The sheep conceptus modulates proteome profiles in caruncular endometrium during early pregnancy

This project was funded by NHS Grampian R&D project number RG05/019Peer reviewedPostprin

Amino acids in the uterine luminal fluid reflects the temporal changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle

In cattle, conceptus-maternal interactions are critical for the establishment and maintenance of pregnancy. A major component of this early interaction involves the transport of nutrients and secretion of key molecules by uterine epithelial cells to help support conceptus development during the peri-implantation period of pregnancy. Objectives were to: 1) analyze temporal changes in the amino acid (AA) content of uterine luminal fluid (ULF) during the bovine estrous cycle; 2) understand conceptus-induced alterations in AA content; 3) determine expression of AA transporters in the endometrium and conceptus; and 4) determine how these transporters are modulated by (Progesterone) P4. Concentrations of aspartic acid, arginine, glutamine, histidine, lysine, isoleucine, leucine, phenylalanine and tyrosine decreased on Day 16 of the estrous cycle but increased on Day 19 in pregnant heifers (P < 0.05). Glutamic acid only increased in pregnant heifers on Day 19 (P,0.001). Asparagine concentrations were greater in ULF of cyclic compared to pregnant heifers on Day 7 (P < 0.05) while valine concentrations were higher in pregnant heifers on Day 16 (P < 0.05). Temporal changes in expression of the cationic AA transporters SLC7A1 SLC7A4 and SLC7A6 occurred in the endometrium during the estrous cycle/early pregnancy coordinate with changes in conceptus expression of SLC7A4, SLC7A2 and SLC7A1 (P < 0.05). Only one acidic AA transporter (SLC1A5) increased in the endometrium while conceptus expression of SLC1A4 increased (P < 0.05). The neutral AA transporters SLC38A2 and SLC7A5 increased in the endometrium in a temporal manner while conceptus expression of SLC38A7, SLC43A2, SLC38A11 and SLC7A8 also increased (P < 0.05). P4 modified the expression of SLC1A1, -1A4, -1A5, -38A2 , -38A4, -38A7, -43A2, -6A14, -7A1, -7A5 and -7A7 in the endometrium. Results demonstrate that temporal changes in AA in the ULF reflect changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle, some of which are modified by P4. © 2014 Forde et al

Deficiency in clonogenic endometrial mesenchymal stem cells in obese women with reproductive failure – a pilot study

The mechanisms of obesity associated reproductive complications remain poorly understood. Endometrial mesenchymal stem-cells are critical for cyclic renewal and uterine function. Recently, W5C5+ cells, with high clonogenicity, capable of producing endometrial stroma in vivo, have been described. We sought to investigate the abundance and cloning efficiency of W5C5+ and W5C5− endometrial cells in relation to Body Mass Index, age and reproductive outcome. Design W5C5+ and W5C5− cells were purified from mid-luteal endometrial biopsies (n = 54) by magnetic bead separation and subjected to in vitro colony-forming assays. Results First trimester pregnancy losses were significantly higher in obese subjects (n = 12) compared to overweight (n = 20) and subjects with normal Body Mass Index (n = 22) (P0.05). Conclusions Our observations suggest that the regenerative capacity and plasticity of the endometrium of obese women is suboptimal, which in turn may account for the increased risk of reproductive complications associated with obesity

Global Transcriptomic Profiling of Bovine Endometrial Immune Response In Vitro. I. Effect of Lipopolysaccharide on Innate Immunity

The dysregulation of endometrial immune response to bacterial lipopolysaccharide (LPS) has been implicated in uterine disease and infertility in the postpartum dairy cow, although the mechanisms are not clear. Here, we investigated whole-transcriptomic gene expression in primary cultures of mixed bovine epithelial and stromal endometrial cells. Cultures were exposed to LPS for 6 h, and cellular response was measured by bovine microarray. Approximately 30% of the 1006 genes altered by LPS were classified as being involved in immune response. Cytokines and chemokines (IL1A, CX3CL1, CXCL2, and CCL5), interferon (IFN)-stimulated genes (RSAD2, MX2, OAS1, ISG15, and BST2), and the acute phase molecule SAA3 were the most up-regulated genes. Ingenuity Pathway Analysis identified up-regulation of many inflammatory cytokines and chemokines, which function to attract immune cells to the endometrium, together with vascular adhesion molecules and matrix metalloproteinases, which can facilitate immune cell migration from the tissue toward the uterine lumen. Increased expression of many IFN-signaling genes, immunoproteasomes, guanylate-binding proteins, and genes involved in the intracellular recognition of pathogens suggests important roles for these molecules in the innate defense against bacterial infections. Our findings confirmed the important role of endometrial cells in uterine innate immunity, whereas the global approach used identified several novel immune response pathways triggered by LPS in the endometrium. Additionally, many genes involved in endometrial response to the conceptus in early pregnancy were also altered by LPS, suggesting one mechanism whereby an ongoing response to infection may interfere with the establishment of pregnancy

