Ovarian Stimulation in Mice Resulted in Abnormal Placentation through Its Effects on Proliferation and Cytokine Production of Uterine NK Cells

Abstract

Ovarian stimulation is associated with an increased incidence of abnormal placentation. Uterine natural killer (uNK) cells are the major subpopulation of decidual immune cells, which are crucial for placentation. In a previous study, we found that ovarian stimulation impairs uNK cell density on gestation day (GD) 8.5 in mice. However, it was not clear how ovarian stimulation led to a reduction in the density of uNK cells. In this study, we constructed two mouse models, an in vitro mouse embryo transfer model and an estrogen-stimulated mouse model. We used HE and PAS glycogen staining, immunohistochemical techniques, q-PCR, Western blot, and flow cytometry to analyze the mouse decidua and placenta, and the results showed that SO resulted in a fetal weight reduction, abnormal placental morphology, decreased placental vascular density, and abnormal density and function of uNK cells. Our results suggest that ovarian stimulation resulted in aberrant estrogen signaling and may contribute to the disorder of uNK cells caused by ovarian stimulation. Together, these results provide new insights into the mechanisms of aberrant maternal endocrine environments and abnormal placentation.

An Estrogen–NK Cells Regulatory Axis in Endometriosis, Related Infertility, and Miscarriage

“…In terms of uNK cell recruitment, Chantakru, S, demonstrated that estrogen could enhance homing (ovariectomized C57Bl/6 mice, 17β-estradiol at 100 ng/day, 3 days) by upregulating the expression of L-selectin and α-integrin on circulatory CD56 bright NK cells [ 47 ]. Conversely, Ma, R et al found that estrogen stimulation (pseudopregnant CD1 mice obtained blastocysts transfer, intraperitoneal injection of 5 IU of Pregnant mare serum gonadotropin (PMSG) followed by 5 IU of hCG 48 h later) significantly reduced the density and maturity of uNK cells in pregnant mice, suggesting an inhibitory effect of upregulated estrogen signaling on the accumulation and maturation of uNK cells [ 48 ]. Regarding NK function, Jaillon et al reported that pro-inflammatory cytokine production and NK cell activity increased after menopause, whereas estrogen therapy (0.625 mg conjugated estrogen + 2.5 mg medroxyprogesterone acetate for 2 months) could reverse this effect [ 49 , 50 ], indicating that estrogen may suppress peripheral NK cell activity and the secretion of pro-inflammatory cytokines.…”

“…Estrogen was also reported to increase the secretion of CCL2 in uNK cells (primary uNK cells, estrone 10 −8 mol/L or estradiol 10 −8 mol/L, cultured for 2 h), which is involved in the construction of blood vessels in the endometrium [ 53 ]. These conflicting findings on NK cell proliferation, recruitment, cytotoxicity, and cytokine secretion [ 46 , 47 , 48 , 49 , 50 , 51 , 52 ] indicate that the influence of estrogen on NK cells may differ based on the cell type, the method of administration, and the duration of exposure to estrogen. This highlights the need for additional research to fully understand these complex interactions.…”

“…In contrast, other studies have indicated that decidual stromal cells with estrogen stimulation (pseudopregnant CD1 mice obtained blastocysts transfer, intraperitoneal injection of 5 IU of PMSG followed by 5 IU of hCG 48 h later) exhibited reduced expression of various chemokines and cytokines, such as CXCL12, CXCL14, CX3CL1, IL-15, PLGF, and TGF-β. These findings suggest the presence of abnormal estrogen signaling during ovarian stimulation prior to assisted reproductive technologies [ 48 ]. HAND2, a transcription factor implicated in the cellular response to estradiol, exerts direct regulatory control over the expression of IL-15 in human ESCs.…”

High estrogen during ovarian stimulation induced loss of maternal imprinted methylation that is essential for placental development via overexpression of TET2 in mouse oocytes

“…The pathogenesis of the increased rate of placental disorders may involve endometrial changes associated with high estradiol levels. In our previous study, we found that OS resulting in aberrant estrogen signaling could lead to disorders in uNK cells, which are crucial for placentation [ 10 ]. In addition, the potential effects of high estradiol levels on oocytes during OS must be considered.…”