Endometrial Receptivity

Function

Embryo implantation requires a receptive endometrium, a functional, normally developing embryo, and synchronized embryo-endometrial cross-talk.[5] 

The preparation of a receptive endometrium is established by sequential exposure to the steroid hormones estrogen and progesterone.[6] Estrogen signals the proliferation of the endometrial lining during the preovulatory phase and induces an increase in progesterone receptor expression.[7] While estrogen is essential for proliferation, excessive estrogen activity can negatively impact endometrial receptivity.[8] Estrogen receptor alpha (ER alpha) is upregulated in response to estrogen during the proliferative phase. The down-regulation of ER alpha by progesterone in the secretory phase is required for successful embryo implantation.[4] After ovulation, progesterone induces major cellular changes within the endometrium that are required to create a receptive endometrium and maintenance of early pregnancy. Progesterone is also suspected of inducing immuno-tolerance in early pregnancy.[6] In natural, ovulatory menstrual cycles, implantation can occur over several days. Through histological dating and transcriptomic studies, the window of implantation has been estimated to occur between cycle days 20 to 24.[2][3] This window of implantation can also be artificially created by the sequential use of exogenous estrogen and progesterone. Since progesterone triggers the cascade of events that lead to endometrial receptivity, endometrial age is commonly dated as the number of days post-ovulation or, in artificial cycles, the number of days of progesterone exposure.

The cross-talk between the synchronized embryo and receptive endometrium is facilitated by estrogen and progesterone and permits the process of embryo implantation. Implantation is defined as the process by which an embryo attaches to the uterine wall and penetrates first the epithelium and then the circulatory system of the mother to form the placenta. The implantation process occurs in three steps - apposition, adhesion, and invasion.

In natural ovulatory menstrual cycles, the fertilized ovum (zygote) travels through the fallopian tube encased in the zona pellucida, a nonadhesive coating. During this journey, the fertilized ovum divides into the morula, a mass of 12 to 16 cells. Approximately 2 to 3 days after fertilization, the morula enters the uterine cavity.[3] The next stage involves the morula becoming a blastocyst, defined by a fluid-filled cavity within the mass of cells. The blastocyst contains an inner cell mass that gives rise to the embryo and the trophectoderm, or surface cells, which become extraembryonic structures, including the placenta. Within 72 hours of being in the uterus, the embryo hatches from the zona pellucida, exposing the trophoblasts to prepare for implantation. In patients undergoing assisted reproductive technology (ART) cycles with in vitro fertilization (IVF), the blastocyst can be inserted into the uterine cavity with unassisted (non-hatched) or assisted hatching.

Apposition involves the hatched blastocyst orienting correctly based on its polarity and weakly adhering to pinopods on the surface of the endometrium.[9] It is hypothesized that chemokines guide the blastocyst to the site of implantation.[10] Pinopod development occurs in the mid-luteal phase, which is upregulated by Leukemia Inhibitory Factor (LIF), progesterone, and beta-3 integrin.[2] The initial contact between the blastocyst trophectoderm and the endometrium triggers the first communication between the two tissues and is driven by heparin-binding epidermal growth-like factor (HB-EGF) signaling.[9]

Adhesion results in a stronger connection between the blastocyst and endometrium. This process involves adhesion molecules, immune cells, and cytokines, namely beta-3 integrin and L selectin.[10] Integrins are a family of transmembrane glycoproteins utilized in cell-cell and cell-matrix interactions, the assembly of which forms focal adhesion sites. Embryonic interleukin 1 causes the upregulation of epithelial beta-3 integrin in the endometrium.[11] Selectins are cell surface lectins that promote adhesion.[12] L-selectin expressed by the trophoblast interacts with oligosaccharide ligands on the maternal endometrium to facilitate adhesion.[11]

During the process of invasion, the blastocyst penetrates the epithelial layer and invades the decidual stroma in order to secure the blood supply.[2] Maternal immune tolerance is required as the embryo expresses paternally derived alloantigens. Both the innate and adaptive immune systems mediate this immune tolerance.[13] During the luteal phase, innate immune cells, including macrophages, dendritic cells (DCs), and uterine natural killer (uNK) cells, are found in the endometrial decidua. These cells support immune regulation and facilitate the support of trophoblast invasion. The adaptive immune system acts through T regulatory and T effector cells. CD4+ regulatory T cells inhibit effector immunity, contain inflammation, and support maternal vascular adaptations. T regulatory cells are found in the uterus in the proliferative phase of each menstrual cycle and remain elevated until the end of pregnancy. The embryo responds through the action of human leukocyte antigen G (HLA-G) expressed by invading trophoblasts. HLA-G modulates trophoblast cytokine secretion to maintain a local immunosuppressive state.[14] The trophoblasts also interact with macrophages in the decidua to remove apoptotic cells. This, in turn, prevents the release of further proinflammatory signals and teaches macrophages to secrete substances that promote trophoblast growth.[15] Trophoblast cells have also been found to secrete Fas Ligand, which protects them from maternal immune recognition.[16]

