John D. Aplin

Human Endometrial Mucin MUC1 Is Up-Regulated by Progesterone and Down-Regulated In Vitro by the Human Blastocyst

Abstract Expression of MUC1 in endometrial epithelium has been suggested to create a barrier to embryo attachment that must be lifted at the time of implantation. In this study, we investigated the hormonal regulation of human endometrial MUC1 in hormone replacement therapy cycles and in the human blastocyst. We also analyzed the embryonic regulation of MUC1 in human endometrial epithelial cells (EECs) during the apposition and adhesion phases of human implantation using two different in vitro models. Our results indicate that endometrial MUC1 mRNA and immunoreactive protein increase in receptive endometrium compared to nonreceptive endometrium. Human blastocysts express MUC1, as demonstrated by reverse transcription-polymerase chain reaction and immunocytochemistry, localized at the trophectoderm. In vitro, MUC1 was present at the surface of primary cultures of human EEC, and presence of a human blastocyst (i.e., apposition phase) increases EEC MUC1 protein and mRNA compared to control EEC lacking embryos. Interestingly, when human blastocysts were allowed to attach to the EEC monolayer (i.e., adhesion phase), MUC1 was locally removed in a paracrine fashion on EEC at the implantation site. These results demonstrate a coordinated hormonal and embryonic regulation of EEC MUC1. Progesterone combined with estradiol priming induces an up-regulation of MUC1 at the receptive endometrium. During the apposition phase, presence of a human embryo increases EEC MUC1. However, at the adhesion phase, the embryo induces a paracrine cleavage of EEC MUC1 at the implantation site. These findings strongly suggest that MUC1 may act as an endometrial antiadhesive molecule that must be locally removed by the human blastocyst during the adhesion phase.

Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy.

Decidual artery remodeling is essential for a healthy pregnancy. This process involves loss of vascular smooth muscle cells and endothelium, which are replaced by endovascular trophoblasts (vEVTs) embedded in fibrinoid. Remodeling is impaired during pre-eclampsia, a disease of pregnancy that results in maternal and fetal mortality and morbidity. Early vascular changes occur in the absence of vEVTs, suggesting that another cell type is involved; evidence from animal models indicates that decidual leukocytes play a role. We hypothesized that leukocytes participate in remodeling through the triggering of apoptosis or extracellular matrix degradation. Decidua basalis samples (8 to 12 weeks gestation) were examined by immunohistochemistry to elucidate associations between leukocytes, vEVTs, and key remodeling events. Trophoblast-independent and -dependent phases of remodeling were identified. Based on a combination of morphological attributes, vessel profiles were classified into a putative temporal series of four stages. In early stages of remodeling, vascular smooth muscle cells showed dramatic disruption and disorganization before vEVT presence. Leukocytes (identified as uterine natural killer cells and macrophages) were apparent infiltrating vascular smooth muscle cells layers and were matrix metalloproteinase-7 and -9 immunopositive. A proportion of vascular smooth muscle cells and endothelial cells were terminal deoxynucleotidyl transferase dUTP nick-end labeling positive, suggesting remodeling involves apoptosis. We thus confirm that vascular remodeling occurs in distinct trophoblast-independent and -dependent stages and provide the first evidence of decidual leukocyte involvement in trophoblast-independent stages.

An immunohistochemical study of human endometrial extracellular matrix during the menstrual cycle and first trimester of pregnancy.

SummaryChanges in the organisation and composition of extracellular matrix in human endometrium during the menstrual cycle and early pregnancy have been assessed by immunofluorescence. Amongst interstitial components, type-III and type V-collagens and fibronectin are present in endometrial stroma throughout the menstrual cycle as well as in first trimester decidua. Type V-collagen epitopes are masked early in the cycle, but become accessible in first trimester decidua. Type VI-collagen is abundant in endometrium in the proliferative phase, but is progressively lost in the secretory phase and decidua, in which it is retained only in blood vessel walls. Vitronectin is present in some blood vessels in decidua. Decidualising stromal cells also produce basement membrane components (type IV-collagen, laminin, heparan sulphate proteoglycan and a glycoprotein family recognised by monoclonal antibody G71) and these become organised into a pericellular aura.

