Alkaline Phosphatase Protects Lipopolysaccharide-Induced Early Pregnancy Defects in Mice

Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as early pregnancy defects/loss and preterm birth. Bacterial toxin lipopolysaccharide (LPS), long recognized as a potent proinflammatory mediator, has been identified as a risk factor for pregnancy complications. Alkaline phosphatase (AP) isozymes have been shown to detoxify LPS by dephosphorylation. In this study, we examined the role of alkaline phosphatase (AP) in mitigating LPS-induced early pregnancy complications in mice. We found that 1) the uterus prior to implantation and implantation sites following embryo implantation produce LPS recognition and dephosphorylation molecules TLR4 and tissue non-specific AP (TNAP) isozyme, respectively; 2) uterine TNAP isozyme dephosphorylates LPS at its sites of production; 3) while LPS administration following embryo implantation elicits proinflammatory cytokine mRNA levels at the embryo implantation sites (EISs) and causes early pregnancy loss, dephosphorylated LPS neither triggers proinflammatory cytokine mRNA levels at the EISs nor induces pregnancy complications; 4) AP isozyme supplementation to accelerate LPS detoxification attenuates LPS-induced pregnancy complications following embryo implantation. These findings suggest that a LPS dephosphorylation strategy using AP isozyme may have a unique therapeutic potential to mitigate LPS- or Gram-negative bacteria-induced pregnancy complications in at-risk women.     

Lipid emulsion therapy in women with recurrent pregnancy loss and repeated implantation failure: The role of abnormal natural killer cell activity

Altered immune and/or inflammatory response plays an important role in cases of recurrent pregnancy loss (RPL) and repeated implantation failure (RIF). Exacerbation of the maternal immune response through increased NK cell activity and inflammatory cytokines can cause embryo rejection leading to abortion or embryo implantation failure. Immunosuppressors or immunomodulators can help or prevent this condition. Currently, lipid emulsion therapy (LET) has emerged as a treatment for RPL and RIF in women with abnormal NK cell activity, by decreasing the exacerbated immune response of the maternal uterus and providing a more receptive environment for the embryo. However, the mechanisms by which the intralipid acts to reduce NK cell activity are still unclear. In this review, we focus on the studies that conducted LET to treat patients with RPL and RIF with abnormal NK cell activity. We find that although some authors recommend LET as an effective intervention, more studies are necessary to confirm its effectiveness in restoring NK cell activity to normal levels and to comprehend the underlying mechanisms of the lipids action in ameliorating the maternal environment and improving the pregnancy rate.     

Bovine peripheral blood MSCs chemotax towards inflammation and embryo implantation stimuli

Mesenchymal stem cells (MSCs) have a great potential in regenerative medicine because of their multipotential and immunoregulatory capacities, while in early pregnancy they could participate in the immunotolerance of the mother towards the embryo. Peripheral blood constitutes an accessible source of MSCs. We successfully isolated peripheral blood MSC (pbMSCs) lines, with or without previous bone marrow mobilization. All pbMSCs lines obtained in both conditions presented classical MSC markers and properties, alkaline phosphatase activity and multipotent capacity to differentiate among adipogenic, osteogenic or chondrogenic lineages, and suppressed the proliferation of T cells. pbMSCs showed migratory capacity without cytokine stimulation while increasing their migration rate in the presence of inflammatory or embryo implantation stimuli. Interestingly, in contrast to MSCs derived from endometrial tissue, three pbMSCs lines also showed increased migration towards the IFN‐τ implantation cytokine. Moreover, the secretome produced by an early implantation stage embryonic trophectoderm cell line showed a chemoattractant effect in pbMSCs. Our results suggest that circulating MSCs are present in the peripheral blood under healthy conditions. The fact that both the inflammation and implantation signals induced pbMSCs chemotaxis highlights MSC heterogeneity and suggests that their migratory capacity may differ according to their tissue of origin and would suggest the possible active recruitment of MSCs from bone marrow during pregnancy to repress the immune response to prevent the embryo rejection by the maternal organism.     

