Profiling the expression and function of oestrogen receptor isoform ER46 in human endometrial tissues and uterine natural killer cells.

Does the oestrogen receptor isoform, ER46, contribute to regulation of endometrial function? ER46 is expressed in endometrial tissues, is the predominant ER isoform in first trimester decidua and is localised to the cell membrane of uterine natural killer (uNK) cells where activation of ER46 increases cell motility. Oestrogens acting via their cognate receptors are essential regulators of endometrial function and play key roles in establishment of pregnancy. ER46 is a 46-kDa truncated isoform of full length ERα (ER66, encoded by ESR1) that contains both ligand- and DNA-binding domains. Expression of ER46 in the human endometrium has not been investigated previously. ER46 is located at the cell membrane of peripheral blood leukocytes and mediates rapid responses to oestrogens. uNK cells are a phenotypically distinct (CD56brightCD16-) population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. We have shown that oestrogens stimulate rapid increases in uNK cell motility. Previous characterisation of uNK cells suggests they are ER66-negative, but expression of ER46 has not been characterised. We hypothesise that uNK cells express ER46 and that rapid responses to oestrogens are mediated via this receptor. This laboratory-based study used primary human endometrial (n = 24) and decidual tissue biopsies (n = 30) as well as uNK cells which were freshly isolated from first trimester human decidua (n = 18). Primary human endometrial and first trimester decidual tissue biopsies were collected using methods approved by the local institutional ethics committee (LREC/05/51104/12 and LREC/10/51402/59). The expression of ERs (ER66, ER46 and ERβ) was assessed by quantitative PCR, western blot and immunohistochemistry. uNK cells were isolated from first-trimester human decidua by magnetic bead sorting. Cell motility of uNK cells was measured by live cell imaging: cells were treated with 17β-oestradiol conjugated to bovine serum albumin (E2-BSA, 10 nM equivalent), the ERβ-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nM) or dimethylsulphoxide vehicle control. ER46 was detected in proliferative and secretory phase tissues by western blot and was the predominant ER isoform in first-trimester decidua samples. Immunohistochemistry revealed that ER46 was co-localised with ER66 in cell nuclei during the proliferative phase but detected in both the cytoplasm and cell membrane of stromal cells in the secretory phase and in decidua. Triple immunofluorescence staining of decidua tissues identified expression of ER46 in the cell membrane of CD56-positive uNK cells which were otherwise ER66-negative. Profiling of isolated uNK cells confirmed expression of ER46 by quantitative PCR and western blot and localised ER46 protein to the cell membrane by immunocytochemistry. Functional analysis of isolated uNK cells using live cell imaging demonstrated that activation of ER46 with E2-BSA significantly increased uNK cell motility. N/A. Expression pattern in endometrial tissue was only determined using samples from proliferative and secretory phases. Assessment of first trimester decidua samples was from a range of gestational ages, which may have precluded insights into gestation-specific changes in these tissues. Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in situ. E2 is an essential regulator of reproductive competence. This study provides the first evidence for expression of ER46 in the human endometrium and decidua of early pregnancy. We describe a mechanism for regulating the function of human uNK cells via expression of ER46 and demonstrate that selective targeting with E2-BSA regulates uNK cell motility. These novel findings identify a role for ER46 in the human endometrium and provide unique insight into the importance of membrane-initiated signalling in modulating the impact of E2 on uNK cell function in women. Given the importance of uNK cells to regulating vascular remodelling in early pregnancy and the potential for selective targeting of ER46, this may be an attractive future therapeutic target in the treatment of reproductive disorders. These studies were supported by Medical Research Council (MRC) Programme Grants G1100356/1 and MR/N024524/1 to PTKS. H.O.D.C. was supported by MRC grant G1002033. The authors declare no competing interests related to the published work.

Gibson DA ，Esnal-Zufiaurre A ，Bajo-Santos C ，Collins F ，Critchley HOD ，Saunders PTK ... -  《-》

Estrogen-dependent regulation of human uterine natural killer cells promotes vascular remodelling via secretion of CCL2.

