Kristina Gemzell-Danielsson, Division of Obstetrics and Gynecology, Department of Woman and Child Health, Karolinska Institutet, Karolinska University Hospital, S-171 76, Stockholm, Sweden. E-mail: firstname.lastname@example.org
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.
Blastocyst implantation is dependent on nonlinear interaction of multiple modules and motifs with specific time and space contexts. These operational characteristics collectively attribute to the emerging property of a robust order in the system.1 Our knowledge about the process underlying the control system of blastocyst implantation is very thin.2 According to the Cartesian paradigm, blastocyst implantation is a process that is determined by linear and direct interaction of three primary modules that are integral to the physiological process: endometrial competence with adequate progesterone priming, viable embryo, and a synchronized dialogue between endometrium and pre-implantation embryo.
The term endometrial ‘receptivity’ was introduced to define the short time window during which uterus allows embryo implantation to occur.3,4 This phenomenon was first established in the rat and later validated in other species.5 Receptivity refers to the physiological property of a state when endometrium allows blastocyst to attach, penetrate, and induce localized changes in the stroma resulting in decidualization.6 Endometrial receptivity is conditional, requiring an escape mechanism sensitive to maternal and embryonic status. It is a self-limited period in which the endometrium acquires a functional and transient ovarian steroid-dependent status that allows a blastocyst to be received and further supports implantation through the mediation by immune cells, cytokines, growth factors, chemokines, and adhesion molecules.7–9 This specific period, known as ‘the implantation window’ opens 4–5 days after endogenous or exogenous progesterone stimulation and closes 9–10 days afterward.10,11 The anatomical change in the surface epithelium is the appearance of microprotrusions from the apical surface of the epithelium, termed pinopodes, which appear 6 days after ovulation and is retained for 24 hr during the implantation window. These exhibit a smooth surface that facilitates the apposition of blastocyst with the endometrium.12,13 During secretory phase of the endometrium, a complex meshwork of capillaries is evident in the upper functionalis layer and this increase in the capillary permeability is one of the earliest detectable responses that lead to stromal edema at the time of implantation in the human.14 Thus, vascular changes appear to be an important factor in establishing endometrial receptivity.
The key to endometrial receptivity is the dynamic and precisely controlled molecular and cellular events that drive implantation and establishment of pregnancy. This dynamic process involves coordinated effects of autocrine, paracrine, and endocrine factors. As it is not possible to study the implantation process in women in vivo because of ethical and technical issues, most of the data in the literature to understand this process have been derived from animal studies and they have provided valuable insights into molecular mechanisms that occur during embryo implantation.
Hormonal Requirement for Endometrial Receptivity and Blastocyst Implantation
In most eutherian mammals, ovarian steroidal hormones, estrogen, and progesterone play very important roles in endometrial receptivity, blastocyst implantation, and maintenance of pregnancy. However, serum levels of these hormones in pregnancy cycles show high degree of variation across the species. It also appears from human IVF-ET reports that the ratio of progesterone to estrogen in circulation is critical for the secretory maturation of the endometrial glands during the mid-luteal phase and high level of estrogen may adversely affect endometrial receptivity and inhibit implantation.15–17 It has also been shown that luteal phase ovarian estrogen is not essential for blastocyst implantation in the human.18 Animal models also prove this concept.19 However, progesterone is essential for blastocyst implantation and has also been well proved by (i) neutralization of circulating progesterone20–23 by administration of specific antibodies; (ii) by inhibiting the biosynthesis of progesterone or indirectly by inhibiting stimulation of these cells by hormones, and (iii) by interfering with the action of progesterone at target organ level through the use of progesterone receptor blockers or antiprogestogens.24–26
Collectively, it appears that progesterone is essential for endometrium to attain the receptive stage, blastocyst implantation, and maintenance of pregnancy, and that mid-luteal-phase ovarian estrogen is not essential for blastocyst implantation and maintenance of pregnancy.
