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Background Angiogenesis is a key feature of endometrial development. Inappropriate endometrial vascular development has been associated with recurrent miscarriage (RM) with increased amounts of perivascular smooth muscle cells surrounding them. Methods In the current study, we have used immunohistochemistry to study temporal and spatial expression of a series of angiogenic growth factors (AGFs) and their receptors; vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2, VEGF-R3, platelet-derived growth factor (PDGF)-BB, PDGF-Rα, PDGF-Rβ, transforming growth factor (TGF)-β1, TGF-βRI, TGF-βRII, angiopoietin (Ang)-1, Ang-2 and Tie-2, in the proliferative, early secretory and mid-late secretory phase endometrium from control women as well as in the mid-late secretory phase of women with a history of RM. The AGFs and their receptors studied were immunostained and assessed separately in stromal, vascular smooth muscle, endothelial and glandular epithelial cells. Laser capture microdissection and real-time RT–PCR were used to confirm expression patterns observed by immunohistochemistry. Results Most AGFs investigated showed both temporal and spatial expression patterns in normal cycling endometrium. In addition, immunostaining intensity for several AGFs was altered in women with a history of RM, particularly in vascular smooth muscle cells (VSMCs). VSMC expression of TGF-β1, VEGF-R1 and VEGF-R2 was increased while expression of PDGF-BB, TGF-βRI, TGF-βRII, Ang-2, VEGF-A and VEGF-C was reduced. Conclusions This study confirms that the cycling endometrium is a highly angiogenic tissue and that this process is likely to be altered in women with a history of RM and may contribute to the aetiology of this condition. [ABSTRACT FROM AUTHOR]

The aim of this work was to isolate, enumerate and attempt the identification of fetal cells recovered from the lower uterine pole. Immediately before elective termination of pregnancy at 7-17 weeks gestation, samples were recovered by transcervical flushing of the lower uterine pole (n = 108) or transcervical aspiration of mucus from just above the internal os (n = 187), and their contents examined using histological, immunohistochemical and molecular techniques. Syncytiotrophoblasts were identified morphologically in 28 out of 89 (31%) and 50 out of 180 (28%) flushings and aspirates respectively (mean 29%). Immunocytochemistry with monoclonal antibodies (mAbs) recognizing trophoblast or epithelial cell antigens on a smaller number of samples (n = 69) identified putative placental cells in 13 out of 19 (68%) and 25 out of 50 (50%) flushings and aspirates respectively (mean 55%). These included groups of distinctive cells with a small, round, hyperchromatic nucleus, strongly reactive with mAbs PLAP, NDOG1 and FT1.41.1. Smaller groups of larger, amorphous cells, usually containing multiple large, pale staining nuclei, reactive with mAb 340 and to a lesser degree with mAb NDOG5 were also observed. Taking cellular morphology and immunophenotype into consideration, the smaller uninucleate cells were likely to be villous mesenchymal cells, while the larger cells were possibly degrading villous syncytiotrophoblast. There was no significant difference in the frequency of fetal cells obtained by the two recovery methods. Squamous or columnar epithelial cells, labelled strongly with antibodies to cytokeratins or human milk fat globule protein, were observed in 97% (29 out of 30) of aspirates. The use of cervagem in a small number of patients prior to termination of pregnancy did not appear to influence the subsequent recovery of placental cells. Y-specific DNA was detected by polymerase chain reaction (PCR) in 13 out of 26 (50%) flushings and 99 out of 154 (64%) aspirates analysed (mean 62%). In-situ hybridization (ISH) revealed Y-specific targets in 40 out of 69 (60%) of aspirates analysed. A comparison of PCR data obtained from transcervical recovered samples and placental tissues showed a concordance of 80% (76 out of 95), with 10 false positives. Comparing the PCR data from tissues with data derived by ISH from 41 aspirates gave a concordance of 90% with two false positives. Although syncytiotrophoblasts were much more likely to be present in samples containing immunoreactive placental cells, the detection rates of fetal-derived DNA were similar regardless of the morphological and/or immunological presence of placental cells. We conclude that the transcervical recovery of fetal cells, while promising, requires considerable additional effort being expended in further research and development, particularly in in the sampling procedure. Keywords:fetal cells/immunohistology/in-situ hybridization/polymerase chain reaction/preimplantation diagnosis-transcervical sampling [ABSTRACT FROM PUBLISHER]

Apoptosis has been implicated in the pathogenesis of several diseases and is partly regulated by bcl-2, which blocks the apoptotic pathway and promotes cell survival. Apoptosis and bcl-2 expression were examined in paired eutopic and ectopic endometrium from women with endometriosis (n = 30 samples) or adenomyosis (n = 15 samples) and compared with control endometrium (n = 30 samples). Apoptotic cells were detected using the dUTP nick-end labelling (TUNEL) assay for DNA fragmentation; bcl-2 expression was demonstrated with a streptavidin-biotin peroxidase immunohistochemical technique. Apoptotic cells were rare in eutopic, ectopic and control endometrium; there were no significant differences between subject groups nor between eutopic and ectopic endometrium. Stromal bcl-2 expression increased in the late secretory phase in control and eutopic endometrium in endometriosis; double labelling studies revealed that most stromal bcl-2 + cells were leukocytes. Stromal bcl-2 expression in endometriotic foci was significantly increased compared with the paired eutopic endometrium, did not vary with menstrual cycle and included a significant population of non-leukocytic bcl-2 + stromal cells. In contrast, stromal bcl-2 expression in adenomyosis remained at low levels and did not show significant cyclical variation. Glandular epithelial bcl-2 expression also varied with menstrual cycle phase and peaked in the proliferative phase; in contrast, surface epithelial bcl-2 expression increased in the late secretory phase. Elevated stromal bcl-2 expression in ovarian endometriotic lesions could have implications for the growth and survival of ectopic endometrial tissue at these sites. [ABSTRACT FROM PUBLISHER]

Intraepithelial leukocytes (IEL) are recognized as an important component of most mucosal surfaces but have received scant attention in the human female reproductive tract. The aim of the present study was to characterize, quantify and compare IEL populations in normal endometrium (n = 30) and in eutopic and ectopic (endometriotic or adenomyotic lesions) endometrium from women with endometriosis (n = 30) or adenomyosis (n = 15) at different menstrual cycle phases in order to assess the role of IEL in these common but poorly understood disorders. IEL populations were examined in formalin-fixed, paraffin-embedded sections using a streptavidin-biotin-peroxidase complex technique and quantified in relation to epithelial cell numbers. IEL in control endometrium and eutopic endometrium in endometriosis and adenomyosis varied during the menstrual cycle, with CD45+, CD43+ and CD56+ cells increasing from the proliferative to the late secretory phase. IEL were elevated in surface compared with glandular epithelium in the proliferative and early secretory phases. Throughout the menstrual cycle there were no significant differences in IEL between eutopic and ectopic endometrium in adenomyosis. Endometriotic foci, however, contained elevated levels of CD45+, CD3+ and CD8+ cells and reduced numbers of CD56+ cells compared with the corresponding eutopic endometrium and these did not vary with menstrual cycle phase. In contrast, ectopic endometrium in adenomyosis showed some cyclical changes with CD56+ cells increasing significantly in the late secretory phase. It is possible these differences may play a role in the pathogenesis of endometriosis and the associated complications. [ABSTRACT FROM PUBLISHER]

