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Proteomics of the human endometrium and uterine fluid: a pathway to biomarker discovery

2012; Elsevier BV; Volume: 99; Issue: 4 Linguagem: Inglês

10.1016/j.fertnstert.2012.09.013

1556-5653

Lois A. Salamonsen, Tracey A. Edgell, Luk Rombauts, Andrew N. Stephens, David Robertson, Adam Rainczuk, Guiying Nie, Natalie J. Hannan,

Endometrial and Cervical Cancer Treatments

Failure of the endometrium to achieve receptivity results in infertility, and it is also a rate-limiting step in in vitro fertilization (IVF) success. The microenvironments provided by the endometrium during the receptive phase and that support implantation are highly complex and constantly changing as implantation progresses. Although a number of gene array studies have defined mRNA changes across the cycle, with infertility, and in IVF cycles, these have not generally been informative due in part to the subsequent regulation of transcription and posttranslational modifications of the proteins. State-of-the-art proteomic technologies now enable analysis of changes in the endometrium and its secretome related to cycle phase and associated with infertility. These techniques include two-dimensional differential in-gel electrophoresis, isobaric tags for relative and absolute quantitation, and multiplex analyses of selected panels of markers. Subsequent definition of cellular location, timing of production of identified proteins, and their regulation by steroid hormones and blastocyst-derived factors provide indications of their functions and their relationship to the establishment of pregnancy. Proteins discovered by proteomic analyses and fully evaluated will provide the differentiative profiles necessary to inform clinical practice and serve as an end point for optimizing stimulation cycles in IVF clinics as well as more clearly defining the molecular mechanisms underlying successful implantation. Failure of the endometrium to achieve receptivity results in infertility, and it is also a rate-limiting step in in vitro fertilization (IVF) success. The microenvironments provided by the endometrium during the receptive phase and that support implantation are highly complex and constantly changing as implantation progresses. Although a number of gene array studies have defined mRNA changes across the cycle, with infertility, and in IVF cycles, these have not generally been informative due in part to the subsequent regulation of transcription and posttranslational modifications of the proteins. State-of-the-art proteomic technologies now enable analysis of changes in the endometrium and its secretome related to cycle phase and associated with infertility. These techniques include two-dimensional differential in-gel electrophoresis, isobaric tags for relative and absolute quantitation, and multiplex analyses of selected panels of markers. Subsequent definition of cellular location, timing of production of identified proteins, and their regulation by steroid hormones and blastocyst-derived factors provide indications of their functions and their relationship to the establishment of pregnancy. Proteins discovered by proteomic analyses and fully evaluated will provide the differentiative profiles necessary to inform clinical practice and serve as an end point for optimizing stimulation cycles in IVF clinics as well as more clearly defining the molecular mechanisms underlying successful implantation. Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/salamonsenl-proteomics-human-endometrium-uterine-fluid-biomarker/ Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/salamonsenl-proteomics-human-endometrium-uterine-fluid-biomarker/ The endometrium is unique among adult tissues in the extent of remodeling that it undergoes during each menstrual cycle. The outer functionalis layer is shed during menstruation while, in parallel, the denuded surface is rapidly reepithelialized. During the proliferative phase, under the influence of estrogen, all the cellular compartments and their supporting extracellular matrices are restored. After ovulation, and driven by rising progesterone levels, the various cell types differentiate in preparation for implantation should conception occur (1Salamonsen L.A. The menstrual and estrous cycles.in: Aplin J.D. Fazleabas A.T. Glasser S.R. Giudice L.C. The endometrium. molecular, cellular, and clinical perspectives. Informa UK, London2008: 25-45Crossref Google Scholar). For most of the menstrual cycle, the endometrium is not receptive for an embryo to implant; indeed, for at least some of the time, specifically in the proliferative phase, it appears to be actively hostile (2Hannan N.J. Paiva P. Meehan K.L. Rombauts L.J. Gardner D.K. Salamonsen L.A. Analysis of fertility-related soluble mediators in human uterine fluid identifies VEGF as a key regulator of embryo implantation.Endocrinology. 