Functional Parameter of Endometrial Receptivity
The ovarian steroids, progesterone, and estrogen have a major regulatory role by mobilizing several molecular modulators in spatiotemporal manner which supports embryo implantation.27 They control the cascade of growth factors and cytokines which are the prime paracrine mediators of the dialogue at the maternal–embryonic interface. It appears that the endometrial receptivity is the result of the synchronized and integrated interaction of ovarian hormones, endometrial factors, and embryonic signals. Various endocrine and paracrine factors correlate relevant to endometrial receptivity and implantation have been documented based on studies in areas directly related to human reproduction as well as from research studies using small animals and non-human primates. Progesterone during the luteal phase is known to modulate the synthesis and secretion of a number of proteins.28–32
Cell adhesion molecules involved in cell–cell and cell–matrix interaction contribute to cell migration and transduction of differentiation signals.33 The co-expression of αvβ3 and alpha4beta1in human endometrium during implantation window has been documented.34–36 Endometrial epithelial cells synthesize a large amount of calcitonin 37 and HOXA-1038 during mid-secretory phase of the menstrual cycle.
It is well documented that prostaglandins (PGs) play an important role in various reproductive processes, including ovulation, implantation, and menstruation.39 Cyclooxygenases (COX-1 and COX-2) are the crucial enzymes responsible for the synthesis of various PGs. PG levels in the uterine fluid undergo cyclic variations in response to P4.40
The importance of many cytokines and growth factors and their receptors in the control of cellular processes and blastocyst formation is well documented.41–43 Cytokines are small multifunctional glycoproteins, whose biological actions are mediated by specific cell-surface receptors and act as potent intercellular signals regulating functions of endometrial cells and embryo–maternal interactions. Entry of blastocyst into the receptive uterine is very important for the production of cytokines by trophoblastic cells and uterine epithelium which can modulate the endometrial receptivity by regulating the expression of various adhesion molecules.44 In mammals, deregulated expression of cytokines and their signaling leads to an absolute or partial failure of implantation and abnormal placental formation.45
The first evidence of the role of LIF, a pleiotropic cytokine, in implantation came from the report that embryos failed to implant in LIF-deficient female mice. However, after LIF supplementation in the same mouse model, normal implantation was restored. Most recently, it has been shown that LIF plays a role in both adhesive and invasive phases of implantation owing to its anchoring effect on the trophoblast.46 These findings suggest that LIF plays a major role in both rodents and primate implantation. Our studies using in vivo human implantation show that an optimum level of LIF is required for blastocyst implantation. Also, it shows that neutralization of LIF in the invitro 3-D culture inhibits blastocyst implantation (unpublished data).
Expression of IL-6 was found to be present during mid-secretary phase and is mostly localized in epithelial glandular cells.47 In humans, IL-6 receptor (IL-6-R) is found to be expressed during menstrual cycle, thus providing evidence that the role of IL-6 in controlling endometrial receptivity and implantation is not species specific.48 Together with LIF and IL-6, IL-11 belongs to the gp130 cytokines (i.e. cytokines which share the gp130 accessory signal-transducing subunit). IL-11 and its receptor (IL-11Rα) have recently been observed in the human endometrium. All the major cell types in endometrium express IL-11 with cyclical variation.
Matrix metalloproteases (MMP) play an important role in tissue remodeling and break down by degrading the components of extracellular matrix. MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-10 are seen in the endometrium.49 The balance between the MMPs and their natural inhibitors is of primary importance in determining the tissue degradation at the implantation site.
Apart from cytokines, there are various growth factors like TGF beta, a cytokine that plays an important role in tissue remodeling, extracellular matrix formation, and immunomodulation depending on its concentration. TGF-beta may act as autocrine-paracrine regulator at the implantation site by expressing proteases and anti-proteases.50 Heparin-binding epidermal growth factor (HB-EGF) plays a crucial role in implantation was given from the studies conducted in mice.51 HB-EGF was found to be expressed in human endometrium at the time of implantation.52 IGF-I was found be immunolocalized in stromal cells at day 5 of pregnancy in rat uterus and was suggested to be involved in decidualization of stromal cells. The function of IGF-II: IGF-BP1 at the feto-maternal interface involve balance between invasion and its suppression to achieve normal implantation and placental development.53,54 Apart from these factors, there are many others which have been studied to be regulated during endometrial receptivity.
