Uterine Receptivity For Implantation Research Articles

46 Adrenomedullin: A Novel Peptide Hormone for Uterine Receptivity and Conceptus Elongation in Pigs?

Abstract Adrenomedullin (ADM) is an evolutionarily conserved multi-functional peptide hormone in mammalian species. The implantation sites of conceptuses (embryo/fetus and its extra-embryonic membranes) in pregnant Adm+/− female mice are abnormally spaced leading to fetal crowding, intrauterine growth restriction (IUGR) and increases in pregnancy loss. As litter-bearing species, pigs exhibit a high incidence of early embryonic death (30-40%) and naturally occurring IUGR (15-25%), and significant numbers of stillborn piglets (3-9%). Therefore, it is imperative that the roles of ADM in regulation of uterine receptivity, as well as growth and development of conceptus in pigs be established. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components in uteri from cyclic and pregnant gilts, as well as conceptuses during the peri-implantation period of pregnancy. ADM receptor components include: calcitonin receptor-like receptor (CALCRL; G protein-coupled receptor bound by ADM), receptor activity modifying protein (RAMP2; dimerized with CALCRL to form ADM1 receptor) and RAMP3 (dimerized with CALCRL to form ADM2 receptor with lower specificity to ADM) and atypical chemokine receptor 3 (ACKR3; a decoy receptor that serves as a cell-autonomous molecular rheostat to dampen ADM signaling). Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, expression of CALCRL, RAMP2 and ACKR3 increased between days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line isolated from elongating porcine conceptuses recovered on day 2 post-breeding, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation and placentation in pigs. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.

Temporal and spatial expression of adrenomedullin and its receptors in the porcine uterus and peri-implantation conceptuses.

Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10-7M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs.

Spatiotemporal expression of endogenous opioid processing enzymes in mouse uterus at peri-implantation.

Successful implantation requires intimate interactions between a competent blastocyst and a receptive uterus. We recently demonstrated that the aberrant activation of opioid signaling by exogenous ligands adversely affects preimplantation embryonic development and subsequent implantation in mice. However, the underlying machinery governing the dynamic homeostasis of the endogenous opioid system in the uterus during early pregnancy remains elusive. We now show that all three major endogenous opioid precursors are spatiotemporally expressed in the uterus during early pregnancy. Moreover, we observe the well-coordinated expression of the synthetic enzyme prohormone convertases 1/3 (PC1/3) at lower levels and of its inhibitor proprotein convertase subtilisin/kexin type 1 inhibitor (Pcsk1n) and the degrading enzyme membrane metallo-endopeptidase (MME) at higher levels in the receptive uterus. Both estrogen and progestin tend to reduce the uterine levels of opioid ligand precursors in the ovariectomized mouse model. This tight regulation of the endogenous opioid system by PC1/3, Pcsk1n and MME has been further confirmed in physiologically related pseudopregnancy and delayed implantation mouse models. The coordinated regulation of opioid precursor biosynthesis and metabolism helps to create appropriate opioid signaling ensuring uterine receptivity for implantation. Thus, endogenous uterine opioid levels are primarily determined by the coordinated expressions of PC1/3, Pcsk1n and MME under the influence of ovarian progestin and estrogen. Our findings raise an additional cautionary note regarding the effects of opioid abuse on early pregnancy events.

Calpain 2 activity increases at the time of implantation in rat uterine luminal epithelial cells and administration of calpain inhibitor significantly reduces implantation sites

The present study investigated the role of calpain 2 in rat uterine luminal epithelial cells during early pregnancy. Calpain 2 is an intracellular calcium-dependent proteolytic enzyme which cleaves numerous focal adhesion proteins. Calpain 2 was concentrated along the basal cell surface of uterine luminal epithelial cells at the predicted site of focal adhesions on day 1 of pregnancy and remained unchanged at the time of implantation as observed by immunofluorescence microscopy. However, Western blotting analysis showed a marked increase in the active form and a significant decrease in the latent form of calpain 2 at the time of implantation. The increase in calpain 2 activity coincides with the disassembly of focal adhesion proteins, talin, paxillin, integrin β1 and β3 from the site of focal adhesions. Intraperitoneal injection of calpain inhibitor, calpain inhibitor l (ALLN), significantly reduced the number of implantation sites, implying that calpain 2 plays an important role in implantation. The present study suggests a role for calpain 2 in the disassembly of focal adhesions, which has been previously shown to play a key role in uterine receptivity for implantation.

Mucin 15 is lost but mucin 13 remains in uterine luminal epithelial cells and the blastocyst at the time of implantation in the rat

The glycocalyx of the uterine luminal epithelium in the rat undergoes considerable reduction before implantation. In particular, the reduction of some mucins is necessary to facilitate blastocyst adhesion and subsequent implantation. The present study investigated the localisation, abundance and hormonal control of two mucin proteins, Muc13 and Muc15, in rat uterine epithelial cells during early pregnancy to determine whether they are likely to play a role in uterine receptivity for implantation. Muc13 and Muc15 are localised to the uterine luminal epithelium but show a presence and an absence, respectively, at the apical cell surface at the time of implantation. This localisation corresponds to changes in the molecular weights of Muc13 and Muc15, as shown with western blotting analysis. Furthermore, the localisation of Muc13 and Muc15 was shown to be controlled by the ovarian hormones, oestrogen and progesterone, and they were also localised in preimplantation rat blastocysts. Our results suggest that Muc15 may operate in an anti-adhesive capacity to prevent implantation while Muc13 potentially functions in either an adhesive or cell-signalling role in the events of implantation.

Novel pathways for implantation and establishment and maintenance of pregnancy in mammals

Uterine receptivity to implantation varies among species, and involves changes in expression of genes that are coordinate with attachment of trophectoderm to uterine lumenal and superficial glandular epithelia, modification of phenotype of uterine stromal cells, silencing of receptors for progesterone and estrogen, suppression of genes for immune recognition, alterations in membrane permeability to enhance conceptus-maternal exchange of factors, angiogenesis and vasculogenesis, increased vascularity of the endometrium, activation of genes for transport of nutrients into the uterine lumen, and enhanced signaling for pregnancy recognition. Differential expression of genes by uterine epithelial and stromal cells in response to progesterone, glucocorticoids, prostaglandins and interferons may influence uterine receptivity to implantation in mammals. Uterine receptivity to implantation is progesterone-dependent; however, implantation is preceded by loss of expression of receptors for progesterone (PGR) so that progesterone most likely acts via PGR-positive stromal cells throughout pregnancy. Endogenous retroviruses expressed by the uterus and/or blastocyst also affect implantation and placentation in various species. Understanding the roles of the variety of hormones, growth factors and endogenous retroviral proteins in uterine receptivity for implantation is essential to enhancing reproductive health and fertility in humans and domestic animals.

Interferons and Uterine Receptivity

This article focuses on the potential roles of interferons (IFNs) in establishing uterine receptivity to implantation. A common feature of the peri-implantation period of pregnancy in most mammals is production of type I and/or type II IFNs by trophoblasts that induce and/or stimulate expression of an array of IFN-stimulate genes (ISGs). These effects range from pregnancy recognition signaling in ruminants through IFN tau to effects on cellular functions of the uterus and uterine vasculature. For actions of IFNs, progesterone (P4) is permissive to the expression of many effects and to the expression of ISGs that are induced directly by an IFN or induced by P4 and stimulated by an IFN in a temporal and/or cell-specific manner. Uterine receptivity to implantation is P4 dependent; however, implantation events are preceded by loss of expression of progesterone (PGR) and estrogen (ESR1) receptors by uterine epithelia. Therefore, P4 likely acts via PGR-positive stromal cells to induce expression of fibroblast growth factors-7 and -10 and/or hepatocyte growth factor (progestamedins) that then act via their respective receptors on uterine epithelia and trophectoderm to affect expression of ISGs. The permissive effects of P4 on the expression of ISGs and the effects of P4 to induce and IFNs to stimulate gene expression raise the question of whether uterine receptivity to implantation requires P4 and IFN to activate unique, but complementary, cell signaling pathways. Uterine receptivity to implantation, depending on species, involves changes in the expression of genes for the attachment of trophectoderm to the uterine lumenal epithelium (LE) and superficial glandular epithelium (sGE), modification of the phenotype of uterine stromal cells, the silencing of PGR and ESR1 genes, the suppression of genes for immune recognition, alterations in membrane permeability to enhance conceptus-maternal exchange of factors, increased vascularity of the endometrium, activation of genes for transport of nutrients into the uterine lumen, and enhanced signaling for pregnancy recognition. Differential expression of genes by uterine LE/sGE, mid- to deep-glandular epithelia (GE), and stromal cells in response to P4 and IFNs is likely to influence uterine receptivity to implantation in most mammals. Understanding the roles of IFNs in uterine receptivity for implantation is necessary to develop approaches to enhance reproductive health and fertility in humans and domestic animals.

Cochaperone immunophilin FKBP52 is critical to uterine receptivity for embryo implantation

Embryo implantation in the uterus is a critical step in mammalian reproduction, requiring preparation of the uterus receptive to blastocyst implantation. Uterine receptivity, also known as the window of implantation, lasts for a limited period, and it is during this period blastocysts normally implant. Ovarian steroid hormones estrogen and progesterone (P(4)) are the primary regulators of this process. The immunophilin FKBP52 serves as a cochaperone for steroid hormone nuclear receptors to govern appropriate hormone action in target tissues. Here we show a critical role for FKBP52 in mouse implantation. This immunophilin has unique spatiotemporal expression in the uterus during implantation, and females missing the Fkbp52 gene have complete implantation failure due to lack of attainment of uterine receptivity. The overlapping uterine expression of FKBP52 with nuclear progesterone receptor (PR) in wild-type mice together with reduced P(4) binding to PR, attenuated PR transcriptional activity and down-regulation of several P(4)-regulated genes in uteri of Fkbp52(-/-) mice, establishes this cochaperone as a critical regulator of uterine P(4) function. Interestingly, ovulation, another P(4)-mediated event, remains normal. Collectively, the present investigation provides evidence for an in vivo role for this cochaperone in regulating tissue-specific hormone action and its critical role in uterine receptivity for implantation.

Role of utero–ovarian vascular impedance: predictor of ongoing pregnancy in an IVF–embryo transfer programme

The objective of this study was to evaluate the role of uterine, endometrial and follicular blood flow in prediction of ongoing pregnancy after assisted conception. A prospective observational study was conducted on 53 women undergoing IVF treatment. Transvaginal colour and pulsed Doppler measurements were performed on the day that pituitary suppression was confirmed, on day 10 of ovarian stimulation and on the day prior to human chorionic gonadotrophin injection. On the last day of ovarian stimulation, blood flow in the ascending uterine artery of the women who would conceive was characterized by significantly lower pulsatility index values. Sub-endometrial vascular impedance was comparable in the pregnant and non-pregnant groups. There were no differences in the perifollicular vascularity between pregnant and non-pregnant women. The chance of achieving pregnancy predicted by uterine artery Doppler and perifollicular blood flow in women whose PI values were higher than 3.26 and 1.08 was very low, with a sensitivity of 1.00 and specificity of 0.59 and 0.82 respectively. The data provide evidence for an association between utero–ovarian perfusion and reproductive outcome following IVF treatment. Uterine and ovarian vascular impedance values identify those women whose pregnancy chances are significantly limited. Measures to decrease vascular impedance in such women might enhance pregnancy rates by improving embryo quality and uterine receptivity for implantation.

Alterations in tight junction molecules of uterine epithelial cells during early pregnancy in the rat

Distribution patterns of the tight junction associated proteins ZO-1, claudin-1 and occludin were investigated in rat uterine epithelial cells during early pregnancy. Light microscopy and immunohistochemical labelling were used to detect these proteins on days 1, 3, 6 and 7 of pregnancy. Intense staining of claudin-1 at the apical region of the lateral plasma membrane accompanied diffuse staining throughout the cytoplasm. ZO-1 was also localised in the apical region, but ZO-1 was not present in the lower two thirds of the lateral plasma membrane or in the cytoplasm. Occludin was present only on days 6 and 7 of pregnancy. Labelling was also localised in the apical region of the lateral plasma membrane where tight junctions are known to be present. Our results show that ZO-1, claudin-1 and occludin are present in the apical region of uterine epithelial cells, and appear to play a role in the very dynamic tight-junctional network of uterine epithelial cells during early pregnancy. In particular, occludin appears only during uterine receptivity for implantation.

Dysregulated Cannabinoid Signaling Disrupts Uterine Receptivity for Embryo Implantation

The mechanisms by which synchronized embryonic development to the blastocyst stage, preparation of the uterus for the receptive state, and reciprocal embryo-uterine interactions for implantation are coordinated are still unclear. We show in this study that preimplantation embryo development became asynchronous in mice that are deficient in brain-type (CB1) and/or spleen-type (CB2) cannabinoid receptor genes. Furthermore, whereas the levels of uterine anandamide (endocannabinoid) and blastocyst CB1 are coordinately down-regulated with the onset of uterine receptivity and blastocyst activation prior to implantation, these levels remained high in the nonreceptive uterus and in dormant blastocysts during delayed implantation and in pregnant, leukemia inhibitory factor (LIF)-deficient mice with implantation failure. These results suggest that a tight regulation of endocannabinoid signaling is important for synchronizing embryo development with uterine receptivity for implantation. Indeed this is consistent with our finding that while an experimentally induced, sustained level of an exogenously administered, natural cannabinoid inhibited implantation in wild-type mice, it failed to do so in CB1(-/-)/CB2(-/-) double mutant mice. The present study is clinically important because of the widely debated medicinal use of cannabinoids and their reported adverse effects on pregnancy.

Molecular signaling in uterine receptivity for implantation

Successful implantation is the result of an intimate ‘cross-talk’ between the blastocyst and uterus in a temporal and cell-specific manner. Thus, both the uterine and embryonic events must be examined to better understand this process. Although various aspects and molecules associated with these events have been explored, a comprehensive understanding of the implantation process is still very limited. In this review, we have highlighted the importance of the blastocyst’s activity state and the receptive state of the uterus in determining the ‘window’ of implantation. In this context, we provide a testable scheme that signifies the important roles of various key molecules in embryo–uterine interactions during implantation.

Multiple roles for heparin-binding epidermal growth factor-like growth factor are suggested by its cell-specific expression during the human endometrial cycle and early placentation.

Embryonic expression of the epidermal growth factor (EGF) receptor as well as embryonic and steroid-dependent uterine secretion of its ligand, heparin-binding EGF-like growth factor (HB-EGF), are temporally associated with the period of blastocyst implantation. We examined the temporal cell type-specific expression of HB-EGF in human endometrium during the menstrual cycle by immunohistochemistry and in situ hybridization. Early first trimester implantation sites were also examined to determine HB-EGF protein levels in decidual and fetal tissues. In the endometrial stroma, HB-EGF protein expression increased markedly during the late proliferative phase and then decreased in the early secretory phase. By contrast, luminal and glandular epithelial cells as well as blood vessel endothelium accumulated the protein between midcycle and cycle day 20, with peak expression observed during the period of uterine receptivity for implantation. HB-EGF expression decreased dramatically at the end of the cycle, before menses. Spatiotemporal expression of HB-EGF messenger ribonucleic acid demonstrated a similar pattern. During early pregnancy, HB-EGF immunostaining was noted in the decidua and in both villous and extravillous trophoblast populations. These findings suggest that HB-EGF promotes implantation and trophoblast invasion through paracrine and autocrine signaling as cells penetrate the stroma and displace the arteriole endothelium.