Endometrial receptivity array: Clinical application.

To identify the contribution of the endometrial factor in RIF To see if PR improved after using the personalized WOI To find out if patients with persistently thin endometrium have a defective WOI. To identify the contribution of the endometrial factor in RIF To see if PR improved after using the personalized WOI To find out if patients with persistently thin endometrium have a defective WOI.

A retrospective analysis of data. Primary end point of the study for group I and II were ongoing PR (OPR). Ongoing pregnancy was defined as pregnancy over 12 weeks. Primary end point for group III was percentage of nonreceptive to receptive endometrium.

Human implantation is a highly complex and multifactorial process. Successful implantation at the very least, requires the presence of a healthy embryo, a receptive endometrium, a synchronized and successful molecular dialogue between the two and immune protection from the host. Due to lack of objective and accurate methods of assessment, endometrial receptivity (ER) is rarely investigated in an infertile patient or even prior to in-vitro fertilization (IVF). Advent of “Omics” that is, the analysis of biological samples using molecular profiling has revived interest in the study of ER particularly in the context of implantation failure (IF) in IVF. The human endometrium is a dynamic tissue; it undergoes changes at multiple levels during the menstrual cycle in response to ovarian hormones and paracrine secretions. The endocrine and paracrine secretions control gene expression of the different endometrial cell types.[ 1 ] The proliferative phase, controlled by estrogen allows for the proliferation of stromal cells and glands and elongation of the spiral arteries. The postovulatory progesterone (P) rise brings about secretory changes and the endometrium acquires a receptive phenotype permitting implantation of the blastocyst. This period of receptivity is known as the “window of implantation” (WOI). The WOI opens on day 19 or 20 of the cycle and remains open for just 4–5 days at the time whenP reaches peak serum concentrations.[ 2 ] During the phase of receptivity, the endometrium undergoes morphological, cytoskeletal, biochemical, and genetic changes to become functionally competent. The ability to identify the endometrial WOI in the clinical setting would enhance the outcome of fertility treatments such as IVF.

Diagnosis of ER has posed a challenge because of the lack of an accurate, noninvasive, and clinically applicable test. Histological, biochemical, and ultrasound markers of ER have been proposed for use to improve implantation rates (IRs) in IVF. Unfortunately, most of these methods are invasive and none have any predictive value. Though limited in value, ultrasound markers of ER are used in the clinical setting. ER is assessed on the basis of endometrial thickness, character, volume, and blood flow patterns.[ 3 4 ]

ERA was non-receptive in 23% (3/13) and receptive in 77% (10/13). The proportion of receptive to NR endometrium was similar to group II ( P = 0.4). Nine patients (69%) went for BET. The overall OPR was 66.7% (6/9). In patients where 1 st ERA was receptive, the OPR was 57.1% (4/7) and in NR group after pET PR was 100% (2/2). There were no miscarriages in this group. Thin endometrium has long been associated with IF. It was reassuring to know that despite the reduced endometrial thickness the changed WOI occurred in the same proportion of patients as the non-RIF group. The knowledge that an embryo is being transferred into a receptive environment in such patients lends comfort to the patient and the physician [ Table 4 ]. Thin endometrium group III

The various molecular approaches for the study of biological samples are collectively called the “Omics” and include - genomics (study of genes), epigenomics (study of epigenetic DNA modifications), transcriptomics (study of gene expression), proteomics (quantification of proteins), metabolomics and lipidomics (composition and quantification of metabolites and lipids). Currently, transcriptomics is considered the most established technology available for evaluation of the endometrial factor.

ERA is the most objective and accurate test available today for diagnosing ER. It has been used to define an altered WOI, and thus establish a personalized WOI for each patient. It has shown benefit in improving reproductive performance in patients with RIF. However, more studies are required to confirm these initial findings. It is limited by its invasive nature and associated costs. Nil. There are no conflicts of interest.

