Densities of decidual high endothelial venules correlate with T-cell influx in healthy pregnancies and idiopathic recurrent pregnancy losses.

Do high endothelial venules (HEVs) appear in the uterus of healthy and pathological pregnancies? Our study reveals that HEVs are present in the non-pregnant endometrium and decidua parietalis (decP) but decline upon placentation in decidua basalis (decB) and are less abundant in decidual tissues from idiopathic, recurrent pregnancy losses (RPLs). RPL is associated with a compromised decidual vascular phenotype. Endometrial (n = 29) and first trimester decidual (n = 86, 6-12th week of gestation) tissue samples obtained from endometrial biopsies or elective pregnancy terminations were used to determine the number of HEVs and T cells. In addition, quantification of HEVs and immune cells was performed in a cohort of decidual tissues from RPL (n = 25). Position and frequency of HEVs were determined in non-pregnant endometrial as well as decidual tissue sections using immunofluorescence (IF) staining with antibodies against E-selectin, intercellular adhesion molecule, von Willebrand factor, ephrin receptor B4, CD34 and a carbohydrate epitope specific to HEVs (MECA-79). Immune cell distribution and characterization was determined by antibodies recognizing CD45 and CD3 by IF staining- and flow cytometry-based analyses. Antibodies against c-c motif chemokine ligand 21 (CCL21) and lymphotoxin-beta were used in IF staining and Western blot analyses of decidual tissues. Functional HEVs are found in high numbers in the secretory endometrium and decP but decline in numbers upon placentation in decB (P ≤ 0.001). Decidua parietalis tissues contain higher levels of the HEV-maintaining factor lymphotoxin beta and decP-associated HEVs also express CCL21 (P ≤ 0.05), a potent T-cell chemoattractant. Moreover, there is a positive correlation between the numbers of decidual HEVs and the abundance of CD3+ cells in decidual tissue sections (P ≤ 0.001). In-depth analysis of a RPL tissue collection revealed a decreased decB (P ≤ 0.01) and decP (P ≤ 0.01) HEV density as well as reduced numbers of T cells in decB (P ≤ 0.05) and decP (P ≤ .001) sections when compared with age-matched healthy control samples. Using receiver-operating characteristics analyses, we found significant predictive values for the ratios of CD3/CD45 (P < 0.001) and HEVs/total vessels (P < 0.001) for the occurrence of RPL. Analyses were performed in first trimester decidual tissues from elective terminations of pregnancy or non-pregnant endometrium samples from patients diagnosed with non-endometrial pathologies including cervical polyps, ovarian cysts and myomas. First trimester decidual tissues may include pregnancies which potentially would have developed placental disorders later in gestation. In addition, our cohort of non-pregnant endometrium may not reflect the endometrial vascular phenotype of healthy women. Finally, determination of immune cell distributions in the patient cohorts studied may be influenced by the different modes of tissue derivation. Pregnancy terminations were performed by surgical aspiration, endometrial tissues were obtained by biopsies and RPL tissues were collected after spontaneous loss of pregnancy. In this study, we propose an inherent mechanism by which the endometrium and in particular the decidua control T-cell recruitment. By demonstrating reduced HEV densities and numbers of T cells in decB and decP tissues of RPL samples we further support previous findings reporting an altered vascular phenotype in early pregnancy loss. Altogether, the findings provide important information to further decipher the etiologies of unexplained RPL. This study was supported by the Austrian Science Fund (P31470 B30 to M.K.) and by the Austrian National Bank (17613ONB to J.P.). There are no competing interests to declare. N/A.

Windsperger K ，Vondra S ，Lackner AI ，Kunihs V ，Haslinger P ，Meinhardt G ，Dietrich B ，Dekan S ，Fiala C ，Knöfler M ，Saleh L ，Pollheimer J ... -  《-》

Extravillous trophoblast invasion of venous as well as lymphatic vessels is altered in idiopathic, recurrent, spontaneous abortions.

