human endometrial epithelial cells (EECs) migration by reducing the CKB expression, which altered endometrial receptivity [ 34 ]. Complementally, our findings showed another negative effect of GnRH-ant on ESCs, which was the repression of ESCs growth capacity. Even so, the whole effects of GnRH-ant protocol on endometrium of women undergoing IVF-ET were still unclarified. Hence, further researches related the molecular mechanism are needed. Conclusion In brief, GnRH-ant attenuated the activation level of c-kit receptor by decreasing its expression in ESCs. Subsequently, this impaired effect could further inhibit the phosphorylation of AKT and lessened the expression of cyclin D1, which resulted in the reduction of growth ability of ESCs. The present study provides a new insight into the role of GnRH-ant and suggests that the adverse effects of GnRH-ant protocol on endometrium should be considered before using in IVF-ET. Abbreviations GnRH-ant: Gonadotropin-releasing hormone antagonist; GnRH-a: Gonadotropin-releasing hormone agonist; GnRHR: Gonadotropin-releasing hormone receptor; ESCs: Endometrial stromal cells; EECs: Endometrial epithelial cells; IVF-ET: In vitro fertilization-embryo transfer; RTK: Receptor tyrosine kinase; BMI: Body mass index; AFC: Antral follicle count; AMH: Anti-mullerian hormone; rhFSH: recombinant human FSH; Declarations Ethics approval and Consent to participate This study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Clinical Ethical Committee of Jiangxi Maternal and Child Health Hospital, and informed consents from patients were obtained before the initiation of the study. All the authors consented to participate in this study Consent for publication All the authors consented for publication. Availability of supporting data All data generated and analyzed in this study are included in this published manuscript. Competing interests The authors declared no potential conflicts of interest with respect tothe research, authorship, and/or publication of this article. Funding This work was supported by the National Natural Science Foundation of China (81960271 and 81960288), the Major Research and Development Project of Science and Technology Department of Jiangxi Province (20192BBGL70005 and 20203BBGL73140) and the China Postdoctoral Science Foundation (2019M662272). Authors’ contributions Conception and design of the study: Ding-Fei Xu and Jun Tan. Cell biology experiments: Pei-Pei Liu, Lu-Fan and Qi Xie. Data analysis and interpretation: Zhi-Qin Zhang. Draft of the manuscript: Ding-Fei Xu and Jun Tan. Supervision and critical revision of the manuscript for important intellectual content: Jun Tan, Qiong-Fang Wu and Li-Qun Wang. Final approval of the version to be published: Ding-Fei Xu, Pei-Pei Liu, Lu-Fan, Qi Xie, Jun Tan, Qiong-Fang Wu and Li-Qun Wang. 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Signaling pathways influencing SLF and c-kit-mediated survival and proliferation. Immunol Res. 2006;35(1–2):1–12. Cardoso HJ, Figueira MI, Correia S, Vaz CV, Socorro S. The SCF/c-KIT system in the male: Survival strategies in fertility and cancer. Mol Reprod Dev. 2014;81(12):1064–79. Kim JO, Kim HN, Kim KH, et al. Development and characterization of a fully human antibody targeting SCF/c-kit signaling. Int J Biol Macromol. 2020;159:66–78. Chen Q, Fan Y, Zhou X, et al. GnRH antagonist alters the migration of endometrial epithelial cells by reducing CKB. Reproduction. 2020;159(6):733–43. Supplementary Files SupplementaryFigure1.tif ESCs was verified by immunohistochemical analysis. As shown in results, the expression of vimentin but not cytokeratin was found in human isolated ESCs. Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2022 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted Editorial decision: Major revision 28 Sep, 2021 Review # 3 received at journal 24 Sep, 2021 Review # 2 received at journal 18 Sep, 2021 Review # 1 received at journal 15 Sep, 2021 Reviewer # 3 agreed at journal 08 Sep, 2021 Reviews received at journal 08 Sep, 2021 Reviewers invited by journal 08 Sep, 2021 Reviewer # 2 agreed at journal 07 Sep, 2021 Reviewer # 1 agreed at journal 07 Sep, 2021 Submission checks completed at journal 27 Aug, 2021 Editor invited by journal 27 Aug, 2021 Editor assigned by journal 26 Aug, 2021 First submitted to journal 26 Aug, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-845752","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":49159556,"identity":"948473ac-632b-461d-8d89-f8f8e1d79040","order_by":0,"name":"Ding-Fei Xu","email":"","orcid":"","institution":"Jiangxi Medical University: Medical College of Nanchang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ding-Fei","middleName":"","lastName":"Xu","suffix":""},{"id":49159557,"identity":"a20202c6-27d6-4c1a-9753-48e47b4a5e8e","order_by":1,"name":"Pei-Pei Liu","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pei-Pei","middleName":"","lastName":"Liu","suffix":""},{"id":49159558,"identity":"2c828b70-ab66-4b82-bc20-9b24754538f9","order_by":2,"name":"Lu Fan","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Fan","suffix":""},{"id":49159559,"identity":"2fd7f63d-83f3-434b-b64c-cfc49a2f4e33","order_by":3,"name":"Qi Xie","email":"","orcid":"","institution":"Jiangxi Medical College: Medical College of Nanchang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Xie","suffix":""},{"id":49159560,"identity":"6e5c55b6-55a1-4ba5-ab56-bc66a8063386","order_by":4,"name":"Zhi-Qin Zhang","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhi-Qin","middleName":"","lastName":"Zhang","suffix":""},{"id":49159561,"identity":"2c57eca8-4da9-4d8c-b20e-c5fc35cd9af8","order_by":5,"name":"Jun Tan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYJACAwaGf3JszMzHf3wwsLEjqJwHouWAMR87W4LkjIK0ZKK0AMGBRDl+HgNpng+HGBsIabFnP3ugmHfHnQQ2ZgYDYxuDA8wM7IePbsBrC09egjHvmWd5QC0JyTkGd/gYeNLSbuB3WI6BMW8bczFQy4HDOQbPmBkkeMzwa+F/A9aS2MbM2NhsYXCYsYGgFgmwLYeBWoCAgSgtN94YGM5tSzNmY2ZjY+wxSEtmI+QX9v4cM4O3bTZy8v3nvzH8+GNjx89++BheLUDAZoDKJaAcBJgfEKFoFIyCUTAKRjIAADclQV/cbdqXAAAAAElFTkSuQmCC","orcid":"","institution":"Jiangxi Provincial Maternal and Child Health Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Tan","suffix":""},{"id":49159562,"identity":"105df0e6-3a91-4836-a043-627db9f449e4","order_by":6,"name":"Qiong-Fang Wu","email":"","orcid":"","institution":"Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiong-Fang","middleName":"","lastName":"Wu","suffix":""},{"id":49159563,"identity":"a902b841-c3ce-4e4f-8c14-4e9add9ea5e2","order_by":7,"name":"Li-Qun Wang","email":"","orcid":"","institution":"Jiangxi Medical University: Medical College of Nanchang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li-Qun","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2021-08-25 