The introduction of microarray technology has enabled rapid progress in the understanding of many biological functions and disease processes computing Omics with bioinformatic predictors has improved the diagnosis and subsequent treatment in diseases such as cancer.[ 35 ] Success in this area coupled with identification of the transcriptomics of the receptive endometrium during natural and stimulated cycles,[ 36 ] led to the development of a molecular diagnostic test to identify the WOI – ERA.[ 27 ] In the era of personalized medicine, a “one size fits all” policy is no longer acceptable. In IVF individualized ovarian stimulation, protocols are being promoted to optimize treatment. So far, for lack of an objective and accurate test ER remained a gray area. ERA is a step forward in improving IVF results through identification of the WOI and personalizing embryo transfer. The test has been shown to be accurate and reproducible and does not have the limitation of inter cycle variability. The clinical application of the test has been applied only in some clinics, and hence larger studies are required to validate it. In our study, we found that 27.5% women with RIF showed a displaced WOI. The paper by Ruiz-Alonso et al . 2013[ 31 ] suggested an increased percentage of NR endometrium in the RIF group, but this did not reach statistical significance. Our results, however, showed that this difference was statistically significant. A larger number of women in the RIF group had a NR endometrium. The PR, OPR, and IR in the RIF group improved after pET with values similar to those seen in our fresh non-RIF IVF cycles. In the sub-group analysis done on 33 RIF patients wherein 1 FET with the transfer of 2 good quality blastocysts had been done at our center, we found that one-third of the patients had a NR endometrium. Personalized ET improved the reproductive outcome in these patients as also patients with 1 previous IVF failure. Another difficult group is the women with thin endometrium as thin endometrium is associated with poor implantation. It was reassuring to know that the proportion of nonreceptive endometrium was not more than our 1 IVF failure group and that after pET the PR and IRs were also similar to that group. Though we saw an improvement of results, the numbers in our study are not high enough to draw definite conclusions. What then is the place of ERA in the treatment of infertile patients given the existing knowledge? In the current setting, ERA is the only test available that can determine ER with accuracy. Since it is reproducible and does not change over a long period of time (1–2 years), it need not be repeated in the event of a delay in treatment. In the clinical setting, ERA definitely has a place in RIF where endometrial factor could be the contributory cause in a quarter of the patients. In women with even 1 IVF-OD failure with the transfer of 2 good quality embryos, it is advisable to rule out an altered WOI. Defining a receptive window would avoid embryo wastage and emotional, physical, and financial distress. Its use in patients with adenomyosis, endometriosis, and chronic endometritis can prove beneficial, as these conditions are associated with an altered ER. Persistent thin or thick endometrium is also an indication for carrying out ERA. ERA is a valuable addition to our diagnostic armamentarium. The invasive nature of the test, the need for embryo vitrification and cost are some of its limitations. Much of the implantation process still remains to be unraveled. It has to be remembered that the embryo remains a major player in this equation and genetic testing of the embryo with array comparative genomic hybridization has shown improved IRs.[ 37 ] However, there are no reports suggesting a 100% success even after doing a pET with a euploid embryo. Maternal factors especially the immune system involvement needs to be understood.

A number of molecules present during the mid-secretory phase have been studied as markers of ER. The ones which have shown significant association with the WOI are the integrins, leukemia inhibitory factor, homeobox A10, mucin 1, calcitonin, and cyclo-oxygenase 2.[ 11 ] Many more are being investigated; however, none have found their place in the clinical setting.

The endometrial factor was studied in patients undergoing IVF treatment at our center once the test became available in India. We performed ERA in patients with RIF, patients with 1 previous IVF failure and patients with persistent thin endometrium <6 mm. RIF was defined as failure of implantation after two consecutive cycles of IVF, ICSI, frozen embryo transfer (FET), IVF-OD, where the cumulative number of transferred embryos was number less than 4 for cleavage stage embryos and number less than 2 for blastocyst. With all embryos being of good quality and of appropriate developmental stage.[ 34 ]