Does intrauterine biosynthesis of estrogen play an important role in early pregnancy by altering the function of uterine natural killer (uNK) cells? Estrogens directly regulate the function of human uNK cells by increasing uNK cell migration and secretion of uNK cell-derived chemokine (C-C motif) ligand 2 (CCL2) that critically facilitates uNK-mediated angiogenesis. uNK cells are a phenotypically distinct population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. Recently we discovered that decidualisation of human endometrial stromal cells results in the generation of an estrogen-rich microenvironment in areas of decidualised endometrium. We hypothesize that intrauterine biosynthesis of estrogens plays an important role in early pregnancy by altering the function of uNK cells. This laboratory-based study used primary human uNK cells which were isolated from first trimester human decidua (n = 32). Primary uNK cells were isolated from first trimester human decidua using magnetic cell sorting. The impact of estrogens on uNK cell function was assessed. Isolated uNK cells were treated with estrone (E1, 10(-8) M) or estradiol (E2, 10(-8) M) alone or in combination with the anti-estrogen ICI 182 780 (ICI, 10(-6) M). uNK cell motility was assessed by transwell migration assay and time-lapse microscopy. Expression of chemokine receptors was assessed by quantitative PCR (qPCR) and immunohistochemistry, and angiogenic factors were assessed by qPCR and cytokine array. Concentrations of CCL2 in supernatants were measured by enzyme-linked immunosorbent assay. Angiogenesis was assessed in a human endometrial endothelial cell network formation assay. Treatment with either E1 or E2 increased uNK cell migration (P = 0.0092 and P = 0.0063, respectively) compared with control. Co-administration of the anti-estrogen ICI blocked the effects of E1 and E2 on cell migration. Concentrations of C-X-C chemokine receptor type 4 (CXCR4) mRNA in uNK cells were increased by E2 treatment. The network formation assay revealed that conditioned media from uNK cells treated with E2 significantly increased human endometrial endothelial cell (HEEC) angiogenesis (P = 0.0029 versus control). Analysis of media from uNK cells treated with E2 using an antibody array identified CCL2 as the most abundant cytokine. Validation assays confirmed concentrations of CCL2 mRNA and protein were increased by E2 in uNK cells (P < 0.05 versus controls). Compared with the control, recombinant human CCL2 was found to increase HEEC network formation (P < 0.05) and neutralization of CCL2 in uNK conditioned media significantly decreased E2-dependent uNK-mediated network formation (P = 0.0006). Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in vivo in humans. Primary human uNK cells were isolated from first trimester decidua at a range of gestations (8-12 weeks), which may be a source of variation. Primary human uNK cells from non-pregnant endometrium were not assessed and therefore the responses of uNK cells to E2 treatment described in this study may be distinct to uNK cells from first trimester decidua. E2 is an essential regulator of reproductive competence. This study demonstrates a critical role for E2 in regulating cellular cross-talk within the endometrium during early pregnancy. We provide the first evidence that E2 directly regulates the function of human uNK cells by altering uNK cell migration and the secretion of uNK-derived angiogenic factors. We describe a novel mechanism of estrogen-dependent secretion of CCL2 which critically mediates uNK-dependent endometrial angiogenesis. Dysregulation of uNK cell function has been implicated in the aetiology of early implantation disorders and disorders of pregnancy. These novel findings provide unique insight into the regulation of uNK cell activity during the establishment of pregnancy in women and highlight key processes which may be targeted in future therapeutic strategies. Studies undertaken in the authors' laboratory were supported by MRC Programme Grant G1100356/1 to P.T.K.S. The authors have no conflicts of interest to disclose.

Gibson DA ，Greaves E ，Critchley HO ，Saunders PT ... -  《-》

Decidual NK cell-derived conditioned medium from miscarriages affects endometrial stromal cell decidualisation: endocannabinoid anandamide and tumour necrosis factor-α crosstalk.