Do extravillous trophoblasts (EVTs) invade non-arterial decidual vessels in healthy and pathological pregnancies? Our results reveal that trophoblast invasion of venous and lymphatic vessels is a frequent event during the first trimester of pregnancy and is compromised in  recurrent spontaneous abortion (RSA). In addition, the present data suggest that EVTs populate regional lymph nodes during pregnancy. Human trophoblasts remodel and invade decidual spiral arteries. In addition, a recent report demonstrates that trophoblasts contact and invade decidual veins. Tissue samples of human first trimester deciduae basalis (n = 54, 6th-13th weeks of gestation) obtained from elective pregnancy terminations were used to study trophoblast invasion into veins and lymphatics, in comparison to arteries. Age-matched cases of idiopathic, recurrent spontaneous abortions tissue samples (n = 23) were assessed for cell numbers of EVTs in these decidual vessels. In addition, lymph nodes of four pregnant women were analysed for the presence of EVTs. Localization, frequency and EVT-mediated targeting and invasion of arterial, venous as well as lymphatic vessels were determined in first trimester decidua basalis tissue sections using immunofluorescence staining with antibodies against CD31, CD34, ephrin B2 (EFNB2), ephrin receptor B4 (EPHB4), HLA-G, podoplanin, prospero-related homeobox 1 (Prox-1), alpha-smooth muscle actin 2 (ATCTA2), von willebrand factor (vWF) and proteoglycan 2 (PRG2). Arterial, venous and lymphatic-associated EVTs were further characterized according to their position in the vascular structure and classified as intramural (im) or intraluminal (il). EVTs, specifically expressing PRG2, target and invade veins and lymphatics in first trimester decidua basalis since HLA-G+ trophoblast were detected in the vascular wall (intramural EVT, imEVTs) and in the lumen of these vessels (intraluminal EVT, ilEVTs). In total, 276 arteries, 793 veins and 113 lymphatics were analysed. While EVTs contact and invade arteries and veins to a similar extent we found that lymphatics are significantly less affected by EVTs (P = 0.001). Moreover, ilEVTs were detected in the lumen of venous and lymphatic vessels, whereas ilEVTs were only found occasionally in the lumen of arteries. Interestingly, RSA tissue sections contained significantly more arterial (P = 0.037), venous (P = 0.002) and lymphatic vessels (P < 0.001), compared to healthy controls. However, while RSA-associated arterial remodeling was unchanged (P = 0.39) the ratios of EVT-affected versus total number of veins (P = 0.039) and lymphatics (P < 0.001) were significantly lower in RSA compared to age-matched healthy decidual sections. Finally, HLA-G+/PRG2+/CD45-EVTs can be detected in regional lymph nodes of pregnant women diagnosed with cervical cancer. N/A. In this study, first trimester decidual tissues from elective terminations of pregnancies have been examined and used as a reference for healthy pregnancy. However, this collective may also include pregnancies which would have developed placental disorders later in gestation. Due to limitations in tissue availability our staining results for EVT-specific marker expression in regional lymph nodes of pregnant women are based on four cases only. In this study, we propose migration of HLA-G+ cells into regional lymph nodes during pregnancy suggesting that the human EVT is capable of infiltrating maternal tissues via the blood stream. Moreover, the description of compromised EVT invasion into the venous and lymphatic vasculature in RSA may help to better understand the pathological characteristics of idiopathic recurrent pregnancy loss. This study was supported by the Austrian Science Fund (grant P-25187-B13 to J.P. and grant P-28417-B30 to M.K.). There are no competing interests to declare.

Windsperger K ，Dekan S ，Pils S ，Golletz C ，Kunihs V ，Fiala C ，Kristiansen G ，Knöfler M ，Pollheimer J ... -  《-》

Newly characterized decidual Tim-3+ Treg cells are abundant during early pregnancy and driven by IL-27 coordinately with Gal-9 from trophoblasts.