In vivo analysis of progesterone receptor action in the uterus during embryo implantation

In order for a successful pregnancy to occur, the embryo must attach to the luminal epithelial cells and invade into the stroma. Then, the surrounding stromal cells need to undergo decidualization in order to establish the vasculature necessary for survival of the embryo. These events in early pregnancy are tightly regulated by the steroid hormones, estrogen (E2) and progesterone (P4), through their cognate receptors, the estrogen receptor (ER) and the progesterone receptor (PR), respectively. Using a mouse model in which the PR has been ablated, it was demonstrated that the PR is necessary for embryo implantation and decidualization. Therefore, understanding the mechanism of PR action in the adult uterus is necessary in order to understand the events of early pregnancy. Insights from both mouse models and human samples have been integral in elucidating uterine PR action. These studies have shown that not only PR target genes, but also mediators of PR action are important for correct PR action in early pregnancy. Many of the genes involved in PR action in early pregnancy have also been shown to have roles in uterine diseases such as endometriosis and endometrial cancer. Therefore, the integration of mouse and human studies on PR action in the uterus will be important for the future understanding of uterine diseases and in the development of treatment for these diseases.     

The role of extracellular vesicles in endometrial receptivity and their potential in reproductive therapeutics and diagnosis

Extracellular vesicles (EVs) are small, nanometre sized, membrane-enclosed structures released by cells and are thought to be crucial in cellular communication. The cargo of these vesicles includes lipids, proteins, RNAs and DNA, and control various biological processes in their target tissues depending on the parental and receiver cell’s origin and phenotype. Recently data has accumulated in the role of EVs in embryo implantation and pregnancy, with EVs identified in the uterine cavity of women, sheep, cows, horses, and mice, in which they aid blastocyst and endometrial preparation for implantation. Herein is a critical review to decipher the role of extracellular vesicles in endometrial receptivity and their potential in reproductive therapies and diagnosis. The current knowledge of the function of embryo and endometrial derived EVs and their cargoes, with regards to their effect on implantation and receptivity are summarized and evaluated. The findings of the below review highlight that the combined knowledge on EVs deriving from the endometrium and embryo have the potential to be translated to various clinical applications including treatment, a diagnostic biomarker for diseases and a drug delivery tool to ultimately improve pregnancy rates.     

In vivo analysis of progesterone receptor action in the uterus during embryo implantation

In order for a successful pregnancy to occur, the embryo must attach to the luminal epithelial cells and invade into the stroma. Then, the surrounding stromal cells need to undergo decidualization in order to establish the vasculature necessary for survival of the embryo. These events in early pregnancy are tightly regulated by the steroid hormones, estrogen (E2) and progesterone (P4), through their cognate receptors, the estrogen receptor (ER) and the progesterone receptor (PR), respectively. Using a mouse model in which the PR has been ablated, it was demonstrated that the PR is necessary for embryo implantation and decidualization. Therefore, understanding the mechanism of PR action in the adult uterus is necessary in order to understand the events of early pregnancy. Insights from both mouse models and human samples have been integral in elucidating uterine PR action. These studies have shown that not only PR target genes, but also mediators of PR action are important for correct PR action in early pregnancy. Many of the genes involved in PR action in early pregnancy have also been shown to have roles in uterine diseases such as endometriosis and endometrial cancer. Therefore, the integration of mouse and human studies on PR action in the uterus will be important for the future understanding of uterine diseases and in the development of treatment for these diseases.     

Enhanced expressions of endometrial tumour necrosis factor alpha and its receptors during early pregnancy in bonnet monkeys

Tumour necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine may play an active role in stimulating inflammatory reactions during pregnancy. However, the expression of endometrial TNF-alpha has not been investigated especially during early pregnancy, a phenomenon invariably accompanied by inflammatory reaction. In the present study, the endometrial expressions of TNF-alpha and its receptors (TNFR1 and TNFR2) during early pregnancy, when the embryo lies free in the zona hatched state in the uterine lumen, were analyzed by immunohistochemistry. The endometrial expressions of TNF-alpha, TNFR1 and TNFR2 were found to be significantly up-regulated (p < 0.05) in the glandular epithelium on day 6 post-ovulation in pregnant animals. The alteration in the expression of these molecules may contribute to the induction of local inflammatory reactions during implantation.     

