Embryonic Regulation of Integrins b3, a4, and a1 in Human Endometrial Epithelial Cells in Vitro*

In the present study, we examined the embryonic regulation of b3 integrin in human endometrial epithelial cells (EEC) at the protein level and analyzed putative embryonic factors responsible for this regulation. The model employed is based on a clinical in vitro fertilization program in which single human embryos were cocultured with EEC until blastocyst stage and then transferred back to the uterus. After embryo transfer, EEC wells were divided according to the embryonic status reached: EEC with embryos that achieved the blastocyst stage, EEC with arrested embryos, and EEC without embryos. Immunostaining for b3 was positive in plasma membrane of EEC. Flow cytometry showed a mean percentage of b3-stained cells of 24.1 6 5.7 in EEC cocultured with embryos that achieved the blastocyst stage (n 5 13) vs. 9.5 6 1.6 (P , 0.05) in those EEC cultured with arrested embryos (n 5 12). Immunostaining for a1 and a4 integrins was negative in EEC monolayers studied, regardless of the presence or absence of embryos, and these findings were confirmed by flow cytometry. The possibility that the embryonic IL-1 system and leukemia inhibitory factor were involved in the endometrial b3 upregulation was investigated by neutralizing experiments demonstrating a significant inhibition of b3-stained cells when EEC monolayers were cultured in the presence of EEC/blastocyst-conditioned media with (n 5 4) vs. without (n 5 8) antihuman interleukin (IL)-1a 1 IL-1b (1.65% vs. 14.6%; P , 0.05). Dose-response experiments further demonstrated an up-regulation of b3 positive cells when IL-1a 1 IL-1b were added to the medium at a concentration of 10 pg/mL compared with control medium without added cytokines (40% vs. 20%, n 5 4). The functional relevance of the EEC b3 up-regulation was tested using a mouse blastocyst adhesion assay. More mouse blastocysts attached to EEC previously in contact with human blastocyst (72.7%) compared with those EEC previously in contact with arrested embryos (40%). Our results demonstrate the selective effect of a developing human embryo on EEC expression of b3, which is maximal when a human blastocyst instead of an arrested embryo is considered. Furthermore, the embryonic IL-1 system seems to be involved in the EEC b3 up-regulation, reinforcing the concept of precise paracrine crosstalk between blastocyst and endometrial epithelium during embryonic implantation. (J Clin Endocrinol Metab 82: 2607–2616, 1997) A of the blastocyst to the maternal endometrium is a progressive phenomenon that binds the embryo to the lumenal epithelium and is necessary for embryonic implantation to proceed (1). It is becoming increasingly apparent that adhesion molecules involved in cellular adhesion to other cells and to the extracellular matrix are crucial to this process (1–3). Specifically, integrins are membrane glycoproteins composed of two subunits (a and b) forming homologous groups. Their primary function is to mediate cell-to-cell and cell-to-extracellular matrix binding by specialized cell attachment sites, such as the tripeptide sequence Arg-Gly-Asp (RGD), which is a target sequence for integrin binding (4). This property provides cells with a number of possibilities to recognize different adhesive substrates. Embryonic implantation occurs in humans from cycle days 20–24 in the so-called implantation window, which is the period of optimal endometrial receptivity (5). During this time, b3, a4, and a1 integrins are considered potential markers of uterine receptivity (6–9). The a1 subunit is present only during the luteal phase (days 15–28) (7–9). The a4 integrin is expressed from days 14–24 (7, 8, 9) whereas the b3 subunit appears only on day 20 of the menstrual cycle and continues in the midluteal phase (7, 9). Therefore coexpression of b3, a4, and a1 integrins occur on the glandular epithelium during the implantation window. It is clear that these endometrial integrins are hormonally regulated (10). Integrins a4 and a1 are progesterone driven; they appear when progesterone production starts and endometrial progesterone receptors are highest (10, 11). In contrast, b3 appears when progesterone production is maximal, and endometrial progesterone receptors are lowest (10, 11). However, there is a lack of information regarding the role of the human embryo on the regulation of these endometrial integrins. It is also evident that paracrine-autocrine cytokine systems such as the interleukin-1 (IL-1) system (12) and leukemia inhibitory factor (LIF) (13) seem to control at least in part the Received February 12, 1997. Revision received April 9, 1997. Accepted April 18, 1997. Address all correspondence and requests for reprints to: Carlos Simon, Instituto Valenciano de Infertilidad, Guardia Civil 23, 46020 Valencia, Spain. E-mail: ivi@futurnet.es. * This work was supported by Fundacion Salud 2000, Instituto Valenciano de Infertilidad Foundation, and Fondo de Investigaciones de la Seguridad Social (FISss) 96/1263 grant from the Spanish Government, Ministerio de Sanidad y Consumo, Madrid. The International Cooperation has been supported by NATO Grant CRG 931614 and NIH Grant HD31575. Presented in part at the 52nd Annual Meeting of the American Fertility Society, Boston, Massachusetts, 1996. 0021-972X/97/$03.00/0 Vol. 82, No. 8 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright © 1997 by The Endocrine Society