Noyes et al .[ 5 ] established morphological criteria to evaluate endometrial development and receptivity. For many decades, these criteria remained the mainstay for defining ER despite the huge inter and intra-cycle variability. The use of Noyes criteria to predict the WOI accurately has been questioned in recent years and randomized studies[ 6 7 8 ] have invalidated their use. Endometrial pinopods identified on electron microscopy also generated interest as a marker of ER.[ 9 ] Pinopods are cytoplasmic projections of the luminal epithelial cells, abundant during the WOI, thought to promote blastocyst adhesion. The presence of pinopods was demonstrated in post receptive endometrium, and this precluded their use as a useful marker of ER.[ 10 ]

DNA microarray technology, allows measurement of thousands of genes simultaneously, and its use to study the level of gene transcription in tissues has revolutionized medicine. The transcriptome reflects genes that are actively expressed at any given time within a specific cell population or tissue. Transcriptomics allows characterization of gene expression at the mRNA level, giving rise to a “sample-specific” molecular profile or its “Transcriptomic Signature.” This permits characterization of tissue function or disease phenotype.[ 12 ] Analysis of transcriptomic profiles or signatures is done using exploratory methods and statistical tests. The exploratory methods used are trees, clustering, or principal component analysis (PCA). PCA is a statistical method that allows projecting higher-dimensional data onto a lower-dimensional space. When PCA is applied to data, samples with similar trends in their gene expression profiles tend to cluster close together in the plot[ 13 ] [ Figure 1 ]. Hierarchical clustering with visual heat map representation and PCA's are the most widely used methods. Specialized software is used to analyze the microarray data. Visual heat map representations show the differential expression patterns between different samples and genes.[ 14 ] For endometrial dating, the phase assignment is made based on previous histological dating by at least two independent pathologists.[ 15 ] Principal component analysis of human endometrium throughout the development of the luteal phase in natural (LH + n) and controlled ovarian stimulation cycles (human chorionic gonadotropin + n). Reprinted with permission Horjcadas et al . J Clin Endocrinol Metab, November 2008, 93(11):4500-10 The transcriptome of the endometrium has been defined in all phases of the menstrual cycle;[ 16 17 ] clustering of genes into four groups was identified and these were consistent with histological phenotypes of proliferative (PE), early secretory (ESE), mid-secretory (MSE), and late-secretory (LSE) phases. The human endometrial transcriptome has been studied in women with a gynecological disease such as endometriosis and cancer.[ 18 19 ] Studies on the transcriptomics of human ER also gained impetus and ER transcriptome was studied in the natural cycle[ 20 ] and recurrent implantation failure (RIF).[ 21 22 ] Gene expression patterns have also been defined during controlled ovarian stimulation (COS) and hormonal replacement therapy (HRT) cycles.[ 23 24 ] Haouzi et al . 2010[ 25 ] compared the gene expression profile of ER between natural and stimulated cycles for the same patients. They observed that the gene transcription profile of the endometrial WOI under COS was defective for biological functions such as transforming growth factor beta signaling, leukocyte transendothelial migration, and the cell cycle. Extensive research on endometrial transcriptomics has allowed a provisional definition of the genomic signature of human ER and an understanding of alterations in the endometrium and ER that are associated with gynecological pathology and infertility. At a molecular level, the prereceptive/early secretory phase is characterized by increased metabolic activity in preparation for implantation. This leads to a predominance of products related to cell metabolism (fatty acids, lipids, eicosanoids, and amino alcohols), transport, and germ cell migration.[ 14 ] There is an inhibition of mitosis during this phase as suggested by the downregulation of several growth factors.[ 17 ] The receptive phase witnesses a “transcriptional awakening” or upregulation of most gene expression. Apart from a high level of metabolic and secretory activity there is also an upregulation of genes involved in the activation of the immune response.[ 17 ] During the late-secretory phase, the WOI closes and in this phase genes related to immune response-both cellular and humoral, blood coagulation, steroid bio-synthesis, and prostaglandin metabolism are regulated[ 26 ] [ Figure 2 ]. Evolution of endometrial tissue over time and the gene expression profile at each given stage. Heat map showing ERA gene expression profiles in each endometrial cycle stage (PE, prereceptive, receptive, and postreceptive) and the major biological functions regulated in each of these phases. Reprinted with permission from Garrido-Goˇmez. Genomics of endometrial receptivity. Fertil Steril 2013