Heparanase expression and function during early pregnancy in mice

Embryo implantation is a complex process that involves interactions between cell-surface and extracellular components of the blastocyst and the uterus, including blastocyst adhesion to the uterine luminal epithelium, epithelial basement membrane penetration and stromal extracellular matrix remodeling, angiogenesis, and decidualization. These processes all involve interactions with heparan sulfate (HS) proteoglycans, which harbor various growth factors and cytokines and support cell adhesion. Heparanase (HPSE) is an endo-beta-glucuronidase that cleaves HS at specific sites. HPSE also can act as an adhesion molecule independent of its catalytic activity. Thus, HPSE is a multifunctional molecule contributing to and modulating HS-dependent processes. Exogenously added HPSE improves embryo implantation in mice; however, no information is available regarding the normal pattern of HPSE expression and activity during the implantation process in any system. Using several approaches, including real-time RT-PCR, in situ hybridization, and immunohistochemistry, we determined that uterine HPSE expression increases dramatically during early pregnancy in mice. Heparanase mRNA and protein were primarily expressed in decidua and were rapidly induced at the implantation site. Uterine HPSE activity was characterized and demonstrated to increase >40-fold during early pregnancy. Finally, we demonstrate that the HPSE inhibitor PI-88 severely inhibits embryo implantation in vivo. Collectively, these results indicate that HPSE plays a role in blastocyst implantation and complements previous studies showing a role for HS-dependent interactions in this process.     

Nodal expression in the uterus of the mouse is regulated by the embryo and correlates with implantation

Nodal, a transforming growth factor beta (TGFB) superfamily member, plays a critical role during early embryonic development. Recently, components of the Nodal signaling pathway were characterized in the human uterus and implicated in the tissue remodeling events during menstruation. Furthermore, the Nodal inhibitor, Lefty, was identified in the mouse endometrium during pregnancy, and its overexpression led to implantation failure. Nonetheless, the precise function and mechanism of Nodal signaling during pregnancy remains unclear. In order to elucidate the potential roles Nodal plays in these processes, we have generated a detailed profile of maternal Nodal expression in the mouse uterus throughout pregnancy. NODAL, although undetectable during the nonpregnant estrus cycle, was localized throughout the glandular epithelium of the endometrium during the peri-implantation period. Interestingly, Nodal expression generated a banding pattern along the proximal-distal axis of the uterine horn on Day 4.5 that directly correlated with blastocyst implantation. Embryo transfer experiments indicate the embryo regulates Nodal expression in the uterus and directs its expression at the time of implantation, restricting NODAL to the sites between implantation crypts. During the later stages of pregnancy, Nodal exhibits a dynamic expression profile that suggests a role in regulating the endometrial response to decidualization and associated trophoblast invasion.     

Endocannabinoid signaling in synchronizing embryo development and uterine receptivity for implantation

There are reports of adverse effects of cannabinoids on pregnancy outcome including retarded embryo development and pregnancy failure. Thus, discoveries of endogenous cannabinoid-like lipid mediators and cannabinoid receptors raise questions about their pathophysiological roles during normal pregnancy. We previously reported that anandamide, an endogenously produced arachidonate derivative (endocannabinoid), is synthesized in the female reproductive tracts, and it acts on cannabinoid receptors expressed on the cell surface of the embryo to regulate the preimplantation embryo development and implantation in mice. This review presents genetic, molecular, physiological and pharmacological evidence that the levels of uterine anandamide and blastocyst CB1 cannabinoid receptors are coordinately regulated to synchronize preimplantation development and uterine receptivity for implantation in mice.     

