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.
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.
Problem In mammals, implantation involves interactions between an activated blastocyst and a receptive endometrium. There are controversies on the role of microRNAs in preimplantation embryo development. The actions of endometrial microRNAs on implantation are beginning to be understood. Method of Study Review of literature on microRNAs in preimplantation embryos and endometrium. Results Emerging evidence suggests a role of microRNAs in blastocyst activation and implantation. Differential expression of microRNAs is found between receptive and non‐receptive endometria. Members of the let‐7, miR‐200, miR‐30 families, and the miR‐17‐92 clusters are more commonly found to be associated with endometrial receptivity. Experimental studies show that the targets of the differentially expressed microRNAs affect endometrial receptivity, decidualization, and embryo implantation. Free and exosome/microvesicle containing microRNAs have been detected in human and ovine uterine luminal fluid (ULF). They may serve as mediators of embryo–endometrium dialog. Some observations suggest that the microRNAs in ULF may be used as biomarkers in infertility treatment. Conclusion MicroRNAs in endometrium and blastocysts are involved in the implantation process.
Extracellular vesicles (EVs) are membrane-bound vesicles, found in biofluids, that carry and transfer regulatory molecules, such as microRNAs (miRNAs) and proteins, and may mediate intercellular communication between cells and tissues. EVs have been isolated from a wide variety of biofluids, including plasma, urine, and, relevant to this review, seminal, follicular and uterine luminal fluid. We conducted a systematic search of the literature to review and present the currently available evidence on the possible roles of EVs in follicular growth, resumption of oocyte development and maturation (meiosis), sperm maturation, fertilization and embryo implantation. MEDLINE, Embase and Web of Science databases were searched using keywords pertaining to EVs, including 'extracellular vesicles', 'microvesicles', 'microparticles' and 'exosomes', combined with a range of terms associated with the period of development between fertilization and implantation, including 'oocyte', 'sperm', 'semen', 'fertilization', 'implantation', 'embryo', 'follicular fluid', 'epididymal fluid' and 'seminal fluid'. Relevant research articles published in English (both animal and human studies) were reviewed with no restrictions on publication date (i.e. from earliest database dates to July 2015). References from these articles were used to obtain additional articles. A total of 1556 records were retrieved from the three databases. After removing duplicates and irrelevant titles, we reviewed the abstracts of 201 articles, which included 92 relevant articles. Both animal and human studies unequivocally identified various types of EVs in seminal, follicular and ULFs. Several studies provided evidence for the roles of EVs in these biofluids. In men, EVs in seminal fluid were linked with post-testicular sperm maturation, including sperm motility acquisition and reduction of oxidative stress. In women, EVs in follicular fluid were shown to contain miRNAs with potential roles in follicular growth, resumption of oocyte meiosis, steroidogenesis and prevention of polyspermy after fertilization. EVs were also detected in the media of cultured embryos, suggesting that EVs released from embryos and the uterus may mediate embryo-endometrium cross-talk during implantation. It is important to note that many of the biologically plausible functions of EVs in reproduction discussed in the current literature have not yet been substantiated by conclusive experimental evidence. A detailed understanding of the contributions of EVs in the series of events from gametogenesis to fertilization and then on to implantation, in both normal and pathological cases, may enable the development of valuable tools to advance reproductive health. Because of the early stage of the field, it is unsurprising that the current literature includes not only growing experimental evidence, but also as-yet unproven hypotheses pertaining to the roles of EVs in key reproductive processes. In this review, we present a comprehensive survey of the rapidly expanding literature on this subject, highlighting both relevant findings and gaps in knowledge.
Human embryo implantation is a three-stage process (apposition, adhesion and invasion) involving synchronized crosstalk between a receptive endometrium and a functional blastocyst. This ovarian steroid-dependant phenomenon can only take place during the window of implantation, a self-limited period of endometrial receptivity spanning between days 20 and 24 of the menstrual cycle. Implantation involves a complex sequence of signalling events, consisting in the acquisition of adhesion ligands together with the loss of inhibitory components, which are crucial to the establishment of pregnancy. Histological evaluation, now considered to add little clinically significant information, should be replaced by functional assessment of endometrial receptivity. A large number of molecular mediators have been identified to date, including adhesion molecules, cytokines, growth factors, lipids and others. Thus, endometrial biopsy samples can be used to identify molecules associated with uterine receptivity to obtain a better insight into human implantation. In addition, development of functional in vitro systems to study embryo-uterine interactions will lead to better definition of the interactions existing between the molecules involved in this process. The purpose of this review was not only to describe the different players of the implantation process but also to try to portray the relationship between these factors and their timing in the process of uterine receptivity.
Macrophages are prominent in the uterus and ovary at conception. Here we utilize the Cd11b-Dtr mouse model of acute macrophage depletion to define the essential role of macrophages in early pregnancy. Macrophage depletion after conception caused embryo implantation arrest associated with diminished plasma progesterone and poor uterine receptivity. Implantation failure was alleviated by administration of bone marrow-derived CD11b(+)F4/80(+) monocytes/macrophages. In the ovaries of macrophage-depleted mice, corpora lutea were profoundly abnormal, with elevated Ptgs2, Hif1a, and other inflammation and apoptosis genes and with diminished expression of steroidogenesis genes Star, Cyp11a1, and Hsd3b1. Infertility was rescued by exogenous progesterone, which confirmed that uterine refractoriness was fully attributable to the underlying luteal defect. In normally developing corpora lutea, macrophages were intimately juxtaposed with endothelial cells and expressed the proangiogenic marker TIE2. After macrophage depletion, substantial disruption of the luteal microvascular network occurred and was associated with altered ovarian expression of genes that encode vascular endothelial growth factors. These data indicate a critical role for macrophages in supporting the extensive vascular network required for corpus luteum integrity and production of progesterone essential for establishing pregnancy. Our findings raise the prospect that disruption of macrophage-endothelial cell interactions underpinning corpus luteum development contributes to infertility in women in whom luteal insufficiency is implicated.
