Endometrial uNK cell counts do not predict successful implantation in an IVF population.

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.

Quantification of spiral artery remodelling using an Adobe Photoshop-based technique.

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.

Uterine spiral artery muscle dedifferentiation.

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-ßRI, TGF-ßRII), 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.

Transcriptomic analysis of vitamin D responses in uterine and peripheral NK cells.

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 ß (TGFß). 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.

Androgen receptors are acquired by healthy postmenopausal endometrial epithelium and their subsequent loss in endometrial cancer is associated with poor survival.

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 ERß) 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 ERß (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 ERß may promote the unrestrained growth of HGEC. Androgen receptor may therefore be a clinically relevant prognostic indicator and a potential therapeutic target in EC.

Placental expression of adenosine A(2A) receptor and hypoxia inducible factor-1 alpha in early pregnancy, term and pre-eclamptic pregnancies: interactions with placental renin-angiotensin system.

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.

Immunological role of vitamin D at the maternal-fetal interface.

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.

Estrogen receptor ß: the guardian of the endometrium.

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 ERß. The main function of the endometrium, embryo implantation, appears unperturbed in ERß knockout mice, which has led researchers to disregard other potentially important functional roles that ERß may have in endometrium. This review focuses on ERß 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 ERß 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 ERß isoforms in restraining ERα-mediated cell-specific trophic/mitotic responses to estrogen in other tissues has allowed appreciation of the important potential role of ERß in the regulation of cell fate in the human endometrium. Our current understanding of ERß expression and function in endometrium is, however, incomplete. ERß 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 ERß and aberrations in ERß expression have been reported in almost all benign and malignant endometrial proliferative disease. CONCLUSIONS: The collective evidence suggests that ERß 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 ERß 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 ERß 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.

IFPA Meeting 2012 Workshop Report III: trophoblast deportation, gestational trophoblastic disease, placental insufficiency and fetal growth restriction, trophoblast over-invasion and accreta-related pathologies, placental thrombosis and fibrinolysis.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of clinical research and pregnancy disorders: 1) trophoblast deportation; 2) gestational trophoblastic disease; 3) placental insufficiency and fetal growth restriction; 4) trophoblast overinvasion and accreta-related pathologies; 5) placental thrombosis and fibrinolysis.

Effects of local decidua on trophoblast invasion and spiral artery remodeling in focal placenta creta - an immunohistochemical study.

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.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

IFPA Meeting 2011 workshop report II: Angiogenic signaling and regulation of fetal endothelial function; placental and fetal circulation and growth; spiral artery remodeling.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2011 there were twelve themed workshops, three of which are summarized in this report. These workshops related to vascular systems and circulation in the mother, placenta and fetus, and were divided in to 1) angiogenic signaling and regulation of fetal endothelial function; 2) placental and fetal circulation and growth; 3) spiral artery remodeling.

Role of interleukin 8 in uterine natural killer cell regulation of extravillous trophoblast cell invasion.

BACKGROUND: Extravillous trophoblast cell (EVT) invasion of maternal tissues is critical for successful pregnancy. Decidual factors, including uterine natural killer (uNK) and T cell derived cytokines play a role in regulating this process. Interleukin (IL) 8 has been implicated as a regulator of EVT invasion. HYPOTHESIS: uNK cell stimulation of EVT invasion is associated with IL-8 levels. METHODS: CD8+, total decidual and CD56(+) uNK cells (8-10 and 12-14 weeks gestational age) were cultured. IL-8 mRNA and protein levels were determined. IL-8 receptors (IL-8RA and IL-8RB) were localised in first trimester placental bed biopsies. The effect of IL-8 +/- IL-8 neutralising antibodies and CD8+ T cell or uNK cell supernatants +/- IL-8 neutralising antibodies on EVT invasion was assessed. EVT secreted levels of MMP-2, MMP-9, uPA, PAI-1 and PAI-2 were assessed by substrate zymography or Western Blot. RESULTS: High levels of IL-8 protein and mRNA were detected in all samples. IL-8RA and IL-8RB were expressed by EVT. Exogenous IL-8 stimulated EVT invasion in a paracrine manner. uNK cell supernatants, but not CD8+ cell supernatants, stimulated EVT invasion. IL-8 neutralising antibody partially abrogated this uNK cell stimulated invasion. IL-8 increased levels of secreted MMP-2, but did not alter any of the other proteases or protease inhibitors tested. CONCLUSION: uNK cell stimulation of EVT invasion is partially mediated by IL-8. Unstimulated CD8+ T cells do not alter EVT invasion despite secreting similar levels of IL-8 as uNK cells.

Expression of AT1R, AT2R and AT4R and their roles in extravillous trophoblast invasion in the human.