T lymphocyte subpopulations were compared in normal first trimester human decidua and in decidua associated with spontaneous abortion. Cryostat sections were labelled using a panel of monoclonal antibodies specific for CD3, CD8, CD4 and for the αβ and γδ heterodimers of the T cell receptor using an avidin-biotin complex peroxidase method. All the endometrial T cell subsets which have been demonstrated in normal early pregnancy were detected in similar numbers and proportions in spontaneous abortion. The findings suggest that adverse pregnancy outcome is not influenced by altered proportions of T cell subpopulations; nevertheless, the possibility remains that these cells may have an altered antigenic phenotype in spontaneous abortion which could contribute to pregnancy success or failure. [ABSTRACT FROM PUBLISHER]

The aetiology of endometriosis, a common and disabling disorder, is presently unknown, although immune dysfunction could allow ectopic endometrial fragments to survive outside the uterine cavity. These studies investigate the relationship between leukocyte populations, steroid hormone receptor expression, proliferative activity, bcl-2 expression and apoptosis in eutopic and ectopic endometrium from women with endometriosis or adenomyosis at different phases of the menstrual cycle. Significantly increased oestrogen receptor expression, bcl-2 expression and numbers of CD8+ leukocytes were found in ectopic compared with eutopic endometrium in endometriosis, and CD56+ endometrial granulated lymphocytes (eGLs) were significantly reduced in ectopic endometrium. Apoptotic cells were rarely found in control and subject endometria. In contrast with endometriosis, adenomyotic lesions showed identical steroid hormone receptor expression, proliferative activity, bcl-2 expression and leukocyte subpopulations to eutopic endometrium, indicating different aetiologies for these disorders. The unusual CD56+ CD16-eGLs present in large numbers in late secretory phase eutopic endometrium were highly purified (>98%) by immunomagnetic separation. Except for a negligible cytotoxic activity of eGLs from early proliferative samples, cytotoxic activity of eGLs from non-pregnant endometrium during the menstrual cycle was comparable with those in peripheral blood, predominantly CD56+ CD16+ natural killer cells. eGLs from non-pregnant endometrium and early pregnancy showed a variable proliferative response to 5 and 100 U/ml interleukin-2 over 48-h and 120-h time courses. eGLs are evidently functionally important in the eutopic endometrium. Their absence in endometriotic lesions together with increased CD+8 T cell numbers and increased oestrogen receptor and bcl-2 expression may have significant effects on the development and progression of endometriosis. [ABSTRACT FROM PUBLISHER]

Research Question: What is the expression pattern of platelet-derived growth factor BB (PDGF-BB), and its receptors, across the menstrual cycle in healthy control women and those with abnormal uterine bleeding-endometrial disorder (AUB-E)?, Design: Immunohistochemical staining for PDGF-BB, platelet-derived growth factor receptor alpha (PDGFRα) and platelet-derived growth factor beta (PDGFRβ) was performed in control and AUB-E endometrium from the proliferative, early, mid- and late secretory phases of the menstrual cycle (n = 5 each group). Control proliferative phase endometrium was cultured in PDGF-BB (0, 10 ng/ml) and vascular maturation assessed (n = 3). Endothelial cell to vascular smooth muscle cell (VSMC) association was assessed after treatment with PDGF-BB (0, 1, 10 ng/ml). Secretion of angiogenic growth factors by endothelial cells or VSMC was determined., Results: Endothelial cell immunoreactivity for PDGF-BB was reduced in the mid and late secretory phases in AUB-E (P = 0.008). PDGFRα was also reduced in mid secretory phase endothelial cells, proliferative and early secretory phase glandular epithelium in AUB-E (P = 0.008). PDGFRβ expression was not altered. Treatment of proliferative phase endometrium with PDGF-BB (10 ng/ml) reduced the percentage of vessels expressing contractile VSMC markers. PDGF-BB had no effect on angiogenic growth factor secretion by endothelial cells or VSMC in vitro and did not affect their association in an in-vitro endothelial cell-VSMC association assay., Conclusions: Reduced endothelial cell expression of PDGF-BB in the AUB-E endometrium may contribute to the reduced vascular maturation previously observed in these women., (Copyright © 2022 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate clinicians' views of managing women with first-trimester Recurrent Miscarriage within the UK compared with RCOG guidance. An online survey of 150 Association of Early Pregnancy Units members was conducted using SurveyMonkey™. Analysis was limited to UK-based respondents (102). Of the three key investigations, 98% performed antiphospholipid antibodies (APA) screening, 93.1% performed karyotyping for subsequent miscarriages and 86.3% performed a pelvic ultrasound routinely. Other routine investigations included inherited thrombophilias (65.7%), thyroid function tests (51.9%), diabetes mellitus screening (35.3%), parental karyotyping (34.3%), androgen profile (25.5%), 3-D ultrasound (17.6%), hysteroscopy (12.7%), hysterosalpingogram (9.8%), Vitamin D (7.8%), peripheral natural killer cells (2.9%) and uterine natural killer cells (2.9%). APA-positive women were offered treatment by 97.1%; however, 23.5% routinely offered treatment for APA-negative women. Other treatments offered routinely included progesterone (27.5%) and metformin (1.9%). Most clinicians managed RM as recommended by RCOG, however we have highlighted considerable deviation from the RCOG guidelines.IMPACT STATEMENT What is already known on this subject? Recurrent miscarriage (RM) can cause significant distress to women and their partners prompting referrals for investigation and management of this condition. Although UK national clinical guidance exists published by RCOG, the adherence to the guidance in clinical practice is not known. What do the results of this study add? This study shows that most clinicians performed investigations recommended by RCOG when managing women with RM. However, we have highlighted considerable variation of practice; many additional investigations were routinely performed and a quarter of clinicians offered treatments outside the RCOG guidance. What are the implications of these findings for clinical practice and/or further research? This paper demonstrates considerable variation of practice across the UK. Clinical practice may continue to vary whilst there are separate guidelines available from different professional organisations worldwide. Collaboration to produce a general consensus could reduce the variation in the care that these women receive.

