Dados do Trabalho

Title

Embryo secretome is essential for endometrium remodelling during the window of implantation: in vitro analyzes of several cytokines

Objective

Successful embryo implantation is paramount in medically assisted reproduction. Defects in the endometrium have been associated with reproductive disorders, may cause implantation failures and these anomalies have been identified as one of the top research priorities in reproductive field. However, given the numerous endometrial functions that collectively allow the embryo to attach, the short period to implant and ethical considerations, makes difficult the full understanding of the molecular network that safeguards the embryo-endometrium crosstalking. This study aimed to evaluate the influence on the embryo secretome in modelling endometrium in vitro, possibly representing the embryo-endometrium crosstalking during the implantation window.

Methods

Endometrium biopsies were collected from fertile women during gynecologic routine procedures after consenting term signature between July-December 2022. Tissue biopsies were digested, and primary endometrial stromal cell cultures (EC) were established as described by D'Amora et al. 2009, with minor modifications. Briefly, cells were seeded onto 12-well plates at a density of 5,000 cells/cm2 and cultured for 48h (70% confluence). EC were hormone primed with estrogen (E2; 100 nmol/L for 24h), progesterone (P4; 100 nmol/L for 24h), mimicking the window of implantation. A pool of conditioned embryo culture medium (CEmbCM) collected from IVF cycles was prepared. E2/P4-primed EC were stimulated with CEmbCM in a volume of 20% of total culture media and then maintained for 24h. As controls, EC were grown in the following conditions: (i) Control: no E2/P4-priming or CEmbCM-stimulation; (ii) Hormone: E2/P4-priming and no CEmbCM-stimulation; (iii) CEmbCM: no E2/P4-priming and with CEmbCM-stimulation. Concentration of molecular factors (EGF, FGF-2, FRACTALKIN, GM-CSF, IL-6, IL-8, IL-15, IL-18, IP-10, TNF-A, VEGF-A, and LIF) were detected in the EC supernatant by a magnetic bead-based assay (MAGPIX® System, Luminex). Experimental procedures used three biological replicates, and experiments were performed in triplicates.

Results

A two-way Generalized Linear Model (GzLM) was used to evaluate the effect of the E2/P4-priming, CEmbCM-stimulation, considering each factor independently and the interaction between them (experimental condition), on secretion of the molecular factors by the EC. Statistical interaction is defined as the combined effects of factors on a measure of interest (dependent variable). If an interaction effect is present, it means that the impact of one factor depends on the other factor. An interaction between the variables was detected, showing a synergistic effect of E2/P4-priming and CEmbCM-stimulation on EC, resulting in a significant increase of molecular factors secretion, compared to control (mean±standard error): FGF-2 (2731.6±526.8 vs. 80.1±1.8; p<0.001), GMC-SF (54.4±3.6 vs. 33.1±3.8; p<0.001), IL-6 (12691.7±5319,4 vs. 13.7±5.3; p<0.001), IL-8 (2145.8±255.0 vs. 8.3±3.4; p<0.001), IL-18 (24.7±0.9 vs. 15.3±0.5; p=0.027), IP10 (60.7±2.6 vs. 51.8±1.9; p=0.016), TNF-a (15.8±0.4 vs. 11.6±0.3; p=0.019), VEGF-a (164.6±6.3 vs. 39.7±6.7; p<0.001) and LIF (87.6±5.2 vs. 28.7±0.2; p<0.001).

Conclusion

E2/P4-primed EC strongly responded to embryo secretome stimulation producing cytokines and growth factors, clearly demonstrating the crosstalk between embryo-endometrium. The factors secreted by the EC may participate in the endometrium receptivity and/or the subsequent implantation process. Our results reinforce that human embryo implantation mainly requires the interaction between the endometrium and the blastocyst secretome to achieve a successful pregnancy. Also this line of research intends to test other conditions, such as infertile patients presenting implantation failure, to assess how EC could be remodeled upon embryo stimulation.

Keywords

embryo-endometrium crosstalk, in vitro model, implantation, cytokines, growth factors