The placental renin-angiotensin system (RAS) is active from early pregnancy and may have a role in placentation. Angiotensin II (AngII) acts via binding to receptor types AT1R and AT2R. Recently smaller peptide members of the angiotensin family have been recognised as having biological relevance. Angiotensin (3-8) (AngIV) has a specific receptor (AT4R) and evokes hypertrophy, vasodilatation and vascular inflammatory response. The aim of this study was to characterise placental expression of AT1R, AT2R and AT4R, and to determine whether AngII and AngIV regulate extravillous trophoblast (EVT) invasion, apoptosis and proliferation. Placental samples were obtained from women undergoing elective surgical termination of pregnancy (TOP) at 8-10 weeks gestation (early TOP), 12-14 weeks gestation (mid TOP) or at delivery following normal pregnancy or with pre-eclampsia (PE). Immunohistochemistry and qRT-PCR were performed to determine placental mRNA and protein expression of AT1R, AT2R and AT4R at all gestational ages. EVT invasion following culture with AngII or AngIV was assessed in early placental tissue using Matrigel invasion assays. Invasion was assessed on day 6 of culture and placental explants were harvested for immunohistochemical analysis of apoptosis and proliferation. The results from qRT-PCR and immunohistochemistry showed placental AT1R expression which did not vary with gestation. The highest levels of expression of AT2R were found in early and mid TOP placentae compared to term pregnancy. Expression of AT4R was increased in term placentae, with a significant reduction in PE placentae. Moreover, culture with AngIV or AngII increased EVT invasion from placental explants, which showed increased trophoblast proliferation and reduced apoptosis. This study has characterised expression of AT4R and AT1R and AT2R in human placenta throughout normal pregnancy and in PE. Both AngIV and AngII may play an important role in normal pregnancy.

Secretion of angiogenic growth factors by villous cytotrophoblast and extravillous trophoblast in early human pregnancy.

Angiogenesis is a key feature of placental and uterine development in early pregnancy. We hypothesized that trophoblast cells produce angiogenic growth factors, and that expression differs between villous cytotrophoblast (CTB) and extravillous trophoblast (EVT) cells. Fourteen angiogenic growth factors were measured by multiplex growth factor analysis or ELISA in tissue culture supernatants from EVT and CTB from pregnancies at 8-10 and 12-14 weeks' gestation. Gestational age and cell type differences were observed. EVT and CTB are major producers of angiogenic growth factors that likely contribute to placental vascular development and spiral artery remodeling.

Expression of thyroid hormone transporters in the human placenta and changes associated with intrauterine growth restriction.

Thyroid hormones (TH) are important for the development of the human fetus and placenta from very early gestation. The transplacental passage of TH from mother to fetus and the supply of TH into trophoblasts require the expression of placental TH plasma membrane transporters. We describe the ontogeny of the TH transporters MCT8, MCT10, LAT1, LAT2, OATP1A2 and OATP4A1 in a large series (n = 110) of normal human placentae across gestation and describe their expression changes with intrauterine fetal growth restriction (IUGR n = 22). Quantitative RT-PCR revealed that all the mRNAs encoding TH transporters are expressed in human placenta from 6 weeks gestation and throughout pregnancy. MCT8, MCT10, OATP1A2 and LAT1 mRNA expression increased with gestation. OATP4A1 and CD98 (LATs obligatory associated protein) mRNA expression reached a nadir in mid-gestation before increasing towards term. LAT2 mRNA expression did not alter throughout gestation. Immunohistochemistry localised MCT10 and OATP1A2 to villous cytotrophoblasts and syncytiotrophoblasts, and extravillous trophoblasts while OATP4A1 was preferentially expressed in the villous syncytiotrophoblasts. Whilst MCT8 protein expression was increased, MCT10 mRNA expression was decreased in placentae from IUGR pregnancies delivered in the early 3rd trimester compared to age matched appropriately grown for gestational age controls. No significant change was found in the mRNA expression of the other transporters with IUGR. In conclusion, several TH transporters are present in the human placenta from early 1st trimester with varying patterns of expression throughout gestation. Their coordinated effects may regulate both transplacental TH passage and TH supply to trophoblasts, which are critical for the normal development of the fetus and placenta. Increased MCT8 and decreased MCT10 expression within placentae of pregnancies complicated by IUGR may contribute to aberrant development of the fetoplacental unit.

Review: Functional role of uterine natural killer (uNK) cells in human early pregnancy decidua.

Leukocytes comprise approximately 30-40% of decidual stromal cells in early human pregnancy. The major leukocyte component is the uterine natural killer (uNK) cells. Despite over 20 years of research the functional role of these cells in situ remains unknown although they have been proposed to play roles in immunotolerance, regulation of trophoblast invasion and remodeling of the spiral arteries. Herein we review the functional roles of this important decidual cell type.

Decidual leucocyte populations in early to late gestation normal human pregnancy.