Leukemia inhibiting factor (LIF) also plays a critical role in implantation. LIF is a pleiotropic cytokine from the interleukin-6 family produced by both the embryo and the endometrium. It is expressed in the luteal epithelium and decidualized stromal cells.[10] LIF is involved in multiple processes associated with successful embryo implantation. LIF promotes decidualization of the endometrium via the recruitment of cytokines and progesterone, promotes pinopod expression, increases growth factors, increases implantation genes, increases trophoblast differentiation and invasiveness, and recruits leukocytes to the endometrium.[17]

Issues of Concern

Implantation failure can be caused by 1) issues related to embryo development and/or aneuploidy, 2) reduced or absent endometrial receptivity, and 3) embryo-endometrial asynchrony.[1] Factors that disturb receptivity include inflammatory events, thin endometria, fibroids, polyps, septa, immunologically mediated disturbances, and endocrine causes.[18] Endometrial receptivity is not an all-or-nothing phenomenon but rather exists on a spectrum. A mild defect in receptivity may cause placental abnormalities, which lead to issues like pre-eclampsia or low birth weight. More severe forms of receptivity aberrations may lead to early pregnancy loss, and in complete loss of receptivity, infertility may rise.[6]

As described above, embryo maturation and endometrial development co-occur independently. In ART cycles, these two processes can diverge, resulting in an asynchronous window of implantation. Ovarian hyperstimulation leads to supraphysiological levels of estrogen and progesterone, which alter gene expression and hormone receptor presence, triggering rapid transformation to a secretory endometrium.[19] This has proven problematic, particularly in fresh IVF cycles, where a premature progesterone elevation shifts the optimal timing of endometrial receptivity to earlier in the cycle while the embryo continues to develop ex-vivo at its usual pace, creating an asynchronous window of implantation.[20] 

A large 2013 meta-analysis demonstrated that the progesterone level at which the phenomena occurs can be as low as 0.8-1.1 ng/ml and that higher progesterone levels on the day of ovulation trigger were associated with a greater reduction in pregnancy. Additionally, in a 2018 threshold analysis by Hill et al., progesterone of 2.0 ng/ml on the day of the trigger was associated with a 20% absolute reduction in a live birth in fresh cycles. They also reported that high progesterone levels on the day of ovulation trigger were positively associated with younger age, higher gonadotropin dosing, higher estrogen (E2) values during stimulation, and higher follicle count >14mm.[21] Lastly, older maternal age has been associated with slower-growing embryos, further widening the embryo-endometrial asynchrony.[22]

Endometrial receptivity can be further impaired by an imbalance of steroid hormones by way of progesterone resistance and estrogen dominance.[18] In both natural and artificial cycles, progesterone resistance can result from a pro-inflammatory state. Conditions that cause inflammation in the endometrium include endometriosis, endometritis, adenomyosis, and hydrosalpinges.[23] Regardless of the etiology, estrogen-progesterone imbalance due to progesterone resistance leads to decreased decidualization and estrogen dominance. Proper decidualization is a key factor in implantation as the decidua provides nutrients to the embryo, shelters the developing embryo from the maternal immune response, and regulates invasion of the trophoblast.[18] 

Estrogen dominance results in increased cell proliferation, inflammation, and angiogenesis, exacerbating the non-receptive endometrial state. In studies of endometriosis, a condition defined by excess estrogen and progesterone resistance, it has been shown that the resulting inflammation of the endometrium is associated with an overexpression of p450 aromatase, which changes the dynamic of progesterone-to-estrogen activity. The overexpression of aromatase has been associated with poor IVF outcomes.[24] 

Endometrial receptivity continues to be a very active area of research. The contributing factors to endometrial receptivity have not all been recognized and continue to be investigated.

Clinical Significance

Understanding the molecular processes responsible for coordinating the window of implantation and facilitating normal embryo implantation is critical to developing diagnostic tests and treatments for receptivity defects that contribute to recurrent pregnancy loss, implantation failure, and infertility.