This retrospective study examined 186 infertile women who underwent ER array (ERA) from August 2013 to November 2014. Patient population was divided into three groups. Group I - 80 patients with RIF, Group II - 93 patients with one IVF failed, and Group III - 13 patients with thin endometrium (≤6 mm). Group I - 80 patients with RIF, Group II - 93 patients with one IVF failed, and Group III - 13 patients with thin endometrium (≤6 mm). Patients were in the age group of 25–46 years (34.8 ± 4.8) in group I, 24–46 years (33.3 ± 4) in group II, and 26–40 years (33.4 ± 4.5) in group III. The average body mass index in the three groups was 26.6 ± 4.3 kg/m 2 , 25.2 ± 3.7 kg/m 2 , and 27.4 ± 4 kg/m 2 , respectively. Number of previous IVF failures ranged from 2 to 6. Patients having a normal ovarian reserve (follicle stimulating hormone 10, and anti-mullerian hormone >2 ng/ml) were included. Only those previous cycles where embryo grading was available for scrutiny were included. These failed cycles had been done in good ART centers with an in-house embryologist and good quality control in the laboratory. Patients with uncorrected uterine and adnexal pathology, e.g., hydrosalpinx, submucous polyps, or myomas previous difficult ET’s, and thin endometrium in group I and II were excluded from the study. Apart from the routine infertility work-up a hysteroscopy (when not done earlier), oral glucose tolerance test, and thyroid profile were done for all patients. For RIF patients karyotypes of both partners, thrombophilia profile, lupus anticoagulant, and anticardiolipin antibodies IgG and IgM, were carried out and were normal. An endometrial biopsy (EB) for Koch's culture and DNA polymerase chain reaction was also performed routinely in all patients before IVF. Patients in this retrospective analysis were negative for endometrial Koch’s. All patients underwent an EB for the ERA test in a hormone replacement cycle (HRT). Transvaginal ultrasound was done on the 2 nd day of the period and after ensuring that the endometrium had shed completely HRT was started. Estradiol valerate was started in a dose of 2 mg, this was increased to 6 mg or more till an appropriate endometrial thickness (≥7 mm) was achieved. Vaginal progesterone suppository 400 mg twice a day was then started for a period of 5 days ( P + 5). In patients with thin endometrium, we tried to achieve the maximum thickness that had been observed on earlier scans, before starting progesterone. Serum progesterone level was checked when the endometrial thickness was adequate and a cut-off value ≤0.9 ng/ml was taken to start progesterone (P) administration. If serum progesterone level was ≥1 ng/ml, ERA testing was canceled because high endogenous progesterone levels compromise result of the test. Endometrial biopsies were collected from the uterine cavity with the use of Pipelle catheters from Gynetics on day P + 5 in an HRT cycle. The day of the EB in HRT cycle is after five full days of P impregnation that is on 6 th day morning. After the biopsy, the endometrial tissue was transferred to a cryotube containing 1.5 mL RNA stabilizing agent (Qiagen), vigorously shaken for a few seconds, and kept at 4°C in refrigerator for 4 h. Care was taken that the tissue was adequate and well-immersed in the fluid provided. If the tissue is too much, there is RNA degradation, and if too little, sufficient RNA is not available for extraction. The samples were then transported at room temperature to IVIOMICS, India and sent for analysis to IVIOMICS Valencia Spain. The test results were available in 2 weeks. ERA test diagnosed the endometrium to be receptive (R) or nonreceptive (NR). Nonreceptive endometrium was further classified as pre or post receptive. A second ERA sample was taken for these patients on the suggested day ( P + 6, P + 7, or P + 4). This result was either receptive or gave a short WOI of 12 h that is, embryo transfer to be done at P + 4.5, 5.5 or 6.5. Patients with a receptive endometrium ( P + 5) underwent FET in a subsequent HRT cycle simulating the ERA cycle. In patients with a changed implantation window FET was done based on the personalized WOI identified by ERA (pET). Two good quality blastocysts were transferred. Written informed consent was obtained from the patients for performing ERA.