Appropriate growth and development of the endometrium across the menstrual cycle is key for a woman's quality of life and reproductive well-being. Recurrent pregnancy loss (RPL) and heavy menstrual bleeding (HMB) affect a significant proportion of the female population worldwide. These endometrial pathologies have a significant impact on a woman's quality of life as well as placing a high economic burden on a country's health service. An underlying cause for both conditions is unknown in approximately 50% of cases. Previous research has demonstrated that aberrant endometrial vascular maturation is associated with both RPL and HMB, where it is increased in RPL but reduced in HMB. TGFβ1 is one of the key growth factors that regulate vascular maturation, by inducing phenotypic switching of vascular smooth muscle cells (VSMCs) from a synthetic phenotype to a more contractile one. Our previous data demonstrated an increase in TGFβ1 in the endometrium of RPL, while others have shown a decrease in women with HMB. However, TGFβ1 bioavailability is tightly controlled, and we therefore sought to perform an extensive immunohistochemical analysis of different components in the pathway in the endometrium of normal controls, women with HMB or RPL. In addition, two in vitro models were used to examine the role of TGFβ1 in endometrial vascular maturation and endothelial cell (EC):VSMC association. Taken all together, the immunohistochemical data suggest a decrease in bioavailability, receptor binding capacity, and signaling in the endometrium of women with HMB compared with controls. In contrast, there is an increase in the bioavailability of active TGFβ1 in the endometrium of women with RPL compared with controls. Endometrial explants cultured in TGFβ1 had an increase in the number of vessels associated with contractile VSMC markers, although the total number of vessels did not increase. In addition, TGFβ1 increased EC:VSMC association in an in vitro model. In conclusion, TGFβ1 is a key regulator of endometrial vascular maturation and could be considered as a therapeutic target for women suffering from HMB and/or RPL., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Sun, Shivhare, Hou, Bulmer, Innes, Hapangama and Lash.)

Spiral artery (SpA) remodelling is essential for a successful pregnancy and is best described by its morphological features; vascular smooth muscle cell separation and loss, vessel dilatation, and invasion by extravillous trophoblast cells (EVT). Current opinion holds that EVT fully replace the endothelial cells (EC) of the SpA and take on EC-like characteristics. Placental bed biopsies (6-20 weeks gestation) were immunostained for EC and EVT, showing transient loss of EC. In vessels containing an endovascular EVT plug (n = 28) 77.6 ± 19.3% of the lumen was covered by EC while in vessels without endovascular EVT (n = 100) it was 100 ± 0%., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Study Question: Are uterine natural killer (uNK) cell numbers and their distribution relative to endometrial arterioles altered in women with recurrent implantation failure (RIF) compared to women with embryo implantation success (IS)?, Summary Answer: uNK cell numbers and their distribution relative to endometrial arterioles are not significantly different in women with RIF compared to women in whom embryo implantation occurs successfully following IVF., What Is Already Known: uNK cells are regulators of decidual angiogenesis and spiral arteriole remodelling during early pregnancy. Although some studies have shown that uNK cell numbers may be altered in women with RIF, the methods used to measure uNK cell numbers have proven inconsistent, making reproduction of these results difficult. It is unclear, therefore, whether the results reported so far are reproducible. Moreover, it is not known how uNK cell numbers may impact IVF outcomes. Despite the lack of conclusive evidence, uNK cell numbers are often evaluated as a prognostic criterion in women undergoing assisted reproductive procedures., Study Design, Size, Duration: Endometrial pipelle biopsies were collected 6-8 days post-LH surge in natural cycles from women with RIF (n = 14), women with IS (n = 11) and women with potential RIF at the time of the study (PRIF; n = 9) from 2013 to 2015., Participants/materials, Setting, Methods: uNK cells (i.e. CD56+ and/or CD16+ phenotypes) and their distribution relative to endometrial arterioles were investigated by standard immunohistochemistry protocols and quantified using Aperio ScanScopeXT images digitized by ImageJ and deconvoluted into binary images for single cell quantification using a Gaussian Blur and Yen algorithm., Main Results and the Role of Chance: There was no significant difference in the cell density of CD56+ or CD16+ uNK cells in women with RIF compared to women with IS or PRIF. There was a higher proportion of uNK cells in the distal regions compared to the regions closest to the arterioles in all patient groups. Further, we identified a significant reduction in uNK cell density in women who had a previous pregnancy compared to those who had not, regardless of their current implantation status., Large Scale Data: Not applicable., Limitations, Reasons for Caution: Spiral arterioles could not always be accurately identified by digital image analysis; therefore, all endometrial arterioles were selected and analysed. Patient numbers for the study were low. However, as the clinical phenotypes of each patient were well defined, and endometrial dating was accurately determined by three independent pathologists, differences between patient groups with respect to the uNK numbers and distribution should have been measurable if uNK cell counts were to be useful as a prognostic marker of RIF., Wider Implications of the Findings: Our findings demonstrate that CD56+ and CD16+ uNK cell numbers are not significantly different in women with RIF in a typical cohort of women undergoing IVF. Further, prior pregnancy was associated with a significantly reduced number of uNK cells in both the RIF and IS patient groups, suggestive of a long-term pregnancy induced suppression of uNK cells. Combined, these findings do not support the clinical value of using uNK cell numbers as a prognostic indicator of implantation success with IVF treatment., Study Funding/competing Interest(s): Funding for this work was provided by Royal Women's Hospital Foundation. P.P. was supported by an NHMRC Early Career Fellowship [TF 11/14] and W.T.T. was supported by an NHMRC Postgraduate Scholarship [1055814]. The authors do not have any competing interests with this study., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Uterine spiral arteries undergo remodelling in normal pregnancy, with replacement of the musculoelastic arterial media by fibrinoid containing extravillous trophoblast cells. Deficient spiral artery remodelling is associated with several adverse pregnancy outcomes. Although there are distinct components of spiral artery remodelling, assessment is subjective and often based on an overall impression of morphology. We aimed to develop a quantitative approach for assessment of uterine spiral artery remodelling. Placental bed biopsies were immunostained using smooth muscle markers, digital images of spiral arteries were captured and Adobe Photoshop was used to analyse positive immunostaining. The method was then used to investigate variation in the same vessel at different levels within a paraffin block, and the effect of parity, pre-eclampsia or miscarriage on vascular smooth muscle cell content. Results were also compared with a more subjective morphology-based assessment system. There was good intra- and interobserver agreement and the method correlated well with the more subjective assessment system. There was an overall reduction in vascular smooth muscle, as detected by caldesmon 1 (h-caldesmon) immunopositivity, with increasing gestational age from 8 weeks to term. A previous pregnancy did not affect the amount of spiral artery smooth muscle. Comparison of pre-eclampsia and late miscarriage samples with controls of the appropriate gestational age demonstrated increased medial smooth muscle in pathological samples. This technique provides a simple, rapid, reproducible and inexpensive approach to quantitative assessment of spiral artery remodelling in normal and pathological human pregnancy, a process which although fundamental for successful pregnancy, is still incompletely understood.

Spontaneous preterm birth (sPTB, delivery <37 weeks gestation), accounts for approximately 10% of births worldwide; the aetiology is multifactorial with intra-amniotic infection being one contributing factor. This study aimed to determine whether asymptomatic women with a history of sPTB or cervical surgery have altered levels of inflammatory/antimicrobial mediators and/or microflora within cervical fluid at 22-24 weeks gestation. External cervical fluid was collected from women with history of previous sPTB and/or cervical surgery at 22-24 weeks gestation (n = 135). Cytokine and antimicrobial peptides were measured on a multiplex platform or by ELISA. qPCR was performed for detection of 7 potentially pathogenic bacterial species. IL-8 and IL-1β levels were lower in women who delivered preterm compared to those who delivered at term (IL-8 P = 0.02; IL-1β P = 0.04). There were no differences in elafin or human beta defensin-1 protein levels between the two groups. Multiple bacterial species were detected in a higher proportion of women who delivered preterm than in those who delivered at term (P = 0.005). Cervical fluid IL-8 and IL-1β and microflora have the potential to be used as biomarkers to predict sPTB in high risk women.