What are the effects of endocannabinoid anandamide (AEA) in uterine natural killer (unK) cells from miscarriage decidua, regarding their cytokine profile and endometrial stromal cell (ESC) crosstalk? uNK-conditioned media from miscarriage samples present high TNF-α levels which inhibit ESC decidualisation. AEA plasma levels are higher in women who have suffered a miscarriage. Moreover, AEA inhibits ESC proliferation and differentiation, although the levels and impact on the uNK cell cytokine profile at the feto-maternal interface remain elusive. This laboratory-based study used human primary uNK cells which were isolated from first-trimester decidua (gestational age, 5-12 weeks) derived from 8 women with elective pregnancy termination and 18 women who suffered a miscarriage. The first-trimester placental tissues were assayed for AEA levels by UPLC-MS/MS and respective enzymatic profile by western blot. The uNK cells were isolated and maintained in culture. The expression of angiogenic markers in uNK cells was examined by quantitative PCR (qPCR). The uNK-conditioned medium was analysed for IFN-γ, TNF-α and IL-10 production by enzyme-linked immunosorbent assay, and the impact on ESC differentiation was assessed by measuring decidual markers Prl, Igfbp-1 and Fox01 mRNA expression using qPCR. AEA levels were higher in miscarriage decidua compared with decidua from elective terminations. The uNK cell-conditioned medium from the miscarriage samples exhibited high TNF-α levels and interfered with the decidualisation of ESCs. Exacerbated inflammation and elevated TNF-α levels at the feto-maternal interface may trigger AEA signalling pathways that, in turn, may impact decidualisation and the angiogenic ability of uNK cells. N/A. Primary uNK cell responses are based on a simple in vitro model. Thus, in complex microenvironments, such as the feto-maternal interface, the mechanisms may not be exactly the same. Also, the inflammatory events of miscarriage that, in this study, have happened prior to processing of the samples may cause different responses to that observed. In addition, the magnitude of the inflammatory response, required to trigger the AEA pathways that impact decidualisation and the uNK angiogenic ability in vivo, is still unclear. The endocannabinoid AEA is a modulator of reproductive competence. AEA not only may contribute to neuroendocrine homeostasis but also can take part in uterine changes occurring during early pregnancy. The work was supported by UID/MULTI/04378/2019 with funding from Fundação para a Ciência e a Tecnologia (FCT)/MCTES through national funds and PORTUGAL 2020 Partnership Agreement, NORTE-01-0145-FEDER-000024. S.C. Cunha acknowledges FCT for the IF/01616/2015 contract. There are no conflicts of interest.

Fonseca BM ，Cunha SC ，Gonçalves D ，Mendes A ，Braga J ，Correia-da-Silva G ，Teixeira NA ... -  《-》

Hormonal stimulation reduces numbers and impairs function of human uterine natural killer cells during implantation.

