Embryologist Media has contacted Dr María Enciso, iGLS scientific director, to ask her about their revolutionary endometrial receptivity test ER Map®. iGLS is an innovative company that provides advanced and integrated genetic services for reproductive medicine. The ER Map® test allows for the identification of the best endometrial moment to perform embryo transfer, thus increasing the probability of implantation and pregnancy. Such an improvement will surely help many couples to achieve their dream of becoming parents.

“The ER Map® test analyses the expression of 40 specific endometrial receptivity-related genes”.

E.M. Dr Enciso, could you briefly explain the different steps required for this test, from the moment of sample collection until 21 days later, when results are available?

M.E. The ER Map® protocol consists of three steps: 1) the clinician collects the endometrial biopsy in the fertility centre; 2) we perform ER Map® gene expression analyses of the sample, which are indicative of the endometrial receptivity; 3) results are reported to the specialist, along with our recommendation on an optimal day for embryo transfer.

E.M. Usually, women are stimulated in order to control both ovarian and endometrial functions. Is this biopsy suitable for a substituted cycle?

M.E. Yes, it is. In such a case, the patient is treated with estrogen and progesterone in order to inhibit the endogenous production of these hormones. Around 7-10 days after oestradiol administration, once it has been confirmed the presence of trilaminar endometrium (>6,5 mm) by ultrasound and that the levels of progesterone in serum are appropriate (<1 ng/mL), progesterone administration is initiated. The first day of administration is called day P+0; biopsy must be performed at day P+5.5, this is five and a half days after the first progesterone administration, which is about 132 hours. For instance, if the first dose takes place on a Tuesday evening, the endometrial tissue must be sampled for a biopsy the following Monday morning.

E.M. What about a natural cycle?

M.E. During a natural cycle there must be a regular control by ultrasound of the total follicular count and size from day 11 of the cycle. LH levels in blood or urine must be measured on a daily basis in order to identify the LH surge. The day of LH surge is considered LH+0, and so tissue sample must be collected at day LH+7, 7 days after the LH surge.
“We can diagnose the receptivity status of the endometrium at the point of the cycle when the biopsy was performed”.

E.M. Are those patients on a natural cycle but treated by hCG administration followed-up by ultrasound just as those monitored during the entire natural cycle?

M.E. Not quite. Regarding treated patients on a natural cycle, count and measure of follicles must be performed, as well as two analyses of LH levels in urine or blood per day. The hCG dose must be administered once follicles reach the appropriate size (<17mm). The day of hCG administration is considered hCG+0, and so biopsy must be performed at hCG+7, seven days post hCG administration.

E.M. What kind of cycle would you recommend in order to perform the endometrial biopsy?

M.E. We recommend biopsying the endometrium either on a substituted cycle or on a modified natural cycle since follow-up of the whole procedure would be more accurate.

E.M. As for the biopsy itself, how is it performed?

M.E. A Pipelle de Cornier® cannula is used to retrieve a sample of about 30 mg from the uterine fundus. This tissue must be immediately transferred into the ER Map® tube we provide, and then sent to our laboratory for analysis.

E.M. Once the sample for biopsy has been acquired, what is the methodology followed to analyze gene expression?

M.E. When the sample reaches our facilities, we perform RNA extraction and expression analysis of about 40 genes involved in endometrial receptivity. Based on the expression profile obtained and by means of specialized software, we can diagnose the receptivity status of the endometrium at the point of the cycle when the biopsy was performed. We then include our results and the recommendation for embryo transfer in a report that will be emailed to the clinician.

E.M. Which specifications are normally included among your recommendations?

M.E. In our report, after the analyses we may classify a particular endometrium as receptive, which would mean biopsy was taken within the window of implantation (WOI). If this is the case, embryo transfer should be performed on the same day and type of cycle on which the ER Map® biopsy was taken.

E.M. What about those cases in which a temporal displacement of the WOI has been observed?

M.E. In such cases, because the WOI is displaced, the endometrium is classified as non-receptive, either post-receptive or pre-receptive. A pre-receptive endometrium has not yet reached the WOI, whereas a post-receptive one has already missed it. About 30% of the patients show some kind of WOI displacement, and these are the patients that can benefit most from the ER Map® diagnosis. In those cases, their endometrium is not prepared to accommodate the embryo at the time most women are, and so regular protocols do not work for them. That is why it is very important to clearly define the moment in which the endometrium becomes receptive and then schedule the embryo transfer accordingly to succeed.

E.M. What was the main motivation for you to develop this test?

M.E. Even though reproductive medicine is a fascinating area that has largely progressed within the last years, its efficacy is still far from ideal. Every day we see more and more tools developed to improve cycles´ outcomes. From the genetic point of view (our background), most developments have been focused on the analysis of the embryo, but pregnancy requires a second element: the mother. We found it interesting in trying to analyze the mother from our field, and we realized there were not too many diagnostic tools for this approach.

E.M. There is many research lines trying to find out the optimal moment to perform embryo transfer. Some of them aim to investigate the molecular environment of the endometrial fluid; others focus on blood biomarkers or endometrial biopsies… One might argue blood studies may not be the best choice because certain biomarkers are specific to the endometrial fluid and absent from blood. However, you chose to study gene expression from endometrial biopsies rather than analyzing the composition of the follicular fluid. Could you tell us a bit about the reasons for this?

M.E. Well, we are geneticists, so we naturally tend to apply these techniques to our approaches. The presence of nucleic acids in the follicular fluid is scarce. Most studies on this environment are focused on the search for protein markers, but for now, we lack both the expertise and the equipment for it. However, we do not rule out the possibility of expanding our approach to other sources. In fact, we are currently participating in projects focused on the analyses of other tissues.