What is the mechanism of Tim-3+ regulatory T (Treg)-cell accumulation in the decidua during early pregnancy and is its disruption associated with recurrent pregnancy loss (RPL)? IL-27 and Gal-9 secreted by trophoblasts activate the Tim-3 signaling pathway in CD4+ T cells and Treg cells and so promote accumulation of Tim-3+ Treg cells, the abnormal expression of IL-27 and Gal-9 is associated with impaired immunologic tolerance in RPL patients. Tim-3+ Treg cells are better suppressors of Teff cell proliferation, and display higher proliferative activity than Tim-3- Treg cells. Tim-3+ Treg cells are tissue-specific promoters of T-cell dysfunction in many tumors. These cells express a unique factor that influences and shapes the tumor microenvironment. The animal study included 80 normal pregnant mice. In human study, decidua tissues in the first trimester for flow cytometry analysis were collected from 32 normal pregnant women and 23 RPL patients. Placenta tissues for immunohistochemistry analysis were collected from 15 normal pregnant women. Placenta tissues for western blot analysis were collected from 5 normal pregnant women, 5 RPL patients and 5 women who have experienced one miscarriage. Blood samples for in vitro experiments were collected from 30 normal pregnant women. This study was performed between January 2017 and March 2019. In this study, we investigated the kinetics of Tim-3+ CD4+ T-cell accumulation, and the proportions of Tim-3+ Treg cells throughout murine pregnancies using flow cytometry. We compared Tim-3 expression on decidual CD4+ T cells and Treg cells during normal pregnancies with expression on the same cell populations in women suffering from RPL. IL-27 and Gal-9 transcription and protein expression in the placenta were determined by RT-PCR and western blot, respectively. An in vitro co-culture model consisting of peripheral CD4+ T cells and primary trophoblasts from early pregnancy was used to mimic the maternal-fetal environment. The percentage of Tim-3+ Treg cells present in mouse uteri fluctuates as gestation proceeds but does not change in the spleen. Levels of Tim3+ Treg cells in uteri peaked at pregnancy Day 6.5 (E 6.5), then progressively diminished, and fell to non-pregnant levels by E18.5. In pregnant mice, Tim-3+ Treg cells constituted 40-70% of Treg cells in uteri but were present at much lower abundance in spleens. About 60% of decidual Treg cells were Tim-3 positive at E6.5. Of these decidual Tim3+ Treg cells, nearly 90% were PD-1 positive. However, only about 16% of Tim3- Treg cells expressed PD-1. Blocking the Tim-3 signaling pathway decreased the proportion of Treg cells and led to embryo resorption. Moreover, much lower Tim-3 expression was observed on CD4+ T cells and Treg cells in women who had suffered from RPL at 6-9 gestational weeks compared with those who had normal pregnancies at matched gestations. In a normal pregnancy, Tim-3 expression on decidual CD4+ T cells is induced initially by IL-27. Then Gal-9-Tim-3 interaction promotes differentiation of decidual Tim-3+ CD4+ T cells into Treg cells. IL-27 and Gal-9 cooperatively induced Tim-3+ Treg cells in vitro. N/A. We did not investigate the kinetics of human decidual Tim-3+ CD4+ T and Tim-3+ Treg cell populations throughout pregnancy due to limited availability of second and third trimester decidua. In addition, functional suppressive data on the decidual Tim-3+ Treg cells are lacking due to limited and low quantities of these cells in decidua. These findings might have therapeutic clinical implications in RPL. This study was supported by research grants from the National Natural Science Foundation of China (No. 81871186) and National Key Research & Developmental Program of China (2018YFC1003900, 2018YFC1003904). The authors declare no conflict of interest.

Hu X ，Zhu Q ，Wang Y ，Wang L ，Li Z ，Mor G ，Liao A ... -  《-》

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 ... -  《-》

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 ... -  《-》