10:41:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-845752/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-845752/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12958-021-00886-y","type":"published","date":"2022-02-04T13:25:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":13017014,"identity":"c69f4737-dd6f-4823-81ef-1d7723db2fe2","added_by":"auto","created_at":"2021-09-02 15:01:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":128691,"visible":true,"origin":"","legend":"Comparison of the growth ability of human isolated ESCs after treating with different concentrations of GnRH-ant and GnRH-a (10-8, 10-5 and 2x10-5 mol/L). (A) The proliferation of ESCs was investigated by using CCK8 assay. No difference was found in the proliferation of ESCs at concentration of 10-8 and 10-5 mol/L between GnRH-ant and GnRH-a group. However, when the concentration was added up to 2x10-5 mol/L, the proliferation capacity of ESCs in GnRH-ant group was significantly restrained compared to those in GnRH-a group. (B) Flow cytometry was used to analyze the apoptosis of ESCs. Similarly, the apoptotic level of ESCs was remarkably increased when the concentration at 2x10-5 mol/L. Each group of experiment was repeated for three times. (Control group: ESCs was treated only with culture medium. GnRH-ant-control group: ESCs was treated with GnRH-ant solvent. GnRH-a-control group: ESCs was treated with GnRH-a solvent.) (*** p \u003c 0.001). ","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-845752/v1/a5ff9b2cfff75be4220b2885.png"},{"id":13017016,"identity":"e950ffa0-8b4d-4f2f-a20b-a614aea737bb","added_by":"auto","created_at":"2021-09-02 15:01:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1323401,"visible":true,"origin":"","legend":"The expression of GnRH receptor (GnRHR) and c-kit receptor was detected by Immunofluorescence (A) and Western Blot (B), respectively. Furthermore, exposure of ESCs to the GnRH-ant at 2x10-5 mol/L significantly decreased the expression of c-kit receptor followed by the attenuated phosphorylation of c-kit receptor (B). Each group of experiment was repeated for three times. (Control group: ESCs was treated only with culture medium. GnRH-ant-control group: ESCs was treated with GnRH-ant solvent. GnRH-a-control group: ESCs was treated with GnRH-a solvent.) (n = 4 for per lane).","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-845752/v1/4c9edfad58ef5d81938a1525.png"},{"id":13017017,"identity":"f2c4e91e-7843-458f-8b69-f3a0dcf29a5c","added_by":"auto","created_at":"2021-09-02 15:01:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":260517,"visible":true,"origin":"","legend":"Investigation of the proliferation (A) and apoptosis (B) of human isolated ESCs after treating with different concentrations of Imatinib (0 μM, 4 μM, 8 μM, 16 μM, 24 μM, 32 μM). With the increase of drug concentration, the proliferation of ESCs was distinctly repressed (A) and the apoptotic level of ESCs was dramatically elevated (B). Each group of experiment was repeated for three times (*** p \u003c 0.001).\n\n","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-845752/v1/277b20f7a95294549edd2eb7.png"},{"id":13017013,"identity":"0c407d8f-b61d-4f8b-9157-ed7f7b949ea6","added_by":"auto","created_at":"2021-09-02 15:01:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":173849,"visible":true,"origin":"","legend":"After inhibiting the activation of c-kit receptor by treating ESCs with Imatinib (24 μM), the phosphorylation of AKT and the expression of Cyclin D1 were significantly diminished. Each group of experiment was repeated for three times (n = 4 for per lane).","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-845752/v1/43865b73eb2f06e4b31b988c.png"},{"id":18161140,"identity":"b45e6ce2-a1f4-4cd8-9eb1-d831571baf49","added_by":"auto","created_at":"2022-02-12 13:27:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1705987,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-845752/v1/bc7427eb-1731-48ce-bc82-f7bca895baa1.pdf"},{"id":13017018,"identity":"f97e21bb-5d71-4216-a2f3-32a84ec6062e","added_by":"auto","created_at":"2021-09-02 15:01:38","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2638240,"visible":true,"origin":"","legend":"ESCs was verified by immunohistochemical analysis. As shown in results, the expression of vimentin but not cytokeratin was found in human isolated ESCs. ","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-845752/v1/f056f1becb9499fbb3720d40.tif"}],"financialInterests":"","formattedTitle":"GnRH antagonist weakens endometrial stromal cells growth ability by decreasing c-kit receptor expression","fulltext":[{"header":"Background","content":"\u003cp\u003eGonadotropin-releasing hormone antagonist (GnRH-ant), which could competitively bind with GnRH receptor (GnRHR) and reversibly inhibited hypothalamic-pituitary-gonadal axis, has been used in assisted reproduction for several decades from the first generation to the recent third generation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Based on the superiority of convenience, flexibility and safety, GnRH-ant protocol has been one of the most commonly therapeutic schedules in \u003cem\u003ein vitro\u003c/em\u003e fertilization-embryo transfer (IVF-ET) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the unsatisfactory clinical outcomes of this protocol, compared to GnRH-a protocol, has restrained its promotion and application in clinic. Many clinical investigations have reported a lower fresh embryo implantation and pregnancy rates in GnRH-ant protocol than those in GnRH-a protocol [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similarly, Bukulmez\u0026rsquo;s research also showed a lower HCG positive and clinical pregnancy rates in GnRH-ant protocol when compared to GnRH-a protocol under the no difference of embryo quality between these two protocols [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, these worse clinical outcomes from GnRH-ant protocol were only occurred in fresh embryo transfer but not in freezing embryo transfer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], suggesting that the adverse effects of GnRH-ant on endometrium might be the key reason.\u003c/p\u003e \u003cp\u003eIn fact, many researches have demonstrated that the endometrial thickness could be an important factor which influenced the pregnancy after embryo transfer [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Patients with endometrial thickness less than 8 mm showed an obviously lower implantation and pregnancy rate [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Significantly, recent studies have proved that patients treated with GnRH-ant protocol showed a remarkably thinner endometrial thickness than patients treated with GnRH-a protocol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These results implied that one of the adverse effects of GnRH-ant on endometrium was to weaken endometrial thickness. Nonetheless, the related molecular mechanism was still unclear.