The endometrial receptivity array test (ERA) for personalization of embryo transfer (pet)

Transcription

1 mt Médecine de la Reproduction, Gynécologie Endocrinologie 2013 ; 15 (3) : The endometrial receptivity array test (ERA) for personalization of embryo transfer (pet) doi: /mte David Blesa 1,2,a Maria Ruiz-Alonso 2,a Carlos Simon 1,2 1 Fundación Instituto Valenciano de Infertilidad (FIVI), and Instituto Universitario IVI/ Incliva, Valencia University. Parc Cientific Universitat de Valéncia. C/Catedrático Agustín Escardino n 9, Edificio 3, Paterna (Valencia), Spain 2 Iviomics S.L. Parc Cientific Universitat de Valéncia. C/Catedrático Agustín Escardino n 9, Edificio 3, Paterna (Valencia), Spain <david.blesa@ivi.es> <maria.ruiz@iviomics.com> <carlos.simon@ivi.es> a These authors contributed equally to this work médecine thérapeutique Reprints : C. Simon Médecine de la Reproduction Gynécologie Endocrinologie Abstract. The endometrium is a factor that has been neglected in the research efforts to improve implantation rates. Here, we summarize previous studies on biomarkers of receptivity and review our publications on the development of the ERA test. This test is based in a customized gene expression microarray, that contains 238 genes differentially expressed in the receptive endometrium, coupled to a computational predictor, trained with a set of menstrual cycle phase controlled samples, that is able to diagnose whether the endometrium of a patient sampled at the assumed WOI is receptive or not, regardless of its histological appearance. The analysis of the transcriptomic profile allows us to suggest a personalized WOI that can be validated with a second ERA test and used for personalized embryo transfer. Accuracy and reproducibility of the test have been analyzed and its clinical application has been tested on recurrent implantation failure patients. Key words: endometrial receptivity, diagnostic test The endometrium and endometrial receptivity The endometrium is the tissue that lines the inside of the endometrial cavity. Morphologically it is divided into the functional and the basal layers. The functional layer presents different cellular compartments: the luminal and glandular epitheliums, the stroma and vascular compartments. The epithelium is composed of epithelial cells on the surface or coating epithelial glands; the stroma consists in an extracellular matrix (ECM) and fibroblasts that differentiate during the decidualization process. In the stroma, blood vessels (spiral arteries) are also present, along with immune resident cells such as macrophages and NK cells [1-3]. The functional layer is responsible for proliferation, secretion and tissue degeneration, while the regenerative capacity of this organ lies in the basal layer [2-4]. The gene expression of the different endometrial cell types is regulated by ovarian steroids and paracrine-secreted molecules from neighboring cells. Due to this regulation, the endometrium goes through cyclic modifications which compose the endometrial cycle. An endometrial cycle can be divided simply into the proliferative phase, corresponding to the follicular phase in the ovary, the secretory phase, corresponding to the luteal phase in the ovary, and the menstrual phase. The first day of the endometrial cycle corresponds to the beginning of the menstrual phase (the first day of menstrual bleeding) and desquamation of the functional layer takes place during this phase. From the menstrual phase to ovulation, the proliferative phase takes place and, at this time, estrogen levels begin to rise, leading to not only a proliferation of stromal cells and glands, but to the elongation of spiral arteries. Finally, the secretory phase is defined as the time between ovulation and menstruation. The beginning of this phase is characterized by a rise in progesterone levels [4]. In the secretory phase the endometrium acquires a receptive phenotype which allows the implantation of the blastocyst. This period of receptivity is known as the window of implantation (WOI). It is generally accepted that the WOI opens on day 19 or 20 of the cycle, and lasts 4-5 days [5]. If implantation does not occur, the progesterone and To cite this article: Blesa D, Ruiz-Alonso M, Simón C. The endometrial receptivity array test (ERA) for personalization of embryo transfer (pet). mt Médecine de la Reproduction, Gynécologie Endocrinologie 2013; 15 (3): doi: /mte

