Enhancing endometrial receptivity: the roles of human chorionic gonadotropin in autophagy and apoptosis regulation in endometrial stromal cells

Abstract

Inadequate endometrial receptivity often results in embryo implantation failure and miscarriage. Human chorionic gonadotropin (hCG) is a key signaling molecule secreted during early embryonic development, which regulates embryonic maternal interface signaling and promotes embryo implantation. This study aimed to examine the impact of hCG on endometrial receptivity and its underlying mechanisms. An exploratory study was designed, and endometrial samples were obtained from women diagnosed with simple tubal infertility or male factor infertile (n = 12) and recurrent implantation failure (RIF, n = 10). Using reverse transcription-quantitative PCR and western blotting, luteinizing hormone (LH)/hCG receptor (LHCGR) levels and autophagy were detected in the endometrial tissues. Subsequently, primary endometrial stromal cells (ESCs) were isolated from these control groups and treated with hCG to examine the presence of LHCGR and markers of endometrial receptivity (HOXA10, ITGB3, FOXO1, LIF, and L-selectin ligand) and autophagy-related factors (Beclin1, LC3, and P62). The findings revealed that the expressions of receptivity factors, LHCGR, and LC3 were reduced in the endometrial tissues of women with RIF compared with the control group, whereas the expression of P62 was elevated. The administration of hCG to ESCs specifically activated LHCGR, stimulating an increase in the endometrial production of HOXA10, ITGB3, FOXO1, LIF and L-selectin ligands. Furthermore, when ESCs were exposed to 0.1 IU/mL hCG for 72 h, the autophagy factors Beclin1 and LC3 increased within the cells and P62 decreased. Moreover, the apoptotic factor Bax increased and Bcl-2 declined. However, when small interfering RNA was used to knock down LHCGR, hCG was less capable of controlling endometrial receptivity and autophagy molecules in ESCs. In addition, hCG stimulation enhanced the phosphorylation of ERK1/2 and mTOR proteins. These results suggest that women with RIF exhibit lower levels of LHCGR and compromised autophagy function in their endometrial tissues. Thus, hCG/LHCGR could potentially improve endometrial receptivity by modulating autophagy and apoptosis.

Fig. 1

Endometrial receptivity is down-regulated in RIF patients. A The mRNA expression levels of HOXA10, ITGB3 and LHCGR in the endometrium of normal controls (n = 12) and RIF patients (n = 10). B WB was used to detect the protein levels of HOXA10, ITGB3 and LHCGR in endometrium of normal control (n = 12) and RIF patients (n = 10). Analyzing gels and western blots with ImageJ. Error bars, mean ± SD. *P < 0.05, **P < 0.01. C Representative IHC images of LHCGR localization in endometrial tissue from normal control (n = 12) and RIF patients (n = 10) in secretory phase. Scale, 100 μm and 50 μm, analyzing relative OD with ImageJ. Error bars, mean ± SD. **P < 0.01

Fig. 2

The level of endometrial autophagy in RIF patients was down-regulated. A mRNA expression levels of LC3-II and P62 in endometrium of normal control (n = 12) and RIF patients (n = 10). B Left: Western blotting was used to detect the protein levels of LC3B and P62 in the endometrium of normal controls (n = 12) and RIF patients (n = 10). Right: analyzing gels and western blots with Image J. Error bars, mean ± SD. *P < 0.05, **P < 0.01. C Representative IHC images of LC3 localization in endometrial tissue from normal control (n = 12) and RIF patients (n = 10) in secretory phase. Scale, 100 μm and 50 μm, analyzing relative OD with Image J. Error bars, mean ± SD. *P < 0.05, **P < 0.01

Fig. 3

Identification and viability determination of primary ESCs. A IF showed the expression levels of VIM and CK-18 in the cytoplasm of primary ESCs, Scale, 20 μm. B hCG compared with the baseline and 0.001,0.01, 0.1 IU/ml hCG, P < 0.05. mean ± SD. C The effect of hCG on the proliferation of ESCs. The curve is the cell growth count of each group, and the line chart is the relative value-added growth curve of each group.*P < 0.05. mean ± SD. Scale: 1000 μm

