BACKGROUND Time-lapse observation presents an opportunity for optimizing embryo selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. The objective of this retrospective study was to identify the morphokinetic parameters specific to embryos that were capable of implanting. In order to compare a large number of embryos, with minimal variation in culture conditions, we have used an automatic embryo monitoring system. METHODS Using a tri-gas IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we have simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 min in five different focal planes for at least 64 h for each embryo. We have monitored the development of transferred embryos from 285 couples undergoing their first ICSI cycle. The total number of transferred embryos was 522, of which 247 either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted. RESULTS A detailed retrospective analysis of cleavage times, blastomere size and multinucleation was made for the 247 transferred embryos with either failed or full implantation. We found that several parameters were significantly correlated with subsequent implantation (e.g. time of first and subsequent cleavages as well as the time between cleavages). The most predictive parameters were: (i) time of division to 5 cells, t5 (48.8–56.6 h after ICSI); (ii) time between division to 3 cells and subsequent division to 4 cells, s2 (≤0.76 h) and (iii) duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc2 (≤11.9 h). We also observed aberrant behavior such as multinucleation at the 4 cell stage, uneven blastomere size at the 2 cell stage and abrupt cell division to three or more cells, which appeared to largely preclude implantation. CONCLUSIONS The image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting. We propose a multivariable model based on our findings to classify embryos according to their probability of implantation. The efficacy of this classification will be evaluated in a prospective randomized study that ultimately will determine if implantation rates can be improved by time-lapse analysis.
BACKGROUND Repeated implantation failure (RIF) is a major problem encountered in IVF. We have previously reported that RIF-IVF patients have a different endometrial gene expression profile during the window of implantation. Considering microRNA (miRNA) function in post-transcriptional regulation of gene expression, the aim of the study was to evaluate the involvement of miRNA in defects of endometrial receptivity. METHODS We used TaqMan miRNA array cards to identify the miRNAs differentially expressed in the secretory endometrium of RIF-IVF patients when compared with fertile women, and bioinformatics tools to identify their predicted targets and the molecular networks they may affect. RESULTS Comparing miRNA expression profiles, we identified 13 miRNAs, differentially expressed in RIF endometrial samples, that putatively regulate the expression of 3800 genes. We found that 10 miRNAs were overexpressed (including miR 145, 23b and 99a) and 3 were underexpressed. Using our previous gene expression analysis, we paralleled miRNA–mRNA expression profiling. By this means, we identified novel and previously characterized miRNA-regulated molecular pathways such as adherens junctions, cell adhesion molecules, Wnt-signaling, p53 signaling and cell cycle pathways. Consistent with the miRNA-predicted targets, mRNA levels of N-cadherin, H2AFX, netrin-4 and secreted frizzled-related protein-4, belonging to the cell adhesion molecules, Wnt signaling and cell cycle pathways were lower in RIF-IVF patients. CONCLUSIONS To our knowledge, this is the first study to evaluate the differential expression of miRNAs in the secretory endometrium of RIF-IVF patients. We suggest that the RIF-associated miRNAs could be exploited as new candidates for diagnosis and treatment of embryo implantation failures.
Citation Rashid NA, Lalitkumar S, Lalitkumar PG, Gemzell‐Danielsson K. Endometrial Receptivity and Human Embryo Implantation. Am J Reprod Immunol 2011; 66 (Suppl. 1): 23–30 Problem The pre‐requisite of successful implantation involves an intricate cascade of molecular interactions which plays a crucial role in preparing receptive endometrium and implanting blastocyst. Method of study Data are hereby presented for a better understanding of endometrial receptivity in women, hoping to provide a comprehensive picture of the process and identify new areas of basic and translational research in the biology of blastocyst implantation. Results Timely regulation of the expression of a number of complex molecules like hormones, cytokines and growth factors, and their crosstalk from embryonic and maternal endometrial side play a major role in determining the fate of the embryo. The molecular basis of endometrial receptivity and the mechanisms by which the blastocyst first adheres to the luminal epithelium and then penetrates into the stroma are only just beginning to be resolved. Conclusion Advances in the development of implantation models and ‘omics’ technologies, particularly proteomics and metabolomics, are set to have a major impact on the development of this field.
► In this review we discuss mediators and effectors of progesterone signaling. ► We collate data and insights gained from many mouse models. ► We discuss progesterone regulation of proliferation and differentiation. ► We examine how progesterone regulates implantation and uterine decidualization. During the early stages of pregnancy, fertilized embryos must attach to the uterine epithelium, invade into the underlying uterine stroma, and the stroma must then differentiate in a process termed decidualization in order for a successful pregnancy to be initiated. The steroid hormone progesterone (P4) is an integral mediator of these early pregnancy events, exerting its effects via the progesterone receptor (PR). Insights gained from the use of mouse models and genomic profiling has identified many of the key molecules enlisted by PR to execute the paradigm of early pregnancy. This review describes several of the molecules through which the PR exerts its pleiotropic effects including ligands, receptors, chaperones, signaling proteins and transcription factors. Understanding these molecules and their concatenation is of vital importance to our ability to clinically treat reproductive health problems like infertility and endometriosis.