\u003c/p\u003e \u003cp\u003ePrevious studies have found the expression of c-kit receptor on animal embryo and endometrium [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], suggesting the crucial role of c-kit receptor in the process of animal embryo implantation. C-kit receptor belonged to a member of III type RTK family which could be stimulated by stem cell factor (SCF). Activated c-kit receptor was associated with multiple kinds of biological events, including proliferation, differentiation, migration and apoptosis, in many types of cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our previous study has found the expression of c-kit receptor on human embryo and demonstrated that added exogenous SCF to activate c-kit receptor could significantly promote embryo development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recently, we surprisingly found the expression of c-kit receptor on human endometrial stromal cells (ESCs). Based on the regulatory function on the growth ability of many kinds of cells, we speculated that c-kit receptor might also influenced the growth ability of human ESCs. Furthermore, whether its regulatory effect is associated with GnRH-ant is still unknown.\u003c/p\u003e \u003cp\u003eTherefore, the present study is to explore the effects of GnRH-ant and c-kit receptor on the growth of human ESCs, and to investigated the regulatory relationship between GnRH-ant and c-kit receptor.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eThe following reagents were used in this study: rabbit anti-GnRHR (Abcam, UK), mouse anti-c-kit (CST, USA), rabbit anti-c-kit (CST, USA), rabbit anti-phospho-c-kit (CST, USA), rabbit anti-AKT (CST, USA), rabbit anti-phospho-AKT (CST, USA), rabbit anti-Cyclin D1 (CST, USA), mouse anti-β-actin (Santa Cruz, USA), Donkey anti rabbit Alexa flour 488 (Thermo Fisher Scientific, USA), Donkey anti rabbit Alexa flour 594 (Thermo Fisher Scientific, USA), GnRH-a (Decapeptyl; Ferring), GnRH-ant (Cetrotide; Serono) and Imatinib (Biovision, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eThe present study retrospectively analyzed the clinical data of 2815 patients undergoing fresh embryo transfer in Reproductive Medicine Center of Jiangxi Maternal and Child Health Hospital from Jan 2016 to Dec 2020. All patients were in good physical and mental condition. The inclusion criteria for all patients included age\u0026thinsp;\u0026le;\u0026thinsp;37 years, body mass index (BMI) of 15\u0026ndash;25 kg/m\u003csup\u003e2\u003c/sup\u003e, 1.1\u0026thinsp;\u0026lt;\u0026thinsp;anti-M\u0026uuml;llerian hormone (AMH)\u0026thinsp;\u0026lt;\u0026thinsp;5.0, 5\u0026thinsp;\u0026le;\u0026thinsp;antral follicle count (AFC)\u0026thinsp;\u0026le;\u0026thinsp;20, and less than twice IVF-ET experiences. Women with a history of the following procedures or disorders were excluded: uterine malformation, ovarian surgery, radiotherapy or chemotherapy, premature ovarian failure, ovarian dysfunction, adenomyosis, polycystic ovarian syndrome, thyroid dysfunction, recurrent implantation failure (failed to achieve a pregnancy more than three times), submucosal fibroids, intrauterine adhesion, hydrosalpinx, and patients (women or men) with abnormal chromosomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and groups\u003c/h2\u003e \u003cp\u003e2815 patients were divided into GnRH-ant group (563) and GnRH-a group (2252) according to the different protocols. In GnRH-ant group, recombinant human FSH (rhFSH, Merck-Serono, German) was used on day 2 or 3 of the menstrual cycle. The initial dosage (112.5\u0026ndash;225 IU/day) was determined based on age, BMI, AFC, and AMH. The dose of rhFSH were adjusted according to ovarian response as monitored by ultrasonography and serum estradiol (E\u003csub\u003e2\u003c/sub\u003e) levels. GnRH antagonist (Cetrorelix, Merck Serono, Switzerland) at a daily dose of 0.25 mg was started when the largest follicle exceeded 12 mm. Both GnRH antagonist and rhFSH were stopped and a single injection of 6000\u0026ndash;8000 IU of hCG (Merck-Serono, German) was administered when the dominant follicle was \u0026ge;\u0026thinsp;19 mm in diameter or at least 2 follicles were \u0026ge;\u0026thinsp;18 mm in diameter. Oocyte retrieval was performed 36\u0026ndash;40 hours later under transvaginal ultrasound guidance. In GnRH-a group, a standard full dose of gonadotropin-releasing hormone agonist (3.75 mg, GnRH agonist, Ipsen, France) was used in the second day of menstrual cycle for down regulation. Pituitary down regulation (Endometrial thickness\u0026thinsp;\u0026le;\u0026thinsp;5 mm, serum FSH\u0026thinsp;\u0026lt;\u0026thinsp;5 mIU/mL, LH\u0026thinsp;\u0026lt;\u0026thinsp;5 mIU/mL, E\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;50 pg/mL) was confirmed with transvaginal ultrasound and endocrine examination after 28\u0026ndash;30 days. The initial dosage (112.5\u0026ndash;225 IU/day) was determined based on age, BMI, AFC, and AMH. The dose of rhFSH were adjusted according to ovarian response as monitored by ultrasonography and serum estradiol levels. The HCG trigger process was the same as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEmbryo assessment\u003c/h2\u003e \u003cp\u003eThe embryo assessment criteria were executed as the previously described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A good quality embryo should consist of 7\u0026ndash;9 blastomeres with a uniform size, and the fragment proportion should be less than 20% at day 3 for human 3PN embryos after fertilization. The good quality embryo rate refers to the number of good quality embryos divided by the total number of all embryos. Blastocyst formation was determined and graded by using the system of Gardner and Schoolcraft [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The blastulation rate refers to the number of blastocysts divided by the total number of all embryos. The good quality blastulation rate refers to the number of good quality blastocyst divided by the total number of all blastocysts. The assessment was made in a blinded manner by two embryologists.