Study Question: Is vascular smooth muscle cell (VSMC) dedifferentiation a feature of uterine spiral artery (SpA) remodelling in early human pregnancy?, Summary Answer: Remodelling of human uterine SpAs is associated with dedifferentiation of VSMCs and can be induced in vitro by uterine natural killer (uNK) cells and extravillous trophoblast cells (EVTs)., What Is Known Already: Uterine SpAs undergo profound morphological changes in normal pregnancy with replacement of the musculoelastic arterial wall structure by fibrinoid containing EVTs. The fate of VSMCs in SpA remodelling is unknown; in guinea pig uterine artery VSMCs dedifferentiate, remain in the vessel wall and differentiate after parturition to restore the arterial wall. There is increasing evidence that uNK cells play a role in SpA remodelling. We hypothesized that SpA remodelling in human pregnancy is associated with VSMC dedifferentiation, initiated by uNK cell-derived growth factors., Study Design, Size, Duration: Formalin fixed, paraffin embedded placental bed biopsies were immunostained for angiogenic growth factor (AGF) receptors and markers of VSMC differentiation. An in vitro model of SpA remodelling using chorionic plate arteries (CPAs) was used to test the effect of different cell types and AGFs on VSMC differentiation., Participants/materials, Setting, Methods: Placental bed biopsies were immunostained for vascular endothelial growth factor receptors 1-3 (VEGF-R1, VEGF-R2, VEGF-R3), transforming growth factor beta 1 receptors I and II (TGF-βRI, TGF-βRII), interferon gamma receptors 1 and 2 (IFN-γR1, IFN-γR2), Tie2, α-smooth muscle actin (α-SMA), H-caldesmon (H-Cal), myosin heavy chain (MyHC), osteopontin and smoothelin. Staining intensity was assessed using a modified quickscore. Expression by VSMCs of the AGF receptors was confirmed by laser capture microdissection and real-time RT-PCR of non-remodelled SpAs, after laser removal of the endothelium. As an in vitro model, VSMC differentiation was assessed in CPAs by immunohistochemistry after culture in uNK cell-conditioned medium (CM), EVT-CM, uNK cell/EVT co-culture CM, Ang-1, Ang-2, IFN-γ, VEGF-A and VEGF-C, and after blocking of both Ang-1 and Ang-2 in uNK-CM., Main Results and the Role of Chance: SpA VSMC expression of Tie-2 (P = 0.0007), VEGF-R2 (P = 0.005) and osteopontin (P = 0.0001) increased in partially remodelled SpAs compared with non-remodelled SpAs, while expression of contractile VSMC markers was reduced (α-SMA P < 0.0001, H-Cal P = 0.03, MyHC P = 0.03, smoothelin P = 0.0001). In the in vitro CPA model, supernatants from purified uNK cell (H-Cal P < 0.0001, MyHC P = 0.03, α-SMA P = 0.02, osteopontin P = 0.03), EVT (H-Cal P = 0.0006, MyHC P = 0.02, osteopontin P = 0.01) and uNK cell/EVT co-cultures (H-Cal P = 0.001, MyHC P = 0.05, osteopontin P = 0.02) at 12-14 weeks, but not 8-10 weeks, gestational age induced reduced expression of contractile VSMC markers and increased osteopontin expression. Addition of exogenous (10 ng/ml) Ang-1 (P = 0.006) or Ang-2 (P = 0.009) also reduced H-Cal expression in the CPA model. Inhibition of Ang-1 (P = 0.0004) or Ang-2 (P = 0.004) in uNK cell supernatants blocked the ability of uNK cell supernatants to reduce H-Cal expression., Limitations, Reasons for Caution: This is an in vitro study and the role of uNK cells, Ang-1 and Ang-2 in SpA remodelling in vivo has not yet been shown., Wider Implications of the Findings: VSMC dedifferentiation is a feature of early SpA remodelling and uNK cells and EVT play key roles in this process by secretion of Ang-1 and Ang-2. This is one of the first studies to suggest a direct role for Ang-1 and Ang-2 in VSMC biology., Study Funding/competing Interest(s): This work was supported by a grant from British Biotechnology and Biosciences Research Council (BB/E016790/1). The authors have no competing interests to declare., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Vitamin D deficiency is prevalent in pregnant women and is associated with adverse pregnancy outcomes, in particular disorders of malplacentation. The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a potent regulator of innate and adaptive immunity, but its immune effects during pregnancy remain poorly understood. During early gestation, the predominant immune cells in maternal decidua are uterine natural killer cells (uNK), but the responsivity of these cells to 1,25(OH)2D3 is unknown despite high levels of 1,25(OH)2D3 in decidua. Transcriptomic responses to 1,25(OH)2D3 were characterised in paired donor uNK and peripheral natural killer cells (pNK) following cytokine (CK) stimulation. RNA-seq analyses indicated 911 genes were differentially expressed in CK-stimulated uNK versus CK-stimulated pNK in the absence of 1,25(OH)2D3, with predominant differentially expressed pathways being associated with glycolysis and transforming growth factor β (TGFβ). RNA-seq also showed that the vitamin D receptor (VDR) and its heterodimer partner retinoid X receptor were differentially expressed in CK-stimulated uNK vs CK-stimulated pNK. Further analyses confirmed increased expression of VDR mRNA and protein, as well as VDR-RXR target in CK-stimulated uNK. RNA-seq analysis showed that in CK-stimulated pNK, 1,25(OH)2D3 induced 38 and suppressed 33 transcripts, whilst in CK-stimulated uNK 1,25(OH)2D3 induced 46 and suppressed 19 genes. However, multiple comparison analysis of transcriptomic data indicated that 1,25(OH)2D3 had no significant overall effect on gene expression in either CK-stimulated pNK or uNK. These data indicate that CK-stimulated uNK are transcriptionally distinct from pNK and, despite expressing abundant VDR, neither pNK nor uNK are sensitive targets for vitamin D.

The presence of unusual natural killer cells in human endometrium has been recognized for 30 years, but despite considerable research effort, the in vivo role of uterine natural killer (uNK) cells in both normal and pathological pregnancy remains uncertain. uNK cells may differentiate from precursors present in endometrium, but migration from peripheral blood in response to chemokine stimuli with in situ modification to a uNK cell phenotype is also possible. uNK cells produce a wide range of secretory products with diverse effects on trophoblast and spiral arteries which may play an important role in implantation and early placentation. Interactions with other decidual cell populations are also becoming clear. Recent evidence has demonstrated subpopulations of uNK cells and the presence of other innate lymphoid cell populations in decidua which may refine future approaches to investigation of the role of uNK cells in human pregnancy., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

During early human pregnancy the uterine mucosa transforms into the decidua, into which the fetal placenta implants and where placental trophoblast cells intermingle and communicate with maternal cells. Trophoblast-decidual interactions underlie common diseases of pregnancy, including pre-eclampsia and stillbirth. Here we profile the transcriptomes of about 70,000 single cells from first-trimester placentas with matched maternal blood and decidual cells. The cellular composition of human decidua reveals subsets of perivascular and stromal cells that are located in distinct decidual layers. There are three major subsets of decidual natural killer cells that have distinctive immunomodulatory and chemokine profiles. We develop a repository of ligand-receptor complexes and a statistical tool to predict the cell-type specificity of cell-cell communication via these molecular interactions. Our data identify many regulatory interactions that prevent harmful innate or adaptive immune responses in this environment. Our single-cell atlas of the maternal-fetal interface reveals the cellular organization of the decidua and placenta, and the interactions that are critical for placentation and reproductive success.

