Human chorionic gonadotropin enhances trophoblast-epithelial interaction in an in vitro model of human implantation

Abstract

Embryo implantation, which is an absolute requirement for reproduction, starts with blastocyst apposition to the uterine endometrium, followed by attachment to the endometrial surface epithelium. Recent clinical studies reported an increase in implantation and pregnancy rates among women receiving intrauterine human chorionic gonadotropin (hCG) prior to embryo transfer suggesting that, at least in some cases, female infertility is a result of inadequate secretion of hCG. In this study, we characterized the effect of hCG on trophoblast-epithelial interaction by further developing our recently described in vitro model of implantation. Here, we confirmed hCG increased attachment of trophoblast to epithelial cells, using a single-cell trophoblast-epithelial coculture system in addition to a blastocyst-like spheroid-epithelial coculture system. Furthermore, we discovered that the source and concentration was pivotal; the first preparation of hCG affected 2 molecules related to implantation, MUC16 and osteopontin, while the second preparation required additional cytokines to mimic the effects. Using this system, we can develop a comprehensive knowledge of the cellular and gene targets of hCG and other factors involved in embryo apposition and implantation and potentially increase the number of therapeutic targets for subfertile patients.

Keywords: hCG; implantation; pregnancy.

Figure 1.

Pregnyl human chorionic gonadotropin (hCG) affects Sw.71 trophoblast cell viability at concentrations higher than 100 IU. A, Sw.71 cells were plated in triplicate and viability was determined 24, 48, and 72 hours post-hCG treatment at concentrations ranging between 2.5 and 500 IU. B, Sw.71 cell growth was further analyzed by measuring cell confluence for 60 hours post-hCG treatment at concentrations ranging between 25 and 1000 IU. Concentrations of hCG less than or equal to 100 IU did not affect viability and cells reached confluence 28 hours posttreatment, similar to cells that received no treatment.

Figure 2.

Pregnyl human chorionic gonadotropin (hCG) causes a decrease in MUC16 messenger RNA (mRNA) while Sigma hCG alone has no effect. There is a negative correlation between Pregnyl hCG concentration and MUC16 mRNA between 5 and 300 IU hCG (A). Sigma hCG does not affect MUC16 mRNA unless supplemented with interleukin 1B which results in a decrease in MUC16 concentration at 750 IU (B, C). Results are represented as fold change from nontreated control Sw.71 cells and represent 3 independent experiments.

Figure 3.

Sw.71 cells secrete higher concentrations of osteopontin (OPN) when treated with Pregnyl human chorionic gonadotropin (hCG). There were higher concentrations of OPN in Sw.71-conditioned medium following 100 IU hCG treatment compared to nontreated controls 48 hours posttreatment (n = 3, P = .038).

Figure 4.

Sw.71 cells treated with 100 IU human chorionic gonadotropin (hCG) have increased attachment to endometrial epithelial cells compared to nontreated controls. Sw.71 cells constitutively expressing green fluorescent protein (GFP) were treated with 100 IU hCG for 24 hours and co-cultured with epithelial carcinoma cell 1 (ECC-1) cells for 1 hour. Once unattached cells were removed, the GFP-positive cells were quantified; one representative experiment is shown (A). Treatment with hCG increased Sw.71 attachment to the ECC-1 monolayer compared to nontreated Sw.71 controls (n = 5, P = .0006; A, B).

Figure 5.

Treatment with human chorionic gonadotropin (hCG) increases blastocyst-like spheroids (BLS) attachment to epithelial carcinoma cell 1 (ECC-1) monolayers. The BLS treated with hCG had more intimate contact with ECC-1 cells; ECC-1 cells form nest-like structures around hCG spheroids and there were more spheroids still attached to the monolayer after 72 hours.