2011; 152: 4948-4956Crossref PubMed Scopus (125) Google Scholar). Embryo transfer studies in animals during the 1970s and 1980s (3Betteridge K.J. An historical look at embryo transfer.J Reprod Fertil. 1981; 62: 1-13Crossref PubMed Scopus (50) Google Scholar, 4Rowson L.E. Moor R.M. Embryo transfer in the sheep: the significance of synchronizing oestrus in the donor and recipient animal.J Reprod Fertil. 1966; 11: 207-212Crossref PubMed Scopus (66) Google Scholar) demonstrated that developmental synchrony between the embryo and endometrium is essential for successful implantation and hence for establishment of pregnancy. In women, this "receptive phase" is of only approximately 4 days in duration, occurring at about 5 to 10 days after the luteinizing hormone (LH) surge (the midsecretory phase) (5Navot D. Bergh P.A. Williams M. Garrisi G.J. Guzman I. Sandler B. et al.An insight into early reproductive processes through the in vivo model of ovum donation.J Clin Endocrinol Metab. 1991; 72: 408-414Crossref PubMed Scopus (190) Google Scholar). During the past decade, many molecular changes associated with endometrial receptivity in women have been identified. These occur in the luminal and glandular epithelium, in the stromal compartment during the initiation of decidualization close to the spiral arterioles, in the vasculature itself, and in the phenotypes of the leukocyte subsets that migrate into the tissue. Implantation is a continuum from first apposition of the blastocyst to the luminal epithelial surface, its attachment and invasion of trophoblast cells between the epithelial cells, and syncytialization and invasion of the extracellular trophoblast through the developing decidua until some trophoblast cells invade and remodel the spiral arterioles (6Salamonsen L.A. Nie G. Hannan N.J. Dimitriadis E. Society for Reproductive Biology Founders' Lecture 2009. Preparing fertile soil: the importance of endometrial receptivity.Reprod Fertil Dev. 2009; 21: 923-934Crossref PubMed Scopus (114) Google Scholar). Preparedness for all facets of this process is initiated within the receptive phase, but the most important changes for the initial stages of implantation are in the endometrial epithelial compartment and on the outer trophectodermal layer of the conceptus (Fig. 1). The focus of this review is on the maternal changes associated with implantation. Molecular changes in the glands and luminal epithelium result in changes in their secretions into the uterine cavity. Typically uterine fluid is complex and contains nutrients, enzymes, cytokines, antiproteases, and transport proteins, along with other biologically active factors (7Hannan N.J. Stephens A.N. Rainczuk A. Hincks C. Rombauts L.J. Salamonsen L.A. 2D-DiGE analysis of the human endometrial secretome reveals differences between receptive and nonreceptive states in fertile and infertile women.J Proteome Res. 2010; 9: 6256-6264Crossref PubMed Scopus (106) Google Scholar, 8Leese H.J. Hugentobler S.A. Gray S.M. Morris D.G. Sturmey R.G. Whitear S.L. et al.Female reproductive tract fluids: composition, mechanism of formation and potential role in the developmental origins of health and disease.Reprod Fertil Dev. 2008; 20: 1-8Crossref PubMed Scopus (146) Google Scholar); it provides support to and can modify certain characteristics of the preimplantation blastocyst. Important changes also occur at the luminal epithelial surface in terms of mucins, integrins, and other adhesion molecules (9Quenby S. Anim-Somuah M. Kalumbi C. Farquharson R. Aplin J.D. Different types of recurrent miscarriage are associated with varying patterns of adhesion molecule expression in endometrium.Reprod Biomed Online. 2007; 14: 224-234Abstract Full Text PDF PubMed Scopus (50) Google Scholar) and within the epithelial cells from which the secretions arise (e.g., changes in junctional complexes, Ca++ regulators, growth factors, cytokines, chemokines, and prostanoids) (10Boomsma C.M. Kavelaars A. Eijkemans M.J. Amarouchi K. Teklenburg G. Gutknecht D. et al.Cytokine profiling in endometrial secretions: a non-invasive window on endometrial receptivity.Reprod Biomed Online. 2009; 18: 85-94Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 11Buck V.U. Windoffer R. Leube R.E. Classen-Linke I. Redistribution of adhering junctions in human endometrial epithelial cells during the implantation window of the menstrual cycle.Histochem Cell Biol. 2012; 137: 777-790Crossref PubMed Scopus (37) Google Scholar, 12Hannan N.J. Evans J. Salamonsen L.A. Alternate roles for immune regulators: establishing endometrial receptivity for implantation.Expert Rev Clin Immunol. 