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePrimary human ESCs isolation, culture and treatment\u003c/h2\u003e \u003cp\u003ePrimary human decidual ESCs were isolated from the decidual tissue of healthy multipara women (aged 25\u0026ndash;32 years) undergoing elective surgical termination of a normal pregnancy at 8 to 10 weeks of gestation. The informed consents from all patients were obtained before the initiation of this study. According to the standard protocol [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], the human decidual tissue was minced and treated with type IV collagenase and DNase type I in a shaking water bath at 37℃ for 90 minutes. The cell digest was then passed through a 70 \u0026micro;m filter, both decidual stromal and epithelial cells were collected in tube. Then, decidual stromal cells were separated from epithelial cells with a 45 \u0026micro;m filter. The stromal cells were subsequently pelleted by centrifugation at 1000 rpm for 5 minutes. The cell pellets were washed once, resuspended, and cultured in Dulbecco modified Eagle medium containing 25 mM glucose, Lglutamine, antibiotics and supplemented with 10% fetal bovine serum at 37℃. ESCs was confirmed by detecting the expression of vimentin protein via immunohistochemical analysis (Supplementary Fig.\u0026nbsp;1). The cells were used after reaching 70\u0026ndash;80% confluence. ESCs were treated for 120 hours with different concentrations of GnRH-a, GnRH-ant (10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, and 2x10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/L) or imatinib (0 \u0026micro;M, 4 \u0026micro;M, 8 \u0026micro;M, 16 \u0026micro;M, 24 \u0026micro;M, 32 \u0026micro;M). Cells were subsequently collected for cell proliferation assay, flow cytometry and western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003e5000 cells/well were planted into 96-well plates in quintuplicate wells. Normally, cells were placed in the culture medium with different concentrations of GnRH analogs for 24, 48, 72, 96 and 120 hours, a total of 10 \u0026micro;L CCK8 reagent (APExBio, USA) was added to each well. After incubating for 2 hours at 37\u0026deg;C, the absorbance at 450 nm per well was measured using microplate reader. Each group of experiment was repeated for three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eThe suspended cells were collected by centrifugation. Centrifuge 1000g, centrifugation time 5 minutes at 2\u0026ndash;8℃. Cultured cells needed to be digested with EDTA-free trypsin, then terminated with serum-containing medium, centrifuged at 1000g for 5 minutes, supernatant removed, and washed with PBS resuspension. After centrifugal precipitation, the PBS was resuspended, transferred into the flow tube, washed once with PBS, centrifuged at 1000g for 5 minutes, and the supernatant was discarded. Cells were suspended with 400 UL \u0026times; Annexin binding solution at a concentration of approximately 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml. 5 UL Annexin V-FITC staining solution (BestBio, China) was added to the cell suspension, gently mixed and incubated for 15 minutes at 2\u0026ndash;8℃ in the dark. After adding 10 UL PI staining solution, gently mixed and incubated for 15 minutes at 2\u0026ndash;8℃ in the dark. Immediately detected by flow cytometry. Each group of experiment was repeated for three times\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eThe methods were described previously [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Cells were fixed with 4% paraformaldehyde at 4℃ for 10 minutes and washed with PBS three times. The cells were sealed with 2% triton-100 solution at room temperature for 30 minutes. The primary antibody diluted by blocking solution was added and incubated overnight at 4\u0026deg;C. The next day, cells were washed with PBS three times, 5 minutes each time. The fluorescent secondary antibodies were added and incubated for 1 hour at room temperature in the dark, then the cells were washed with PBS for 3 times, 5 minutes each time. Fluoroshield mounting medium (containing DAPI) with 1:10 dilution was added and stored at 4\u0026deg;C in the dark for fluorescence microscopy observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eTotal proteins were extracted from cells using the RIPA lysis buffer containing protease inhibitors (Applygen, China) and phosphatase inhibitors (Sigma, USA). The protein concentrations were determined by NanoDrop 2000c spectrophotometer using BCA protein assay kit (Applygen, China). After loading equal amount of protein samples, SDS-PAGE (12% sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed. The proteins were then transferred to a PVDF membrane (Merck-Millipore, USA). After blocking with Tris buffered saline containing 0.05% Tween-20 (TBST) and 5% non-fat dry milk or 5% BSA for 1 hour, the membrane was incubated with corresponding antibodies at 4℃ overnight, washed in TBST, followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies for 1 hour. Visualization of the proteins was detected with ECL chemiluminescence. Beta-actin was used as a loading control. The intensity values were assessed and analyzed with Image J software. (n\u0026thinsp;=\u0026thinsp;4 for per lane)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted by using SPSS 24.0 software (SASInstitute Inc.), and all data were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the means (s.e.m.s) or percentage (%). Results among experimental groups were analyzed by student\u0026rsquo;s t-test or one-way ANOVA. For all tests, P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGeneral characteristics\u003c/h2\u003e \u003cp\u003eNo differences were found in general characteristics between these two groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the general information of the two groups of patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGnRH-ant (n\u0026thinsp;=\u0026thinsp;563)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGnRH-a (n\u0026thinsp;=\u0026thinsp;2252)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (y)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.99\u0026thinsp;\u0026plusmn;\u0026thinsp;4.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.01\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (Kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of infertility (y)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntral follicle count (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.94\u0026thinsp;\u0026plusmn;\u0026thinsp;3.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.95\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndometrial thickness (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntrauterine adhesions rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScar uterus rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMH (ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal FSH (mIU/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.19\u0026thinsp;\u0026plusmn;\u0026thinsp;21.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.48\u0026thinsp;\u0026plusmn;\u0026thinsp;15.