Diagnosis of Endometrial-Factor Infertility: Current Approaches and New Avenues for Research.

! Over the last decade, research to improve success rates in reproductive medicine has focused pre- dominantly on the understanding and optimiza- tion of embryo quality. However, the emergence of personalized medicine in ovulation induction and embryology has shifted the focus to assessing the individual status of the endometrium. The en- dometrium is considered receptive during an in- dividually defined period, the window of implan- tation (WOI), when the mother permits a blasto- cyst to attach and implant. This individual recep- tivity status can now be objectively diagnosed us- ing the endometrial receptivity array (ERA) devel- oped in 2011. The ERA, together with a computa- tional algorithm, detects the unique transcrip- tomic signature of endometrial receptivity by an- alyzing 238 differentially expressed genes and reliably predicting the WOI. We and others have illustrated the utility of this personalized diag- nostic approach to discriminate between individ- ual physiological variation in endometrial recep- tivity and unknown endometrial pathology, deemed as causal in recurrent implantation fail- ure (RIF). An international randomized controlled trial (“The ERA as a diagnostic guide for personal- ized embryo transfer. ” ClinicalTrials.gov Identi- fier: NCT01954758) is underway to determine the clinical value of this endometrial diagnostic intervention in the work-up for reproductive care. In this review, we analyse the current clini- cal practice in the diagnosis of the endometrial factor together with new avenues of research. Zusammenfassung ! Die Verbesserung der Erfolgsraten der assistierten Reproduktion hat sich bisher vor allem auf das Verständnis sowie die Optimierung der Embryo- qualität konzentriert. Erst durch die Einführung der personalisierten Medizin im Rahmen der Ovulationsinduktion und in der Embryologie hat sich der Fokus auch auf den individuellen Status des Endometriums erweitert. Die endometriale Rezeptivität definiert den persönlichen Zeitraum, in dem sich der im Blastozystenstadium befindli- che Embryo an das hormonell regulierte Endome- trium einer Frau anheften und einnisten kann. Durch den 2011 patentierten Endometrial Recep- tivity Array (ERA) steht erstmals ein objektiver diagnostischer Test zur Bestimmung der rezepti- ven Phase des Endometriums zur Verfügung. ERA identifiziert das transkriptomische Profil des En- dometriums anhand der Signatur von 238 unter- schiedlich exprimierten Genen in Verbindung mit einem computerbasierten Prädiktor und klassifi- ziert darauf beruhend den rezeptiven Status der Patientin. Die Bedeutung dieser personalisierten Untersuchung des Endometriums, die zwischen individueller physiologischer Variabilität der endometrialen Rezeptivität und unbekannter Pa- thologie des Endometriums unterscheiden kann, konnte sowohl durch uns als auch durch andere Gruppen bisher bei Patientinnen mit rezidivie- rendem Implantationsversagen (RIF) gezeigt wer- den. Auf diesen Ergebnissen aufbauend, wird der Nutzen von ERA in der Routinediagnostik bei un- erfülltem Kinderwunsch aktuell in einer interna- tionalen, randomisierten, kontrollierten Studie („The ERA as a diagnostic guide for personalized embryo transfer. “ ClinicalTrials.gov Identifier: NCT01954758) untersucht. Diagnosis of Endometrial-Factor Infertility: Current Approaches and New Avenues for Research Die Bedeutung des Endometriums in der Infertilitätsdiagnostik: aktueller Stand und neue Forschungsansätze Authors N. Katzorke 1, 2, F. Vilella 1, M. Ruiz 1, J.-S. Krüssel 2, C. Simón 1, 3 Affiliations 1 Fundación Instituto Valenciano de Infertilidad, Department of Obstetrics and Gynecology, Valencia University and Instituto Universitario IVI/INCLIVA, Valencia University, Valencia, Spain 2 Department of Obstetrics and Gynecology, Heinrich Heine University Medical Center, Düsseldorf, Germany 3 Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University, Stanford, California, United States of America Key words l" endometrial receptivity array (ERA) l " microRNA l" window of implantation l" implantation failure l" assisted reproduction Schlüsselwörter l" endometriale Rezeptivität l" Implantationsfenster (WOI) l" assistierte Reproduktions- techniken (ART) l " micro‑Ribonucleinsäure (RNA) received 8. 1. 2016 revised 21. 2. 2016 accepted 23. 2. 2016

