Endometrial receptivity: The journey so far

¹K Osazee and ²A Omorogiuw

ABSTRACT

Implantation is an act of coordinated interaction between the nascent blastocyst and a receptive endometrium mediated by the molecular and cellular interplay in a spatiotemporal manner. Though the advent of modern technology has enhanced the availability of relatively good quality embryos; the implantation rate has not positively correlated. Unlike the human embryo, the study of implantation is laden with ethical and technical challenges. Hence, most of the data on the process of implantation derived from animal studies. Unfortunately, there is wide variation in implantation process among animal species. Thus, cannot be transposed for a human. Hence, the in-vivo model remained the basis for the study of the mechanism of implantation. Research directed towards this direction may help in optimising the outcome of Assisted Reproduction Technology (ART).

KEYWORDS: Endometrial receptivity, Blastocyst, Implantation, Infertility.

INTRODUCTION

Implantation is the process of complementary interaction of the endometrium and the nascent blastocyst often achieved in a stepwise fashion of apposition, adhesion, and invasion predicated on genetic and cellular signals . Though, the viability of the embryo is essential, the receptivity of the endometrium has been shown to be pivotal given its propensity to create a barrier to implanting blastocyst². For optimal receptivity, there must be synchronous molecular and cellular interplay between the endometrium and the blastocyst guaranteed by the ovarian steroid-primed endometrium within a time frame in the mid-secretory phase termed Window of Implantation (WOI)³. An attempt at synchronizing embryo transfer within the period of optimal endometrial receptivity has led to several types of research aim at improving the success rate of ART as well as unravel the causes of unexplained infertility and recurrent implantation failure (RIF). Unfortunately, there is no consensus on the biomarker to establish it.

Success has been made in the study of human embryos in- vitro. Unfortunately, the uterus is not accessible to demonstrate the exact site of implantation for technical and ethical reasons. The situation is even compounded by the heterogeneity associated with implantation process in different species. Thus, make it difficult to develop the right animal model for research. Hence, the in-vivo model has remained the basis for the study of the physiological and pathological mechanism of implantation.

MENSTRUAL CYCLE

Menstrual cycle involves series of organized events comprising the hypothalamus, anterior pituitary, Ovary, and endometrium. Commonly referred to as the hypothalamic pituitary ovarian axis. The morphological and physiological modifications involved in these organs in the course of the menstrual cycle are subject to the autocrine, paracrine and endocrine effect associated with the axis . At the onset of the cycle, the gonadotropins are secreted under the influence of hypothalamus, by the anterior pituitary. The hormone impact on the ovary to secrete the steroid hormones (estrogen, progesterone and other peptides) in the course of folliculogenesis. These steroid hormones are responsible for the structural and functional changes associated with the endometrium in anticipation for conception or menstruation to mark the beginning of another cycle in the absence of pregnancy.

The dynamics of the endometrium is initiated by the estrogen secretion resulting in increase production and stimulation of estrogen receptor alpha and progesterone receptor isoforms. Thus triggers the expression of relevant genes and cell division and proliferation in what is term proliferative phase of the endometrium^9,10. The phase is characterized by the hypertrophy of the stroma and glandular cells as well as the elongation of the spiral vessels. Following ovulation, the progesterone is secretion by the corpus luteum. This terminates the proliferative process through the disappearance of the estrogen receptor alpha1 and heralds the onset of the early secretory phase of the endometrium. The phase is characterized by the secretion of mucus and glycogen from the glandular cells¹. In the mid-secretory phase, the progesterone, through its receptor acts on the stroma tissue. Thus, makes the stromal cells render paracrine function by stimulating the expression of epithelial genes necessary for the implantation of the embryo. In light of the associated decidualization and other respective potentials for implantation, the phase is often related to the window of implantation.
The period termed WOI is characterized by epithelial luminal transformation and changes for the trophoblast attachment and apposition^13,14 and associated with elaborate stromal density and epithelial projections called pinapodes¹⁵. These receptive features for which WOI is the hallmark is related to several biomarkers such as transcription factors, cytokines, integrin, and as well as growth factors¹. However, the prognostic value of these biomarkers through genetic profiling of endometrial cells has been subject for debate in the literature^17,18. With pregnancy, the contact of the blastocyst and the endometrium commence the process of attachment, invasion of the trophoblast culminating the formation of the placenta.