Study Question: Are there any phenotypic and structural/architectural changes in the vessels of endometrium and superficial myometrium during the normal menstrual cycle in healthy women and those with heavy menstrual bleeding (HMB)?, Summary Answer: Spatial and temporal differences in protein levels of endothelial cell (EC) markers and components of the extracellular matrix (ECM) were detected across the menstrual cycle in healthy women and these are altered in HMB., What Is Known Already: HMB affects 30% of women of reproductive age with ~50% of cases being idiopathic. We have previously shown that the differentiation status of endometrial vascular smooth muscle cells (VSMCs) is altered in women with HMB, suggesting altered vessel maturation compared to controls. Endometrial arteriogenesis requires the co-ordinated maturation not only of the VSMCs but also the underlying ECs and surrounding ECM. We hypothesized that there are spatial and temporal patterns of protein expression of EC markers and vascular ECM components in the endometrium across the menstrual cycle, which are altered in women with HMB., Study Design, Size, Duration: Biopsies containing endometrium and superficial myometrium were taken from hysterectomy specimens from both healthy control women without endometrial pathology and women with subjective HMB in the proliferative (PP), early secretory (ESP), mid secretory (MSP) and late secretory (LSP) phases (N = 5 for each cycle phase and subject group). Samples were fixed in formalin and embedded in paraffin wax., Participants/materials, Setting, Methods: Serial sections (3μm thick) were immunostained for EC markers (factor VIII related antigen (F8RA), CD34, CD31 and ulex europaeus-agglutinin I (UEA-1) lectin), structural ECM markers (osteopontin, laminin, fibronectin and collagen IV) and for Ki67 to assess proliferation. Immunoreactivity of vessels in superficial myometrium, endometrial stratum basalis, stratum functionalis and luminal region was scored using either a modified Quickscore or by counting the number of positive vessels., Main Results and the Role of Chance: In control samples, all four EC markers showed a dynamic expression pattern according to the menstrual cycle phase, in both endometrial and myometrial vessels. EC protein marker expression was altered in women with HMB compared with controls, especially in the secretory phase in the endometrial luminal region and stratum functionalis. For example, in the LSP expression of UEA-1 and CD31 in the luminal region decreased in HMB (mean quickscore: 1 and 5, respectively) compared with controls (3.2 and 7.4, respectively) (both P = 0.008), while expression of F8RA and CD34 increased in HMB (1.4 and 8, respectively) compared with controls (0 and 5.8, respectively) (both P = 0.008). There was also a distinct pattern of expression of the vascular structural ECM protein components osteopontin, laminin, fibronectin and collagen IV in the superficial myometrium, stratum functionalis and stratum basalis during the menstrual cycle, which was altered in HMB. In particular, compared with controls, osteopontin expression in HMB was higher in stratum functionalis in the LSP (7.2 and 11.2, respectively P = 0.008), while collagen IV expression was reduced in stratum basalis in the MSP (4.6 and 2.8, respectively P = 0.002) and in stratum functionalis in the ESP (7 and 3.2, respectively P = 0.008)., Limitations, Reasons for Caution: The protein expression of vascular EC markers and ECM components was assessed using a semi-quantitative approach in both straight and spiral arterioles. In our hospital, HMB is determined by subjective criteria and levels of blood loss were not assessed., Wider Implications of the Findings: Variation in the protein expression pattern between the four EC markers highlights the importance of choice of EC marker for investigation of endometrial vessels. Differences in expression of the different EC markers may reflect developmental stage dependent expression of EC markers in endometrial vessels, and their altered expression in HMB may reflect dysregulated vascular development. This hypothesis is supported by altered expression of ECM proteins within endometrial vessel walls, as well as our previous data showing a dysregulation in VSMC contractile protein expression in the endometrium of women with HMB. Taken together, these data support the suggestion that HMB symptoms are associated with weaker vascular structures, particularly in the LSP of the menstrual cycle, which may lead to increased and extended blood flow during menstruation., Study Funding/competing Interest(s): This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare., Trial Registration Number: Not applicable.

Successful remodeling of the uterine spiral arteries is essential for a complication-free pregnancy and is best described in terms of its morphologic features. The molecular mediators and cellular sources of spiral artery remodeling are not known, although a role for uterine leukocytes has been proposed. Immunohistochemical assessment of placental bed biopsies demonstrated uterine NK cells, macrophages, and T lymphocytes in the wall and adventitia of spiral arteries at different stages of remodeling, regardless of the presence of extravillous trophoblast cells. Leukocytes were more prevalent in vessel adventitia than wall, and macrophages were the most abundant leukocyte population. Macrophages, separated from early pregnancy decidua, did not alter extravillous trophoblast cells invasion or vascular smooth muscle cell organization or differentiation status but did induce extracellular matrix breakdown (reduced immunostaining of laminin, P = 0.05; fibronectin, P = 0.02) and were able to phagocytose apoptotic vascular smooth muscle cells. Decidual macrophages were shown to secrete a wide range of cytokines (IL-1β, -2, -4, -5, -6, -8, -10, and -13 and TNF-α), proteases (matrix metalloproteinase-1, -2, -7, -9, and -10), and angiogenic growth factors (angiogenin, keratinocyte growth factor, fibroblast growth factor B, vascular endothelial growth factor A, and angiopoietin-1 and -2). We conclude that spiral artery remodeling involves the coordinated activity of a range of cell types, including extravillous trophoblast cells, decidual uterine NK cells, and macrophages in a carefully, spatiotemporally regulated manner., (© Society for Leukocyte Biology.)

