What is endometrial receptivity?

In order to understand endometrial receptivity, it is important to first understand the endometrium, which is the lining of the uterus. The endometrium experiences a pattern of molecular and structural changes during every menstrual cycle to prepare the uterus for a potential pregnancy. Some key players that are important for endometrial development and maturation include the ovarian hormones estrogen and progesterone, and the pituitary luteinizing hormone (LH) surge or peak (important for triggering ovulation and endometrial maturation).ⁱⁱⁱ In women, the period when the endometrial environment is most ideal for embryo implantation is an approximately 4-day window at days 20-24 of a 28-day menstrual cycle.^iv In a menstrual cycle, this window of time is exactly when the embryo is migrating from the fallopian tube into the uterine cavity. This period is commonly known as the window of implantation (WOI).^v However, it is theorized that in some patients, the window of implantation may be shifted by hours or days and that this dyssynchrony between the embryo and the endometrium could be a potential cause of infertility in these patients.

Endometrial receptivity describes the physiological state of the endometrium and whether it is mature enough to allow the implantation of a transferred embryo and to maintain its development towards a healthy pregnancy.^vi When considering its structural and molecular maturity, the endometrium is considered the most receptive during the WOI.^vii Conversely, outside of this WOI, the endometrium is not receptive to implantation of an embryo.  

Testing receptivity of the endometrium

There have been significant advances in the methods and protocols used for testing receptivity of the endometrium over time. For decades, the gold standard testing to assess the endometrial environment were biopsies of the endometrium and review of these tissue samples under a microscope (histologic evaluation).^viii However, the accuracy and clinical functionality of histological dating to predict endometrial receptivity and fertility has been questioned by several multicenter trials.^ix,x

As such, ERA was introduced as a more accurate way to determine endometrium receptivity.^xi,xii ERA determines whether the necessary genes to facilitate implantation are being adequately expressed at the expected time of embryo transfer.^xiii It is postulated that if an endometrium is deemed to lack receptivity at the expected time of embryo transfer based on the ERA results, a shift in the timing of the embryo transfer could be made to better synchronize the embryo transfer with endometrial receptivity. This tailoring of transfer timing based on a specific patient’s unique receptive window is called personalized embryo transfer, or pET.

What is gene expression and how is it evaluated?

Gene expression is the process by which DNA is converted into mRNA (messenger RNA) and other proteins that create specific cellular functions. In a simpler sense, it is how the genetic information in our cells gets converted into the specific functions these cells perform. The genetic information within a cell starts from the coding genes (DNA), and when these genes are activated, the cell produces mRNA that is analyzed by other cellular machinery to produce functional proteins. Depending on the cell type, tissue type, and biological needs, specific proteins are expressed at different levels and different times.^xiv

Microarray is one way to measure gene expression. It involves quantification of mRNA and can simultaneously evaluate the expression of thousands of genes with high accuracy. DNA microarrays are small slides which can have hundreds to tens of thousands of DNA molecules attached. The RNA (isolated from tissue samples) is quantified after it is bound to complementary sequences of DNA attached to the microarray slide.^xv ERA was originally performed using microarray technology, which is dependent on the different number of genes researchers opted to test and the timing of the biopsy. Different research groups utilized a different number of genes and these genes had had higher or lower expressions during the WOI. This makes microarray-based ERA highly dependent on the method of testing and the number of genes selected for it.^xvi

Next-generation sequencing (NGS), which includes RNA sequencing, is a high-efficiency technique that quantifies RNA expression levels by a different method than DNA microarrays. RNA molecules are converted into DNA fragments that are then passed through a specialized machine to count each fragment. The data is then analyzed by cross-referencing the counts to a genome database to identify the activated/expressed genes.^xvii NGS ERA is not subject to the same limits of microarray testing in that the test does not require technicians to select a certain number of genes to test. Because NGS rapidly allows for measurement of gene expression throughout the whole genome, it is more expansive in its scope.^xviii

What is ERA?