DOI http://dx.doi.org/ 10.1055/s-0042-103752 Geburtsh Frauenheilk 2016; 76: 699–703 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0016‑5751 Correspondence Prof. Jan Steffen Krüssel UniKiD Department of Obstetrics and Gynecology Heinrich-Heine-University Moorenstraße 5 40225 Düsseldorf [email protected] 699 Katzorke N et al. Diagnosis of Endometrial-Factor … Geburtsh Frauenheilk 2016; 76: 699 –703 Review

! Successful implantation of the embryo in the maternal endome- trium is the result of a perfect synchrony between a viable blas- tocyst, the receptive endometrium, and appropriate communica- tion between them [1]. The most investigated element in the im- plantation triad is the embryo, which seeks to adhere to the en- dometrial epithelium and invade the decidualized stroma, ini- tiating trophoblast invasion and placentation. Indeed, the under- standing of human pre-implantation development is critical (for review see [2]), as are the soluble ligands produced and received by their receptors to mediate this fundamental process (for re- view see [3]). However, research to develop an understanding of the endometrial component of implantation has been largely ne- glected. The maternal endometrium is receptive to an embryo only dur- ing the specific period of time in the menstrual cycle known as the window of implantation (WOI). Classically, this period is con- sidered as occurring 8 to 10 days after ovulation and lasting 2 or 3 days, during which time a functional and transient ovarian ste- roid-dependent status is acquired to enable the blastocyst to im- plant. This classical definition was established on the grounds of a relevant clinical study [4] but without basic research support- ing it. In this important contribution, published by Wilcox et al. in 1999 [4], the day of ovulation was defined on the basis of changes in urinary excretion of the estradiol metabolite estrone 3-glucuronide and the progesterone metabolite pregnanediol 3- glucuronide, which were measured by radioimmunoassay. The authors developed an algorithm to identify the day of ovulation based on the ratio of these urinary hormone metabolites, and claimed the test was similar to measurement of the luteinizing hormone (LH) peak [4]. However, 26 years later, the method pro- posed by these authors to identify ovulation has not been clini- cally adopted. Further, we now recognize limitations of the use of LH measurements in urine or even in blood to predict ovula- tion [5]. Nevertheless, the clinical community has since assumed that the endometrium in all patients becomes receptive during the indicated time frame (8 to 10 days after ovulation), regardless of individual characteristics or hormonal treatments received (i.e., natural cycles or controlled ovarian stimulation). Human Endometrial Receptivity ! To date no single molecular or histological biomarker has been identified to objectively and reliably diagnose endometrial recep- tivity. In the absence of such a diagnosis, the endometrium has been supported by progesterone or human chorionic gonadotro- pin (hCG) as the only “endometrial treatment” in patients under- going assisted reproductive techniques (ART). Accordingly, em- bryo transfer (ET) has been guided only by the quality and devel- opmental stage of the embryo and the thickness of the endome- trial layer. However, we have demonstrated that in 25 % of cases repeated implantation failure is attributable to endometrial ori- gin [6], which is consistent with the clinical relevance of endome- trial receptivity in successful pregnancy [7]. In 1950 Noyes et al. histologically defined the endometrial dating criteria for evaluating the endometrium [8]. However, multiple randomized [9, 10] and prospective studies [11 – 17] questioned the accuracy and reproducibility of the Noyes method to diag- nose endometrial receptivity or fertility status. Subsequent research has focused on discovering biochemical markers to assess endometrial status. Although myriad molecular