Citation Robertson SA, Chin PY, Glynn DJ, Thompson JG. Peri‐Conceptual Cytokines – Setting the Trajectory for Embryo Implantation, Pregnancy and Beyond. Am J Reprod Immunol 2011; 66 (Suppl. 1): 2–10 Problem The peri‐conceptual environment influences the early embryo to impart long‐term consequences for the fetus and neonate; however, the underlying mechanisms are not well defined. Method of Study We argue that the cytokine network acting in the female reproductive tract during the pre‐ and peri‐implantation period integrates environmental information to program the embryo and fine‐tune the maternal immune response and endometrial remodelling to determine implantation success. Results As well as sex steroid hormones and male seminal fluid factors, female tract cytokines are influenced by agents signalling via the Toll‐like receptors including the microbiome and a plethora of metabolic, chemical and other stressors. In mouse models, an altered peri‐conceptual cytokine environment induced by cytokine deficiency, inflammatory insults or dysregulated seminal fluid signalling is associated with adverse effects on embryo development, pregnancy viability and reproductive outcome. Conclusion The cytokine network provides a pivotal mechanism through which environmental factors influence both embryo development and receptivity of the uterus.
The implantation rate is reduced in the human species and a great number of embryos present developmental anomalies. The past, present, and future of artificial reproductive technology (ART) strategies to improve the chances of implantation are discussed, in addition to the social, clinical, and epidemiological aspects of a progressively lower human fertility potential. The increasing age of couples looking to become pregnant for the first time, and the general tendency of deferring motherhood, are aspects that only a long‐term social policy can address. A general proposal for oocyte and sperm cryopreservation is discussed as an option to reduce the impact of age and the possible risks of unethical use of gamete donation. Investing in gamete banking resources may be a unique option for the future.
A subset of imprinted genes in the mouse have been reported to show imprinted expression that is restricted to the placenta, a short-lived extra-embryonic organ. Notably, these so-called “placental-specific” imprinted genes are expressed from both parental alleles in embryo and adult tissues. The placenta is an embryonic-derived organ that is closely associated with maternal tissue, and as a consequence, maternal contamination can be mistaken for maternal-specific imprinted expression. The complexity of the placenta, which arises from multiple embryonic lineages, poses additional problems in accurately assessing allele-specific repressive epigenetic modifications in genes that also show lineage-specific silencing in this organ. These problems require that extra evidence be obtained to support the imprinted status of genes whose imprinted expression is restricted to the placenta. We show here that the extra-embryonic visceral yolk sac (VYS), a nutritive membrane surrounding the developing embryo, shows a similar “extra-embryonic–lineage-specific” pattern of imprinted expression. We present an improved enzymatic technique for separating the bilaminar VYS and show that this pattern of imprinted expression is restricted to the endoderm layer. Finally, we show that VYS “extra-embryonic–lineage-specific” imprinted expression is regulated by DNA methylation in a similar manner as shown for genes showing multi-lineage imprinted expression in extra-embryonic, embryonic, and adult tissues. These results show that the VYS is an improved model for studying the epigenetic mechanisms regulating extra-embryonic–lineage-specific imprinted expression. ► Maternal contamination exaggerates imprinted expression in placenta. ► The visceral yolk sac (VYS) endoderm shows widespread imprinted expression. ► New technique to separate VYS reveals masked endoderm imprinted expression. ► Expression of imprinted genes in VYS is regulated by DNA methylation. ► VYS is an improved model for extra-embryonic imprinted expression.
Abstract This study investigated the expression pattern of galectin-3 (Gal-3) in mouse endometrium during early pregnancy and its function during embryo implantation. The expression of Gal-3 was measured at the mRNA level using real-time PCR and at the protein level using immunohistochemistry and Western blotting. The expression of Gal-3 mRNA and protein in the pregnant group was higher than in the non-pregnant group, and mRNA and protein expression reached their maximum levels on days 4 and 2, respectively. Immunohistochemistry results showed that Gal-3 protein presented in luminal epithelium and glandular epithelium and reached its maximum on days 6–8 and days 2–4 after pregnancy, respectively. The number of embryos implanted decreased substantially when Gal-3 was knocked down in mouse endometrium. In conclusion, increased Gal-3 expression after pregnancy is required for embryo implantation. Galectin-3 (Gal-3), a β-galactoside-binding protein, plays important roles in many physiological and pathological processes. Previous studies on Gal-3 mainly focused on its role in cancer cells, its role in embryonic implantation is largely unknown. Although some in-vitro studies suggested that it might be a factor related to endometrial receptivity, it needs further investigation, such as identifying the importance of Gal-3 in vivo . In this paper, we detected Gal-3 expression in mice uterus at the peri-impanation stage and further identified the role of Gal-3 in embryonic implantation by injecting silencing RNA into uteri in vivo . Our study indicated that Gal-3 was differently expressed in mice glandular epithelium and stromal cells at the time of implantation. The expression of Gal-3 was maximum on days 2–6 of pregnancy, which is the crucial period of embryonic implantation. Knock-down of Gal-3 significantly down-regulated the implanted embryos in mice. Our results indicate that Gal-3 is necessary for the embryonic implantation and provide new hints on the cause of infertility due to a defective endometrium.