Expression and localization of nodal in bovine oviduct and uterus during different functional stages of oestrus cycle and pregnancy

Members of TGF-β superfamily play a major role in the endometrial changes involved in the establishment and maintenance of pregnancy. Their deregulated expression and action could lead to absolute or partial failure of embryo implantation. Nonetheless, the precise function and mechanism of many of these cytokines remain unclear. Nodal, a transforming growth factor beta (TGF-β) superfamily member, was characterized in the human and rodent uterus and implicated in the tissue remodeling events during menstruation and embryo implantation. In order to study its possible role in the cattle reproductive process, we have analyzed Nodal expression pattern and localization in the oviduct and uterine horn during the oestrus cycle and early pregnancy (day 20). Nodal was detected both in oviduct and uterus during either the oestrus cycle or pregnancy; however, it shows a differential expression profile in the uterine horn at dioestrus and pregnancy, decreasing 1.5 and 1.4 folds in comparison with oestrus. Nodal immunostaining intensity was observed in stromal and in epithelial cells of the surface and the glandular epithelium. The staining pattern correlates with the RT-qPCR expression profile. This work is the first to evidence the presence of Nodal in the bovine reproductive tract; our data suggest that Nodal is a novel cytokine that would be involved in the remodelling occurring in the endometrium of cattle during the oestrus cycle and in the embryo implantation. The identification of new molecules that participate in endometrium cycling and/or pregnancy may be useful for predicting the ability of the uterine tissue to establish and maintain pregnancy or for detecting the infertility processes. These results highlight Nodal as a possible novel marker of the fertility process, nevertheless further studies should be done to determine its role in the reproductive system.     

Physiological and molecular determinants of embryo implantation

Embryo implantation involves the intimate interaction between an implantation-competent blastocyst and a receptive uterus, which occurs in a limited time period known as the window of implantation. Emerging evidence shows that defects originating during embryo implantation induce ripple effects with adverse consequences on later gestation events, highlighting the significance of this event for pregnancy success. Although a multitude of cellular events and molecular pathways involved in embryo–uterine crosstalk during implantation have been identified through gene expression studies and genetically engineered mouse models, a comprehensive understanding of the nature of embryo implantation is still missing. This review focuses on recent progress with particular attention to physiological and molecular determinants of blastocyst activation, uterine receptivity, blastocyst attachment and uterine decidualization. A better understanding of underlying mechanisms governing embryo implantation should generate new strategies to rectify implantation failure and improve pregnancy rates in women.     

Important aspects of placental-specific gene transfer

The placenta is a unique and highly complex organ that develops only during pregnancy and is essential for growth and survival of the developing fetus. The placenta provides the vital exchange of gases and wastes, the necessary nutrients for fetal development, acts as immune barrier that protects against maternal rejection, and produces numerous hormones and growth factors that promote fetal maturity to regulate pregnancy until parturition. Abnormal placental development is a major underlying cause of pregnancy-associated disorders that often result in preterm birth. Defects in placental stem cell propagation, growth, and differentiation are the major factors that affect embryonic and fetal well-being and dramatically increase the risk of pregnancy complications. Understanding the processes that regulate placentation is important in determining the underlying factors behind abnormal placental development. The ability to manipulate genes in a placenta-specific manner provides a unique tool to analyze development and eliminates potentially confounding results that can occur with traditional gene knockouts. Trophoblast stem cells and mouse embryos are not overly amenable to traditional gene transfer techniques. Most viral vectors, however, have a low infection rate and often lead to mosaic transgenesis. Although the traditional method of embryo transfer is intrauterine surgical implantation, the methodology reported here, combining lentiviral blastocyst infection and nonsurgical embryo transfer, leads to highly efficient and placental-specific gene transfer. Numerous advantages of our optimized procedures include increased investigator safety, a reduction in animal stress, rapid and noninvasive embryo transfer, and higher a rate of pregnancy and live birth.     