Cell adhesion molecules on the oocyte and preimplantation human embryo

The presence of cell adhesion molecules on human oocytes, early embryos, and pre-hatched blastocysts was examined by indirect immunofluorescence and compared to the distribution found on first trimester villous placenta with the same antibodies. Six integrin subunits (alpha 3, alpha V, beta 1, beta 3, beta 4, beta 5) were observed consistently throughout preimplantation development. Evidence was also obtained for the presence of integrin subunits alpha 2, alpha 4, alpha L, beta 2, and beta 7 on a small number of oocytes. A more restricted developmental analysis of E-cadherin, ICAM-1, NCAM, and VCAM-1 demonstrated that these cell adhesion molecules are also present on oocytes and early embryos. L-selectin was detected on oocytes but was not found on 8-cell embryos. The oocyte and early blastomeres have complex surfaces in which the integrin and CAM families are represented.

Epithelial-Mesenchymal Transition during Trophoblast Differentiation

Embryo implantation in higher primates is mediated by trophoblast: in the earliest phases by syncytiotrophoblast, then by both cytotrophoblast and syncytiotrophoblast. In the course of placentation three main trophoblast populations can be identified: cytotrophoblast stem cells and two differentiated derivative cell types: the syncytiotrophoblast and the extravillous cytotrophoblast. The syncytiotrophoblast remains mainly epithelial while the extravillous cytotrophoblast undergoes an epithelial-mesenchymal transition (EMT), initially forming multilayered cell columns and then, in human, infiltrating deeply the maternal decidual stroma and blood vessels. Finally, some infiltrating cells differentiate further to become giant cells of the placental bed and myometrium. During the course of these events the extravillous cytotrophoblast acquires a distinct phenotype, losing some typical epithelial components (e.g. E-cadherin, integrin alpha 6 beta 4), but retaining others (e.g. cytokeratins). The signals that trigger this EMT are not well understood but its realisation is of critical importance for pregnancy success.

Uterine natural killer cells initiate spiral artery remodeling in human pregnancy

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.—Robson, A., Harris, L. K., Innes, B. A., Lash, G. E., Aljunaidy, M. M., Aplin, J. D., Baker, P. N., Robson, S. C., Bulmer, J. N. Uterine natural killer cells initiate spiral artery remodeling in human pregnancy. FASEB J. 26, 4876–4885 (2012). www.fasebj.org

Trophoblast-uterine interactions at implantation.

Implantation of the embryo in the uterus is a critical and complex event and its failure is widely considered an impediment to improved success in assisted reproduction. Depending on whether placentation is invasive or superficial (epitheliochorial), the embryo may interact transiently or undergo a prolonged adhesive interaction with the uterine epithelium. Numerous candidate interactions have been identified, and there is good progress on identifying gene networks required for early placentation. However no molecular mechanisms for the epithelial phase are yet firmly established in any species. It is noteworthy that gene ablation in mice has so far failed to identify obligatory initial molecular events.