Impact of sitagliptin on endometrial mesenchymal stem-like progenitor cells : a randomised, double-blind placebo-controlled feasibility trial

Background: Recurrent pregnancy loss (RPL) is associated with the loss of endometrial mesenchymal stem-like progenitor cells (eMSC). DPP4 inhibitors may increase homing and engraftment of bone marrow-derived cells to sites of tissue injury. Here, we evaluated the effect of the DPP4 inhibitor sitagliptin on eMSC in women with RPL, determined the impact on endometrial decidualization, and assessed the feasibility of a full-scale clinical trial. Methods: A double-blind, randomised, placebo-controlled feasibility trial on women aged 18 to 42 years with a history of 3 or more miscarriages, regular menstrual cycles, and no contraindications to sitagliptin. Thirty-eight subjects were randomised to either 100 mg sitagliptin daily for 3 consecutive cycles or identical placebo capsules. Computer generated, permuted block randomisation was used to allocate treatment packs. Colony forming unit (CFU) assays were used to quantify eMSC in midluteal endometrial biopsies. The primary outcome measure was CFU counts. Secondary outcome measures were endometrial thickness, study acceptability, and first pregnancy outcome within 12 months following the study. Tissue samples were subjected to explorative investigations. Findings: CFU counts following sitagliptin were higher compared to placebo only when adjusted for baseline CFU counts and age (RR: 1.52, 95% CI: 1.32–1.75, P<0.01). The change in CFU count was 1.68 in the sitagliptin group and 1.08 in the placebo group. Trial recruitment, acceptability, and drug compliance were high. There were no serious adverse events. Explorative investigations showed that sitagliptin inhibits the expression of DIO2, a marker gene of senescent decidual cells. Interpretation: Sitagliptin increases eMSCs and decreases decidual senescence. A large-scale clinical trial evaluating the impact of preconception sitagliptin treatment on pregnancy outcome in RPL is feasible and warranted. Funding: Tommy's Baby Charity. Clinical trial registration: EU Clinical Trials Register no. 2016-001120-54

TGF beta 1 attenuates expression of prolactin and IGFBP-1 in decidualized endometrial stromal cells by both SMAD-dependent and SMAD-independent pathways

Background: Decidualization (differentiation) of the endometrial stromal cells during the secretory phase of the menstrual cycle is essential for successful implantation. Transforming Growth Factor beta 1 (TGF beta 1) canonically propagates its actions via SMAD signalling. A role for TGF beta 1 in decidualization remains to be established and published data concerning effects of TGF beta 1 on markers of endometrial decidualization are inconsistent. Methodology/Principal Findings: Non-pregnant endometrial stromal cells (ESC) and first trimester decidual stromal cells (DSC) were cultured in the presence or absence of a decidualizing stimulus. Incubation of ESCs with TGF beta 1 (10 ng/ml) down-regulated the expression of transcripts encoding the decidual marker proteins prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP-1) and tissue factor (TF). TGF beta 1 also inhibited secretion of PRL and IGFBP-1 proteins by ESCs and surprisingly this response preceded down-regulation of their mRNAs. In contrast, DSCs were more refractory to the actions of TGF beta 1, characterized by blunted and delayed down-regulation of PRL, IGFBP-1, and TF transcripts, which was not associated with a significant reduction in secretion of PRL or IGFBP-1 proteins. Addition of an antibody directed against TGF beta 1 increased expression of IGFBP-1 mRNA in decidualised cells. Knockdown of SMAD 4 using siRNAs abrogated the effect of TGF beta 1 on expression of PRL in ESCs but did not fully restore expression of IGFBP-1 mRNA and protein. Conclusions/Significance: TGF beta 1 inhibits the expression and secretion of decidual marker proteins. The impact of TGF beta 1 on PRL is SMAD-dependent but the impact on IGFBP1 is via an alternative mechanism. In early pregnancy, resistance of DSC to the impact of TGF beta 1 may be important to ensure tissue homeostasis

A Combination of Two Human Monoclonal Antibodies Limits Fetal Damage by Zika Virus in Macaques

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement in vitro (ADE) and extend their half-lives. Here we report on prophylactic co-administration of the Fc-modified antibodies to pregnant rhesus macaques challenged 3 times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV