The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. Because microRNAs (miRNAs) have major roles in regulating gene expression, we speculated that they participate in directing the highly regulated spatiotemporally expressed genetic network during implantation. Here, we show that two miRNAs, mmu-miR-101a and mmu-miR-199a*, are spatiotemporally expressed in the mouse uterus during implantation coincident with expression of cyclooxygenase-2, a gene critical for implantation. More interestingly, our in vitro gain- and loss-of-function experiments show that cyclooxygenase-2 expression is posttranscriptionally regulated by these two miRNAs. We report on miRNA-mediated regulation of uterine gene expression in the context of implantation. We believe that many other critical genes related to this process are also regulated by miRNAs. Thus, elucidating the physiological roles of uterine miRNAs will help us better understand the genetic control of implantation, the gateway to a successful pregnancy.
Uterine glands are essential for pregnancy establishment. By employing forkhead box A2 (FOXA2)-deficient mouse models coupled with leukemia inhibitory factor (LIF) repletion, we reveal definitive roles of uterine glands in embryo implantation and stromal cell decidualization. Here we report that LIF from the uterine glands initiates embryo-uterine communication, leading to embryo attachment and stromal cell decidualization. Detailed histological and molecular analyses discovered that implantation crypt formation does not involve uterine glands, but removal of the luminal epithelium is delayed and subsequent decidualization fails in LIF-replaced glandless but not gland-containing FOXA2-deficient mice. Adverse ripple effects of those dysregulated events in the glandless uterus result in embryo resorption and pregnancy failure. These studies provide evidence that uterine glands synchronize embryo-endometrial interactions, coordinate on-time embryo implantation, and impact stromal cell decidualization, thereby ensuring embryo viability, placental growth, and pregnancy success.
The anti-implantation effects of high oestradiol (E-2) concentrations could be mediated by E-2 metabolites. Herein, we examined whether 2-methoxyoestradiol (2ME) impairs embryo implantation via its target protein F-spondin. Mice on Day 3 of pregnancy were treated with E-2 concomitantly with the cathecol-O-methyl transferase inhibitor OR486 and the number of implanted embryos was recorded 5 days later. The effect of 2ME or 4-methoxyoestradiol (4ME) on embryo implantation was also investigated. Plasma and uterine levels of 2ME were measured 0.5, 1 or 3h after E-2 treatment while the mRNA for spondin 1 (Spon1) and F-spondin were determined in the uterus 3, 6, 12 or 24h after 2ME treatment. Finally, the effect of a neutralising F-spondin antibody on the anti-implantation effect of 2ME was explored. OR486 blocked the anti-implantation effect of E-2; 2ME, but not 4ME, affected embryo implantation. The 2ME concentration was increased after 0.5 and 1h in plasma and 3h in uterine fluid following E-2 treatment. 2ME increased levels of Spon1 at 12 and 24h although F-spondin was increased at 12h. F-spondin antibody blocked the effect of 2ME on embryo implantation. We conclude that 2ME impairs mouse embryo implantation via activation of F-spondin in the uterus.
Abstract Computer-automated time-lapse analysis has been shown to improve embryo selection by providing quantitative and objective information to supplement traditional morphology. In this multi-centre study, the relationship between such computer-derived outputs (High, Medium, Low scores), embryo implantation and clinical pregnancy were examined. Data were collected from six clinics, including 205 patients whose embryos were imaged by the EevaTM System. The Eeva scores were blinded and not considered during embryo selection. Embryos with High and Medium scores had significantly higher implantation rates than those with Low scores (37% and 35% versus 15%; P < 0.0001; P = 0.0004). Similar trends in implantation rates were observed in different IVF centres each using their own protocols. Further analysis revealed that patients with at least one High embryo transferred had significantly higher clinical pregnancy rates than those with only Low embryos transferred (51% versus 34%; P = 0.02), although patients' clinical characteristics across groups were comparable. These data, together with previous research and clinical studies, confirm that computer-automated Eeva scores provide valuable information, which may improve the clinical outcome of IVF procedures and ultimately facilitate the trend of single embryo selection.
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 ) 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 binding to PR, attenuated PR transcriptional activity and down-regulation of several P -regulated genes in uteri of Fkbp52 mice, establishes this cochaperone as a critical regulator of uterine P function. Interestingly, ovulation, another P -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.