2011; 7: 789-802Crossref PubMed Scopus (23) Google Scholar, 13Luu K.C. Nie G.Y. Salamonsen L.A. Endometrial calbindins are critical for embryo implantation: evidence from in vivo use of morpholino antisense oligonucleotides.Proc Natl Acad Sci USA. 2004; 101: 8028-8033Crossref PubMed Scopus (84) Google Scholar). The incidence of infertility is on the increase worldwide, with around 1 in 30 pregnancies in Australia and other developed countries now achieved by assisted reproductive technologies (14Wang Y.A. Chambers G.M. Sullivan E.A. Assisted reproductive technology in Australia and New Zealand 2008. Assisted reproduction technology series no 14. Australian Institute of Health and Welfare, Canberra2010Google Scholar). While infertility clinics have focused on providing high-quality embryos for transfer, the other side of the equation, endometrial receptivity, has been largely ignored. Indeed, the endometrium is far from normal during IVF cycles as a result of ovarian stimulation (15Devroey P. Bourgain C. Macklon N.S. Fauser B.C. Reproductive biology and IVF: ovarian stimulation and endometrial receptivity.Trends Endocrinol Metab. 2004; 15: 84-90Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 16Kolibianakis E.M. Bourgain C. Platteau P. Albano C. Van Steirteghem A.C. Devroey P. Abnormal endometrial development occurs during the luteal phase of nonsupplemented donor cycles treated with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonists.Fertil Steril. 2003; 80: 464-466Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar; Evans J, Hincks NJ, Rombauts LJ, Salamonsen LA, unpublished data). Ovarian stimulation protocols have a marked effect on endometrial differentiation, at least in part due to elevated progesterone levels and prematurely high levels of human chorionic gonadotropin (hCG) (17Papanikolaou E.G. Bourgain C. Fatemi H. Verpoest W. Polyzos N.P. De Brabanter A. et al.Endometrial advancement after triggering with recombinant or urinary hCG: a randomized controlled pilot study.Reprod Biomed Online. 2010; 21: 50-55Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 18Van Vaerenbergh I. Fatemi H.M. Blockeel C. Van Lommel L. In't Veld P. Schuit F. et al.Progesterone rise on hCG day in GnRH antagonist/rFSH stimulated cycles affects endometrial gene expression.Reprod Biomed Online. 2011; 22: 263-271Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 19Evans J, Salamonsen L. Inflammation, leukocytes and menstruation. Rev Endocrine Metabol Disord. Published online August 5, 2012.Google Scholar), which is administered in place of LH as an ovulation stimulus. Furthermore, there is growing evidence that IVF pregnancy rates are better in frozen cycles (unstimulated) compared with those when fresh embryos are transferred to a stimulated cycle (20Pinborg A. To transfer fresh or thawed embryos?.Semin Reprod Med. 2012; 30: 230-235Crossref PubMed Scopus (26) Google Scholar, 21Shapiro B.S. Daneshmand S.T. Garner F.C. Aguirre M. Hudson C. Thomas S. Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders.Fertil Steril. 2011; 96: 344-348Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar). Thus, inability of the endometrium to achieve receptivity is a likely reason for the failure of some IVF treatments. Markers of receptivity are urgently needed if we are to readily identify the underlying cause of infertility in many women, to improve the success rate of IVF, and ultimately to treat infertility of endometrial origin without expensive reproductive technologies. Genomewide analyses applied to the endometrium have generated distinct molecular profiles in terms of cycle stage (22Ponnampalam A.P. Weston G.C. Susil B. Rogers P.A. Molecular profiling of human endometrium during the menstrual cycle.Aust NZ J Obstet Gynaecol. 2006; 46: 154-158Crossref PubMed Scopus (15) Google Scholar, 23Punyadeera C. Dassen H. Klomp J. Dunselman G. Kamps R. Dijcks F. et al.Oestrogen-modulated gene expression in the human endometrium.Cell Mol Life Sci. 2005; 62: 239-250Crossref PubMed Scopus (81) Google Scholar, 24Talbi S. Hamilton A.E. Vo K.C. Tulac S. Overgaard M.T. Dosiou C. et al.Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.Endocrinology. 2006; 147: 1097-1121Crossref PubMed Scopus (455) Google Scholar); disease status, such as endometriosis (25Burney R.O. Talbi S. Hamilton A.E. Vo K.C. Nyegaard M. Nezhat C.R. et al.Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis.Endocrinology. 2007; 148: 3814-3826Crossref PubMed Scopus (552) Google Scholar); fertility status; and after various ovarian stimulation protocols (26Horcajadas J.A. Riesewijk A. Polman J. van Os R. Pellicer A. Mosselman S. et al.Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles.Mol Hum Reprod. 2005; 11: 195-205Crossref PubMed Scopus (231) Google Scholar, 27Martinez-Conejero J.A. Simon C. Pellicer A. Horcajadas J.A. Is ovarian stimulation detrimental to the endometrium?.Reprod Biomed Online. 2007; 15: 45-50Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 28Van Vaerenbergh I. Van Lommel L. Ghislain V. In't Veld P. Schuit F. Fatemi H.M. et al.In GnRH antagonist/rec-FSH stimulated cycles, advanced endometrial maturation on the day of oocyte retrieval correlates with altered gene expression.Hum Reprod. 