Maria Ruiz-Alonso

The endometrial receptivity array for diagnosis and personalized embryo transfer as a treatment for patients with repeated implantation failure

OBJECTIVE To demonstrate the clinical value of the endometrial receptivity array (ERA) in patients with repeated implantation failure (RIF), for guiding their personalized embryo transfer (pET) as a novel therapeutic strategy. DESIGN Prospective interventional multicenter clinical trial. SETTING University-affiliated infertility and private clinics. PATIENT(S) Eighty-five RIF patients and 25 comparison patients. INTERVENTION(S) Endometrial sampling and pET guided by ERA. MAIN OUTCOME MEASURE(S) A receptive (R) or nonreceptive (NR) endometrial status according to ERA. Pregnancy (PR) and implantation (IR) rates after pET. RESULT(S) The ERA test gave an R result of 74.1% in RIF patients versus 88% in control subjects. Clinical follow-up was possible in 29 RIF patients, in whom pET was performed, resulting in 51.7% PR and 33.9% IR. The IRs and PRs in the 6 months after the biopsy showed that pregnancy was not related to the local injury. Twenty-two RIF patients (25.9%) were NR, and in 15 of them a second ERA validated a displacement of the window of implantation (WOI). In eight of them, pET was performed on the day designated by the ERA, resulting in 50.0% PR and 38.5% IR. These results should be considered as preliminary. CONCLUSION(S) There is an increased percentage of WOI displacement in RIF patients compared with comparison group patients, leading to the concept of pET as a therapeutic strategy. Rescue of NR patients by pET in a displaced WOI results in similar PR and IR.

The accuracy and reproducibility of the endometrial receptivity array is superior to histology as a diagnostic method for endometrial receptivity

OBJECTIVE To compare the accuracy and reproducibility of the endometrial receptivity array (ERA) versus standard histologic methods. DESIGN A comparative prospective study (May 2008-May 2012). SETTING University-affiliated infertility clinic. PATIENT(S) Eighty-six healthy oocyte donors, regularly cycling, aged 20-34 years with a body mass index (BMI) of 19-25 kg/m(2). INTERVENTION(S) Endometrial biopsies were collected throughout the menstrual cycle. For the accuracy study, 79 samples were grouped into two cohorts: the training set (n = 79) for ERA machine-learning training and dating, and a dating subset (n = 49) for comparison between histologic and ERA dating. For the reproducibility study, seven women underwent ERA testing and it was repeated in the same patients on the same day of their cycle 29-40 months later. MAIN OUTCOME MEASURE(S) Concordance of histologic and ERA dating related to LH as a reference, and interobserver variability between pathologists were statistically analyzed by the quadratic weighted Kappa index. The ERA reproducibility was tested and its gene expression visualized by principal component analysis. RESULT(S) For each pathologist, concordance against LH peak yielded values of 0.618 (0.446-0.791) and 0.685 (0.545-0.824). Interobserver variability between pathologists yielded a Kappa index of 0.622 (0.435-0.839). Concordance for ERA dating against LH peak showed a value of 0.922 (0.815-1.000). Reproducibility of the ERA test was 100% consistent. CONCLUSION(S) The ERA is more accurate than histologic dating and is a completely reproducible method for the diagnosis of endometrial dating and receptivity status.

MicroRNAs miR-30b, miR-30d, and miR-494 Regulate Human Endometrial Receptivity

MicroRNAs (miRNAs) act as important epigenetic posttranscriptional regulators of gene expression. We aimed to gain more understanding of the complex gene expression regulation of endometrial receptivity by analyzing miRNA signatures of fertile human endometria. We set up to analyze miRNA signatures of receptive (LH + 7, n = 4) versus prereceptive (LH + 2, n = 5) endometrium from healthy fertile women. We found hsa-miR-30b and hsa-miR-30d to be significantly upregulated, and hsa-miR-494 and hsa-miR-923 to be downregulated in receptive endometrium. Three algorithms (miRanda, PicTar, and TargetScan) were used for target gene prediction. Functional analyses of the targets using Ingenuity Pathways Analysis and The Database for Annotation, Visualization and Integrated Discovery indicated roles in transcription, cell proliferation and apoptosis, and significant involvement in several relevant pathways, such as axon guidance, Wnt/β-catenin, ERK/MAPK, transforming growth factor β (TGF-β), p53 and leukocyte extravasation. Comparison of predicted miRNA target genes and our previous messenger RNA microarray data resulted in a list of 12 genes, including CAST, CFTR, FGFR2, and LIF that could serve as a panel of genes important for endometrial receptivity. In conclusion, we suggest that a subset of miRNAs and their target genes may play important roles in endometrial receptivity.