Inhibiting uterine PC6 blocks embryo implantation: an obligatory role for a proprotein convertase in fertility

Successful embryo implantation involves complex interactions between the embryo and the uterus and is critical in establishing pregnancy. Proprotein convertase (PC) 6 (PC6) is one of the PC endoproteases regulating protein function through posttranslational activation of precursor proteins, including growth and differentiation factors. Here we show that PC6 protein is induced in the uterine stromal cells specifically at the site of embryo attachment during early pregnancy in mice. In vivo blocking of uterine production of PC6 protein using morpholino antisense oligonucleotides in mice resulted in total inhibition of implantation, revealing a vital role for PC6 in modulating the uterus for embryo implantation. Studies in primates (rhesus monkey and human) showed a dramatic upregulation of endometrial PC6 during the phase of uterine receptivity and at implantation, particularly during a critical uterine cell differentiation process termed decidualization. Thus, the current studies have demonstrated that PC6 is an essential molecule in modulating uterine function to support the establishment of embryo implantation. Interestingly, PC6 is one of the PCs identified to be important in processing the coat protein of HIV; inhibition of PCs has been suggested to be an effective approach to reduce HIV transmission. We therefore propose the novel concept that PC6 could be a potential nonhormonal target in the female reproductive tract for dual protection for women, both in preventing pregnancy and reducing HIV infection.     

Transient {beta}2-adrenoceptor activation confers pregnancy loss by disrupting embryo spacing at implantation

Pregnancy loss is a serious social and medical issue, with one important cause associated with aberrant embryo implantation during early pregnancy. However, whether and how the process of embryo implantation is affected by environmental factors such as stress-induced sympathetic activation remained elusive. Here we report an unexpected, transient effect of β(2)-adrenoreceptor (β(2)-AR) activation (day 4 postcoitus) in disrupting embryo spacing at implantation, leading to substantially increased midterm pregnancy loss. The abnormal embryo spacing could be prevented by pretreatment of β(2)-AR antagonist or genetic ablation of β-AR. Similar β(2)-AR activation at day 5 postcoitus, when implantation sites have been established, did not affect embryo spacing or pregnancy outcome, indicating that the adverse effect of β(2)-AR activation is limited to the preimplantation period before embryo attachment. In vitro and in vivo studies demonstrated that the transient β(2)-AR activation abolished normal preimplantation uterine contractility without adversely affecting blastocyst quality. The contractility inhibition is mediated by activation of the cAMP-PKA pathway and accompanied by specific down-regulation of lpa3, a gene previously found to be critical for uterine contraction and embryo spacing. These results indicated that normal uterine contraction-mediated correct intrauterine embryo distribution is crucial for successful ongoing pregnancy. Abnormal β(2)-AR activation at early pregnancy provided a molecular clue in explaining how maternal stress at early stages could adversely affect the pregnancy outcome.     

The hamster as a model for embryo implantation: insights into a multifaceted process

Defects in preimplantation embryonic development, uterine receptivity, and implantation are the leading cause of infertility, pregnancy problems and birth defects. Significant progress has been made in our basic understanding of these processes using the mouse model, where implantation is ovarian estrogen-dependent in the presence of progesterone. However, an animal model where implantation is progesterone-dependent must also be studied to gain a full understanding of the embryo and uterine events that are required for implantation. In this regard, the hamster is a useful model and this review summarizes the information currently available regarding mechanisms involved in synchronous preimplantation embryo and uterine development for implantation in this species.     

Lipid signaling in embryo implantation

A reciprocal interaction between the implantation-competent blastocyst and the receptive uterus is required for successful implantation. Although various molecular pathways are known to participate in this cross-talk, a comprehensive understanding of the implantation process is still missing. Gene expression studies and genetically engineered mouse models have provided evidence that lipid mediators serve as important signaling molecules in coordinating the series of events during early pregnancy including preimplantation embryo formation and development, implantation and postimplantation growth. This review focuses on the roles of two groups of lipid mediators, prostaglandins (PGs) and endocannabinoids, during early pregnancy. Our laboratory has shown that while PGs generated by the cPLA2-cyclooxygenase (COX) system are essential to ovulation, fertilization, and implantation, endocannabinoids are important for synchronizing preimplantation embryo development with uterine receptivity for implantation. A better understanding of these molecular signaling pathways is hoped to generate new strategies to correct implantation failure and improve pregnancy rates in women.