Corin, an enzyme with a putative role in spiral artery remodeling, is up-regulated in late secretory endometrium and first trimester decidua.

What is the nature of cellular Corin expression in human gestational tissues? CORIN is expressed in non-pregnant late secretory phase endometrium, first trimester human implantation sites and is up-regulated with decidualization ex vivo. Adequate trophoblast invasion and spiral artery remodeling/transformation is critical for successful implantation. CORIN, best known for its role in activating atrial natruietic peptide (ANP) to regulate blood pressure, has recently been proposed to be centrally involved in trophoblast invasion and spiral artery remodeling. It is postulated that ANP, activated by CORIN, promotes trophoblast invasion and that a deficiency causes pre-eclampsia. Mice deficient in either Corin or ANP displayed poor trophoblast invasion, impaired spiral artery remodeling and phenocopied human pre-eclampsia. However, the precise cellular localization of CORIN within human gestational tissues has not been well characterized. We measured CORIN protein localization in a number of human gestational tissues relevant to early embryo/placental implantation: non-pregnant (NP) endometrial biopsies (n = 5 per phase of the menstrual cycle), first trimester placental bed biopsies (n = 12) and pre-term control (n = 10) and severe early onset preeclamptic placentas (n = 15). Endometrial stromal cells were isolated from human endometrial biopsies (n = 5) and induced to decidualize ex vivo. Finally, CORIN concentrations were measured in serum obtained from pregnant women during the first trimester of whom, 56 subsequently ended up with a healthy term delivery (controls), 18 developed fetal growth restriction (FGR) and 21 had a miscarriage. We performed immunohistochemistry to assess CORIN localization. Changes in Corin mRNA expression in human endometrial stromal cells decidualized ex vivo were measured by quantitative RT-PCR, and levels of CORIN within human sera were measured by ELISA. CORIN was expressed in both NP late secretory phase endometrium and first trimester decidua within placental bed biopsies. Importantly, decidualization of primary human endometrial cells ex vivo significantly increased Corin expression (P < 0.05). CORIN was also detected within the villous cytotrophoblast, but there was no change in mRNA levels in placentas complicated by severe preterm pre-eclampsia when compared with pre-term controls. Although CORIN was detected in first trimester serum, levels did not change across gestation, nor could they predict miscarriage or FGR (other disorders of impaired placental invasion). Owing to the fact that we utilized early pregnancy human specimens, this is mainly a descriptive study with a limited amount of functional experiments. This is the first study to thoroughly characterize Corin mRNA and protein expression in human gestational tissue. Our findings support recent data from murine studies collectively suggesting that CORIN plays a critical role in trophoblast migration and spiral artery remodeling during early pregnancy in humans. Therefore, further studies of CORIN biology in early pregnancy may identify new therapeutic targets to improve implantation quality in early pregnancy and potentially reduce the rates of pregnancy complications caused by inadequate implantation (pre-eclampsia, FGR and miscarriage). This study was supported by The National Health and Medical Research Council of Australia (Salary support #490970, #490995). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors declare that no competing interests exist.

Kaitu'u-Lino TJ ，Ye L ，Tuohey L ，Dimitriadis E ，Bulmer J ，Rogers P ，Menkhorst E ，Van Sinderen M ，Girling JE ，Hannan N ，Tong S ... -  《-》

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 ... -  《-》

Dynamic changes in hyperglycosylated human chorionic gonadotrophin throughout the first trimester of pregnancy and its role in early placentation.