How does an altered maternal hormonal environment, such as that seen during superovulation with gonadotropins in ART, impact human uterine immune cell distribution and function during the window of implantation? Hormonal stimulation with gonadotropins alters abundance of maternal immune cells including uterine natural killer (uNK) cells and reduces uNK cell ability to promote extravillous trophoblast (EVT) invasion. An altered maternal hormonal environment, seen following ART, can lead to increased risk for adverse perinatal outcomes associated with disordered placentation. Maternal immune cells play an essential role in invasion of EVTs, a process required for proper establishment of the placenta, and adverse perinatal outcomes have been associated with altered immune cell populations. How ART impacts maternal immune cells and whether this can in turn affect implantation and placentation in humans remain unknown. A prospective cohort study was carried out between 2018 and 2021 on 51 subjects: 20 from natural cycles 8 days after LH surge; and 31 from stimulated IVF cycles 7 days after egg retrieval. Endometrial biopsies and peripheral blood samples were collected during the window of implantation in subjects with regular menstrual cycles or undergoing superovulation. Serum estradiol and progesterone levels were measured by chemiluminescent competitive immunoassay. Immune cell populations in blood and endometrium were analyzed using flow cytometry. uNK cells were purified using fluorescence-activated cell sorting and were subjected to RNA sequencing (RNA-seq). Functional changes in uNK cells due to hormonal stimulation were evaluated using the implantation-on-a-chip (IOC) device, a novel bioengineered platform using human primary cells that mimics early processes that occur during pregnancy in a physiologically relevant manner. Unpaired t-tests, one-way ANOVA, and pairwise multiple comparison tests were used to statistically evaluate differences. Baseline characteristics were comparable for both groups. As expected, serum estradiol levels on the day of biopsy were significantly higher in stimulated (superovulated) patients (P = 0.0005). In the setting of superovulation, we found an endometrium-specific reduction in the density of bulk CD56+ uNK cells (P < 0.05), as well as in the uNK3 subpopulation (P = 0.025) specifically (CD103+ NK cells). In stimulated samples, we also found that the proportion of endometrial B cells was increased (P < 0.0001). Our findings were specific to the endometrium and not seen in peripheral blood. On the IOC device, uNK cells from naturally cycling secretory endometrium promote EVT invasion (P = 0.03). However, uNK cells from hormonally stimulated endometrium were unable to significantly promote EVT invasion, as measured by area of invasion, depth of invasion, and number of invaded EVTs by area. Bulk RNA-seq of sorted uNK cells from stimulated and unstimulated endometrium revealed changes in signaling pathways associated with immune cell trafficking/movement and inflammation. Patient numbers utilized for the study were low but were enough to identify significant overall population differences in select immune cell types. With additional power and deeper immune phenotyping, we may detect additional differences in immune cell composition of blood and endometrium in the setting of hormonal stimulation. Flow cytometry was performed on targeted immune cell populations that have shown involvement in early pregnancy. A more unbiased approach might identify changes in novel maternal immune cells not investigated in this study. We performed RNA-seq only on uNK cells, which demonstrated differences in gene expression. Ovarian stimulation may also impact gene expression and function of other subsets of immune cells, as well as other cell types within the endometrium. Finally, the IOC device, while a major improvement over existing in vitro methods to study early pregnancy, does not include all possible maternal cells present during early pregnancy, which could impact functional effects seen. Immune cells other than uNK cells may impact invasion of EVTs in vitro and in vivo, though these remain to be tested. These findings demonstrate that hormonal stimulation affects the distribution of uNK cells during the implantation window and reduces the proinvasive effects of uNK cells during early pregnancy. Our results provide a potential mechanism by which fresh IVF cycles may increase risk of disorders of placentation, previously linked to adverse perinatal outcomes. Research reported in this publication was supported by the University of Pennsylvania University Research Funding (to M.M.), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P50HD068157 to M.M., S.S., and S.M.), National Center for Advancing Translational Sciences of the National Institutes of Health (TL1TR001880 to J.K.), the Institute for Translational Medicine and Therapeutics of the Perelman School of Medicine at the University of Pennsylvania, the Children's Hospital of Philadelphia Research Institute (to S.M.G.), and the National Institute of Allergy and Infectious Diseases (K08AI151265 to S.M.G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. All authors declare no conflict of interest. N/A.

Kanter J ，Gordon SM ，Mani S ，Sokalska A ，Park JY ，Senapati S ，Huh DD ，Mainigi M ... -  《-》

Periconceptional exposure to lopinavir, but not darunavir, impairs decidualization: a potential mechanism leading to poor birth outcomes in HIV-positive pregnancies.