2 estrogen levels decrease, accompanying a vasoconstriction of the spiral arteries and leading to involution of the endometrium; then the cycle starts again [4]. Historical methods for identifying receptivity Endometrial receptivity markers are all those parameters or characteristics that can allow to identifying a receptive endometrium [6]. These markers can be used to date the endometrium receptive phase and evaluate the status of endometrial receptivity. There are different types of markers, morphological markers based on histology, classic molecular markers as single genes or molecules, and omic molecular markers when a whole kind of molecules characterize the biological process of receptivity [7]. Changes in endometrial tissue morphology have been described by optical microscopy studies of histological sections of the endometrium. These are obtained by paraffin embedding and staining with hematoxylin-eosin the endometrial tissue. During the luteal phase of the natural cycle morphological changes in the endometrium can be observed that, day by day, allow for dating the status of the endometrium. This method was developed by Noyes according to criteria that were obtained from 8,000 endometrial biopsies from infertile patients [8, 9]. Noyes criteria considered eight basic histology features of the endometrium: glandular mitosis degree, nuclear pseudo stratification, basal vacuoles, secretion, stromal edema, pseudodecidual reaction, stromal mitosis and leukocyte infiltration. The accuracy of this method depend on multiple factors. These factors included the method for determining ovulation, the biopsy sampling, the quality of the biopsy sample, fixation techniques and interpretation of the pathologist. Accuracy and functional relevance of this method, based exclusively on histological observations performed by one or several pathologists, has been questioned both in retrospective clinical studies [10-12], prospective studies [13-15], as well as in randomized controlled trials [16, 17]. Many other variables have been identified to determine the endometrial receptivity trying to correlate with the degree of pregnancy rates. Some were related to the thickness and endometrial echo pattern or the endometrial perfusion. Soares et al., after analyzing a total of 3,089 cycles, found no statistical difference between endometrial thickness and pregnancy rate [18]. Until recently there have been two tests available for molecular endometrial dating, based on the analysis of a molecule or a group of them, the E-tegrity Test using antibodies to 3 integrin [19] and the EFT Quick Guide, that evaluates the expression of cyclins throughout the menstrual cycle [20]. However, these methods have not been able to replace molecular histological method based on the morphological criteria of Noyes, not being used routinely for endometrial evaluation. Development of the ERA test The transition from anatomical to molecular medicine has led to the genome-wide screening for all transcribed genes and the analysis of their interactions [21-24]. Evidence from the past decade on endometrial gene expression profile research suggests that it is possible to accurately catalogue and diagnose the human endometrial cycle and specifically its receptive status based on its transcriptomic profile, for a review see [25]. The gene signature of human endometrial receptivity has been investigated in natural cycles [24, 26-33] and in the refractory endometrium induced by the insertion of an intra-uterine device (IUD) in otherwise fertile patients [34]. On these grounds, our group developed a molecular diagnostic tool [35] based on the specific transcriptomic signature that identifies the receptive endometrium at LH+7 in a natural cycle or at day 5 of progesterone (P+5) after proper estradiol priming in a hormonal replacement therapy (HRT) cycle. This test was named the endometrial receptivity array (ERA) and consists of a customized array containing 238 differentially expressed genes that are coupled to a computational predictor which is able to identify endometrial samples that are within the window of implantation regardless of their histological appearance. The gene set used in this test was obtained by selecting those genes whose expression was consistent among three different models of endometrial receptivity: the natural cycle as the optimal model, the controlled ovarian stimulated cycle as suboptimal, and the refractory endometrium as a negative control [35, 36]. The endometrial samples for the ERA gene selection (n = 20, gene selection set) were a pre-receptive group LH+1, LH+3, LH+5, and a receptive group LH+7 of samples, five in each LH day. The LH measurement was performed by assessing the serum LH levels on a daily basis and endometrial biopsies were collected as previously described [33]. Endometrial samples to train the predictor to date the endometrium and to define the transcriptomic signature (n = 68, training set) were obtained from fertile women during their menstrual cycle, and were classified as receptive (R), pre-receptive (PR), or proliferative (P). Group R was represented by those samples taken on day LH+7 (days of the menstrual cycle, n = 40); in the PR group, samples were collected on days LH+1 to LH+5 (n = 13); the P group samples were collected on days 8 12 of the menstrual cycle (n = 15). The best result for prediction in all cases was obtained with a SVM algorithm. 250