Yoga for chronic low back pain: A randomized trial

BACKGROUND Previous studies indicate that yoga may be an effective treatment for chronic or recurrent low back pain. OBJECTIVE To compare the effectiveness of yoga and usual care for chronic or recurrent low back pain. DESIGN Parallel-group, randomized, controlled trial using computer-generated randomization conducted from April 2007 to March 2010. Outcomes were assessed by postal questionnaire. (International Standard Randomised Controlled Trial Number Register: ISRCTN 81079604) SETTING 13 non-National Health Service premises in the United Kingdom. PATIENTS 313 adults with chronic or recurrent low back pain. INTERVENTION Yoga (n = 156) or usual care (n = 157). All participants received a back pain education booklet. The intervention group was offered a 12-class, gradually progressing yoga program delivered by 12 teachers over 3 months. MEASUREMENTS Scores on the Roland-Morris Disability Questionnaire (RMDQ) at 3 (primary outcome), 6, and 12 (secondary outcomes) months; pain, pain self-efficacy, and general health measures at 3, 6, and 12 months (secondary outcomes). RESULTS 93 (60%) patients offered yoga attended at least 3 of the first 6 sessions and at least 3 other sessions. The yoga group had better back function at 3, 6, and 12 months than the usual care group. The adjusted mean RMDQ score was 2.17 points (95% CI, 1.03 to 3.31 points) lower in the yoga group at 3 months, 1.48 points (CI, 0.33 to 2.62 points) lower at 6 months, and 1.57 points (CI, 0.42 to 2.71 points) lower at 12 months. The yoga and usual care groups had similar back pain and general health scores at 3, 6, and 12 months, and the yoga group had higher pain self-efficacy scores at 3 and 6 months but not at 12 months. Two of the 157 usual care participants and 12 of the 156 yoga participants reported adverse events, mostly increased pain. LIMITATION There were missing data for the primary outcome (yoga group, n = 21; usual care group, n = 18) and differential missing data (more in the yoga group) for secondary outcomes. CONCLUSION Offering a 12-week yoga program to adults with chronic or recurrent low back pain led to greater improvements in back function than did usual care. PRIMARY FUNDING SOURCE Arthritis Research UK.

Trophoblast differentiation during formation of anchoring villi in a model of the early human placenta in vitro.

In human implantation sites, formation of the cytotrophoblast shell and morphogenesis of anchoring villi occur during the 2nd and 3rd week of pregnancy. When placental villous tissue from 8-12 weeks was co-cultured with decidua parietalis, morphogenetic changes were observed specifically at sites of heterotypic contact, generating structures that closely resembled first trimester anchoring villi. Local breakdown of the syncytium and cytotrophoblast proliferation occurred, producing columns of cytotrophoblast. These columns showed complex changes of cell surface phenotype that replicate precisely the extravillous trophoblast differentiation pathway seen in vivo, including induction of integrin alpha 5 beta 1, loss of integrin alpha 6 beta 4 and expression of the HLA class I framework epitope recognized by monoclonal antibody W6/32. Extracellular matrix components including laminin, collagen type IV, fibrin and fibronectin were detected in intercytotrophoblastic spaces in the nascent columns. Spreading over the surface of the decidua and infiltration by cytotrophoblast were observed. We conclude that: (i) fist trimester floating villi retain the capacity to differentiate into anchoring villi; (ii) contact with decidua stimulates local breakthrough of the syncytium and cytotrophoblast proliferation; (iii) the resulting cytotrophoblast columns show phenotypic changes characteristic of these structures in vivo; and (iv) parietal decidua is capable of supporting implantation site-specific changes in a manner similar to the basal decidua.

Insulin-like growth factor I and II regulate the life cycle of trophoblast in the developing human placenta

The main disorders of human pregnancy are rooted in defective placentation. Normal placental development depends on proliferation, differentiation, and fusion of cytotrophoblasts to form and maintain an overlying syncytiotrophoblast. There is indirect evidence that the insulin-like growth factors (IGFs), which are aberrant in pregnancy disorders, are involved in regulating trophoblast turnover, but the processes that control human placental growth are poorly understood. Using an explant model of human first-trimester placental villus in which the spatial and ontological relationships between cell populations are maintained, we demonstrate that cytotrophoblast proliferation is enhanced by IGF-I/IGF-II and that both factors can rescue cytotrophoblast from apoptosis. Baseline cytotrophoblast proliferation ceases in the absence of syncytiotrophoblast, although denuded cytotrophoblasts can proliferate when exposed to IGF and the rate of cytotrophoblast differentiation/fusion and, consequently, syncytial regeneration, increases. Use of signaling inhibitors suggests that IGFs mediate their effect on cytotrophoblast proliferation/syncytial formation through the MAPK pathway, whereas effects on survival are regulated by the phosphoinositide 3-kinase pathway. These results show that directional contact between cytotrophoblast and syncytium is important in regulating the relative amounts of the two cell populations. However, IGFs can exert an exogenous regulatory influence on placental growth/development, suggesting that manipulation of the placental IGF axis may offer a potential therapeutic route to the correction of inadequate placental growth.