HISTORICAL PERSPECTIVE

The concept of endometrial receptivity is dated back to the work of Rock and Bartlett in 1950 in the course of trying to design the concept of endometrial dating. While Noyes et al. defined the secretory endometrium through histological examination of the endometrial biopsy. Over time, it became apparent that the histological determination of endometrial receptivity provides irrelevant information and has little or no benefit in the clinical entity². Subsequently, effort at developing biomarkers led to the evaluation of the morphological features associated with endometrial receptivity.

Furthermore, Hertig and Rock² revealed through an examination of the uterus of hysterectomized women intended to get pregnant, that the early event of implantation occurs at about day 19 of the cycle. The corroboration of the findings by other studies^27-29 led to the concept of WOI put at day^19-23 in the human menstrual cycle.

Also, the period has shown to coincide with the time serum progesterone is at its peak suggestive of the central role of progesterone in implantation³⁰. The background knowledge from the in-vivo model has been explored towards the right timing of the embryo and endometrium interaction as a prelude to successful implantation in rodent^31,32 and in human.^33,34 Furthermore, the study of WOI at the molecular level has revealed its association with changes in the steroid receptors and as well as expression of other factors such as the integrin.

MORPHOLOGICAL CHANGES

The endometrial changes before the onset of implantation process are the accelerated cellularity of the luminal and the glandular epithelium often localized at the apex of the proposed area for the implantation³⁶. The luminal change is associated with the formation of nuclear clumps epithelial plaque regulated by steroid hormones³⁷. Though the function is not well known, it is believed to have some nutritional benefit for the proposed implanted embryo through the provision of glycogen^38,39. Also, the basal lamina become thin with the separation of the gap junction⁴⁰ to pave the way for apposition and the subsequent invasion of the trophoblast⁴¹.
Another characteristic feature associated with the epithelial surface is the presence of Mucin 1 (MUC 1). It is a glycoprotein that forms a layer of glycocalyx upregulated during WOI. It is believed to determine the site of attachment and adhesion of the blastocyst through the process of shedding by the blastocyst secreted metalloproteinase enzyme^19,42. The mechanism by which it promotes attachment of blastocyst during implantation has been established in mouse and non-human primates studies⁴³ poorly understood in the human^44,45, L-Selectin, Heparin-binding growth factor (HBGF)^46,22.
The stromal undergoes extensive decidualization by glandular secretion and proliferation of specialized uterine Natural Killer cells and vascular permeability. The transformation creates an environment for optimal trophoblast invasion and subsequent access to maternal vascular bed⁴⁷ to promote adequate perfusion for the nascent embryo. The physiological process determines the quality of the placentation and has a lot of clinical implications⁴⁸. With the onset of implantation, decidualization is maintained by the steroid hormones and the cellular signals⁴⁹. Also, there is the formation of neovascularization regulated by the hormones. This is believed to be a prerequisite for the infiltrations of the immune cells and subsequent differentiation⁵⁰.
The endometrium endowed with several infiltrated immune cells. Of significance, are the uterine Natural killer cells (uNK)⁵¹ and tends to increase during the period of implantation. During pregnancy, the uNK cells differentiate into decidual NK cells. Though the role of uNK cells is lace with controversies, a study in the mouse has shown that reduction in the decidual NK cells results in pregnancy failure⁵². Other immune cells are T-lymphocytes and Macrophages.

MOLECULAR TRANSFORMATION

Cytokines and Growth Factors
The most important cytokines involved in the implantation process are Leukemia Inhibitory Factor (LIF), interleukins- 6 (IL-6) and inter leukin-11 (IL-11) with common receptor protein gp- 130 in carrying their functions⁵³. The central role of LIF in implantation was first established in a knockout female mouse resulting in implantation failure due to downregulation of STAT3 signaling in the endometrial epithelium⁵⁴. Subsequently, in infertile women following demonstration of LIF and its protein expression in endometrial biopsy throughout the menstrual cycle and the association of RIF and unexplained infertility with mutation of LIF gene⁵⁵. A Recent study has shown that LIF is involved in both adhesion and invasion of the blastocyst^30,56.
Furthermore, study with mice has shown that its receptor tends to increase expression in decidualized stromal cells close to the site of implantation and administration of antagonist resulted in the loss of pregnancy in primates and mice⁵⁷. Expression of the IL-6 is mainly in the glandular cells⁵⁸ promoting the decidualization of the endometrium creating better access for the invasion of the trophoblast³⁰. The IL-11 is expressed by all cell type in the endometrium in a cyclical manner⁵⁹ and in combination with IL-6, is involved in the decidualization of the endometrium⁶⁰. Unlike the IL-6 whose regulation is under the influence of ovarian steroid hormones, there is no consensus about IL-11⁶¹.
In addition to cytokines, growth factors like Transforming Growth Factors (TGF) beta promote the implantation process by its regulatory impact on the immune system⁶². Its receptors are predominantly in the glandular epithelial cells and enhance the decidualization for adequate trophoblastic invasion. While the Tumour Necrosis Factor (TNF) alpha and Epidermal Growth Factor (EGF) has their receptors in the glandular and stromal cells with more expression in the stromal cell during early pregnancy in animals⁶³. The increased level in the stromal cell is suggestive of its important role in decidualization³⁶.