“Application of the ER Map® can increase pregnancy rates up to three times”.

E.M. In your test you look at 48 genes related to endometrial receptivity. Why exactly 48?

M.E. The ER Map® test analyses the expression of 40 endometrial receptivity-related genes, plus 8 additional control genes. For the development of the test, we decided to select those genes whose expression during the WOI and embryo implantation had been previously reported in the literature. This was important, since other tools had been developed from commercial microarrays containing a series of genes involved in several physiological processes, but not specifically related to the endometrium. We selected 184 genes based on scientific literature. Out of those 184, 85 displayed significant differences in their expression from donor samples collected within and out of the WOI. By applying principal component analysis (PCA), we were able to conclude that 40 out of those 85 genes were sufficient to discriminate between samples with different receptivity status.

E.M. This seems to be a significant breakthrough in the field. Are you planning on publishing your results eventually?

M.E. We have just published the details of the technical development of this tool in one of the most relevant journals in the field of human reproduction. The manuscript is already available online, so we hope this paper can be useful for scientists and clinicians interested in the field.

E.M. What are the advantages of this technique of yours to analyze gene expression?

M.E. We use high-throughput quantitative PCR (qPCR) to analyze gene expression. qPCR is currently the most sensitive method for gene expression available, and it is considered the reference methodology for this kind of analyses. In fact, qPCR is also used to confirm results on gene expression from other techniques, such as microarrays or next-generation sequencing (NGS).

E.M. What is the actual reliability of this test and what is the percentage of success?

M.E. We obtain results in 100% of the cases. Out of the 1,000 cases analyzed to date, we have found that 30% of patients with previous IVF failure presented some sort of WOI displacement. Our preliminary results indicate that application of the ER Map® to these patients significantly improves IVF outcomes. ER Map® results can increase pregnancy rates up to three times in these patients, reaching a similar value to that from patients with no endometrial receptivity issues.

E.M. Gene expression “ages” like us, too. Clinical profiles of women with endometriosis also present different transcriptomic profiles. Is this test equally reliable for all age ranges and for women with different clinical history?

M.E. That is a very interesting question. Regarding age, we have found no differences in the expression of the genes analyzed by ER Map®  between patients. Not even a trend in WOI displacement that could be related to age. This might be due to the fact that we are analyzing samples from a very specific set of women, who are infertile and with the previous history of recurrent implantation failure. This is the general clinical profile of the women referred for endometrial receptivity evaluation with ER Map®. It would be interesting to carry out this study on fertile women of different ages.

E.M. If the patient is under some sort of pharmacological treatment or suffering from any specific pathology, is there any effect on the final result of the test compared to the rest of patients?

M.E. We have no actual data on the potential effect of pharmacological treatments and/or pathologies, but it is possible that the transcriptomic profile might result altered under certain circumstances. It would be very informative to be able to conduct some research in this direction.

E.M. Once the test and the corresponding embryo transfer have been performed for a specific patient, would it be necessary to repeat it in case pregnancy is not achieved?

M.E. In principle, test results are invariable. This means that a woman diagnosed with WOI displacement in which endometrial receptivity has been confirmed at a specific moment of the cycle will benefit from the customized synchronization of the transfer. A different situation would be a patient who has suffered from any kind of event that might have affected the endometrium, like curettage or oncological treatment. We suspect such situations may alter the WOI; so, if a woman has been subjected to the ER Map® prior to the occurrence of such an event, it would be advisable to repeat the test.

“Being able to identify the very moment of optimal endometrial receptivity is essential”.

E.M. Besides the results from this test and the corresponding recommendation for embryo transfer, are there other factors to be considered when it comes to achieving pregnancy?

M.E. Correct. For pregnancy to occur there are other factors different from the endometrium. Embryo competence plays an essential role in the outcome. So, if a woman does not get pregnant through embryo transfer after ER Map®, it does not mean the test is not reliable.

It must be highlighted that, in those cases of WOI displacement for which embryo transfer has been performed following ER Map® recommendations with good quality euploid embryos, pregnancy rates after the first attempt in patients with no previous positive beta reach 80%. This is quite encouraging for us. We believe these patients would not be pregnant otherwise since embryos are being transferred when the endometrium is not ready to receive them. Therefore, being able to identify the very moment of optimal endometrial receptivity for these patients is essential.

E.M. The so-called Immunology Map® may result beneficial in some cases. When would you recommend applying the ER Map® along with this test?

M.E. The ImMap® (Immunology Map) is indicated for those patients in which it is suspected that reproduction failure is due to immunological causes. Some studies suggest that the presence of abnormal levels of immune cells in the endometrium is responsible for an abnormal response of the maternal immune system, which recognizes the embryo as a foreign element, and thus it is rejected. The combined application of both tests is recommended in those cases of implantation failure with an unknown origin so that the cause can be found out as soon as possible. In most cases, alterations are found in either test; cases of women with both WOI displacement and altered levels of endometrial immune cells are very rare, although we do have come across them.

E.M. If both tests were performed, what would be the success rate?

M.E. The application of both tests per se does not increase the success rate, but it reduces the time to obtain an answer about the cause of IVF failure. By doing so, the treatment can be more accurately defined, as well as the odds for the couple to achieve pregnancy.

Dear Embryologist Media reader, should you want to find more information on the subject, you can now read their new manuscript online following the link here.

From Embryologist Media, we would like to give special thanks to Dr María Enciso and the entire team from iGLS for sharing their knowledge and expertise on this topic.