Profiling the gene signature of endometrial receptivity: clinical results

This article highlights the need for methods to objectively diagnose endometrial receptivity as a factor contributing to infertility in female patients. The correct identification of the appropriate window of implantation in a given patient, by using endometrial receptivity biomarkers, can help to prevent reproductive failure resulting from misplaced timing of the endometrial window of implantation (WOI). Although to date no single, clinically relevant morphologic, molecular, or histologic marker capable of indicating endometrial receptivity status has been identified, global transcriptomic analysis of human endometria performed in the last decade has given us insights into a genomic signature that is capable of identifying endometrial receptivity. As a consequence, a genomic tool named the Endometrial Receptivity Array (ERA), based on a customized microarray, was developed, and along with it a specially trained bioinformatic prediction computer algorithm was created to identify WOI timing in the endometrium. This tool has proven more accurate and consistent than histologic (Noyes) dating at identifying the personalized WOI day, thus leading to the new clinical concept of personalized ET on the optimum day of endometrial receptivity, identified individually case by case.

The genomics of the human endometrium

The endometrium is a complex tissue that lines the inside of the endometrial cavity. The gene expression of the different endometrial cell types is regulated by ovarian steroids and paracrine-secreted molecules from neighbouring cells. Due to this regulation, the endometrium goes through cyclic modifications which can be divided simply into the proliferative phase, the secretory phase and the menstrual phase. Successful embryo implantation depends on three factors: embryo quality, the endometriums state of receptivity, and a synchronised dialogue between the maternal tissue and the blastocyst. There is a need to characterise the endometriums state of receptivity in order to prevent reproductive failure. No single molecular or histological marker for this status has yet been found. Here, we review the global transcriptomic analyses performed in the last decade on a normal human endometrium. These studies provide us with a clue about what global gene expression can be expected for a non-pathological endometrium. These studies have shown endometrial phase-specific transcriptomic profiles and common temporal gene expression patterns. We summarise the biological processes and genes regulated in the different phases of natural cycles and present other works on different conditions as well as a receptivity diagnostic tool based on a specific gene set profile. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

What a difference two days make: “personalized” embryo transfer (pET) paradigm: A case report and pilot study

Embryo implantation requires that the blastocyst will attach during the receptive stage of the endometrium, known as window of implantation (WOI). Historically, it has been assumed that the WOI is always constant in all women. However, molecular analyses of endometrial receptivity demonstrates a personalized WOI (pWOI) that is displaced in one out of four patients suffering from recurrent implantation failure (RIF) of endometrial origin and illustrates the utility of a personalized endometrial diagnostic approach. Here, we report a clinical case of successful personalized embryo transfer (pET) after four IVF and three oocyte donation failed attempts in which different embryo transfer strategies were attempted. This case report is complemented by a pilot study of 17 patients undergoing oocyte donation and who suffered failed implantations with routine embryo transfer (ET) but were then treated with pET after the personalized diagnosis of their WOI.

Is endometrial receptivity transcriptomics affected in women with endometriosis? A pilot study

Endometrial receptivity is still questioned today in women with endometriosis. The aim of this study was to assess the endometrial receptivity gene signature in patients with different stages of endometriosis by investigating transcriptomic modifications of their endometrium using the endometrial receptivity array (ERA) test. A prospective, interventional multicentre pilot trial was designed and implemented in two university-affiliated infertility units from Belgium and Spain. Gene expression microarray was used to diagnose the receptivity status by quantifying the expression of 238 specific genes directly related to human endometrial receptivity. Unsupervised hierarchical clustering showed no clustering of samples based on endometriosis stages. Two subgroups of samples clustered together corresponding on the day of the cycle in which the biopsy was taken (day 18 versus days 19-20). None of the 238 genes present in the ERA array were significantly over- or under- expressed in any of different stages of the disease compared with controls. Minimal differences were found when looking at the functional profile, suggesting that the possible effect from a clinical point of view may be meaningless. Endometrial receptivity gene signature during the implantation window does not vary significantly among patients with endometriosis even considering different stages compared with healthy women.

Transcriptomics of the human endometrium.