As noted above, ERA is a relatively new form of testing endometrial receptivity and was first introduced in 2011 with the goal of enabling providers to personalize IVF treatment by coordinating the time of embryo transfer with a receptive uterine environment.^xix This is the first type of genetic diagnostic testing developed to identify a woman's receptive status as being “receptive” or “non-receptive" based on the known signature of genes expressed during a normal fertile WOI.^xx In assessing the expression of genes coding for specific molecules known to facilitate implantation, ERA aims to identify which days in the cycle a patient will have optimal endometrial receptivity to synchronize the embryo transfer with their personalized WOI.^xxi  

Despite its purported benefits, ERA remains controversial as there is emerging evidence that ERA testing does not improve IVF outcomes for most patients. A recent randomized controlled trial of 767 women found that those undergoing ERA to time embryo transfer did not have a higher rate of ongoing pregnancy than those who underwent embryo transfer based on standard timing.^xxii

ERA biopsy

It is important to know that ERA requires an endometrial biopsy (tissue sample) for analysis. Endometrial biopsies are usually obtained from the uterine lining by a physician, using an endometrial suction curette or pipelle (long, skinny tubes placed through the cervix, into the uterus) to sample some of the endometrial tissue. The tissue sample is placed in a sterile test tube before being sent off for analysis.^{xxiii,xxiv,xxv}

The biopsy is performed during a mock embryo transfer cycle, during which the patient undergoes the same transfer protocol they would during a transfer cycle. The only difference is that there is no embryo transfer. If the patient is planned for a medicated cycle, the same hormone replacement therapy (HRT, i.e., transfer meds) is given during the mock trial. If the patient is planning a natural frozen embryo transfer (FET), then no medications are used.^xxvi  

Typically, providers collect the endometrial biopsy on the day the FET would normally be performed. For medicated cycles, the biopsy is recommended to be done five days (~120 hours) after starting progesterone medication (P+5). The timing of biopsy during this 4-day window enables providers to identify a receptive WOI in up to 70 percent of patients in published studies.^xxvii The purpose of using the mock cycle to time endometrial biopsy for ERA enables providers to easily replicate the same timing for future biopsies and transfers. The cycle for embryo transfer should have the same endometrial preparation as the cycle where the biopsy was obtained for ERA testing.^xxviii

Analysis of the biopsy

Once the biopsy is collected, the tissue is usually frozen until processing. Tissue processing is then performed to extract genetic material (DNA or RNA) from the sample. Gene expression analysis is performed on the extracted genetic fragments (examples include Microarray and Next Generation Sequencing [NGS] analysis).^xxix Gene expression analysis involves assessing a variety of different genes that code for proteins necessary for embryo implantation to see if these necessary genes are upregulated (increased) or downregulated (decreased) in the sample.^xxx An endometrium with upregulated gene expression for the target proteins would be considered more receptive to embryo implantation than an endometrium where these genes are downregulated. Hundreds or thousands of different genes are involved with endometrial receptivity and are assessed in ERA. It is important to note that the technology for ERA has rapidly changed with time, and recent research suggests that ERA results from previous and current studies involving DNA microarray technology may not be accurate.^{xxxi,xxxii,xxxiii} As previously noted, some ERA tests have transitioned from microarray or PCR-based clinical tests to NGS and other RNA sequencing technologies. NGS techniques involve fully sequencing either fragments of the genome or the entire genome, which allows for evaluation of many more genes and dramatically increases the complexity of how many genes can be assessed.^xxxiv  

The most used commercially available ERA is a patented test developed by Igenomix, which evaluates the expression of 238 genes using microarray.^xxxv,xxxvi Another ERA type test recently developed by CooperSurgical and CooperGenomics is called the ERPeak®_.^xxxvii ERPeak® uses a different gene expression analysis technology called real-time quantitative polymerase chain reaction (RT-qPCR) to analyze 48 selected genes that are mostly distinct from the Igenomix ERA®.^xxxviii

Who should consider getting an ERA?