Does HIV protease inhibitor (PI)-based combination antiretroviral therapy (cART) initiated at periconception affect key events in early pregnancy, i.e. decidualization and spiral artery remodeling? Two PIs, lopinavir and darunavir, currently offered as cART options in HIV-positive pregnancies were evaluated, and we found that lopinavir-based cART, but not darunavir-based cART, impaired uterine decidualization and spiral artery remodeling in both human ex vivo and mouse in vivo experimental models. Early initiation of cART is recommended for pregnant women living with HIV. However, poor birth outcomes are frequently observed in HIV-positive pregnancies exposed to PI-based cART, especially when it is initiated prior to conception. The correlation between early initiation of PI-cART and adverse birth outcomes is poorly understood, due to lack of data on the specific effects of PI-cART on the early stages of pregnancy involving uterine decidualization and spiral artery remodeling. Lopinavir and darunavir were evaluated in clinically relevant combinations using an ex vivo human first-trimester placenta-decidua explant model, an in vitro human primary decidual cell culture system, and an in vivo mouse pregnancy model. The first-trimester (gestational age, 6-8 weeks) human placenta-decidua tissue was obtained from 11 to 15 healthy women undergoing elective termination of pregnancy. C57Bl/6 female mice (four/treatment group) were administered either lopinavir-cART, darunavir-cART or water by oral gavage once daily starting on the day of plug detection until sacrifice. Human: Spiral artery remodeling was assessed by immunohistochemical analysis of first-trimester placenta-decidua explant co-culture system. Trophoblast migration was measured using a placental explant culture. A primary decidual cell culture was used to evaluate the viability of immune cell populations by flow cytometry. Soluble factors, including biomarkers of decidualization and angiogenesis, were quantified by ELISA and Luminex assay using decidua-conditioned media. Mouse: In the mouse pregnancy model, gestational day 6.5 or 9.5 implantation sites were used to assess decidualization, spiral artery remodeling and uterine natural killer (uNK) cell numbers by immunohistochemistry. Transcription factor STAT3 was assayed by immunohistochemistry in both human decidua and mouse implantation sites. Lopinavir-cART, but not darunavir-cART, impaired uterine decidualization and spiral artery remodeling in both experimental models. Lopinavir-cART treatment was also associated with selective depletion of uNK cells, reduced trophoblast migration and defective placentation. The lopinavir-associated decidualization defects were attributed to a decrease in expression of transcription factor STAT3, known to regulate decidualization. Our results suggest that periconceptional initiation of lopinavir-cART, but not darunavir-cART, causes defective maturation of the uterine endometrium, leading to impairments in spiral artery remodeling and placentation, thus contributing to the poor birth outcomes. N/A. The human first-trimester placenta/decidua samples could only be obtained from healthy females undergoing elective termination of pregnancy. As biopsy is the only way to obtain first-trimester decidua from pregnant women living with HIV on PI-cART, ethics approval and participant consent are difficult to obtain. Furthermore, our animal model is limited to the study of cART and does not include HIV. HIV infection is also associated with immune dysregulation, inflammation, alterations in angiogenic factors and complement activation, all of which could influence decidual and placental vascular remodeling and modify any cART effects. Our findings provide mechanistic insight with direct clinical implications, rationalizing why the highest adverse birth outcomes are reported in HIV-positive pregnancies exposed to lopinavir-cART from conception. We demonstrate that dysregulation of decidualization is the mechanism through which lopinavir-cART, but not darunavir-cART, use in early pregnancy leads to poor birth outcomes. Although lopinavir is no longer a first-line regimen in pregnancy, it remains an alternate regimen and is often the only PI available in low resource settings. Our results highlight the need for reconsidering current guidelines recommending lopinavir use in pregnancy and indicate that lopinavir should be avoided especially in the first trimester, whereas darunavir is safe to use and should be the preferred PI in pregnancy.Further, in current times of the COVID-19 pandemic, lopinavir is among the top drug candidates which are being repurposed for inclusion in clinical trials world-over, to assess their therapeutic potential against the dangerous respiratory disease. Current trials are also testing the efficacy of lopinavir given prophylactically to protect health care workers and people with potential exposures. Given the current extraordinary numbers, these might include women with early pregnancies, who may or may not be cognizant of their gestational status. This is a matter of concern as it could mean that women with early pregnancies might be exposed to this drug, which can cause decidualization defects. Our findings provide evidence of safety concerns surrounding lopinavir use in pregnancy, that women of reproductive age considering participation in such trials should be made aware of, so they can make a fully informed decision. This work was supported by funding from the Canadian Institutes of Health Research (CIHR) (PJT-148684 and MOP-130398 to L.S.). C.D. received support from CIHR Foundation (FDN143262 to Stephen Lye). S.K. received a TGHRI postdoctoral fellowship. The authors declare that there are no conflicts of interest. L.S. reports personal fees from ViiV Healthcare for participation in a Women and Transgender Think Tank.

Kala S ，Dunk C ，Acosta S ，Serghides L ... -  《-》