Integrin and Ligands
Integrin and ligands (Osteopontin, Fibronectin, and Collagen) are glycoproteins that mediate the adhesion of blastocyst and endometrium. Its receptors exhibit variation in time and location during WOI. Depending on the ligand, it tends to be more expressed in the glandular epithelium at the apical region of the endometrium during the WOI and decreases in the early pregnancy⁶⁴. However, a study with Ishikawa cells has shown that alpha4beta3 is the main receptor for osteopontin⁶⁵. Moreover, play a central role in the process of adhesion during implantation. For example, a knockout mouse study of osteopontin (SPP1) and its receptor alpha4beta3 resulted in implantation failure⁶⁶.
Osteopontin was first recovered from the bone matrix⁶⁷ and associated with several tissues. Its expression in the secretory phase endometrium was first noted by Young et al.¹⁴ and it coincided with the period of blastocyst attachment regulated by progesterone⁶⁸. While progesterone regulates the expression of Osteopontin, its main receptor integrin alpha4beta3 is differentially regulated by EGF and HOXA10 in a paracrine mode of action guaranteed by the progesterone receptor in the stromal cells⁶⁹. The modulation also related to the association of elevated estrogen receptor alpha and downregulation of integrin during WOI. Thus, explains the correlation of elevated estrogen receptor alpha during WOI and implantation failure⁹.
While the Fibronectin and its receptor alpha4beta1 are expressed mainly in the glandular cells, Osteopontin and its receptor alpha4beta3 are expressed in the glandular and stromal cells⁷⁰. Unlike the fibronectin and Osteopontin, the Collagen and its receptor alpha1beta1 expression is lost in early pregnancy⁷¹. A Recent study revealed differential expression of integrin at the site of attachment of the embryo in the upper zone of the luminal epithelium about the basal distribution in the non-attached area⁶³. The finding was corroborated by another study with mouse embryo and Ishikawa cells⁶⁵ suggesting the role of embryo signaling of endometrial epithelial cells in the process of implantation.

Genomics and Endometrial Receptivity
An attempt at the use of the regulatory potential of molecular expression and gene targeting to evaluate abnormalities, has led to the development of genomic sequencing of the various endometrial genes involve in the process of implantation. Hence, different Omics technologies are available, and transcriptomics by microarray or RNA sequencing have been used to look at changes in a large number of transcripts in the endometrium^42,72. The concept involves the use of Omics to analyze the genes, lipids, and proteins of the endometrium to (or “intending to”) generating biomarkers that may be useful to predict endometrial receptivity.
Ponnampalam et al. first applied the technique⁷³ to determine the various stages of the menstrual cycle irrespective of the morphological state through the evaluation of the transcription profile of endometrial genes. As a result, verify the expression of genes during the receptive period of the endometrium.
The concept was facilitated against the backdrop of the impact of ovarian stimulation during IVF treatment on the endometrial morphology²². It has been shown that ovarian stimulation results in unwarranted endometrial morphological changes⁷⁴. Thus, resulting in compromised receptivity. Subsequently, the use of endometrial gene profiling became a valuable tool to determine the receptive status of the endometrium in IVF cycle prior to embryo transfer⁷⁵. Despite the perceived benefits, the gene pool generated could not define the ideal or specific biomarker due to the heterogeneity of the sample and variation in the cycle. So, it became unfeasible for routine clinical use⁷⁶. Furthermore, the invasive nature of getting the endometrial sample for analysis as well as the weak correlation of gene profiling with the secreted proteins makes it less appealing. Consequently, emphasis shifted to the use of proteomics^72,77.