The original intent of developing the ERA test was to help patients with a history of embryo transfers without implantation.  However, there is conflicting data as to the clinical benefits of ERA testing and personalized embryo transfer (pET) to increase pregnancy outcomes. While several studies have shown no benefits from ERA testing.^xxxix others report its advantages for specific patient populations, such as those with a history of recurrent implantation failure (RIF) .^{xl,xli,xlii,xliii,xliv,xlv}

RIF due to endometrial problems, sometimes referred to as “RIF of endometrial origin”, is recognized by many clinicians as a concern associated with repeated failure of euploid embryo transfers. At the same time, some recognize that RIF of endometrial origin is so rare that few patients are likely to benefit from specifically targeting problems with the endometrium. It is therefore up to the physician and patient to discuss the recent research and consider if ERA testing is appropriate for their IVF treatment.^xlvi Due to its potential but unclear benefit in specific populations, some clinics promote the use of ERA, while others do not recommend or use it. Other providers may restrict its use to those with RIF or other specific conditions.  

Understanding ERA results

When patients get their ERA results, they receive a report documenting whether the endometrium was “receptive” or “not receptive” at the time of the biopsy based on the gene expression. Gene expression results are analyzed by an algorithm (ERA computational predictor) which will compute whether the biopsied endometrium was receptive or not at the time of biopsy.^xlvii Based on this ERA gene expression profile, the endometrium is labeled as receptive if the molecular markers expressed are like a normal WOI (around day 20 of a fertile natural cycle), or non-receptive. The non-receptive samples can be further divided into being “pre-receptive”, or “post-receptive” in relation to the gene profile during the WOI.^xlviii

Table 1: The categories of results that are obtained from ERA and their meaning

*Results from table adapted from interventional multicenter clinical trial^xlix,l

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If the ERA test identifies the biopsy’s gene expression profile as pre- or post-receptive (table 1), a certain number of hours is recommended to advance or delay the embryo transfer in the following cycle. This process is called a pET guided by ERA.^li

ERA success rates

Published clinical studies on ERA and outcomes are inconsistent and it can be confusing to patients wondering if they should use ERA. The clinical utility of ERA is even highly debated within the medical community, with views ranging from REIs adopting it for most patients to REIs that believe it is harmful.^lii Here we present available evidence, which shows the varying results and lack of consensus.

The first study to investigate the clinical application of the ERA was a multicenter clinical study by Ruiz-Alonso et al (2013).^liii,liv They reported preliminary findings suggesting that the WOI was displaced more often in patients with RIF (15/22, 25.9 percent non-receptive) compared to controls (3/25, 12 percent non-receptive).  Eight of the RIF non-receptive patients underwent pET, which resulted in an implantation rate of 38.5 percent and pregnancy rate of 50.0 percent. It is also worth noting that changing the number of hours of progesterone administration according to ERA did not always correct the WOI (3/18 patients), according to a second ERA.^lv This was a small preliminary study, by the developers of the Igenomix ERA, to show its clinical application.  

Another retrospective study by Mahajan (2015) examined 186 infertile women who underwent Igenomix ERA testing. The patient groups were divided into 80 women with RIF, 93 women with one failed IVF cycle, and 13 women with a thin endometrium (≤6 mm in thickness). They reported that poor endometrial receptivity was the cause of implantation failure in 27.5 percent of RIF patients compared to 15 percent in the non-RIF group. Utilizing pET, the ongoing pregnancy rate was 42.4 percent and implantation rate was 33 percent.^lvi Interestingly, they found that 75 percent of the thin endometrium patients had a receptive endometrium and a pregnancy rate of 66.7 percent, despite having a persistent endometrial thickness of 6mm or less.^lvii It should be noted, though, that the sample size for the thin endometrium was smaller than that of the other groups.