First and foremost, when an endometrial receptivity defect is due to an endometrial polyp or submucosal myoma, there is good evidence that removing these structures improves implantation and pregnancy outcomes.[25]

When an asynchronous window of implantation is suspected based on a premature progesterone rise in fresh IVF cycles, conversion to a freeze-all cycle with a plan for a frozen embryo transfer (FET) is an efficient option to maximize the chance of pregnancy. This is supported by evidence that elevated progesterone in donor egg cycles did not affect the implantation rate in the recipient.[26] In a recent cost-effective analysis, using progesterone of >1.45 ng/ml as the threshold for conversion to a FET cycle improved pregnancy rates compared to those proceeding with a fresh transfer. Conversion to FET was associated with a low number needed to treat (NNT) to see the benefit and a maximum added expense of $3900.[21] 

Regarding the prevention of a premature progesterone rise, researchers have suggested that using a mixed FSH/LH stimulation protocol is associated with a lower incidence of premature progesterone rise as compared to FSH-only protocols, likely due to LH activation of CYP 17 in theca cells, allowing for the conversion of rising progesterone to androgens, which are then subsequently aromatized to estrogen by granulosa cells.[27][28] Others have proposed that in patients with polycystic ovarian syndrome, IVF pretreatment with metformin might prevent a premature progesterone rise by decreasing ovarian steroid synthesis.[29] For patients committed to proceeding with a fresh embryo transfer, these strategies may be helpful in those at risk for premature progesterone rise, although further studies are needed to assess the generalizability of these findings.

Methods for assessing endometrial receptivity include transvaginal ultrasound imaging (TVUS), histologic evaluation by endometrial biopsy, endometrial receptivity array (ERA), and ReceptivaDx test (BCL6).

TVUS is a widely available tool that can be used to assess endometrial receptivity. TVUS can be used to measure the endometrial thickness, volume, and pattern. A recent meta-analysis found that endometrial thickness >7mm, endometrial volume >2mL, and trilaminar pattern had 99%, 93%, and 87% respective sensitivity for assessing endometrial receptivity.[30]

Histologic evaluation of endometrial receptivity requires endometrial sampling. In the 1950s, the Noyes criteria were established to evaluate luteal phase deficiency (LPD). LPD is described as an abnormal luteal phase with a duration of less than or equal to 10 days and was one of the first known descriptions of endometrial receptivity.[31] Biopsies for the evaluation of endometrial receptivity were traditionally performed within three days of the anticipated onset of menses and evaluated based on the histologic criteria set forth by Noyes. In 2004, Murray et al. refined the histologic criteria to define better endometrial receptivity or LPD using a prospective randomized controlled trial. Ultimately they found that histologic sampling does not provide an accurate or reliable assessment of endometrial receptivity and is not currently used to assess endometrial receptivity in clinical practice.[32]

The ERA is a molecular diagnostic tool used to identify a receptive endometrium via a specific transcriptomic signature present in both natural and hormone replacement therapy cycles. The technology has been applied clinically to identify a patient-specific window of implantation, which is then used to guide a personalized timing of embryo transfer for patients with recurrent implantation failure. This is done by taking an endometrial biopsy at specific times during the mid-luteal phase (LH surge+7 days in natural cycles, progesterone starts +5 days in hormone replacement/”artificial” cycles. The results of the ERA are then used to guide shifts in the timing of progesterone administration before embryo transfer in a future cycle. Most data evaluating ERA-timed embryo transfers and associated pregnancy rates have failed to see an improvement in the live birth rate in these cycles.[33][34] Most recently, a 2022 RCT by Doyle et al. evaluated live birth after standard vs. ERA-timed euploid embryo transfer and found no difference in the live birth rates.[35] However, the study designs of these investigations have been heavily critiqued, bringing the validity of their findings into question.

The ReceptivaDx test identifies endometrial receptivity defects associated with progesterone resistance. The BCL6 protein is overexpressed in women with endometriosis.[36] BCL6 protein overexpression is associated with lower clinical pregnancy rates in women undergoing IVF. It is hypothesized that these women could be treated with GnRH agonists or surgery to improve fertility outcomes.[37] While the ReceptivaDx test is available for clinical use, its ability to improve IVF outcomes is still undergoing investigation.

Enhancing Healthcare Team Outcomes

Patients in the care of infertility practices are cared for by an interprofessional team of medical professionals, including clinicians (MDs, DOs, NPs, and PAs), nurses, embryologists, lab technicians, and other health professionals. It is essential for all team members to be aware of the optimal time for the patient to achieve pregnancy to generate a thoughtful treatment plan with the highest likelihood of success; this requires open communication channels between team members and accurate, updated patient record keeping. This interprofessional approach will yield the best patient outcomes.