PROTEOMICS
Beier and Beier-Hellwig in 199878 were the first to demonstrate the concept of proteomics based on the significant amount of secreted fluid in the endometrial cavity in the secretory phase. Though several genes identified, the function of most the genes was not known. In one study^3,79, progesterone receptors were noted to be downregulated in the luminal epithelium during the secretory phase of the endometrium in mice and sheep. In spite, the observations, the nonuniformity of the sample constituents per sample collection renders the method unacceptable⁸⁰.
Data from animal studies have shown the importance of lipid in the endometrial receptivity^81,82. Though, yet to be established in the human⁸⁰, a study has shown a correlation of reduced serum Lysophosphatidic acid (PLA) and Cyclooxygenase 2 (COX 2) with recurrent implantation failure in IVF Patient⁸³. In the contest of lipidomics, a study has shown only high value in the PGE2 and PGF2 alpha during WOI while other parameters remained unchanged⁸⁴. While its role in the animals seems promising, the relevance in human is still subject to debate²².
Over the years, the concept and its related diagnostic tool of endometrial receptivity array (ERA) have demonstrated the critical role of relevant gene expression during WOI for successful implantation⁸⁵. Despite its drawbacks about specificity⁸⁶, it has established the fact that some genes expressions are involved in the endometrial receptivity and implantation process⁸⁷. In light of this, therefore, MicroRNAs have been considered potential regulatory elements in the concept of endometrial receptivity^4,88.

MicroRNAs
MicroRNAs are minute non-coding RNA molecules of about 18-25 nucleotides involve in the modulation of many target genes⁸⁵. The regulatory process is either by direct degradation of mRNA or inhibition of post-translation expression. Thus, influence range of biological processes⁸⁹.
The regulatory role of microRNAs in the uterine gene expression demonstrated in mouse⁹⁰. In human, Dominguez et al.⁹¹ showed the differential expression of 24 ovarian hormone-dependent microRNAs in the menstrual cycle. The finding corroborated by other studies^85,88 showed that twelve microRNAs are differentially expressed during the secretory phase of the endometrium and were mainly in the glandular and endothelial cells of the epithelial lining of the endometrium.
Even within the mid-secretory phase, differential expression was noted within the pre-receptive and receptive period⁹².For example, microRNAs 30b and 30d are upregulated while microRNAs 494 and 932 downregulated in the receptive phase. Also, the differential expression of microRNAs 22 and 145 has been noted infertile women and those with recurrent implantation failure (RIF) due to altered endometrial microRNA profile resulting in poor endometrial receptivity^88,93. Though, several microRNAs have been demonstrated in the process of implantation, the role of significant number is still not known⁹⁴.

Hormones (Progesterone)
The molecular interplay involved in the receptivity of the endometrium is modulated by several factors and gene expressions regulated by progesterone⁸⁵. As a consequence of a defect in progesterone production or resistance to its receptors, implantation failure may result from altered expression of the relevant genes during WOI⁶⁹. In light of the absolute requirement of progesterone in endometrial receptivity and maintenance of the corpus luteum of pregnancy⁹⁵, the name progesterone was borne out from the Latin word PRO and GESTURE in 1930⁹⁶. Studies by knockout and anti-progesterone (RU 486) Mifepristone, have demonstrated the relationship of its receptors in the genetic and phenotype expression of endometrial receptivity^97,98. The assertion has been heightened by Labarta et al.⁹⁹, who showed the alteration of 140 endometrial genes expression with elevated progesterone level and consequent adverse effect on the endometrial receptivity in non-human primate in-vivo. Thus, give credence to the central role of progesterone in endometrial function.
Progesterone carries out its function through the receptors. The two isoforms A and B are from different slice variant in the same gene. The B isoform had an extra 164 amino acid residues at the N-terminus of the protein⁹⁵.
Despite the difference in size, a knockout study in the mouse has shown that isoform A tends to exhibit more functional attribute in the uterus⁹⁶. Unlike in the mouse, the human isoforms, A and B function in a comparable manner, and their levels tend to vary with the menstrual cycle¹⁰⁰. Furthermore, the B-isoform plays a dominant role in a situation where both isoforms determine the expression of a gene¹⁰¹.
Under the influence of estrogen, at the onset of the cycle, these receptors are expressed on the epithelial lining and in the stromal cells of the endometrium^102,103. At the secretory phase, PR-B is down-regulated while the PR-A remain only in the stromal cells for decidualization^104,105. The expression is closely associated with the expression of Insulin Growth Factor Binding Protein-1 (IGFBP-1), a marker involved in decidualization100.