Embryo Endometrial Dialogue
The first step in implantation is a dialogue between free-floating blastocyst and the receptive endometrium, which is mediated by hormones and growth factors55 is followed by apposition, where the trophoblast cells adhere to the receptive luminal epithelium. Micro protrusions present on the surface of uterine epithelium known as pinopodes may have a role in this process.56 Consequently, blastocyst adheres to the endometrial basal lamina and stromal extracellular matrix through local paracrine signaling between the embryo and endometrium. Finally the invasion process, which involves penetration of the embryo through the luminal epithelium into the stroma thereby establishing a vascular relationship with the mother. Thus, success of implantation depends on a receptive endometrium, a functionally normal blastocyst and a synchronized cross-talk between embryonic and maternal tissues.57,58
Endometrium is known to become receptive only for short periods, and beyond this period of receptivity, embryo is unable to successfully establish contact with refractive endometrium. Therefore, timely arrival of embryo in a receptive endometrium is very much crucial for successful implantation.59 In addition to the physical interaction of the embryonic tissue with the uterine cells, this process is undoubtedly influenced by maternal steroid hormones, growth factors, and cytokines in paracrine manner thus playing a crucial role in embryonic signaling.60,61 The intricate process of implantation also requires other key molecules in addition to hormones like progesterone and estrogen.62 Embryo co-cultured with endometrial epithelial cells demonstrated up-regulation of integrin-beta3 on the surface of epithelial cells suggesting that it could be mediated by embryonic IL-1alpha.63 Co-culture of embryo and human endometrial stromal cells enhances the expression of various isoforms of insulin-like growth factor (IGF) and IGF-1 receptor on preimplantation stage embryos.64 Interestingly, the presence of embryo also upregulates the production of insulin-like growth factor binding proteins (IGF-BPs) by human endometrial stromal cells.65
Thus, it appears that endometrial receptivity during the mid-luteal phase of cycle is likely to be associated with differential activation and repression of various cohorts of genes in endometrium under progesterone action following signal inputs from viable, synchronously developing embryo.
Models to Study Embryo Implantation
To understand the complex phenomenon of human embryo implantation, several research groups have come up with many in vivo models. The simple ones being monolayer cultures with stromal cell show that Rho GTPases Rac1 and RhoA in human endometrial stromal cells modulate invasion of the human embryo through the endometrial stroma.66 Attempts have been made to use human embryonic stem cells to understand the mechanism of trophoblast cell adhesion and invasion in vivo.67 Also, the usage of different endometrial cell lines as substrate for embryonic adhesion and trophoblast invasion have been tested.68 Having monolayer cell culture model have their own limitations as we have evidenced that paracrine signals are involved in leading the endometrium to the matured state. Using intact endometrial strips in vivo cultures were not successful as they lead to necrotic changes at the center of the tissue.69 Our group has developed a 3D culture model using endometrial stromal and epithelial cells from receptive phase to study the human endometrial and embryo interaction. This 3D culture model expresses steroid receptors ER and PR as well as receptivity markers like VEGF, LIF, MUC1, and integrin αvβ3.70 The model was tested for its progesterone regulation of receptivity markers and the process of embryo implantation.71 The schematic representation of the model is shown in Fig. 1. Using this model, we have shown that optimum level of LIF is required for human embryo implantation (unpublished data). Interestingly, the human preimplantation embryo expresses LIF -R reinforcing the dialogue between the embryo and the maternal tract through LIF.72 This model may be applied to study the embryo-endometrial dialog and the process of human embryo implantation; as such, investigative studies are not possible to do in vivo because of the ethical and technical limitations. Improving this method may help us to understand the early gene expression and may lead to more information about some of the genetic disorders. Also, this model may help to screen for teratogenic effects of various drugs or environmental factors.