mediators, including growth factors, cytokines, chemokines, lip- ids, and adhesion molecules, have been identified in the endome- trium [1, 7], so far, none of these molecules has been established as an endometrial biomarker in clinical practice [18]. Developments in molecular biology techniques, along with glob- al transcriptomic analyses, have enabled the investigation of the genomics of human endometrial development [19]. Transcrip- tomic analyses identify actively expressed genes at the mRNA level at any given time [20]. Human endometrial transcriptomic analyses reveal that differential gene expression patterns exist during different phases of the menstrual cycle [21, 22], including during the receptive phase [19, 23]. Further, differential tran- scriptomic profiles have been uncovered in patients with repeti- tive implantation failure [24 – 26] as well as endometrial patholo- gies such as endometriosis or endometrial cancer [27, 28], and gene expression patterns have been defined during controlled ovarian stimulation (COS) and hormonal replacement therapy (HRT) cycles [29, 30]. These efforts enabled the discovery of the unique genomic signature of endometrial receptivity that be- came the basis of the endometrial receptivity array (ERA) [31]. This assay diagnoses the molecular status of the receptive endo- metrium according to its transcriptomic signature, regardless of its histological appearance [31]. Endometrial Receptivity Array ! The ERA is a novel diagnostic method clinically available world- wide that classifies the endometrium as receptive, pre-receptive, or post-receptive [6]. The test requires a small biopsy of endome- trial tissue taken during scheduled treatment at either 7 days after the luteinizing hormone peak (LH + 7) in a natural cycle, or at the end of 5 days of progesterone administration after estro- gen priming in a hormonal replacement therapy cycle (P + 5). RNA extracted from the tissue is applied to a microarray to deter- mine the transcriptomic profile of 238 genes. This transcriptomic profile, when coupled to a computational predictor, objectively identifies whether this endometrium is receptive, pre-receptive or post-receptive by clustering analysis against sample training sets [6, 31]. The 238 genes analyzed by ERA were chosen accord- ing to the expression data of 14 previous papers by our group searching for the transcriptomic signature of endometrial recep- tivity in natural cycles, COS, HRT and even in patients with intra- uterine device (IUD) (for review see [19]). Although these genes were selected by t-test with an absolute fold change > 3 and a false discovery rate < 0.05, the clinical validation was done with a training set in real patients [31]. Importantly, the result ob- tained by ERA is independent of the histological appearance of the endometrium, and has been demonstrated to be more accu- rate than histological dating [32] and completely reproducible even with up to 40 months between samples [32]. This finding is consistent with the idea that the receptivity status remains the same within an individual woman throughout her lifetime, but that different hormonal treatments and states such as preg- nancy may change the endometrium since it is a hormonally reg- ulated organ. Analysis of over 6000 ERA results, performed by our group, indi- cates that, in approximately 30 % of patients, the endometrial bi- opsy is classified as non-receptive. In these instances, the predic- tor describes whether the tissue is pre-receptive (85.0 %) or post- 700 Katzorke N et al. Diagnosis of Endometrial-Factor … Geburtsh Frauenheilk 2016; 76: 699 –703 GebFra Science receptive (12.6 %). Based on these findings, the algorithm then recommends the timing of progesterone treatment for the indi- vidual patient to find her personalized WOI, thereby obtaining an optimal chance of successful implantation through personal- ized embryo transfer (pET) ( l " Fig. 1). Personalized Embryo Transfer ! The clinical application of the ERA has been studied in a prospec- tive, interventional, multicenter, clinical trial in 85 patients with recurrent implantation failure (RIF) versus 25 controls under- going IVF for the first time [6]. The endometrial biopsy was clas- sified as receptive in 74.1 % of patients with RIF; when embryo transfer was performed according to the timing indicated by ERA diagnosis, patients achieved a 33.9 % implantation rate and a 51.7 % pregnancy rate. However, displacement of the WOI was observed in one out of four patients with RIF as diagnosed by ERA [6]. In these 26.3 % of patients, when embryo transfer was performed according to ERA-diagnosed timing of the WOI, preg- nancy and implantation rates rose to the level of normally recep- tive controls, in this initial study 7 patients underwent pET. A clinical case of a successful personalized embryo transfer in a patient having experienced four IVF and three oocyte donation failures has been reported [34]. This patient was diagnosed with a displacement of the WOI using the ERA. Therefore, personalized embryo transfer of two blastocysts was performed after 7 days of progesterone (P + 7) in an HRT cycle, resulting in a successful twin pregnancy after 7 previous repeated implantation failures. Simi- larly, in a pilot study of 17 patients undergoing oocyte donation who had experienced failed implantations with routine embryo transfer, the implantation rate was increased from 12.9 to 34.5 % and the pregnancy rate from 23.5 to 52.9 % when pET was per- formed following ERA diagnosis [33]. All 17 patients were initial- ly diagnosed with a displaced WOI, whereby the endometrium biopsy was classified pre-receptive in 16 patients and post-re- ceptive in one patient [33]. The value of the diagnosis of endometrial receptivity during the routine infertility work-up of patients undergoing assisted re- productive technology is currently being explored in an interna- tional, multicenter, prospective, randomized, interventional and controlled study –“ The ERA as a diagnostic guide for personal- ized embryo transfer ”– comparing fresh embryo transfer versus elective delayed embryo transfer or pET (Clinical trails.gov, Iden- tifier: NCT01954758). MicroRNAs: New Molecules Advancing Our Reproductive Knowledge ! Despite the wealth of information uncovered in recent years, technologies continue evolving to discover all transcripts across the transcriptome. In 2014, Hu et al. reported the first global gene expression profile of the human endometrium using next-gener- ation, high-throughput RNA sequencing (RNA ‑seq) [34]. This RNA‑seq-based transcriptome comparison of pre-receptive and receptive human endometrium revealed a total of 2372 differen- tially expressed genes, including metallothionein family mem- 6000 patients pET pET Endometrial biopsy ( L H + 7o rP + 5 ) 71.4% receptive 2nd endometrial biopsy Change and evaluation of a new kind of cycle 91.5% receptive 5.2% receptivity between both biopsies 3.3% same result as 1st biopsy 28.6% non-receptive 2.4% proliferative 12.6% post-receptive (delayed WOI) 85.0% pre-receptive (advanced WOI) Fig. 1 Clinical algorithm for personalized embryo transfer (pET), including the percentage probability (unpublished data provided by C. Simon). 701 Katzorke N et al. Diagnosis of Endometrial-Factor … Geburtsh Frauenheilk 2016; 76: 699 –703 Review bers, HAP1, ZCCHC12, MRAP2, OVGP1, regulatory factors (GLI2, CDC25A, TLR9, MT1G and SLC5A1), and transcription factors (AP2 and SP1) that have not previously been linked to endometrial re- ceptivity. In addition, the discovery of microRNAs (miRNAs) as potential post-transcriptional regulators of gene expression [35] represents a breakthrough in biology during the last ten years and has become an extremely active research field [36 – 39]. MiRNAs are small, non-coding RNA sequences of 18 to 25 nucleo- tides that regulate gene expression post-transcriptionally [40]. These molecules do not encode proteins; instead, miRNAs target mRNAs through complementary base pairing to the 3 ′-untrans- lated region for degradation or repression, thereby functioning as gene silencers [41]. Based on the degree of sequence homol- ogy, one miRNA can potentially target a broad range of genes and one gene can be regulated by several miRNAs [40]. Initially, long precursors (pri-miRNAs) are transcribed and processed to shorter precursors (pre-miRNAs) in the nucleus [37]; these pre- cursors are exported into the cytoplasm and incorporated into the RNA-induced silencing complex (RISC) to bind an mRNA tar- get [42], as shown in l " Fig. 2. As has been found for mRNAs, miRNAs are differentially ex- pressed in the endometrium across the menstrual cycle [43]. Fur- ther, the endometrial epithelium releases miRNAs that are se- creted into the endometrial fluid [43]. Profiling of miRNA and mRNA transcripts in human endometrium suggests that the hor- monal regulation of miRNAs leads to a suppression of cell prolif- eration by down-regulating the expression of some cell cycle genes in the endometrial epithelium during the secretory phase [44]. By isolating endometrial epithelial cells from endometrial biopsies of 14 fertile women in the late-proliferative and mid-se- cretory phases, Kuokkanen et al. identified miRNA-29B, miRNA- 29C, miRNA-30B, miRNA-30D, miRNA-31, miRNA-193A-3P, miRNA-203, miRNA-204, miRNA-200C, miRNA-210, miRNA- 582-5P, and miRNA-345 as significantly increased in the secre- tory endometrium. Indeed, a subset of miRNAs, namely hsa- miR‑30b and hsa-miR‑30d, are significantly upregulated, where- as hsa-miR ‑494 and hsa-miR ‑923 are downregulated, in recep- tive endometrium (LH + 7) versus pre-receptive endometrium (LH + 2) in fertile women [45]. These findings support the previ- ously reported upregulation of hsa-miR ‑30b and hsa-miR ‑30-d during the acquisition of endometrial receptivity [46]. The in- volvement of miRNAs in failed embryo implantation has been sug- gested in patients with recurrent implantation failure [47]. The existence of 13 differentially expressed miRNAs (miRNA-145, miRNA-23b, miRNA-99a, miRNA-27b, miRNA-652, miRNA-139- 5p, miRNA-195, miRNA-342-3p, miRNA-150, miRNA-374b, miRNA-32, miRNA628-5b, miRNA-874) has been described in pa- tients with recurrent implantation failure; these may regulate the expression of up to 3800 genes [47]. Recently, our group has demonstrated that hsa-miR ‑30d is se- creted by the human endometrial epithelium into the endome- trial fluid either free or in exosome-associated form, and can be incorporated into the pre-implantation embryo to potentially modify its transcriptome [43]. The internalization of this miRNA

in an indirect overexpression of genes encoding for cer- tain molecules involved in embryonic adhesion, such as ITGB3, ITGA7,a n d CDH5 [43]. Furthermore, it has been suggested that miRNAs can be secreted by the human embryo [48]; hsa- miR‑191, hsa-mi-372, and hsa-miR ‑645 are differentially ex- pressed according to the fertilization method, chromosomal sta- tus, and pregnancy outcome. Together, these findings reinforce the concept of maternal-embryonic cross-talk that uses many different languages, with miRNAs as one of them.

! The receptivity status of the endometrium can now be diagnosed reliably by the ERA test, an objective molecular tool based on the transcriptomic signature of human endometrial receptivity, to identify the WOI. The ERA can guide and improve our clinical practice by introducing and enabling a personalized diagnosis of the WOI and, accordingly, a personalized embryo transfer. In the near future, the challenge will be to identify biomarkers of en- dometrial receptivity that could be assessed by non-invasive methods. MiRNAs may be interesting candidate molecules to consider, particularly with the potential role of maternal endo- metrial miRNAs as transcriptomic modifiers of the preimplanta- tion embryo. Machinery of biosynthesis and delivery of miRNAS Transcriptome and epigenetic effects on early embryo Pre- miRNA Endometrium Blastocyst Processing through drosha Pri-miRNA Transcription miRNA gene Processing through dicer Transport to cytosol through exportin-5 Mature miRNA Fig. 2 Diagram showing the process of miRNA synthesis and the potential role of miRNAs in the embryo-maternal dialogue. 702 Katzorke N et al. Diagnosis of Endometrial-Factor … Geburtsh Frauenheilk 2016; 76: 699 –703 GebFra Science Conflict of Interest ! CS is inventor of the ERA patent and holds shares in Igenomix, the company commercializing the ERA test. MR & FV are employees of Igenomix.

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