Pig Pregnancies after Transfer of Allogeneic Embryos Show a Dysregulated Endometrial/Placental Cytokine Balance: A Novel Clue for Embryo Death?

Abstract

Pig embryo transfer (ET) is burdened by high embryo mortality, with cytokines playing a significant role in recruitment of immune cells during embryo attachment and placentation. We hereby tested if their levels in endometrium and placenta from sows carrying hemi-allogeneic (artificially inseminated sows; C+ positive control) or allogeneic embryos (sows subjected to ET; ET) during peri-implantation (D18) or post-implantation (D24) are suitable mirrors of embryo rejection or tolerance after ET. Non-pregnant sows (C−) were used as negative controls. A set of cytokines was assayed in the tissues through multiplexed microsphere-based flow cytometry (Luminex xMAP, Millipore. USA). Fewer (58.7%. p < 0.003) conceptuses were recovered at D24 after ET compared to C+ (80.9%); with more than 20% of the ET conceptuses being developmentally delayed. Cytokine levels shifted during implantation. Anti-inflammatory IL-10 levels were significantly (p < 0.05) lower in ET sows compared to C+ at D24 of pregnancy. The C+ controls (carrying hemi-allogeneic embryos) consistently showed higher levels of pro-inflammatory TNF-α, IFN-γ, and IL-2 cytokines at D18 and IL-1α at D24, compared to the ET group. This clear dysregulation of pro- and anti-inflammatory cytokine levels in sows subjected to ET could be associated with an impaired maternal immune tolerance, explaining the high embryonic mortality of ET programs.

Allogeneic Embryos Disregulate Leukemia Inhibitory Factor (LIF) and Its Receptor in the Porcine Endometrium During Implantation

“…This means that embryos (allogeneic in the case of ETs employing donor embryos and hemi-allogeneic in the case of AIs) have the potential to influence the uterine milieu at the time of implantation, and subsequently determine the fate of pregnancy. This dysregulation could be responsible for the differences in embryo survival at D24 found in a previous study conducted in our laboratory (8) where allogeneic ET sows presented higher percentages of embryo losses and delayed fetuses (54.5 and 22.2%, respectively) in comparison to hemiallogeneic AI sows (23.4 and 5.3%, respectively) at D24 of pregnancy. These results are not surprising, given the pivotal role of LIF during pregnancy.…”

“…tissue samples consisted of pools of three different portions of the endometrium including the epithelial surface and endometrial glands (8). At D24, endometrium was collected from the site of implantation (IMP) and between implantation areas (Non-IMP).…”

“…Of interest, this process is also relevant for humans considering the increasing use of surrogate recipients, involving ET into a recipient who is immunologically distant from the biological parents, i.e., receiving an allogeneic embryo. In this respect, a study in pigs might be of interest, particularly during the process of implantation, being, in contrast to human, central and non-invasive in porcine (7), suggesting that an adequate balance between pro-and anti-inflammatory cytokines during the implantation period is essential in pigs to reach a satisfactory immune response to the allografts (8). The Leukemia inhibitory factor (LIF), part of the Interleukine-6 (IL-6) family of cytokines (9) appears to be relevant during this period due to its role as an uterine immune modulator, as proven in mice (10).…”

Natural Mating Differentially Triggers Expression of Glucocorticoid Receptor (NR3C1)-Related Genes in the Preovulatory Porcine Female Reproductive Tract

“…For example, STAT3 is downregulated in the uterus and upregulated in the ampulla (q < 0.05), infundibulum, and in the UTJ (by seminal plasma only). The downregulation of STAT3 in the uterus (and UTJ by SP-AI) may be also related to its role in the immune response, which is mediated by IL-6 and IL-10 [76], and recently shown to be associated with an increase in embryonic mortality in pigs after embryo transfer [81]. In addition, STAT1 and STAT2 key mediators of the innate immune response providing a first-line defense against pathogens [82], are downregulated by natural mating in the cervix and the uterus and might decrease the inflammation produced by semen deposition.…”

Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM.

“…Despite this, the percentage of non-pregnant animals after embryo transfer in our study was still higher than 64% in both groups. As it has been recently proposed, this could be associated to a dysregulation of pro and anti-in ammatory cytokine levels in recipient sows that, in turn, induce embryonic mortality [31]. However, many other factors related to either the quality of the embryos or the recipient's status and age could have been affecting these rates of unsuccessful transfers [32].…”