3 The results for the endometrial dating indicated an ACC of 95% and an AUC of 0.94, with a specificity and sensitivity of and , respectively. unlike histological data, transcriptomic signature variations between cycles, even 3 years later, are quite rare [37]. Accuracy and reproducibility of the ERA test In order to identify the accuracy of the test, a second training set, composed of 79 endometrial biopsies, was analyzed by the ERA microarray and used to train the machine-learning algorithms [37]. Samples were collected throughout the menstrual cycle and classified into four different classes: receptive (R) (n=39) was formed by samples obtained at day LH+7; pre-receptive (PR) (n=14) obtained from days LH+1 to LH+4; proliferative (PF) group (n=14) which includes samples collected on days 8 to 12 of the menstrual cycle, and the post-secretory group (n=12) consisting of samples from LH+11 to LH+13. The dating set was formed by a subset of the training set samples for comparison between histological and ERA dating. It consisted of 49 endometrial biopsies divided as follows: group R (n=24), PR (n=8), PF (n=6) and the PS group (n=11). The reproducibility set was composed of a total of 7 endometrial samples obtained at LH+7 (R) (n= 4), and 3 outside the WOI (PF n=2; PS n=1), which were diagnosed with the ERA as receptive or non-receptive and analyzed again from the same patients, at the same day of their cycle, between 29 to 40 months later. Our histological study involved the evaluation of 20 distinct histological features, each of which was scored by two pathologists using criteria established by consensus before the study was conducted. Comparison between the histological dating performed by each pathologist against a LH peak reference yielded the Kappa index values of ( ) and ( ), respectively. Interobserver variability, defined as the concordance between both pathologists, was ( ). The algorithm used in the ERA test, showed a Kappa index of ( ) when compared to the LH peak [37]. This shown that the ERA predictor dating was clearly superior to that of the pathologists in the receptive, pre-receptive and post-receptive phases. Histological endometrial dating outperformed the ERA predictor only in the proliferative phase. Regarding the reproducibility of the test, a second endometrial biopsy was obtained from 7 women from the training set on the same day of their menstrual cycle between 29 to 40 months after the first study cycle. The inter-cycle variation observed in our study population showed total consistency in the ERA test diagnosis between the first and second analysis in the same patient with the same diagnostic probability. A principal component analysis showed that pairs of samples cluster consistently closer to each other suggesting that, Clinical application of ERA test in patients with implantation failure Repeated implantation failure (RIF) is an unaddressed major cause of infertility in otherwise healthy women, and one which has remained poorly characterized [38, 39]. Although various definitions of RIF exist [40], it is agreed that after the failure of three IVF cycles in which one or two morphologically high-grade embryos were transferred, special protocols must be enforced. The clinical diagnosis of endometrial receptivity as the temporal window of opportunity in which the endometrial epithelium becomes adhesive to the blastocyst remains uncertain and subjective [16, 17]. In fact, this is the main reason why the endometrial factor, in terms of its receptivity status, is not investigated during the infertility work-up: it is assumed that the WOI is constant in time in all women, including RIF patients. Evidence indicates that there is an endometrial receptivity alteration in patients with RIF. Classical morphometric analysis revealed that women with RIF undergoing insemination have retarded endometria in relation to their cyclical timing [41]. More recently, genomic studies have demonstrated the deregulation of gene expression cycle in RIF versus fertile women [42, 43]. These data suggest the hypothesis that transcriptomic modification of the endometrium occurs in RIF patients during the WOI which could be due to a displacement of the WOI and/or pathological alteration. To demonstrate the clinical value of the endometrial receptivity array (ERA) in patients with repetitive implantation failure (RIF), guiding their personalized embryo transfer (pet) as a novel therapeutic strategy, a prospective interventional multicentre clinical trial was performed analyzing 85 RIF patients and 25 comparison patients [44]. In this study, we identified the receptive (R) or nonreceptive (NR) endometrial status according to the ERA test and pregnancy (PR) and implantation rates (IR) after pet were measured. We designated RIF in IVF patients who were 40 years old or younger, or in ovum donation (OD) patients who were 51 years old or younger, that underwent three or more previous failed cycles in which no less than four morphologically high-grade embryos were transferred in total, and in which there was no other explanation for repeated implantation failures after a through infertility work-up. The comparison group was composed of IVF and OD patients with the same age inclusion criteria, undergoing treatment within the same time period as the RIF patients included in this study, but who had had one or no previous failed cycles [44]. 251