Regulating factors of Progesterone Receptor
Various factors influence the activities of the Progesterone Receptors (PR) and its ability to manipulate the expression of the target genes. Some of these factors include the estrogen and progesterone⁹⁵. A knockout mouse study has shown that estrogenic influence on the PR is through the presence of estrogen receptor (ERAlpha) in the stromal cells106 and progesterone impact by negative feedback mechanism¹⁰⁷. Other factors involve its combination with the immunophilins¹⁰⁸. For example, immunophilins such as FKbp4 has been showed to promote the expression of a gene involves in the optimal decidualization through the suppression of estrogen-primed gene Lactoferrin (Ltf) that promote epithelial proliferation¹⁰⁶.So, the proliferative action of estrogen on the luminal epithelial cells needs to be suppressed during the mid-secretory phase to allow for decidualization during implantation.

In the presence of progesterone, PR disengages from the immunophilins and its activity become modulated by P160/SRC (Steroid Receptor Coactivator)109. SR-1 and SR-2 expressed in the epithelial and stromal cells. While, the SR-1 may be complementary. Knockout study 12 and microarray study^95,110 have shown that SR-2 plays a prominent role in progesterone mediated gene expression, and the place of SR-3 is not well established⁹⁵. Also, Kruppel-like Factor (KLf 9)^111,112 and Bone Morphogenic Protein (BMP2)¹¹³ are cofactors for PR towards optimal implantation.

Effectors of Progesterone Receptors
Base on the background knowledge that progesterone impact through its receptors on the endometrial receptivity by the expression of various genes in diverse signaling routes⁸⁶. It has become imperative to gear efforts towards the determination of the exact genes influenced during the WOI. Studies^12,95 have shown that Indian hedgehog gene (Ihh) is one of such gene expressed on the epithelial lining. This mediates the expression of Patched-1 (Ptch-1) and COUP-TF11 in the stromal during implantation¹¹⁴. These mediators have been shown to be vital in decidualization by their suppression/downregulating ERalpha, preventing epithelial proliferation during WOI, culminating in successful implantation¹⁰⁶. Thus, suggesting the important role of Ihh gene.
Similarly, COUP-TF11 has been noted to promote endometrial receptivity through the expression of BMP2; a critical element expressed near the site of implantation due to its role in decidualization in murine and in the human endometrium¹¹⁴. The impact of which has been demonstrated to be through the induction of Wnt4, which promotes cell development and differentiation¹¹⁵. In addition to receptor signaling, progesterone can directly induce the expression of some genes such as Mig6 and cyclooxygenase 2 (COX-2). Mig6 produced in both epithelial as well as stromal cells can regulate the impact of estrogen and progesterone by feedback mechanism⁹⁵.
The COX-2 mediates the production of Prostaglandin (PG)^86,116 while COX-1 is more of complementary. Knockout mice have shown that COX-2 is associated with angiogenesis due to its involvement in the signaling mechanism of vascular endothelial growth factor (VEGF)¹¹⁷. Another gene under the direct influence of the progesterone is the HOX10 with the unique space and time of expression in the endometrial lumen. Knockout mice, microarray, and siRNA with human endometrial culture (HESC) studies have demonstrated the role in the attachment of blastocyst as well as decidualization^95,100 and optimal function of other progesterone modulated genes like the COX 2 and PG activities.

Furthermore, the importance of anti-estrogenic proliferation through the downregulation of ERalpha has been demonstrated in RU 486 study. The study revealed the role of Hand 2 and STAT 3 mediated progesterone activity in endometrial receptivity and enhancement of blastocyst attachment^98,100. Thus, further emphasize the anti-proliferative role of progesterone in the luminal epithelial cells during WOI.

CONCLUSION
Embryo implantation results from a well-coordinated sequence of molecular and cellular events guaranteed by the endometrial receptivity within a time frame termed window of implantation. (WOI). Endometrial receptivity appears to pose a stumbling block in the context of reproductive process as the only limited number of pregnancy rates have resulted from various treatment modalities aim at failures of conception, despite the availability of relatively quality embryos. Studies done on endometrial receptivity have been on an animal model and cannot transpose to human because of wide species variation. The evaluation of the endometrial biomarkers in the window of implantation could serve as an adjunct to the morphological changes associated with endometrial receptivity. Therefore, research should gear towards the functional components of the endometrial receptivity. Such concept could help to develop a therapeutic intervention for recurrent implantation failure and by extension, generate a novel fertility regulation method.

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