What is the in situ localization and function of hyperglycosylated hCG (hCG-H) in first trimester pregnancy tissues? HCG-H localizes to the syncytiotrophoblast, cytotrophoblast and invasive extravillous trophoblast within the maternal decidua and promotes invasion during the first trimester of pregnancy. Serum levels of hCG-H decline dramatically throughout the first trimester of pregnancy. As hCG-H is produced by choriocarcinoma cells, it is proposed to regulate trophoblast invasion. Tissues were collected from elective first trimester pregnancy terminations. Placental villous and decidua basalis were collected from Week 6 to Week 12 of gestation (n = 49). Tissues were collected from elective first trimester surgical pregnancy terminations to determine localization, abundance and function of hCG-H. Placental villous outgrowth studies determined the impact of neutralizing endogenous hCG-H on trophoblast function. Real-time proliferation, migration and invasion assays using JEG-3 choriocarcinoma cells further elucidated the role of hCG-H in trophoblast function. HCG-H localized to syncytiotrophoblast layer of the placental villous from gestational weeks 6-9; thereafter hCG-H localized as a discrete layer between syncytio- and cyto-trophoblast layers. Immunoreactive hCG-H was also observed within the cytotrophoblast layer in Week 7-8 of gestation. HCG-H abundance decreased within placental villous from Weeks 6-12 of gestation (n = 3 placentas per gestational weeks 6-12). HCG-H also localized to anchoring villi within maternal decidua, extravillous trophoblasts invading into the maternal decidua and endovascular trophoblasts remodeling maternal blood vessels. Treatment of primary first trimester villous explants with hCG-H neutralizing antibody reduced trophoblast outgrowth (n = 3 placentas, P < 0.05). Treatment of a trophoblast cell line with neutralizing antibody reduced trophoblast invasion (n = 4, P < 0.05) but did not affect migration or proliferation. Functional invasion and migration assays performed using cell lines. Not possible to perform such assays with primary human material. HCG-H is an important autocrine factor facilitating trophoblast invasion in the first trimester of pregnancy. Targeting hCG-H may prove useful in the treatment of pathologic pregnancies, such as ectopic pregnancies, or pregnancy complications including pre-eclampsia and gestational trophoblast diseases. This work was supported by the Victorian Government Operational Infrastructure Support Program. J.E. is supported by NHMRC project grant #1047756, L.A.S. and E.D. by NHMRC Fellowships #1002018 and #550905 respectively and E.M. by an NHMRC Early Career Fellowship #611827. The authors have no conflicts of interest relating to this work.

Evans J ，Salamonsen LA ，Menkhorst E ，Dimitriadis E ... -  《-》

Altered expression of interleukin-6, interleukin-8 and their receptors in decidua of women with sporadic miscarriage.

Are alterations in decidual expression of interleukin (IL)-6 and IL-8 associated with sporadic miscarriage? IL-6 and IL-8 secretion from decidual uterine natural killer (uNK) cells and macrophages isolated from women with spontaneous miscarriage was reduced compared with normal controls. Miscarriage is a common gynaecological problem with huge financial and personal implications. Eleven to twenty per cent of all clinically recognized pregnancies are lost before the 20th week of gestation, with miscarriages often being divided into early (≤ 12 completed weeks from last menstrual period) and late (≥ 13 weeks). Spiral artery remodelling is a key feature of early pregnancy; failure of this process has been implicated in sporadic miscarriage. The molecular triggers that initiate spiral artery remodelling are not clear, although cytokines such as IL-6 and IL-8 may play a role. This was a laboratory-based study using decidual and placental bed biopsy samples from women with sporadic miscarriage (n = 30) and termination of pregnancy controls (n = 30). Total adherent decidual cells, CD10(+) stromal cells, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from apparently normal pregnancies that were terminated at either 8-10 or 12-14 weeks' gestation. In addition, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from sporadic miscarriage at 8-10 weeks' gestation. Secreted IL-8 was measured in all isolated cell populations, while IL-6 was measured in CD14(+) macrophages and CD56(+) uNK cells from both sporadic miscarriage and normal controls. Placental bed biopsies were taken from women after sporadic miscarriage or termination of pregnancy at ≤ 12 completed weeks' or >13 weeks' gestational age, formalin-fixed, paraffin-embedded and immunostained for IL-6, IL-6Rα, GP130, IL-8, CXCR1, CXCR2 and CD13 (aminopeptidase N). Staining intensity for each factor was assessed in extravillous trophoblast cell populations, myometrial and decidual stroma, myometrial and decidual spiral arteries and decidual glandular epithelium. A CPA model was used to assess the potential role of IL-6 and IL-8 in spiral artery remodelling. IL-8 was secreted by total adherent decidual cells, CD10(+) stromal cells and CD14(+) macrophages at both 8-10 and 12-14 weeks' gestation, with CD14(+) cells secreting the highest levels. Both CD14(+) and CD56(+) cells isolated from decidua of early sporadic miscarriage produced lower IL-6 (P = 0.04, P = 0.01, respectively) and IL-8 levels (P = 0.0007, P = 0.002, respectively) compared with normal cases. In addition, altered expression of IL-6, IL-8 and their receptors was observed in various cell types in placental bed (myometrial stroma, glandular epithelium, interstitial extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells) in sporadic miscarriage, particularly from later gestational ages. IL-6 and IL-8 disrupted vascular smooth muscle morphology and organization in an in vitro model of spiral artery remodelling. By the nature of sampling at the time of miscarriage, it was not possible to ascertain the cause or effect in the observed alterations of levels of IL-6 and IL-8 in sporadic miscarriage. Alterations in the expression of IL-6, IL-8 and their receptors may be associated with the aetiology of sporadic miscarriage, especially given the potential role of these cytokines in the regulation of trophoblast invasion and spiral artery remodelling. This project was supported by funding from Wellbeing of Women (RG1000). The authors have no competing interests to declare. Not applicable.