Melatonin, a superior antioxidant, is an important molecule which regulates female reproduction due to its receptor‐mediated and receptor‐independent antioxidant actions. In this study, we investigated the effect of melatonin on early gestation in a mouse model. During early gestation, the expression of the melatonin's rate‐limiting enzyme, AANAT, gradually increased – in the uterus while the MT2 melatonin receptor was only expressed at day 2 of gestation and no MT1 was detected. Based on these findings, we conducted a melatonin injection experiment which demonstrated that 15 mg/kg melatonin significantly improved the number of implantation sites and the litter size. Also, the blastocyst and uterus were collected to identify the local action of melatonin. In the melatonin‐treated mice, the endometrium was thicker than in the control mice; melatonin also caused an increase in density of uterine glands, and the uterine gland index (UGI) was significantly elevated over that of the control. Serum steroid hormone measurements revealed that at day 6 of gestation (postimplantation), melatonin significantly downregulated the E2 level, with no obvious effects on progesterone. Gene expression assay revealed that melatonin significantly upregulated expression of HB‐EGF, a crucial gene involved in implantation as well as its receptor ErbB1 in the blastocyst. In addition, PRA, an important gene which influences the decidual response and luminal cell differentiation, p53, which regulates uterine through leukaemia inhibitory factor (LIF), were both increased after melatonin treatment. These data suggest that melatonin and its MT2 receptor influence early gestation. Exogenous melatonin treatment can improve mouse embryo implantation and litter size, which may have important applications in human reproductive health and animal husbandry.
Objective To develop a diagnostic tool for embryo implantation potential with the use of proteomic fingerprinting combined with time-lapse morphokinetic analysis. Design Retrospective cohort study. Setting University-affiliated private in vitro fertilization center. Patient(s) Seventeen infertile patients undergoing intracytoplasmic sperm injection (ICSI) from our ovum donation program. Intervention(s) No patient intervention. We examined morphokinetic data and proteomic data from the spent media of 16 embryos that implanted and 12 embryos that did not implant. Main Outcome Measure(s) We analyzed seven proteins in the embryo spent media—SCF, TNFR1, PIGF-1, IFN-α2, IL-6, CXCL13, and GM-CSF—with the use of a bead-based multiplexing technology and combined this data with the exact timing (in hours) of cell cycle duration (cc2), blastomere synchrony (s2), and 5-blastomere cleavage (t5) with the use of an incubator equipped with time-lapse videography. Result(s) Logistic regression analysis with the use of the forward-step likelihood selection method revealed that the presence/absence of interleukin (IL) 6 and the duration of cc2 were the most relevant embryo features for embryo selection. We combined these two parameters to obtain a hierarchic model that established four categories (A/B/C/D), based on the presence of IL-6 and a cc2 range of 5–12 hours. A direct relationship was observed between the morphologic categories and implantation rates: Those with the presence of IL-6 and 5–12 h cc2 had significantly higher implantation rates. Conclusion(s) The strategy we report here combines time-lapse and proteome analysis to improve embryo selection while minimizing handling and monitoring by the embryologist. Our results describe the utility of a combined biochemical/morphokinetic approach to select embryos for transfer according to their implantation potential. Clinical validation with larger sample sizes is mandatory to confirm the effectiveness of this initial study.
Cytokine-like protein 1 (CytI1), originally described as a protein expressed in CD34+ cells, was recently identified as a functional secreted protein involved in chondrogenesis and cartilage development. However, our knowledge of CytI1 is still limited. Here, we determined the CytI1 expression pattern regulated by ovarian hormones at both the mRNA and protein levels. We found that the endometrial expression of CytI1 in mice was low before or on the first day of gestation, significantly increased during embryo implantation, and then decreased at the end of implantation. We investigated the effects of CytI1 on endometrial cell proliferation, and the effects on the secretion of leukemia inhibitory factor (LIF) and heparin -binding epidermal growth factor (HB-EGF). We also explored the effect of CytI1 on endometrial adhesion properties in cell-cell adhesion assays. Our findings demonstrated that CytI1 is an ovarian hormone-dependent protein expressed in the endometrium that enhances the proliferation of HEC-1-A and RL95-2 cells, stimulates endometrial secretion of LIF and HB-EGF, and enhances the adhesion of HEC-1-A and RL95-2 cells to JAR spheroids. This study suggests that CytI1 plays an active role in the regulation of embryo implantation.
MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy.
MicroRNAs (miRNAs) are emerging as important in human embryo implantation, and we present here a review of the literature from a clinical perspective. Implantation involves complex interactions between the blastocyst and endometrium. miRNAs have been shown to be differentially expressed in implanted compared with non-implanted blastocysts and euploid compared with aneuploid blastocysts. Further, miRNAs are differentially expressed in proliferative compared with decidualized endometrium, and in receptive compared with pre-receptive endometrium. miRNAs are also differentially expressed in endometrium of women who failed implantation, and in endometrium of women with recurrent implantation failure. Due to the complexity of miRNA signaling, studies have suffered from inconsistency in reproducibility of results. However, miRNAs show potential as biomarkers in the pursuit of more reliable prediction of embryo implantation.