Fig. 4

The expression level of LHCGR in ESCs. A The ESCs treated with hCG (0.1IU/mL, 72 h) was analyzed by immunofluorescence to determine the subcellular localization and protein expression level of LHCGR (red). The nuclei were stained with DAPI (blue). Scale, 50 μm and 20 μm. B The qRT-PCR analysis of LHCGR gene in ESCs after hCG treatment. *P < 0.05, **P < 0.01 (Student’s t-test). C Western blot analysis of LHCGR protein in ESCs after hCG treatment. *P < 0.05, **P < 0.01 (Student’s t-test)

Fig. 5

The expression levels of HOXA10, ITGB3, FOXO1, LIF and L-selectin ligand (MECA-79) in uterine ESCs after hCG intervention. A The mRNA expression levels of HOXA10, ITGB3, FOXO1 and LIF in ESCs were stimulated with hCG for 72 h. B The protein expression levels of HOXA10, ITGB3, FOXO1, LIF and L-selectin ligand (MECA-79) in ESCs were stimulated by hCG for 72 h. The data were shown as mean ± SD (n = 3): *P < 0.05, **P < 0.01, ***P < 0.001. C IF Representative images of ITGB3, LIF and L-selectin ligand (MECA-79) in ESCs treated with 0.1IU/mL hCG for 72 h. Scale: 100 μm

Fig. 6

The expression levels of LHCGR, FOXO1, ITGB3 ,HOXA10, LIF and L-selectin ligand in ESCs after the knockdown of LHCGR. A The mRNA expression levels of LCGR, HOXA10, ITGB3, FOXO1 and LIF in ESCs after the knockdown of LHCGR. B The protein expression levels of LHCGR in ESCs were stimulated by hCG and knockdown of LHCGR. C The protein expression levels of FOXO1, ITGB3, HOXA10, LIF and L-selectin ligand in ESCs after the knockdown of LHCGR. The data were shown as mean ± SD (n = 3): *P < 0.05, **P < 0.01

Fig. 7

hCG intervention changes the level of autophagy in endometrial cells. A The mRNA expression levels of Beclin1, LC3-II and P62 in ESCs were stimulated by hCG for 72 h. B The protein expression levels of Beclin1, LC3 and P62 in ESCs were stimulated by hCG for 72 h. C Representative images of LC3-II in ESCs treated with hCG for 72 h. Scale: 50 μm. D The mRNA expression levels of Beclin1, LC3-II and P62 in ESCs after the knockdown of LHCGR. E The protein expression levels of Beclin1, LC3 and P62 in ESCs were stimulated by hCG and knockdown of LHCGR. Data were presented as mean ± SD (n = 3): *P < 0.05, **P < 0.01

Fig. 8

The level of endometrial cell apoptosis after hCG intervention. A The mRNA expression levels of Bax and Bcl-2 in ESCs stimulated with hCG for 72 h. B The protein expression levels of Bax and Bcl-2 in endometrial stromal cells after hCG stimulation of ESCs for 72 h. Data were presented as mean ± SD (n = 3): *P < 0.05, **P < 0.01, ***P < 0.001. C ESCs apoptotic cells were detected by flow cytometry after treatment with 0.1 IU/mL of hCG for 72 h. D Immunofluorescence staining was used to determine the localization of Beclin1 and Bcl-2, and DAPI solution for the nucleus staining.Scale: 50 μm. E Co-IP experiment was performed to confirm the effect of hCG on the interactions of Bcl-2 and Beclin1

Fig. 9

Changes of ERK and mTOR phosphorylation levels in endometrial cells after hCG intervention. A The protein expression levels of p-mTOR and p-ERK1/2 in endometrial stromal cells after hCG stimulation of ESCs for 72 h. B Representative images of p-mTOR and p-ERK in ESCs treated with hCG for 72 h. Scale: 50 μm. C The protein expression levels of p-ERK1/2 and p-mTOR in ESCs after knockdown of LHCGR. Data were presented as mean ± SD (n = 3): *P < 0.05

Fig. 10

The schematic presentation of the roles of human chorionic gonadotropin in autophagy and apoptosis regulation in endometrial stromal cells