Pitman H ，Innes BA ，Robson SC ，Bulmer JN ，Lash GE ... -  《-》

Sphingosine signalling regulates decidual NK cell angiogenic phenotype and trophoblast migration.

Is sphingosine-1-phosphate (S1P) signalling involved in the regulation of the angiogenic function of decidual (d)NK cells during human pregnancy? Human dNK cells, characterized by S1P receptor 5 (S1PR5) expression, are reactive to microenvironmental S1P to modify their VEGF expression and to regulate trophoblast migration and endothelial angiogenesis. S1P signalling can modulate peripheral (p)NK cells migration and function. As a unique NK population, human dNK can produce multiple cytokines and angiogenic growth factors to mediate extravillous trophoblast (EVT) invasion and spiral artery remodelling during pregnancy. The study was designed to examine S1PR expression and function by freshly isolated human dNK cells in response to different S1P scenarios, created by FTY720, an S1P analogue and S1PR modulator. Ex vivo and in vitro experiments were performed to evaluate the functions of dNK cells. The study was performed between September 2011 and June 2013. Human peripheral blood and decidual samples were collected and the S1PR expression by the decidual leukocytes population was examined. FTY720-induced dNK phenotypic and functional changes (including VEGF and IL-8 expression) were evaluated by multi-colour flow cytometric assays and transwell migration studies. Human placental explant culture and wound healing assays were performed to investigate whether S1P-activated dNK mediated trophoblast migration while angiogenesis was assessed by human umbilical vein endothelial cells (HUVEC) tube formation assays. Both first and second trimester dNK cells were studied to compare the difference in S1PR expression over time at the fetal-maternal interface. Freshly isolated NK cells (CD45(+)CD56(+)CD16(-)) from blood (pNK) and decidua (dNK) had low S1PR1 reactivity while S1PR5 was prominently expressed by dNK (40%) and, to a lesser extent, by pNK (18%; P < 0.05) cells. S1PR5 expression by dNK was significantly down-regulated by FTY720 treatment, which also impaired decidual leukocyte mobility and cellular contact with invasive EVT. FTY720 significantly reduced VEGF expression by dNK, both in the numbers of VEGF(+) cells and in fluorescence intensity (P < 0.05). IL-8 expression by dNK was not changed by FTY720 and remained low at 8% positivity. Trophoblast migration and HUVEC tube formation were stimulated by control leukocytes, enriched CD56(+) dNK or their conditioned medium, respectively, but this effect was markedly abrogated once they were pretreated with FTY720 (P < 0.05). There was a significant decrease in S1PR5 expression in second trimester dNK cells, compared with those from first trimester (P < 0.05). No significant differences in the levels of angiogenic factors (VEGF or IL-8) were detected between first and second trimester dNK cells. Our ex vivo and in vitro experimental samples were from healthy women undergoing elective pregnancy termination. FTY720 is a chemical ligand for the S1PRs; little is known regarding the levels or actions of the naturally occurring ligand S1P in human gestational tissues. The in vivo function of S1PR5(+) dNK may be further investigated by using a genetically modified animal model. This is the first study to investigate the role of S1PR and S1P interaction on dNK cell physiology and their downstream effects on trophoblast migration. We suggest that S1PR5 may represent a potential target for cellular targeted treatments for gestational diseases such as pre-eclampsia and intrauterine growth restriction that are characterized by inadequate dNK/trophoblast-coordinated uterine spiral artery transformation. This study was supported by Canadian Institutes of Health Research (CIHR), MOP82811 to Dr S.J.L.

Zhang J ，Dunk CE ，Lye SJ 《-》