In 2017, Hashimoto et al published a retrospective two-center study where they performed ERA on fifty patients with a history of RIF (with FET). ERA suggested that 76 percent (38/50) of the samples were receptive, and 24 percent (12/50) were non-receptive. The twelve non-receptive patients were also labeled as pre-receptive (8/12), post-receptive (3/12), or proliferative (1/12). Forty-four of these RIF patients underwent pET following ERA testing. In the receptive group, pregnancy rate was 58.8 percent per patient with 35.3 percent of these patients becoming pregnant after the first pET. In the non-receptive group, pregnancy rate was 50.0 percent per patient with 50.0 percent of these patients becoming pregnant after the first pET. The authors concluded that transferring euploid embryos in a personal WOI (as determined by ERA testing) resulted in better pregnancy rates for patients with unexplained RIF.^lviii

The largest and most robust clinical study evaluating the Igenomix ERA was performed by Doyle et al in 2022.  This was a 2-year multicenter RCT involving 767 IVF patients aged 30 to 40 years at 30 clinics, which investigated the live birth rate following pET guided by ERA testing compared to frozen embryo transfer (FET) or at the first attempt. The live birth rate in the group undergoing pET guided by ERA was 58.5 percent compared to 61.9 percent undergoing standard timing FET; there was no statistically significant difference between these values. Thus, ERA-guided pET did not improve live birth rate in this cohort.^lix  

A recent study by Richter and Richter (2023) re-analyzed the data from the randomized controlled trial discussed above.^lx Their findings suggest that ERA was unable to identify the window of implantation accurately and that conducting pET based on the ERA results reduced the live birth rates.^lxi

A meta-analysis by Arian et al (2022) did not show any improvement in success rates following ERA.^lxiiThe authors investigated the impact of ERA testing on patients undergoing IVF and receiving FETs, with or without prior ERA testing. Eight studies were included for the meta-analysis (2 784 patients; 831 had undergone ERA and 1 953 without ERA), and they reported that there was not a statistically significant difference for live birth rate or ongoing pregnancy rate for the ERA group compared to the non-ERA group.^lxiii They concluded that it remains unclear whether ERA testing can increase pregnancy or live birth rates. 

Another recent study by Doyle et al (2022) did not support ERA for improvement of live birth rates in patients with and without a history of failed FETs. This study only included FET of single euploid embryos (determined by PGT-A) and compared patients with and without ERA testing. A total of 307 ERA-timed FETs and 2 284 standard FET protocols (without ERA) were analyzed. Of the 307 ERA patients, 40.7 percent were diagnosed as receptive and 59.3 percent were “non-receptive”; however, the difference in live birth rate was not statistically significant between the ERA-receptive (48.8 percent) and ERA-nonreceptive (41.7 percent) patients.^lxiv There were also no statistically significant differences in the live birth rate of patients with ERA-adjusted FET (live birth rate 44.6 percent) versus FET without ERA (live birth rate51.3 percent). This study also showed that patients with an increasing number of previous failed euploid FETs were not at an increased risk of having a displaced WOI (non-receptive ERA result). Overall, patients who underwent ERA testing and ERA-guided FET had similar success rates as those who underwent standard FET protocols with no ERA. These researchers suggested that more RCTs are required.^lxv

Finally, a group in Japan (Ohara et al.) recently published a study to evaluate the effectiveness of a new type of endometrial test, called the ERPeak® endometrial receptivity test. This study investigated its use for pET in patients with RIF. They compared clinical pregnancy rate (CPR) and live birth rate (LBR) between 244 RIF patients who received pET and 306 control/non-pET patients, with an additional comparison between advanced maternal age (AMA) of ≥38 years old and non-AMA (<38 years old). They reported a significantly higher CPR (37.7 percent) and LBR (20 percent) in the pET group compared to the non-pET group (29.9 percent and 9.7 percent respectively). Further comparison between maternal age showed significantly higher CPR and LBR following ERPeak® testing pET for both the AMA (34.1 percent and 24.8 percent respectively) and non-AMA (42.7 percent and 35.4 percent respectively) compared to the non-pET group. The authors concluded that the new ERPeak® endometrial receptivity test is a potentially useful alternative diagnostic tool for RIF patients, regardless of maternal age.^lxvi

Conclusion

The endometrium and the window of implantation are key players in fertility. Endometrial receptivity testing has long been studied as a potential diagnostic tool for patients with infertility or recurrent implantation failure. Unfortunately, the best available research does not currently support ERA as a useful test for patients with infertility. It remains possible that there are certain populations of infertile patients, such as those with RIF, that may benefit from ERA testing and pET. However, further studies into this population are needed before a definitive conclusion can be made.  

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