LETTER TO THE EDITOR

Response to editorial entitled ‘‘Biomarkers ofendometrial receptivity through a minimallyinvasive approach’’

TO THE EDITOR: The editorial by Dr. Robert Norman in thisissue of Fertility and Sterility (1) succinctly summarizes thevarious approaches currently used to identify biomarkers ofendometrial receptivity toward development of a viablepredictive/assessment test for clinical application. As Dr.Norman points out, multiple approaches have been appliedto identify candidate biomarkers, including proteomic andlipidomic assessments of uterine aspirate fluid and geneexpression profiling of endometrial biopsies. In his editorial,Dr. Norman introduces our paper in which we demonstratethe feasibility of performing genome-wide gene expressionprofiling on uterine aspirations (2). Using unsupervised hier-archic clustering, we demonstrate that the phase of sampling(LHþ2 vs. LHþ7) affects gene expression more than individ-ual differences in gene expression between patients. Weidentified and verified robust differences in expression of245 genes due to phase of sampling and determined thatexpression of 53 of these genes efficiently separated ourtwo groups and those of a publically available dataset ofgene expression signatures obtained from endometrial biopsysamples. Because LHþ2 coincides with a prereceptive phaseand LHþ7 with a receptive phase in fertile women, the differ-entially expressed genes identified in our study encode forcandidate biomarkers of endometrial receptivity. The 53-gene list overlaps significantly with those indicated in otherstudies, including the 238-gene list comstituting the endome-trial receptivity assay used by Diaz-Gimeno et al. (3) in theiralgorithm for predicting the receptive period from endome-trial biopsy samples.

We are excited by the potential of our approach becauseuterine aspiration is less traumatic to the endometrium thanbiopsy. Unlike endometrial biopsy, uterine aspiration iscompatible with evaluation within an active IVF or naturalcycle in which a patient is attempting pregnancy (4, 5),enabling us and other researchers to directly associatealtered gene expression with implantation success. Thisapproach will facilitate our future identification of thosecandidate biomarkers for further development of point-of-care assays for clinical use.

Although our 53-gene cassette includes several inter-esting candidate biomarkers, including those encodingsecreted products, we do not propose these as a clinical test.Rather, we propose that our approach enables further testingand reduction of this signature to identify those genes that aremost predictive. We agree with Dr. Norman that protein-based assays would be preferable to transcript measurementsfor clinical assays; however, gene expression data arenecessary to direct proteomic discovery assays to improvethese approaches.

Dr. Norman points out that we did not fully characterizethe cell types present in the uterine aspirations and thatchanges in cell types would affect gene expression signatures.However, this criticism applies equally to proteomic

VOL. 100 NO. 3 / SEPTEMBER 2013

approaches and to gene expression studies of endometrialbiopsies and misses the point. Alterations in infiltratingimmune cells undoubtedly contribute to the gene expressionprofiles, just as they contribute to differences in proteinspresent in the aspirated fluid and likely affect receptivity ofthe endogenous intrauterine environment. Our discoveryapproach mirrors the situation one would encounter in aclinical testing situation, with multiple cell types contributingto the proteome or transcriptome.

Dr. Norman also raises an issue of blood contaminationand an inability to obtain uterine fluid from patients. Wedid not encounter either of these issues. Before adopting thetechnique, we tested various catheters in patients undergoingsurgery. We selected the catheter used in our study because itwas cost-effective, reliably acquired fluid and cells, and didnot inflict pain, and only very few red blood cells were notedon histology as reviewed by a pathologist.

Finally, Dr. Norman seems to suggest that we are propos-ing our 53-gene signature as a clinical test for endometrialassessment. It is important to stress that this is not our claimin the paper. Rather, we recommend adoption of our less inva-sive sampling approach, which would allow the furthervalidation of gene expression changes associated with theclinically receptive period. We point out that this approachcan be used to directly associate biomarker expression withimplantation success, and that those findings can guideproteomic discovery and assay development. Such studiesare presently underway in our laboratory.

Crystal Chan, M.D.a,b,c,d

Theodore J. Brown, Ph.D.a,b,c

Ellen M. Greenblatt, M.D.a,da Department of Obstetrics and Gynaecology, and b Institute

of Medical Sciences, University of Toronto; and c SamuelLunenfeld Research Institute and d Centre for Reproductive

Health and Fertility, Mount Sinai Hospital, Toronto,Ontario, Canada

May 30, 2013

http://dx.doi.org/10.1016/j.fertnstert.2013.05.051

REFERENCES1. Norman RJ. Biomarkers of endometrial receptivity through a minimally

invasive approach. Fertil Steril 2013;100:654–5.2. Chan C, Virtanen C, Winegarden NA, Colgan TJ, Brown TJ, Greenblatt EM.

Discovery of biomarkers of endometrial receptivity through a minimally-invasive approach: a validation study with implications for assisted reproduc-tion. Fertil Steril 2013;100:810–7.

3. Diaz-Gimeno P, Ruiz-Alonso M, Blesa D, Bosch N, Martinez-Conejero JA,Alama P, et al. The accuracy and reproducibility of the endometrial receptivityarray is superior to histology as a diagnostic method for endometrial recep-tivity. Fertil Steril 2013;99:508–17.

4. Boomsma CM, Kavelaars A, Eijkemans MJ, Amarouchi K, Teklenburg G,Gutknecht D, et al. Cytokine profiling in endometrial secretions: a noninvasivewindow on endometrial receptivity. Reprod Biomed Online 2009;18:85–94.

5. van der Gaast MH, Beier-Hellwig K, Fauser BC, Beier HM, Macklon NS.Endometrial secretion aspiration prior to embryo transfer does not reduce im-plantation rates. Reprod Biomed Online 2003;7:105–9.

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