What is endometrial receptivity?

To understand endometrial receptivity, it is important to first understand the function of the endometrium, which is the lining of the uterus. The endometrium undergoes 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 “surge” or peak of the pituitary hormone LH (luteinizing hormone), which are important for triggering ovulation and endometrial maturation.ⁱⁱⁱ

The period of time when the endometrial environment is most ideal for embryo implantation is an approximately four-day window on days 20 to 24 of a typical 28-day menstrual cycle.^iv This window of time, known as the window of implantation (WOI), takes place when an embryo is typically migrating from the fallopian tube into the uterine cavity in females conceiving naturally.^v However, it is theorized that in some people the WOI may be shifted by hours or days and that the embryo and the endometrium being out of sync with each other could be a potential cause of infertility.

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 The endometrium is considered the most receptive during the WOI.^vii Conversely, outside of this WOI, the endometrium is less receptive to implantation of an embryo.

What is endometrial receptivity analysis (ERA)?

There have been significant advances in the methods and protocols used for testing receptivity of the endometrium over time. For decades, the gold standard was taking biopsies of the endometrium and reviewing these tissue samples under a microscope.^viii However, the accuracy and clinical practicality of this method were questioned by several multicenter trials.^ix,x

As such, endometrial receptivity analysis (ERA) was first introduced in 2011 with the goal of enabling providers to personalize in vitro fertilization (IVF) treatment by coordinating embryo transfer with a receptive uterine environment.^xi It is the first type of genetic diagnostic testing developed to identify the uterine lining as being “receptive” or “non-receptive” based on the known signature of genes expressed during a fertile window of implantation (WOI).^xii 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. Then, their doctor can synchronize the embryo transfer with that unique window^xiii — known as personalized embryo transfer (pET).

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

What is gene expression and how is it evaluated?

Gene expression is the process by which DNA is converted into ribonucleic acid (RNA). A type of RNA, called messenger RNA (mRNA) is in turn converted into proteins that have specific cellular functions. Essentially, it is how the genetic information in our cells gets converted into the molecules needed for the cells to perform their specific functions.

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.^xv

Microarray

Microarray is one way to measure gene expression. It allows for quantification of the RNA, such as mRNA, expressed by each gene of interest with high accuracy. The more RNA that is produced, the higher that gene’s expression.

DNA microarrays are small slides which can have hundreds to tens of thousands of DNA molecules attached. The RNA (isolated from the tissue samples) is quantified after it is bound to complementary sequences of DNA attached to the microarray slide.^xvi

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 used a different number of genes, and these genes had higher or lower expressions during the window of implantation (WOI). Therefore, microarray-based ERA is highly dependent on the method of testing and the selection and number of genes.^xvii

Next-generation sequencing (NGS)

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.^xviii

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.^xix

What does endometrial receptivity analysis involve?

There are two primary steps for ERA: biopsy and biopsy analysis.

Endometrial receptivity analysis biopsy

ERA requires an endometrial biopsy (tissue sample) for analysis. Physicians usually obtain endometrial biopsies from the uterine lining using an endometrial suction curette or pipelle (a long, skinny tube 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 analyzed.^xx,xxi

The biopsy is performed during a mock embryo transfer cycle, during which the patient undergoes the same transfer protocol as a regular transfer cycle. The only difference is that an embryo is not actually transferred to the uterus in the mock cycle. If the patient has planned for a medicated cycle, the same hormone medication is given during the mock cycle. If the patient is planning a natural frozen embryo transfer (FET), then no medications are used.^xxii

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 after starting progesterone medication (called day P+5). The timing of the biopsy during this four-day window enables providers to identify a receptive window of implantation (WOI) in up to 70 percent of patients in published studies.^xxiii

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 the actual embryo transfer should have the same endometrial preparation as the cycle in which the biopsy was obtained for ERA testing.^xxiv

Analysis of the biopsy

Once the biopsy is collected, the tissue is usually frozen until processing, which then extracts genetic material (DNA or RNA) from the sample. Gene expression analysis (by microarray or NGS, for example) is performed on the extracted genetic fragments.^xxv

Gene expression analysis involves assessing a variety of different genes that code for the proteins necessary for embryo implantation. The analysis checks to see if these necessary genes are upregulated (increased) or downregulated (decreased) in the sample.^xxvi For example, an endometrium that shows upregulated gene expression for target proteins known to promote embryo implantation would be considered more receptive to embryo implantation than an endometrium where these genes are downregulated. ERA assesses hundreds of genes to examine if they are being expressed at the correct time.