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.
Abstract A cross-sectional study was carried out at the Australian Concept Infertility Medical Centre from June 2014 to June 2015 to relate serum kisspeptin levels on human chorionic gonadotrophin (HCG) day with pregnancy outcome after intracytoplasmic sperm injection (ICSI). A total of 176 women aged 20 to 42 years, with regular menstrual cycles, normal thyroid function and prolactin levels selected for ICSI were included in the study. Patients with uterine fibroids, metabolic disorders, short agonist and antagonist protocol were excluded. Long protocol for down-regulation of ovaries was observed and kisspeptin levels were estimated on HCG day. Results were categorized into groups: Group A, non-pregnant with β-HCG 25 mIU/ml and cardiac activity on transvaginal scan. Kisspeptin levels were significantly higher in Group B versus Group A ( P < 0.001), independently associated with positive pregnancy ( r = 0.388; P < 0.001), but just borderline with endometrial thickness ( r = 0.294; P = 0.05). The area under the curve was highest for kisspeptin, 0.784 (95% CI, 0.681 to 0.886) for positive pregnancy, which indicated that kisspeptin measured on HCG day can be used as a marker for success of treatment in women after ICSI.
Implantation is a key stage during pregnancy, as the fate of the embryo is often decided upon its first contact with the maternal endometrium. Around this time, DCs accumulate in the uterus; however, their role in pregnancy and, more specifically, implantation, remains unknown. We investigated the function of uterine DCs (uDCs) during implantation using a transgenic mouse model that allows conditional ablation of uDCs in a spatially and temporally regulated manner. Depletion of uDCs resulted in a severe impairment of the implantation process, leading to embryo resorption. Depletion of uDCs also caused embryo resorption in syngeneic and T cell-deficient pregnancies, which argues against a failure to establish immunological tolerance during implantation. Moreover, even in the absence of embryos, experimentally induced deciduae failed to adequately form. Implantation failure was associated with impaired decidual proliferation and differentiation. Dynamic contrast-enhanced MRI revealed perturbed angiogenesis characterized by reduced vascular expansion and attenuated maturation. We suggest therefore that uDCs directly fine-tune decidual angiogenesis by providing two critical factors, sFlt1 and TGF-beta 1, that promote coordinated blood vessel maturation. Collectively, uDCs appear to govern uterine receptivity, independent of their predicted role in immunological tolerance, by regulating tissue remodeling and angiogenesis. Importantly, our results may aid in understanding the limited implantation success of embryos transferred following in vitro fertilization.
Abstract Mechanical endometrial injury (biopsy/scratch or hysteroscopy) in the cycle preceding ovarian stimulation for IVF has been proposed to improve implantation in women with unexplained recurrent implantation failure (RIF). This is a systematic review and meta-analysis of studies comparing the efficacy of endometrial injury versus no intervention in women with RIF undergoing IVF. All controlled studies of endometrial biopsy/scratch or hysteroscopy performed in the cycle preceding ovarian stimulation were included and the primary outcome measure was clinical pregnancy rate. Pooling of seven controlled studies (four randomized and three non-randomized), with 2062 participants, showed that local endometrial injury induced in the cycle preceding ovarian stimulation is 70% more likely to result in a clinical pregnancy as opposed to no intervention. There was no statistically significant heterogeneity in the methods used, clinical pregnancy rates being twice as high with biopsy/scratch (RR 2.32, 95% CI 1.72–3.13) as opposed to hysteroscopy (RR 1.51, 95% CI 1.30–1.75). The evidence is strongly in favour of inducing local endometrial injury in the preceding cycle of ovarian stimulation to improve pregnancy outcomes in women with unexplained RIF. However, large randomized studies are required before iatrogenic induction of local endometrial injury can be warranted in routine clinical practice. Some women undergoing IVF treatment fail to conceive despite several attempts with good-quality embryos and no identifiable reason. We call this ‘recurrent implantation failure’ (RIF) where the embryo fails to embed or implant within the lining of the womb. Studies have shown that inducing injury to the lining of the womb in the cycle before starting ovarian stimulation for IVF can help improve the chances of achieving pregnancy. Injury can be induced by either scratching the lining of the womb using a biopsy tube or by telescopic investigation of the womb using a camera. We performed a collective review of the available good-quality studies that used the above two methods in the cycle prior to starting ovarian stimulation for IVF. We pooled results from seven studies, which included 2062 women with RIF and assessed the difference in clinical pregnancy rates for those undergoing injury to the womb lining compared with no injury prior to IVF. The results suggest that inducing injury is 70% more likely to result in a clinical pregnancy as opposed to no treatment. Furthermore, scratching of the lining was 2-times more likely to result in a clinical pregnancy compared with telescopic evaluation of the lining of the womb. This study suggests that in women with RIF, inducing local injury to the womb lining in the cycle prior to starting ovarian stimulation for IVF can improve pregnancy outcomes. However, large studies are required before this can be warranted in routine clinical practice.