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal E2 (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.86\u0026thinsp;\u0026plusmn;\u0026thinsp;163.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.28\u0026thinsp;\u0026plusmn;\u0026thinsp;238.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal P (ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;4.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal LH (mIU/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.49\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: The data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Abbreviations: BMI, Body Mass Index; AMH, anti-mullerian hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; P, progesterone;\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eComparison of the IVF outcomes between GnRH-ant and GnRH-a groups\u003c/h2\u003e \u003cp\u003eFirstly, we compared the effects of ovulation promotion between GnRH-ant and GnRH-a groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among the two groups, the levels of E\u003csub\u003e2\u003c/sub\u003e and P on HCG day were similar (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e), but the initial dose of Gn, duration of Gn used, total dose of Gn and endometrial thickness on HCG Day were significantly lower in GnRH-ant group than those in GnRH-a group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), whereas the LH level on HCG Day was higher in GnRH-ant group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). Then, we investigated the clinical outcomes from these two groups (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). There were no differences in the number of oocytes retrieved, 2PN fertilization rate, 2PN cleavage rate, high quality embryo rate and high quality blastulation rate between these two groups (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). However, GnRH-ant group showed remarkably decreased rate of HCG positive, embryonical implantation and clinical pregnancy when compared to GnRH-a group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), implying the worse clinical outcomes in GnRH-ant group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the effect of ovulation promotion between two groups of patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGnRH-ant (n\u0026thinsp;=\u0026thinsp;563)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGnRH-a (n\u0026thinsp;=\u0026thinsp;2252)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial dose of Gn (IU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e212.92\u0026thinsp;\u0026plusmn;\u0026thinsp;75.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e177.83\u0026thinsp;\u0026plusmn;\u0026thinsp;69.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.01\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of Gn used (d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.01\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal dose of Gn (IU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2132.1\u0026thinsp;\u0026plusmn;\u0026thinsp;631.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2391.2\u0026thinsp;\u0026plusmn;\u0026thinsp;857.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.01\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndometrial thickness on\u003c/p\u003e \u003cp\u003eHCG day (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.09\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.01\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE2 on HCG day (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2041.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1170.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2080.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1022.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLH on HCG day (mIU/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.01\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP on HCG day (ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eNote: The data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Abbreviations: HCG, Human Chorionic Gonadotropin; LH, luteinizing hormone; E2, estradiol; P, progesterone;\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of clinical outcomes between two groups of patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGnRH-ant (n\u0026thinsp;=\u0026thinsp;563)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGnRH-a (n\u0026thinsp;=\u0026thinsp;2252)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP- value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of oocytes retrieved\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.26\u0026thinsp;\u0026plusmn;\u0026thinsp;3.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2PN fertilization rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2PN cleavage rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh quality embryo at day 3 rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh quality blastulation rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of transfer embryos\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCG positive rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.01\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImplantation rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.01\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical pregnancy rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.01\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGnRH-ant reduced the proliferation and induced the apoptosis of human isolated ESCs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCCK8 and Flow cytometry were used to investigated the effects of GnRH-ant on the growth ability of human isolated ESCs, which was confirmed by immunohistochemical analysis (Supplementary Fig.\u0026nbsp;1). Little differences were found in the concentrations of GnRH-ant and GnRH-a at 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e and 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/L. When the concentration was elevated to 2x10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/L, GnRH-ant obviously reduced the proliferation and induced the apoptosis of ESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGnRH-ant decreased the expression of c-kit receptor in human isolated ESCs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs shown in the results of immunofluorescence staining, GnRH receptor and c-kit receptor were co-expressed on human isolated ESCs, indicating a possible potential regulatory relationship between these two receptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After treating human isolated ESCs with the concentration of GnRH-ant and GnRH-a at 2x10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/L, we found significantly reduced expression of c-kit receptor and phosphorylation c-kit receptor in GnRH-ant group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting c-kit signaling was attenuated by GnRH-ant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eC-kit receptor regulated the growth ability of human isolated ESCs through AKT signaling pathway.