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.^{xxvii,xxviii,xxix} Some ERA tests have transitioned away from older microarray technology to NGS and RNA sequencing technologies. Since NGS techniques involve fully sequencing either fragments of the genome or the entire genome, they can evaluate many more genes and dramatically increase the complexity of how many genes can be assessed.^xxx

The most common commercially available ERA is a patented test developed by Igenomix, which evaluates the expression of 238 genes using microarray.^xxxi,xxxii Another ERA type test recently developed by CooperSurgical and CooperGenomics is called the ERPeak^®.^xxxiii This test 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^®.^xxxiv

Who should consider getting an endometrial receptivity analysis?

The original intent of the ERA test was to help patients who have a history of embryo transfers without implantation. In some cases, recurrent implantation failure (RIF) may be due to a non-receptive endometrium, which the ERA was designed to detect. However, there is conflicting data as to the clinical benefits of ERA testing and personalized embryo transfer (pET) to increase pregnancy outcomes. Several studies have shown no benefits from ERA testing, yet others report its advantages for specific patient populations, such as individuals with a history of RIF.^{xxxv,xxxvi,xxxvii,xxxviii,xxxix,xl}

A recent comprehensive meta-analysis including eight studies with 2,784 patients found no benefit of using ERA testing for people with RIF (defined as more than two previous failed embryo transfers) in improving live birth or ongoing pregnancy rates.^xli In contrast, another 2023 meta-analysis showed that ERA improved clinical pregnancy rates (but not live birth rates) in patients with RIF.^xlii The conflicting evidence can make decision-making challenging for individuals with RIF.

As of 2024, the most up-to-date recommendations from the European Society of Human Reproduction and Embryology (ESHRE) state that the evidence is insufficient to recommend the use of ERA testing routinely to diagnose the cause of RIF, but it can be considered in certain cases.^xliii

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 females with RIF or other specific conditions. Therefore, it is up to the physician and patient to discuss the recent research (summarized below) and consider if ERA testing is appropriate.^xliv

Understanding endometrial receptivity analysis 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.^xlv The endometrium is labeled as receptive if the molecular markers expressed are like a normal window of implantation (around day 20 of a fertile natural cycle).

Non-receptive samples can be further categorized as pre-receptive or post-receptive.^xlvi In these cases, the embryo transfer in the following cycle can be scheduled a certain number of hours or days earlier or later than when the biopsy was taken in the mock cycle. This process is called a personalized embryo transfer (pET) guided by ERA.^xlvii

Here are the different results that may be received after ERA:

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

Endometrial receptivity analysis success rates

Published clinical studies on ERA and outcomes are inconsistent, which can be confusing to patients wondering if they should use ERA. The clinical utility of ERA is highly debated with the medical community, with reproductive endocrinologists’ views ranging; some experts have adopted it for most patients while others believe it is harmful.^l Based on the most recent evidence, many reproductive endocrinologists and infertility specialists (REIs) have shifted away from its use.

Here we present available evidence, which shows the varying results and lack of consensus:

Positive outcomes

The first study to investigate the clinical application of ERA was a multicenter clinical study by Ruiz-Alonso et al (2013).^li,lii They reported preliminary findings suggesting that the window of implantation (WOI) was displaced more often in patients with recurrent implantation failure (RIF) (22 out 85 patients, 25.9 percent non-receptive) compared to controls (3 out of 25 patients, 12 percent non-receptive). Eight of the non-receptive patients with RIF underwent personalized embryo transfer (pET), which resulted in an implantation rate of 38.5 percent and pregnancy rate of 50 percent. It is also worth noting that changing the number of hours the patients took progesterone according to ERA did not always correct the WOI (it did so in only 3 out of 18 patients), according to a second ERA. This study was small and preliminary, by the developers of the Igenomix ERA, to show its clinical application.

Another retrospective study by Mahajan (2015) examined 186 infertile females who underwent Igenomix ERA testing.^liii The patient groups were divided into 80 females with RIF, 93 females with one failed IVF cycle, and 13 females with a thin endometrium (6 mm in thickness or less). The researchers 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. Interestingly, they found that in 75 percent of the patients with a thin endometrium, it was receptive. The pregnancy rate in these patients was 66.7 percent, despite their having a persistent endometrial thickness of 6 mm or less. It should be noted, though, that the sample size for the thin endometrium group was smaller than that of other groups.