4 The ERA test gave an R result in 74.1% RIF patients versus 88% in controls showing that RIF patients have higher proportion of patients with a displacement of their WOI. The proportion of RIF patients undergoing IVF versus OD was 61/39 versus 40/60 in the comparison group patients which indicates that most implantation failure problems related to the embryo have been solved. The proportion of R/NR results in OD and IVF patients seemed to be similar in the comparison group (87/13 and 90/10 respectively), but was different in the RIF group, which had a high percentage of NR results in the OD group compared to the IVF group (67/33 and 79/21 respectively). Endometrial samples classified by the ERA predictor as non-receptive were pre-receptive (n = 21; 84%) or postreceptive (n=4; 16%). In these patients, a second ERA test was suggested to confirm the suspected displacement of the WOI and it was performed in 18 cases. In ERA prereceptive results, the second biopsy was recommended on LH+9 in natural cycles or P+6 or P+7 for HRT cycles, depending on the specific expression profile of each sample. In ERA post-receptive results, the second test was recommended on P+4 or P+3. The results for these second biopsies were receptive in 15 of them whereas in 3 of them anon-receptive profile remain and further refinement was necessary. Clinical follow-up was possible in 8 patients on whom an embryo transfer at their personalized WOI (pet) was performed: if delayed, in P+7 (n = 5) or P+6 (n = 2); and if advanced, in P+4 (n = 1). Day three embryos were transferred with this strategy in HRT cycles after four or five days of progesterone administration, or day five blastocysts were transferred in HRT cycles after 4, 6, or 7 days of progesterone administration resulting in a 50.0% PR, and a 38.5% IR [44]. We showed that there is an increased percentage of WOI displacement in RIF patients compared to comparison group patients, leading to the concept of pet as a therapeutic strategy. Rescue of NR patients by pet in a displaced WOI resulted in comparable PR and IR. A revised understanding of implantation timing and personalization of embryo transfer Today, is embryo development timing the factor which guides the moment of the embryo transfer in ART. It is generally accepted that the window of implantation is open in every woman around day 21 to 23 of their menstrual cycle and that it last for three to four days. The research carried out by our group in this field for the last ten years has shown that it is possible to identify the status of the endometrium using the transcriptomic profile of a selected group of genes. The model and the diagnostic test we have developed based in this approach has a very high specificity and sensitivity in detecting the receptive phase of the endometrium. Noyes criteria methods to analyze the phase of the menstrual cycle, or other tests based on molecular approaches, have not been widely used or its functional relevance questioned in retrospective, prospective or randomized controlled trials. We demonstrated that the accuracy of the ERA test for dating the endometrium is much higher than pathologists using Noyes criteria and the test results are reproducible even on samples taken 30 to 40 months apart. The analyses of expression profiles of patient samples have taught us that a large proportion of RIF patients have an out-of-phase endometrium at the moment that they should be at the WOI. We argue that this can be the cause of their RIF because when these patients have embryo transfer at a personalized WOI (pet), guided by the ERA test results, they get pregnant at normal rates. But interestingly, also a noticeable proportion of first visit patients have an out-of-phase endometrium in which no embryo implantation is expected. We have also learned that non-receptive results correspond to endometrial samples that have a delayed or advanced development. We have proved that sampling the same woman a few days (or even hours) sooner or later than the first non-receptive ERA test can be sufficient to find her personal WOI. All this is telling us that some women do not respond normally to the factors that regulate the development of the endometrium. This can make them infertile because the necessary synchronization between the embryo and endometrium development has been lost. In a similar manner, patients will not respond equally to the hormonal treatments used for ART and, for some of them, their endometrium will not be prepared when the embryo transfer is scheduled. Conclusion Acknowledging the need for an objective, reliable, and reproducible diagnostic tool for assessing endometrial status in the work-up for infertility patients, our group has developed the ERA test. This test is based in a customized gene expression microarray, that contains 238 genes differentially expressed in the receptive endometrium, coupled to a computational predictor, trained with a set of menstrual cycle phase controlled samples, that is able to diagnose whether the endometrium of a patient sampled at the assumed WOI is receptive or not, regardless of its histological appearance. The analysis of the transcriptomic profile allows us to suggest a personalized WOI that can be validated with a second ERA test and used for personalized embryo transfer. Conflicts of interest : Carlos Simón is co-inventor of the ERA patent. All authors work part or full time for IVIOMICS which commercializes the ERA test. 252

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