Advanced Tools to Understand Endometrial Receptivity
The current knowledge of pre-implantation and implantation physiology is the result of observations gathered by many researchers and physicians through these years. Collectively, present literature suggests the role of a variety of molecules as potential mediators of embryo–uterine interactions during implantation. Despite its simplistic and deterministic edges, the major problem of this model is that it fails to delineate the following observations like occurrence of successful implantation despite progesterone starvation,73 delayed implantation74 and cases of implantation failure with no anomaly in endometrium and ovarian functions.75
Over a century, the biological research has provided a wealth of knowledge about individual cellular component and its function. Thus, in the pre-genomic era, a one-by-one approach was adopted to investigate several genes and gene products during the window of implantation. Despite this, it is clear that a discrete and robust biological function like endometrial receptivity and blastocyst implantation can rarely be attributed to a single molecule and a small group of molecules, rather they arise from a complex interaction between cells and among numerous molecules. A key aim of post-genomic biomedical research is to adopt an inductive approach toward systematically catalogue all molecules and their interaction within the living cells.76 There are indeed a few human reports wherein transcripts profile of mid-luteal stage endometrium has been compared with that of proliferative stage or early luteal stage endometrium to delineate the role of progesterone in establishing endometrial receptivity for embryo implantation.77–82 Recently, major advances in the genomics of the endometrium83 and oocytes84 have been achieved with the microarray and bioinformatics technologies available, to provide a vast amount of information regarding gene expression in endometrium. All the aforementioned inductive studies give information on the differences in relative gene expression between two fixed time points of menstrual cycles, considering mid-secretory phase as the putative window of implantation. All these studies offer the opportunity to develop an endometrial database of genes expressed during the window of implantation resulting in the contribution in gaining an insight into the complexity of endometrial receptivity. However, gene expression is only one aspect of the complex regulatory network that allows cells to respond to intracellular and extracellular signals. The pre-requisite in this direction is to describe an expression pattern of a molecule followed by more mechanistic approaches such as gene-targeting to correct the abnormality. Previous studies have established the role of various molecules as implantation regulators, but the list is still expanding each year. While significant advancement has been made in this area, there have been limited attempts to systematically analyze and integrate the cohorts of factors in the context of embryo, endometrium, and the hormonal milieu in an integrated manner using systems biology approach and robust tools like high throughput transcriptomics and proteomics, and computational modeling. However, gene expression is only one aspect of the complex regulatory network that allows cells to respond to intracellular and extracellular signals. Unlike the genome, the proteome itself is dynamic, complex, and variable. Furthermore, it depends upon the developmental stage of the cells, reflecting the impact of both internal and external environmental stimuli. Proteomics is often considered the next step in the study of biological systems and is more complicated than genomics. Garrido-Gomez et al.85 compared the proteomes of pre-receptive (day LH+2) vs. receptive (LH+7) endometrial biopsies obtained from the same fertile woman (n = 6) in the same menstrual cycle. Biopsies were analyzed using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Dominguez et al. 86 also investigated the secretome profile of implanted blastocysts which developed after performing an embryo biopsy for pre-implantation genetic diagnosis and were subsequently grown in a sequential system or cocultured with endometrial epithelial cells (EEC). In another study, endometrial fluid obtained transcervically by aspiration immediately prior to embryo transfer was analyzed and the protein profile in each sample was determined.87 Recently, Van der Gaast et al.88 investigated the effect of ovarian stimulation in IVF on endometrial secretion and markers of receptivity in the mid-luteal phase. All these studies gives us the clues to study the protein profile along with genomics which will help us in providing a better understanding of the embryo implantation in relation to endometrial receptivity.
The events during implantation are the result of regulated changes in gene transcriptions that ultimately control the expression of embryonic and endometrial proteomes. In recent years, ‘omics’ techniques have advanced to such an extent that rapid identification of genes or proteins of interest can be readily secured. However, technical limitations still exist which compromise the uniqueness of the results obtained. Proteomics together with genomics and metabolomics are complementary approaches which will improve our understanding of the complexity of the implantation process. All these approaches and information needs to be integrated into a system biology approach to understand endometrial receptivity and the embryo–endometrial dialog in holistic way.
The studies performed by the authors at Karolinska Institutet were supported by grants from the Swedish Research Council (2003-3869, K2007-54X-14212-06-3, K2010-54X-14212-09-3) and Stockholm City County/Karolinska Institutet (ALF). Kristina Gemzell-Danielsson acts occasionally as an ad hoc invited speaker at scientific meetings for MSD.