In 2017, Hashimoto et al published a retrospective two-center study where they performed ERA on 50 patients with a history of RIF (with frozen embryo transfer).^liv ERA suggested that 76 percent (38 out of 50) of the samples were receptive, and 24 percent (12 out of 50) were non-receptive. The 12 non-receptive patients were subcategorized as pre-receptive (8 out of 12), post-receptive (3 out of 12), and proliferative (1 out of 12). Forty-four of these RIF patients underwent pET following ERA testing. In the receptive group, the 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, the pregnancy rate was 50 percent per patient, with half of these patients becoming pregnant after the first pET. The authors concluded that transferring chromosomally normal embryos in a personal WOI (as determined by ERA testing) resulted in better pregnancy rates for patients with unexplained RIF.

Neutral outcomes

The largest and most robust clinical study evaluating the Igenomix ERA was performed by Doyle et al in 2022.^lv It was a two-year multicenter randomized controlled trial 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) without ERA. 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 the two. Thus, ERA-guided pET did not improve live birth rate in this cohort.

A meta-analysis by Arian et al (2023) did not show any improvement in success rates following ERA.^lvi The 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 had not). The researchers 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.^lvii They concluded that it remains unclear whether ERA testing can increase pregnancy or live birth rates.

Negative outcomes

A recent study by Richter and Richter (2023) re-analyzed the data from the randomized controlled trial discussed above (Doyle et al, 2022).^lviii Their findings suggest that ERA was unable to identify the WOI accurately and that conducting pET based on the ERA results reduced the live birth rates.

Controversy

Recent controversy regarding the ERA test has been brought to light by the results published by Cozzolino et al (2022) in a retrospective study, which concluded that using the ERA test to guide embryo transfer timing in patients with a previous failed transfer did not improve reproductive outcomes, but rather led to worse outcomes in this patient population.^lix The study included patients undergoing both own-egg and donor embryo transfers, yielding a total of 5,372 patients from which 574 females underwent pET guided by ERA.

This study was criticized by the authors of a previously published study (Simón et al) that had reached opposite conclusions using data from the same center; they claimed that the difference in results was due to flaws in the experimental design.^lx,lxi The multicenter randomized controlled trial by  Simón et al (2020) included 458 patients from the same clinic network that were randomized either to a pET protocol (following ERA timing) or a frozen embryo transfer (FET) or fresh embryo transfer protocol.^lxii This study showed improved outcomes in the pET group, including an increase in cumulative live birth rates after 12 months:

• ‍Personalized embryo transfer (pET) group: 71.2 percent live birth rate
• ‍Frozen embryo transfer (FET) group: 55.4 percent live birth rate (P=0.04)
• ‍Fresh embryo transfer group: 48.9 percent live birth rate (P=0.003)

Although the authors of the Cozzolino et al (2022) study published a response justifying their experimental methodology, the question of whether the ERA test improves reproductive outcomes during IVF remains open.^lxiii

The new ERPeak® endometrial receptivity test

Finally, a group in Japan recently published a study to evaluate the effectiveness of a new type of endometrial test, called the ERPeak^® endometrial receptivity test.^lxiv This study investigated the test’s use for pET in patients with RIF. The researchers compared clinical pregnancy rate and live birth rate between 244 RIF patients who received pET and 306 control/non-pET patients, with an additional comparison between patients under age 38 and those age 38 and older (considered “advanced maternal age,” or AMA).

They found that the pET group had higher clinical pregnancy and live birth rates compared to the non-pET group:

• ‍pET group: Clinical pregnancy rate of 37.7 percent; live birth rate of 20 percent
• ‍Non-pET group: Clinical pregnancy rate of 29.9 percent; live birth rate of 9.7 percent

Further comparison of maternal age showed significantly higher clinical pregnancy rate and live birth rate following ERPeak testing and pET for patients aged 38 and older (34.1 percent and 24.8 percent respectively) and patients under age 38 (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.

Conclusion

Endometrial receptivity testing has long been studied as a potential diagnostic tool for patients dealing with infertility or recurrent implantation failure (RIF). Unfortunately, the best available research does not currently support endometrial receptivity analysis (ERA) as a useful test. It remains possible that there are certain populations of infertile patients, such as individuals with RIF, that may benefit from ERA testing and personalized embryo transfer (pET). However, further studies into this population are needed before a definitive conclusion can be made.