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCultured human isolated ESCs were treated with different concentrations of imatinib (0 \u0026micro;M, 4 \u0026micro;M, 8 \u0026micro;M, 16 \u0026micro;M, 24 \u0026micro;M, 32 \u0026micro;M) to confirm the ideal dosage required to suppress the c-kit receptor activation. The results showed that with the increase concentration of imatinib, the reduction of proliferation and induction of apoptosis in ESCs were increasingly notable (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Due to most of ESCs were dead at 32 \u0026micro;M, we selected 24 \u0026micro;M concentration as the ideal dosage to treat ESCs. The results showed that after inhibiting the c-kit receptor activation by 24 \u0026micro;M imatinib in ESCs, the phosphorylation of AKT and the expression of cyclin D1 were significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the normal menstrual cycle, endometrium would go through three phases: menstrual period, proliferative period and decidual period. During the decidual phase, the endometrium transformed into a receptive tissue that was suitable for embryo implantation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this process, ESCs were the main cell type providing nutrition for the implanting embryos [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, the abundant numbers of ESCs would directly decide the success of embryo implantation. In the present study, although there were no differences in embryo quality between GnRH-ant and GnRH-a group, GnRH-ant group showed unsatisfied clinical outcomes including lower rates of HCG positive, embryo implantation and clinical pregnancy. It was worth noting that a thinner endometrial thickness was observed in GnRH-ant group, suggesting that the appropriate endometrial thickness might be conducive to embryo implantation. Similar with our findings, many recent studies also found that the thinner endometrium was evidently related to the worse clinical outcomes in fresh and frozen-thaw embryo transfer [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These results reminded that one of the reasons resulting in worse clinical outcomes in GnRH-ant group might be the adverse effect of GnRH-ant on growth ability of human ESCs. Accordingly, we isolated human ESCs from human endometrial tissues and treated cells with different concentrations of GnRH-analog to investigated the effects of GnRH-ant on the growth ability of ESCs. As we expected that GnRH-ant significantly depressed the proliferation and promoted the apoptosis of ESCs, confirming the adverse influences of GnRH-ant on ESCs growth. However, how GnRH-ant negatively regulates the growth of ESCs is still unclear.\u003c/p\u003e \u003cp\u003ePrevious studies have reported the positive effects of c-kit receptor on growth ability of multiple kinds of cells [\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Furthermore, recent studies have found the expression of c-kit receptor on animal embryo and endometrium, and proposed its important role in the process of embryo implantation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Surprisingly, we found the expression of c-kit receptor on human ESCs, indicating that c-kit receptor might also regulate the growth ability of human ESCs. Whereafter, we treated human ESCs with imatinib (c-kit receptor inhibitor) and found that after inhibiting the activation of c-kit receptor by imatinib, ESCs showed remarkably decreased proliferation capacity and increased apoptosis level. Moreover, the phosphorylation of AKT and the expression of cyclin D1, which were closely associated with cell growth, were significantly weakened. These findings supported that activating c-kit receptor could accelerate the growth ability of human ESCs.\u003c/p\u003e \u003cp\u003eBased on the above findings, it is reasonable to speculate that whether the adverse effects of GnRH-ant on human ESCs growth ability is correlated with the inactivation of c-kit receptor? To further verify our hypothesis, we subsequently treated ESCs with GnRH-ant at 2x10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e mol/L for 120 hours. The results showed that GnRH-ant significantly suppressed the expression of c-kit receptor followed by the attenuated phosphorylation of c-kit receptor, implying that GnRH-ant could weaken the growth ability of human ESCs by impairing the activation level of c-kit receptor through suppressing c-kit receptor expression. Notably, recent study directed by Chen et al have demonstrated that GnRH-ant could disrupt human endometrial epithelial cells (EECs) migration by reducing the CKB expression, which altered endometrial receptivity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Complementally, our findings showed another negative effect of GnRH-ant on ESCs, which was the repression of ESCs growth capacity. Even so, the whole effects of GnRH-ant protocol on endometrium of women undergoing IVF-ET were still unclarified. Hence, further researches related the molecular mechanism are needed.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn brief, GnRH-ant attenuated the activation level of c-kit receptor by decreasing its expression in ESCs. Subsequently, this impaired effect could further inhibit the phosphorylation of AKT and lessened the expression of cyclin D1, which resulted in the reduction of growth ability of ESCs. The present study provides a new insight into the role of GnRH-ant and suggests that the adverse effects of GnRH-ant protocol on endometrium should be considered before using in IVF-ET.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eGnRH-ant: Gonadotropin-releasing hormone antagonist; GnRH-a: Gonadotropin-releasing hormone agonist; GnRHR: Gonadotropin-releasing hormone receptor; ESCs: Endometrial stromal cells; EECs: Endometrial epithelial cells; IVF-ET: In vitro fertilization-embryo transfer; RTK: Receptor tyrosine kinase; BMI: Body mass index; AFC: Antral follicle count; AMH: Anti-mullerian hormone; rhFSH: recombinant human FSH; \u0026nbsp; \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Clinical Ethical Committee of Jiangxi Maternal and Child Health Hospital, and informed consents from patients were obtained before the initiation of the study. All the authors consented to participate in this study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors consented for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of supporting data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed in this study are included in this published manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest with respect tothe research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis\u0026nbsp;work was supported by the National Natural Science Foundation of China (81960271 and 81960288), the Major Research and Development Project of Science and Technology Department of Jiangxi Province (20192BBGL70005 and 20203BBGL73140) and the China Postdoctoral Science Foundation (2019M662272).