Most research on human decidual leucocytes to date has focused on the predominant CD56+ uterine natural killer (uNK) cell population in early pregnancy. Few reports have documented decidual leucocyte populations after 13 weeks gestation and in late pregnancy. Placental bed (decidua basalis) and non-placental bed (decidua parietalis) biopsies from normal pregnancies were taken from women undergoing termination of pregnancy in the 1st and 2nd trimesters and following Caesarean section in the 3rd trimester. Immunohistochemistry was used to quantify the numbers of decidual cells expressing CD56, CD3, CD8, CD94, NKG2A and CD14 and double labelled CD161+CD3+ NKT-like cells. Although a significant reduction in CD56+ uNK cells was found in 3rd trimester samples compared with 1st and 2nd trimester decidua, a substantial residual CD56+ leucocyte population was identified in 3rd trimester decidua. Expression of the KIR CD94/NKG2A mirrored that of CD56 at all gestational ages, providing an explanation for the absence of cytotoxic responses at the fetal-maternal interface. There was no difference in leucocyte populations between decidua basalis and decidua parietalis. Double immunohistochemical labelling revealed small numbers of decidual CD3+CD56+ and CD8+CD56+ cells, which decreased in number at term, and CD161+CD3+ cells, which increased in number at term. No differences in leucocyte populations were detected between decidua parietalis and decidua basalis. In contrast to previous reports, a substantial residual CD56+ cell population was demonstrated in 3rd trimester decidua. Decidual cytotoxic T-lymphocytes did not alter in number during gestation, while in contrast CD14+ macrophages decreased at term, representing the smallest decidual population assessed.

The urokinase plasminogen activator (uPA) system in uterine natural killer cells in the placental bed during early pregnancy.

The urokinase plasminogen activator (uPA) system plays pivotal roles in cell invasion, adhesion and migration. Roles for uterine natural killer (uNK) cells in regulating extravillous trophoblast (EVT) invasion and spiral artery remodeling have been proposed. Placental bed biopsies from early pregnancy were obtained from three gestational age groups (8-10, 12-14 and 15-20 weeks). Total caseinase activity in the placental bed was studied using casein in situ zymography. Localisation of uPA, uPA receptor (uPAR), plasminogen activator inhibitor (PAI)-1 and -2 in the placental bed was investigated by immunohistochemistry. CD56(+) uNK cells were separated from collagenase-digested decidual cells using an immunomagnetic technique, and uPA activity was measured in isolated cell culture supernatants by casein/plasminogen gel zymography (8-10 and 12-14 weeks' gestation, n=10 each group). uPAR in cell lysates and PAI-1 and -2 secretion in supernatants were measured by Western blotting. Caseinase activity was stronger in decidua than myometrium as shown by in situ zymography. uPA localised strongly to uNK cells, especially at 8-10 weeks. Moderate uPAR localisation on uNK cells also observed. There was very weak immunostaining of uNK cells for PAI-1 and PAI-2. In casein gel zymography, uPA activity was similar in uNK cell culture supernatant compared with total unseparated decidual cells. uPAR in uNK cell lysates was significantly stronger than in total decidual cell lysates. PAI-1 and PAI-2 were not detected in uNK cell culture supernatants by Western blot analysis. These results suggest that uNK cells may regulate EVT invasion and spiral artery remodeling via the uPA system.

Endometrial carcinoma detected with SurePath liquid-based cervical cytology: comparison with conventional cytology.

INTRODUCTION: Conventional Pap smears (CPS) have little impact on the detection of endometrial carcinoma. Although liquid-based cytology (LBC) is replacing CPS in the UK, experience with identification of endometrial cancers with this technique is limited. AIM: To compare the accuracy of the SurePath LBC with that of CPS for detection of endometrial cancers. METHODS: Our study group comprised SurePath LBC samples reported as atypical endometrial cells and endometrial adenocarcinoma (classified respectively as borderline, code 8 and ?glandular neoplasia, code 6 for the NHS Cervical Screening Programme statistics) in 2004-2005. CPS reported as atypical endometrial cells or adenocarcinoma in 1993-1998 comprised the control group. Histological follow-up was obtained. RESULTS: Endometrial abnormalities were reported in 95 (0.073%) of 130 352 LBC samples, comprising 75 (0.058%) atypical endometrial cells and 20 (0.015%) endometrial adenocarcinoma reports. Of 409 495 CPS, 117 (0.029%) were diagnosed as endometrial abnormalities, comprising 59 (0.014%) atypical endometrial cells and 58 (0.014%) endometrial adenocarcinoma reports. Thus, the endometrial adenocarcinoma reporting rate was similar in both groups, but that for atypical endometrial cells was higher with LBC (P < 0.001). The positive predictive value for endometrial cancer of endometrial adenocarcinoma and atypical endometrial cell reports in the LBC group was 73.3 and 18.8%, respectively, compared with 42.3 and 6.7% in the CPS group. The endometrial adenocarcinoma patients in CPS group were older (mean age 62.5 years versus 56.5 years) and most (22/25) were symptomatic, whereas most (13/17) patients in the LBC group were asymptomatic at the time of sampling (P < 0.001). CONCLUSION: SurePath LBC is at least as accurate a method for detecting endometrial cancer as CPS. SurePath LBC demonstrates enhanced identification of endometrial pathology in asymptomatic women in the cervical screening programme.