During the mid-secretory phase, the endometrium acquires the receptive phenotype, which corresponds to the only period throughout the endometrial cycle in which embryo implantation is viable. Endometrial receptivity is a crucial process and even more important in Assisted Reproductive Technologies (ART) where embryo-endometrial synchronization is coordinated through embryo transfer timing. Over the last decade, transcriptomic analyses performed on the human endometrium have shown that specific genomic signatures can be used to successfully phenotype different phases of the menstrual cycle including the receptive stage, independently of the histological appereance of the endometrial tissue. In this paper, we review current evidence demonstrating that endometrial transcriptomics objectively identifies the implantation window in a personalized manner, opening the field for the diagnosis of the endometrial factor in ART and moving to stratified medicine at this level, using microarray technology and soon high-throughput next generation sequencing coupled with functional and systems genomics approach.

Clinical Management of Endometrial Receptivity

The endometrial window of implantation (WOI), the cycle days during which normal embryo implantation can occur, has generally been assumed to begin on cycle day 19 or 20 of an idealized 28 days cycle and last for 4 to 5 days. Noyes et al took the first steps in defining the WOI by establishing a set of morphological criteria to evaluate endometrial development and receptivity, but recent studies have invalidated their use in the routine evaluation of infertility. Based on greater than 10 years of extensive research, our group has developed a molecular diagnostic tool (the endometrial receptivity array [ERA] test) based on the specific transcriptomic signature that identifies the receptive endometrium in natural and artificial (hormonal replacement therapy) cycles. The ERA test has shown that some patients have a delayed WOI, others have an advanced WOI, and others can have unusually short windows of receptivity. This identification and characterization of the WOI allows the personalization of the embryo transfer. In this review, we describe the ERA and our experience with its use in assessment of the endometrial receptivity in patients undergoing assisted reproduction.

The impact of using the combined oral contraceptive pill for cycle scheduling on gene expression related to endometrial receptivity

STUDY QUESTION Does the combined oral contraceptive pill (COCP) change endometrial gene expression when used for cycle programming? SUMMARY ANSWER COCP used for scheduling purposes does not have a significant impact on endometrial gene expression related to endometrial receptivity. WHAT IS KNOWN ALREADY Controversy exists around COCP pretreatment for IVF cycle programming, as some authors claim that it might be detrimental to the live birth rate. Microarray technology applied to the study of tissue gene expression has previously revealed the behavior of genes related to endometrial receptivity under different conditions. STUDY DESIGN, SIZE, AND DURATION Proof-of-concept study of 10 young healthy oocyte donors undergoing controlled ovarian stimulation (COS) recruited between June 2012 and February 2013. PARTICIPANTS/MATERIALS, SETTING, AND METHODS Microarray data were obtained from endometrial biopsies from 10 young healthy oocyte donors undergoing COS with GnRH antagonists and recombinant FSH. In group A (n = 5), COCP pretreatment was used for 12-16 days, and stimulation began after a 5-day pill-free interval. Stimulation in group B (n = 5) was initiated on cycle day 3 after a spontaneous menses. Endometrial biopsies were collected 7 days after triggering with hCG. MAIN RESULTS AND THE ROLE OF CHANCE No individual genes exhibited increased or decreased expression (fold change (FC) >2) in patients with prior COCP treatment (group A) compared with controls (group B). However, the results of the functional analysis showed a total of 11 biological processes that were significantly enriched in group A compared with group B (non-COCP). LIMITATIONS, REASONS FOR CAUTION The Endometrial Receptivity Array (ERA) has only been validated on endometrial samples obtained in natural cycles and after hormonal replacement treatment (HRT). Therefore, it was not possible in this study to classify the endometrial samples as receptive or non-receptive. We used the ERA to focus on 238 genes that are intimately related to endometrial receptivity, thus simplifying the analysis and understanding of the data. WIDER IMPLICATIONS OF THE FINDINGS Cycle scheduling is common in IVF units and is used to avoid weekend retrievals and/or to distribute evenly the workload for better efficiency. Our failure to detect any relevant changes in the genes related to the window of implantation when cycles were programmed with COCP pretreatment suggests that, despite controversial clinical results in previous studies, the use of COCPs in this way does not affect uterine receptivity adversely. STUDY FUNDING/COMPETING INTEREST(S) Funding for this study was provided by an unrestricted grant from Merck Sharp & Dohme. C.S. and A.P. are co-inventors (with Patricia Diaz-Gimeno) of the Endometrial Receptivity Array and hold the patent. The other authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER EudraCT registration number is 2011-003250-34.