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design of the study:\u0026nbsp;Ding-Fei Xu\u0026nbsp;and Jun Tan. Cell biology experiments:\u0026nbsp;Pei-Pei Liu, Lu-Fan and Qi Xie.\u0026nbsp;Data analysis and interpretation:\u0026nbsp;Zhi-Qin Zhang.\u0026nbsp;Draft of the manuscript:\u0026nbsp;Ding-Fei Xu\u0026nbsp;and Jun Tan. Supervision and critical revision of the manuscript for important intellectual content: Jun Tan,\u0026nbsp;Qiong-Fang Wu and\u0026nbsp;Li-Qun Wang. Final approval of the version to be published:\u0026nbsp;Ding-Fei Xu, Pei-Pei Liu, Lu-Fan, Qi Xie, Jun Tan, Qiong-Fang Wu and Li-Qun Wang.\u0026nbsp;All authors have read, and confirm that they meet the authorship criteria. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank all the staff, nurses, and physicians at the Reproductive Medicine Center for their support in generating this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGobello C. New GnRH analogs in canine reproduction. Anim Reprod Sci. 2007;100(1):1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDevroey P, Aboulghar M, Garcia-Velasco J, et al. Improving the patient\u0026rsquo;s experience of IVF/ICSI: a proposal for an ovarian stimulation protocol with GnRH antagonist co-treatment. Hum Reprod. 2009;24:764\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Inany HG, Abou-Setta AM, Aboulghar M. Gonadotrophinreleasing hormone antagonists for assisted conception: a Cochrane review. Reprod Biomed Online. 2007;14:640\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrvieto R, Patrizio P. GnRH agonist versus GnRH antagonist in ovarian stimulation: an ongoing debate. Reprod Biomed Online. 2013;26:4\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLambalk CB, Banga FR, Huirne JA, et al. GnRH antagonist versus long agonist protocols in IVF: a systematic review and meta-analysis accounting for patient type. Hum Reprod Update. 2017;23:560\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBukulmez O, Carr BR, Doody KM, et al. Serum cetrorelix concentrations do not affect clinical pregnancy outcome in assisted reproduction. Fertil Steril. 2008;89:74\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEftekhar M, Dehghani Firouzabadi R, Karimi H, et al. Outcome of cryopreserved-thawed embryo transfer in the GnRH agonist versus antagonist protocol. Iran J Reprod Med. 2012;10:297\u0026ndash;302.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao G, Cui X, Li S, Ding P, Zhang S, Zhang Y. Endometrial thickness and IVF cycle outcomes: a meta-analysis. Reprod Biomed Online. 2020;40(1):124\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJigal H, Ramsey S, Eran Z, et al. Endometrial compaction (decreased thickness) in response to progesterone results in optimal pregnancy outcome in frozen-thawed embryo transfers. Fertil Steril. 2019;112(3):503\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang T, Li Z, Ren X, et al. Endometrial thickness as a predictor of the reproductive outcomes in fresh and frozen embryo transfer cycles: A retrospective cohort study of 1512 IVF cycles with morphologically good-quality blastocyst. Med (Baltim). 2018;97(4):e9689.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGallos ID, Khairy M, Chu J, et al. Optimal endometrial thickness to maximize live births and minimize pregnancy losses: Analysis of 25,767 fresh embryo transfers. Reprod Biomed Online. 2018;37(5):542\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang R, Cai L, Xiong F, Chen J, Yang W, Zhao X. The effect of endometrial thickness on the day of HCG administration on pregnancy outcome in the first fresh IVF/ICSI cycle. Gynecol Endocrinol. 2016;32(6):473\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Y, Niu Y, Wang Y, et al. Optimal Candidates to Do Fresh Embryo Transfer in Those Using Oral Contraceptive Pretreatment in IVF Cycles. Front Physiol. 2021;12:576917.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen JZ, Han DM, Sha AG, et al. The correlation between endometrial thickness and pattern in predicting outcome of in vitro fertilization and embryo transfer. Chinese Journal of Birth Health Heredity. 2013;21(2):107\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabezas J, Lara E, Pacha P, et al. The endometrium of cycling cows contains populations of putative mesenchymal progenitor cells. Reprod Domest Anim. 2014;49(4):550\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang J, Wu YL, Chen BJ, et al. The C-Kit Receptor-Mediated Signal Transduction and Tumor-Related Diseases. Int J Biol Sci. 2013;9(5):435\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStankov K, Popovic S, Mikov M. C-KIT signaling in cancer treatment. Curr Pharm Des. 2014;20(17):2849\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan J, Zou Y, Huang ZH, et al. C-kit signaling promotes human preimplantation 3PN embryonic development and blastocyst formation. Reprod Biol Endocrinol. 2019;17(1):75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGardner DK, Schoolcraft WB. In vitro culture of human blastocyst. In: Jansen R, Mortimer D, editors. Towards reproductive certainty: infertility and genetics beyond. Carnforth: Parthenon Press; 1999. pp.\u0026nbsp;378\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChou CS, MacCalman CD, Leung PC. Differential effects of gonadotropin-releasing hormone I and II on the urokinase-type plasminogen activator/plasminogen activator inhibitor system in human decidual stromal cells in vitro. J Clin Endocrinol Metab. 2003;88:3806\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu HM, Huang HY, Lee CL, et al. Gonadotropin-Releasing Hormone Type II (GnRH-II) Agonist Regulates the Motility of Human Decidual Endometrial Stromal Cells: Possible Effect on Embryo Implantation and Pregnancy. Biol Reprod. 2015;92(4):98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan J, Yang S, Shen P, et al. C-kit signaling promotes proliferation and invasion of colorectal mucinous adenocarcinoma in a murine model. Oncotarget. 2015;6(29):27037\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl Chami A, Saridogan E. Endometrial Polyps and Subfertility. J Obstet Gynaecol India. 