Considerable work is being carried out on endometrial NK cells to determine whether they play a role in successful pregnancy outcome. In addition there is debate about whether measurements of uNK should be included in the clinical assessment for women with recurrent implantation failure or recurrent miscarriage. A hindrance to taking this forward is the fact that the density of uNK cells reported by different centres is very different. The aim of this study was to determine the reason for these differences and to develop a standardised method. Three centres participated in the study. Each centre exchanged five formalin fixed, wax embedded sections of endometrium from five women. Sections were immunostained for CD56. Images were taken of 10 random fields at ×400 magnification; total stromal and uNK cells were counted using Image J. Results were expressed as % positive uNK cells and the variation in counts obtained in each centre was compared. After initial analysis a standardised protocol was agreed and the process repeated. Significant variation was seen in the counts obtained after initial analysis (Centre A vs.B, mean difference=-0.72 P<0.001; A vs.C mean difference=-0.47 P<0.001; B vs.C, mean difference=0.25 P=0.085). Analysis suggested that differences may be due to duration of tissue fixation, the embedding and sectioning processes, selection of areas for assessment, definition of immunopositive cells and inclusion or exclusion of blood vessels. Adoption of a standardised protocol reduced the variation (Centre A vs.B mean difference=-0.105 P=0.744; A vs.C mean difference=0.219 P=0.150; B vs.C mean difference=0.32 P=0.031). Use of a standardised method is needed to establish a normal range for uNK cells and to develop a meaningful clinical test for uNK cell measurements., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Endometrial cancer (EC) is a hormone-driven disease, and androgen receptor (AR) expression in high-grade EC (HGEC) and metastatic EC has not yet been described., Methods: The expression pattern and prognostic value of AR in relation to oestrogen (ERα and ERβ) and progesterone (PR) receptors, and the proliferation marker Ki67 in all EC subtypes (n = 85) were compared with that of healthy and hyperplastic endometrium, using immunohistochemisty and qPCR., Results: Compared with proliferative endometrium, postmenopausal endometrtial epithelium showed significantly higher expression of AR (P < 0.001) and ERα (P = 0.035), which persisted in hyperplastic epithelium and in low-grade EC (LGEC). High-grade EC showed a significant loss of AR (P < 0.0001), PR (P < 0.0001) and ERβ (P < 0.035) compared with LGEC, whilst maintaining weak to moderate ERα. Unlike PR, AR expression in metastatic lesions was significantly (P = 0.039) higher than that in primary tumours. Androgen receptor expression correlated with favourable clinicopathological features and a lower proliferation index. Loss of AR, with/without the loss of PR was associated with a significantly lower disease-free survival (P < 0.0001, P < 0.0001, respectively)., Conclusions: Postmenopausal endometrial epithelium acquires AR whilst preserving other steroid hormone receptors. Loss of AR, PR with retention of ERα and ERβ may promote the unrestrained growth of HGEC. Androgen receptor may therefore be a clinically relevant prognostic indicator and a potential therapeutic target in EC.

Heavy menstrual bleeding (HMB) is a common gynecological complaint with multiple etiologies and diverse pathophysiological origins. This review discusses HMB with reference to the recently proposed PALM-COEIN classification system for abnormal uterine bleeding, initially describing the endometrial events in normal menstruation followed by discussion of the perturbations of normal endometrial shedding that can result in HMB. Our present understanding of the mechanisms of menstrual bleeding as well as many of the pathological aberrations of HMB is incomplete. Further research into the pathophysiology of HMB is urgently needed, as clear knowledge of the mechanisms of this disorder will provide new therapeutic targets to formulate more effective treatments.

Heavy menstrual bleeding (HMB) affects 30% of women of reproductive age and significantly interferes with quality of life. Altered endometrial vascular maturation has been reported in HMB and recurrent miscarriage, the latter associated with increased uterine natural killer (uNK) cell numbers. This study compared endometrial leukocyte populations in controls and women with HMB. Formalin-fixed paraffin-embedded endometrial biopsies from controls (without endometrial pathology) and HMB were immunostained for CD14 (macrophages), CD56 (uNK cells), CD83 (dendritic cells), FOXP3 (regulatory T cells/Tregs), CD3 and CD8 (T cells). Leukocyte numbers were analysed as a percentage of total stromal cells in five randomly selected fields of view in the stratum functionalis of each sample. In control women across the menstrual cycle, 2-8% of total stromal cells were CD3(+) cells, 2-4% were CD8(+) T cells and 6-8% were CD14(+) macrophages. Compared with controls, CD3(+) cells were reduced during the mid-secretory phase (4%, P<0.01) and increased in the late secretory phase (12%, P=0.01) in HMB. CD83(+) dendritic cells and FOXP3(+) Tregs were scarce throughout the menstrual cycle in both groups. In controls, 2% of stromal cells in proliferative endometrium were CD56(+) uNK cells, increasing to 17% during the late secretory phase. In HMB, CD56(+) uNK cells were increased in the proliferative (5%, P<0.01) and early secretory (4%, P<0.02) phases, but reduced (10%, P<0.01) in the late secretory phase. This study demonstrates dysregulation of uNK cells in HMB, the functional consequence of which may have an impact on endometrial vascular development and/or endometrial preparation for menstruation., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Histological chorioamnionitis (HCA) is an established marker of ascending infection, a major cause of preterm birth. No studies have characterised the global change in expression of genes involved in the toll-like receptor (TLR) signalling pathways in the presence of HCA in the setting of preterm birth (pHCA). Fetal membranes were collected immediately after delivery and underwent histological staging for inflammation to derive 3 groups; term spontaneous labour without HCA (n = 9), preterm birth <34 weeks gestation without HCA (n = 8) and pHCA <34 weeks (n = 12). Profiling arrays ran in triplicate for each group were used to determine the expression of 84 genes associated with TLR signalling and screen for genes of interest (fold change >2; p<0.1). Expression of identified genes was validated individually for all samples, relative to GAPDH, using RT-PCR. Expression of TLR 1, TLR 2, lymphocyte antigen 96, interleukin 8 and Interleukin-1 receptor-associated kinase-like 2 was increased in pHCA (p<0.05). Degree of expression was positively associated with histological staging of both maternal and fetal inflammation (p<0.05). The inflammatory expression profile at the maternal/fetal interface associated with pHCA, a reflection of ascending infection, is extremely heterogeneous suggesting polymicrobial involvement with activation of a common pathway. Antagonism of TLR 1 and TLR 2 signalling in this setting warrants further assessment.

Hypoxia-inducible factors (HIFs), adenosine and tissue renin-angiotensin-system (RAS) promote angiogenesis and vascularisation. We investigated the temporal expression placental adenosine A2AR receptor and HIF-1α in early pregnancy and at delivery in normotensive (NT) and pre-eclamptic (PE) women. Results were compared to our previously reported angiotensin receptor data. Expression of A2AR and HIF-1α was highest at ≤10 weeks, positively correlated through pregnancy and was higher in PE than NT at delivery. The A2AR associated with the AT4R only in early pregnancy. We suggest adenosine and RAS may interact to promote placentation with a potential adaptation to poor placental perfusion in PE., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

During pregnancy, immune activity is tightly regulated so that antimicrobial protection of the mother and fetus is balanced with the need for immune tolerance to prevent fetal rejection. In this setting, the maternal-fetal interface, in the form of the uterine decidua, provides a heterogeneous immune cell population with the potential to mediate diverse activities throughout pregnancy. Recent studies have suggested that vitamin D may be a key regulator of immune function during pregnancy, with the fetal-maternal interface representing a prominent target. Among its non-classical actions are potent immunomodulatory effects, including induction of antibacterial responses and modulation of T-lymphocytes to suppress inflammation and promote tolerogenesis. Thus, vitamin D may play a pivotal role in normal decidual immune function by promoting innate responses to infection, while simultaneously preventing an over-elaboration of inflammatory adaptive immunity. Research to date has focused upon the potential role of vitamin D in preventing infectious diseases such as tuberculosis, as well as possibly suppressing of autoimmune disease. Nevertheless, vitamin D may also influence facets of immune function not immediately associated with primary innate responses. This review summarises our current understanding of decidual immune function with respect to the vitamin D metabolism and signalling, and as to how this may be affected by variations in maternal vitamin D status. There has recently been much interest in vitamin D supplementation of pregnant women, but our knowledge of how this may influence the function of decidua remains limited. Further insight into the immunomodulatory actions of vitamin D during pregnancy will help shed light upon this., (© 2015 Society for Endocrinology.)