2017;67(1):9\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkada H, Tsuzuki T, Murata H. Decidualization of the human endometrium. Reprod Med Biol. 2018;17(3):220\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu RW, Fazleabas AT. Implantation and establishment of pregnancy in human and nonhuman primates. Adv Anat Embryol Cell Biol. 2015;216:189\u0026ndash;213.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu KE, Hartman M, Hartman A, et al. The impact of a thin endometrial lining on fresh and frozen\u0026ndash;thaw IVF outcomes: an analysis of over 40000 embryo transfers. Hum Reprod. 2018;33(10):1883\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKasius A, Smit JG, Torrance HL, et al. Endometrial thickness and pregnancy rates after IVF: a systematic review and meta-analysis. Hum Reprod Update. 2014;20(4):530\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmir W, Micha B, Ariel H, et al. Predicting factors for endometrial thickness during treatment with assisted reproductive technology. Fertil Steril. 2007;87(4):799\u0026ndash;804.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichter K, Bugge K, Bromer J, et al. Relationship between endometrial thickness and embryo implantation, based on 1294 cycles of in vitro fertilization with transfer of two blastocyst-stage embryos. Fertil Steril. 2007;87(1):53\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFigueira MI, Cardoso HJ, Correia S, Maia CJ, Socorro S. Hormonal regulation of c-KIT receptor and its ligand: implications for human infertility? Prog Histochem Cytochem. 2014;49(1\u0026ndash;3):1\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerger SA. Signaling pathways influencing SLF and c-kit-mediated survival and proliferation. Immunol Res. 2006;35(1\u0026ndash;2):1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCardoso HJ, Figueira MI, Correia S, Vaz CV, Socorro S. The SCF/c-KIT system in the male: Survival strategies in fertility and cancer. Mol Reprod Dev. 2014;81(12):1064\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JO, Kim HN, Kim KH, et al. Development and characterization of a fully human antibody targeting SCF/c-kit signaling. Int J Biol Macromol. 2020;159:66\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Q, Fan Y, Zhou X, et al. GnRH antagonist alters the migration of endometrial epithelial cells by reducing CKB. Reproduction. 2020;159(6):733\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"GnRH-ant, c-kit receptor, ESCs, growth ability","lastPublishedDoi":"10.21203/rs.3.rs-845752/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-845752/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSeveral surveys have reported that patients treated with gonadotropin-releasing hormone antagonist (GnRH-ant) protocol showed a significantly lower rate of implantation and clinical pregnancy compared to GnRH agonist (GnRH-a) protocol during in vitro fertilization-fresh embryo transfer. Subsequent studies imputed this poor outcome to the negative effects of GnRH-ant on endometrial receptive. However, the mechanisms were not fully understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe clinical data of 2815 patients undergoing fresh embryo transfer in our center were analyzed. Human endometrial stromal cells (ESCs) from healthy women undergoing elective pregnancy termination of a normal pregnancy at 8\u0026ndash;10 weeks gestation were treated with GnRH-analogs or imatinib (c-kit receptor inhibitor).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe clinical data showed that the endometrial thickness on HCG Day were significantly lower in GnRH-ant group. Although no difference of embryo quality in these two groups, GnRH-ant group showed remarkably decreased rate of HCG positive, embryo implantation and pregnancy. Moreover, GnRH-ant significantly reduced the proliferation and induced the apoptosis of ESCs. Furthermore, the expression and activation of c-kit receptor, which played pivotal roles during embryo implantation, were observably decreased by GnRH-ant. Inhibiting the activation of c-kit by imatinib remarkably suppressed the proliferation and promoted the apoptosis of ESCs. Additionally, the phosphorylation of AKT and expression of Cyclin D1, which were closely related with cellular growth, were distinctly lessened after treating with imatinib.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn summary, our study showed that GnRH-ant weakened the activization of c-kit receptor by decreasing its expression, causing the impaired growth ability of ESCs. Our findings provided a new insight into the effects of GnRH-ant on endometrium.\u003c/p\u003e","manuscriptTitle":"GnRH antagonist weakens endometrial stromal cells growth ability by decreasing c-kit receptor expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-09-02 15:01:23","doi":"10.21203/rs.3.rs-845752/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-09-29T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-09-25T00:00:00+00:00","index":3,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-09-19T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-09-16T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-09-09T00:00:00+00:00","index":3,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-09-08T15:34:26+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-09-08T15:25:18+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-09-08T01:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-09-08T00:00:00+00:00","index":1,"fulltext":""},{"type":"checksComplete","content":"","date":"2021-08-27T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-08-27T23:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-08-27T03:27:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Reproductive Biology and Endocrinology","date":"2021-08-26T04:46:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"91345a1f-362f-4c34-a418-ae13f0d5a505","owner":[],"postedDate":"September 2nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":6894388,"name":"Endocrinology \u0026 Metabolism"}],"tags":[],"updatedAt":"2022-02-12T13:25:37+00:00","versionOfRecord":{"articleIdentity":"rs-845752","link":"https://doi.org/10.1186/s12958-021-00886-y","journal":{"identity":"reproductive-biology-and-endocrinology","isVorOnly":false,"title":"Reproductive Biology and Endocrinology"},"publishedOn":"2022-02-04 13:25:37","publishedOnDateReadable":"February 4th, 2022"},"versionCreatedAt":"2021-09-02 15:01:23","video":"","vorDoi":"10.1186/s12958-021-00886-y","vorDoiUrl":"https://doi.org/10.1186/s12958-021-00886-y","workflowStages":[]},"version":"v1","identity":"rs-845752","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-845752","identity":"rs-845752","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}