Background: The endometrium is the primary target organ for the 'female' sex steroid hormone estrogen, which exerts effects in the endometrium via two main classical estrogen receptor (ER) isoforms, ERα and ERβ. The main function of the endometrium, embryo implantation, appears unperturbed in ERβ knockout mice, which has led researchers to disregard other potentially important functional roles that ERβ may have in endometrium. This review focuses on ERβ in the human endometrium and its protective role from the undesired effects of ERα., Methods: We conducted a systematic search using PubMed and Ovid for publications between January 1996 and February 2014. All studies that examined ERβ expression or function in non-pregnant endometrium or cells derived from the endometrium were considered, including human and animal studies., Results: Studies of the basic function of ERβ isoforms in restraining ERα-mediated cell-specific trophic/mitotic responses to estrogen in other tissues has allowed appreciation of the important potential role of ERβ in the regulation of cell fate in the human endometrium. Our current understanding of ERβ expression and function in endometrium is, however, incomplete. ERβ is dynamically expressed in healthy premenstrual endometrium, persists in post-menopausal atrophic endometrium and may play an important role in endometrial disease. All endometrial cell types express ERβ and aberrations in ERβ expression have been reported in almost all benign and malignant endometrial proliferative disease., Conclusions: The collective evidence suggests that ERβ has an important role in normal endometrial function and also in most, if not all, benign and malignant endometrial diseases. However, the conduct of studies of endometrial ERβ expression needs to be standardized: agreement is needed regarding the most appropriate control tissue for endometrial cancer studies as well as development of standardized methods for the quantification of ERβ immunohistochemical data, similar to those scoring systems employed for other hormonally regulated tissues such as breast cancer, since these data may have direct clinical implications in guiding therapy., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The human endometrium contains a substantial population of leucocytes which vary in distribution during the menstrual cycle and pregnancy. An unusual population of natural killer (NK) cells, termed uterine NK (uNK) cells, are the most abundant of these cells in early pregnancy. The increase in number of uNK cells in the mid-secretory phase of the cycle with further increases in early pregnancy has focused attention on the role of uNK cells in early pregnancy. Despite many studies, the in vivo role of these cells is uncertain. This chapter reviews current information regarding the role of uNK cells in healthy human pregnancy and evidence indicating their importance in various reproductive and pregnancy problems. Studies in humans are limited by the availability of suitable tissues and the limitations of extrapolation from animal models.

Study Question: How does the smooth muscle content and differentiation stage of vascular smooth muscle cells (VSMCs) in endometrial blood vessels change according to the different phases of the menstrual cycle and is this altered in women with menorrhagia?, Summary Answer: The smooth muscle content (as a proportion of the vascular cross-sectional area) of endometrial blood vessels remained unchanged during the normal menstrual cycle and in menorrhagia; however, expression of the VSMC differentiation markers, smoothelin and calponin, was dysregulated in endometrial blood vessels in samples from women with menorrhagia compared with controls., What Is Known Already: Menorrhagia affects 30% of women of reproductive age and is the leading indication for hysterectomy. Previous studies have suggested important structural and functional roles for endometrial blood vessels, including impaired vascular contractility. Differentiation of VSMC from a synthetic to contractile state is associated with altered cellular phenotype that contributes to normal blood flow and pressure. This vascular maturation process has been little studied in endometrium both across the normal menstrual cycle and in menorrhagia., Study Design, Size, Duration: Endometrial biopsies were taken from hysterectomy specimens or by pipelle biopsy prior to hysterectomy in controls without endometrial pathology and in women with menorrhagia (n = 7 for each of proliferative, early-secretory, mid-secretory and late-secretory phases for both groups). Biopsies were formalin fixed and embedded in paraffin wax., Participants/materials, Setting, Methods: Paraffin-embedded sections were immunostained for α smooth muscle actin (αSMA), myosin heavy chain (MyHC), H-caldesmon, desmin, smoothelin and calponin (h1 or basic). VSMC content was measured in 25 αSMA(+) vascular cross sections per sample and expressed as a ratio of the muscular area:gross vascular cross-sectional area. VSMC differentiation was analysed by the presence/absence of differentiation markers compared with αSMA expression. Smoothelin and calponin expression was also analysed in relation to total number of blood vessels by double immunostaining for endothelial cell markers., Main Results and the Role of Chance: Study of VSMC differentiation markers revealed decreased expression of calponin both in αSMA(+) vessels (P = 0.008) and in relation to total number of vessels (P = 0.001) in late secretory phase endometrium in menorrhagia compared with controls. Smoothelin expression in αSMA(+) vessels was increased (P = 0.03) in menorrhagia, although this was not significant in relation to the total number of vessels. In normal endometrium, the proportion of blood vessels expressing αSMA increased from 63% in proliferative endometrium to 81% in the late secretory phase (P = 0.002). The overall arterial muscle content did not differ between control and menorrhagia at any phase of the menstrual cycle, occupying 78-81% of gross vascular cross-sectional area during the different menstrual cycle phases., Limitations, Reasons for Caution: This study included both straight and spiral arterioles and analysed only stratum functionalis. The VSMC differentiation with respect to αSMA expression is an observational study and the data are presented as presence or absence of the differentiation markers in each field of view, corresponding with the vascular cross sections included in the study of vascular muscle content., Wider Implications of the Findings: Smoothelin and calponin have been widely implicated as important regulators of vascular tone, vascular contractility and rate of blood flow. Our results have uncovered a disparate pattern of calponin expression, potentially indicating a dysfunctional contraction mechanism in the endometrial blood vessels in menorrhagia, thus implicating calponin as a potential therapeutic target., Study Funding/competing Interests: This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare., Trial Registration Number: Not applicable., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Failure to transform uteroplacental spiral arteries is thought to underpin disorders of pregnancy, including preeclampsia and fetal growth restriction (FGR). In this study, spiral artery remodeling and extravillous-cytotrophoblast were examined in placental bed biopsies from normal pregnancy (n = 25), preeclampsia (n = 22), and severe FGR (n = 10) and then compared with clinical parameters. Biopsies were immunostained to determine vessel wall integrity, extravillous-cytotrophoblast location/density, periarterial fibrinoid, and endothelium. Muscle disruption was reduced in myometrial spiral arteries in preeclampsia (P = 0.0001) and FGR (P = 0.0001) compared with controls. Myometrial vessels from cases with birth weight <5th percentile (P<0.001), abnormal uterine Doppler (P<0.01), abnormal umbilical artery Doppler (P<0.001), and preterm delivery (P<0.001) had less muscle destruction compared with >5th percentile. Fewer extravillous-cytotrophoblast surrounded both decidual and myometrial vessels in the normal group and preeclampsia group compared with the FGR group (P = 0.001). For myometrial vessels, the normal group contained more intramural extravillous-cytotrophoblast than in preeclampsia (P = 0.015). Decidual vessels in the FGR group had less fibrinoid deposition compared with controls (P = 0.013). For myometrial vessels, less fibrinoid was deposited in both the preeclampsia group (P = 0.0001) and the FGR group (P = 0.01) when compared with controls, and less fibrinoid was deposited in the preeclampsia group when compared with FGR group (P<0.001). Myometrial vessels obtained from birth weights <5th percentile had less periarterial fibrinoid than those with >5th percentile (P<0.02). A major defect in myometrial spiral artery remodeling occurs in preeclampsia and FGR that is linked to clinical parameters. Interstitial extravillous-cytotrophoblast is not reduced in preeclampsia but is increased in FGR.

Study Question: Are alterations in decidual expression of interleukin (IL)-6 and IL-8 associated with sporadic miscarriage?, Summary Answer: 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., What Is Known Already: 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., Study Design, Size, Duration: 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)., Participants/materials, Setting, Methods: 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., Main Results and the Role of Chance: 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., Limitations, Reasons for Caution: 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., Wider Implications of the Findings: 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., Study Funding/competing Interest(s): This project was supported by funding from Wellbeing of Women (RG1000). The authors have no competing interests to declare., Trial Registration Number: Not applicable.

Uterine spiral artery remodeling is required for successful human pregnancy; impaired remodeling is associated with pregnancy complications, including late miscarriage, preeclampsia, and fetal growth restriction. The molecular triggers of remodeling are not known, but it is now clear that there are "trophoblast-independent" and "trophoblast-dependent" stages. Uterine natural killer (uNK) cells are abundant in decidualized endometrium in early pregnancy; they surround spiral arteries and secrete a range of angiogenic growth factors. We hypothesized that uNK cells mediate the initial stages of spiral artery remodeling. uNK cells and extravillous trophoblast (EVT) cells were isolated from early pregnancy decidua and placenta. Chorionic plate arteries from full-term placentas and spiral arteries from nonpregnant myometrium were cultured with angiogenic growth factors or conditioned medium (CM) from uNK cells or EVT or uNK cell/EVT cocultures. In both vessel models, uNK cell CM induced disruption of vascular smooth muscle cells (VSMCs) and breakdown of extracellular matrix components. Angiopoietin (Ang)-1, Ang-2, interferon-γ, and VEGF-C also disrupted VSMC integrity with an Ang-2 inhibitor abrogating the effect of uNK cell CM. These results provide compelling evidence that uNK cells contribute to the early stages of spiral artery remodeling; failure of this process could contribute to pregnancy pathology.

Objectives: Placenta creta is an increasingly prevalent cause of maternal morbidity/mortality. Decidua is at least focally defective and extravillous trophoblast (EVT) may be abnormal. The study aims to compare differences in migratory trophoblast and spiral artery remodeling between areas with and without decidua at the placental implantation site., Study Design: Sixteen (12 creta, 4 non-creta) caesarean hysterectomy specimens were studied immunohistochemically. Invasive EVT and multinucleate trophoblast giant cells (MTGC) were quantified; confluent EVT (>5 opposed EVTs) and spiral artery remodeling were assessed semi-quantitatively., Results: In 6 cases, placenta creta was focal. Compared to placenta creta with local decidua, cases without local decidua had increased interstitial EVT cells (×200 field) (SEM 45.6 [4.9] vs. 80.5 [3.9], p < 0.0001), fewer multinucleate trophoblast giant cells (expressed as a percentage of total EVT) (0.8 [0.3] vs. 31.5 [2.2]% p < 0.0001) and EVT was more confluent (p < 0.0001). In contrast, placenta creta cases with local decidua had a greater degree of spiral artery remodeling (mean remodeling score 1.65 [0.07] vs. 1.13 [0.05], p < 0.0001) associated with increased intramural trophoblast (p = 0.0008). The only difference between placenta creta with local decidua and normal placentation cases was an increased number of interstitial EVT cells in creta cases (45.6 [4.9] vs. 24.8 [3.2], p = 0.04)., Conclusions: Numbers of interstitial EVT are increased in placenta creta, more so in cases without local decidua. Despite this spiral artery modeling is reduced in placenta creta cases with no decidua. The results emphasize the crucial role of decidua in control of trophoblast invasion and spiral artery remodeling., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Invasion of uterine tissues by extravillous trophoblast cells (EVT) is essential for successful human pregnancy. EVT invasion is tightly regulated by a number of factors, including growth factors and cytokines, but the mechanisms that underlie their regulatory effect remain poorly understood. Interleukin (IL)-6 has been suggested to play a role in controlling EVT invasion. We hypothesized that IL-6 produced by cells in uterine decidua would regulate EVT invasiveness via IL-6Rα and gp130 receptors expressed by trophoblast cells. The effect of IL-6 on EVT signalling and cytokine production was also studied. Supernatants from disaggregated 'total' decidual cells, CD8(+) T cells, CD10(+) decidual stromal cells, CD14 macrophages, CD56(+) uterine natural killer cells, cytotrophoblast and EVT cells contained large quantities of IL-6 protein at both 8-10 and 12-14 weeks gestational age. IL-6Rα and gp130 were immunolocalized to EVT in placental bed biopsies from 8 to 20 weeks gestation and IL-6Rα expression was confirmed by western blotting. IL-6 had no effect on the invasive potential of EVT from chorionic villi or the immortalized EVT cell line HTR-8/SVneo in a Matrigel(®) invasion assay. IL-6 stimulated phosphorylation of several cell signalling proteins in EVT (8-14 weeks' gestation), although significance was lost after correction for multiple comparisons. Incubation with IL-6 decreased secretion of regulated upon activation, normal T-cell expressed and secreted (RANTES) by EVT cells. In conclusion, although IL-6 did not affect trophoblast cell invasion, it stimulated EVT cellular cascades and inhibited secretion of RANTES involved in a number of cellular processes.

During human uterine spiral artery (SpA) remodeling, vascular smooth muscle cells (VSMCs) are lost and replaced by fibrinoid, incorporating extravillous trophoblast (EVT) cells. The aim of the current study was to determine the relative contributions of apoptosis and migration to VSMC loss during SpA remodeling. Immunohistochemistry (Apoptag, active caspase 3, lamin) of placental bed biopsies (8-20 wk gestation) demonstrated apoptotic cells in all samples; double immunolabeling identified these as trophoblasts, leukocytes, and endothelial cells. In total, 294 SpAs were studied, and only one apoptotic VSMC was identified. H-caldesmon-immunopositive VSMCs were observed surrounding and separate from SpA walls in partially remodeled vessels; the highest level of VSMC migration was observed in vessels with associated EVT cells (number of migrated cells 6.4 ± 1.2; distance migrated 3.5 ± 0.3 pixels) compared with those without (number of migrated cells 3.6 ± 0.5, P<0.001; distance migrated 2.8 ± 0.1 pixels, P<0.0001). VEGF-A, VEGF-C, TGF-β1, and Ang-2 all stimulated human aorta VSMC invasion in vitro, although EVT cell culture supernatants did not. In summary, apoptosis is unlikely to play a major role in loss of VSMCs from SpAs during remodeling in normal pregnancy, but VSMCs appear to migrate away from the wall of the SpA, an effect enhanced by the presence of EVT cells.