2009; 24: 1085-1091Crossref PubMed Scopus (78) Google Scholar). However, changes in gene expression are not necessarily reflected in changes in translated proteins, nor does gene analysis take into account posttranscriptional, translational, or posttranslational changes that relate to cyclical transitions. Indeed, of the hundreds of gene expression changes typically identified by microarray, relatively few are common to more than two studies (29Haouzi D. Dechaud H. Assou S. De Vos J. Hamamah S. Insights into human endometrial receptivity from transcriptomic and proteomic data.Reprod Biomed Online. 2012; 24: 23-34Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 30Horcajadas J.A. Sharkey A.M. Catalano R.D. Sherwin J.R.A. Dominguez F. Burgos L.A. et al.Effect of an intrauterine device on the gene expression profile of the endometrium.J Clin Endocrinol Metab. 2006; 91: 3199-3207Crossref PubMed Scopus (60) Google Scholar). Comparison of proteomic data with published gene expression data in similar cohorts of women (25Burney R.O. Talbi S. Hamilton A.E. Vo K.C. Nyegaard M. Nezhat C.R. et al.Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis.Endocrinology. 2007; 148: 3814-3826Crossref PubMed Scopus (552) Google Scholar, 29Haouzi D. Dechaud H. Assou S. De Vos J. Hamamah S. Insights into human endometrial receptivity from transcriptomic and proteomic data.Reprod Biomed Online. 2012; 24: 23-34Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 31Chen J.I. Hannan N.J. Mak Y. Nicholls P.K. Zhang J. Rainczuk A. et al.Proteomic characterization of midproliferative and midsecretory human endometrium.J Proteome Res. 2009; 8: 2032-2044Crossref PubMed Scopus (82) Google Scholar) also have revealed an overall lack of correlation between the two, suggesting that posttranscriptional or translational regulation is an important feature of endometrial remodeling. This view is further supported by more recent information on the contribution of microRNAs in endometrial disorders (32Ohlsson Teague E.M. Van der Hoek K.H. Van der Hoek M.B. Perry N. Wagaarachchi P. Robertson S.A. et al.MicroRNA-regulated pathways associated with endometriosis.Mol Endocrinol. 2009; 23: 265-275Crossref PubMed Scopus (271) Google Scholar, 33Revel A. Achache H. Stevens J. Smith Y. Reich R. MicroRNAs are associated with human embryo implantation defects.Hum Reprod. 2011; 26: 2830-2840Crossref PubMed Scopus (150) Google Scholar). Our laboratory has therefore focused on proteomic analyses with the hypothesis that functionally important protein changes, mediated at least in part through posttranslational regulatory mechanisms, will identify biomarkers of relevance to endometrial receptivity. A number of studies (31Chen J.I. Hannan N.J. Mak Y. Nicholls P.K. Zhang J. Rainczuk A. et al.Proteomic characterization of midproliferative and midsecretory human endometrium.J Proteome Res. 2009; 8: 2032-2044Crossref PubMed Scopus (82) Google Scholar, 34DeSouza L. Diehl G. Yang E.C. Guo J. Rodrigues M.J. Romaschin A.D. et al.Proteomic analysis of the proliferative and secretory phases of the human endometrium: protein identification and differential protein expression.Proteomics. 2005; 5: 270-281Crossref PubMed Scopus (77) Google Scholar, 35Parmar T. Sachdeva G. Savardekar L. Katkam R.R. Nimbkar-Joshi S. Gadkar-Sable S. et al.Protein repertoire of human uterine fluid during the mid-secretory phase of the menstrual cycle.Hum Reprod. 2008; 23: 379-386Crossref PubMed Scopus (24) Google Scholar) have applied modern proteomic techniques to analysis of human endometrium, sampled by either biopsy or curettage, and across different cycle phases. In our studies, analysis by differential two-dimensional gel electrophoresis (2D DiGE) and MALDI-TOF-MS/MS has enabled the identification of 196 differentially expressed spots between cycle phases (31Chen J.I. Hannan N.J. Mak Y. Nicholls P.K. Zhang J. Rainczuk A. et al.Proteomic characterization of midproliferative and midsecretory human endometrium.J Proteome Res. 2009; 8: 2032-2044Crossref PubMed Scopus (82) Google Scholar). Not unexpectedly, the vast majority of increases in protein abundance were observed in secretory phase endometrium (196 vs. 39 in the secretory vs. proliferative phase, respectively). Of these, 42 sequences identified separate gene products, and 34 sequences were isoforms of either different pI (suggesting differential phosphorylation or glycosylation and presenting as protein spot "trains") or different size and charge, indicating differences in processing (31Chen J.I. Hannan N.J. Mak Y. Nicholls P.K. Zhang J. Rainczuk A. et al.Proteomic characterization of midproliferative and midsecretory human endometrium.J Proteome Res. 2009; 8: 2032-2044Crossref PubMed Scopus (82) Google Scholar). It is interesting that only 50% of the proteins identified in our proteomic analysis (31Chen J.I. Hannan N.J. Mak Y. Nicholls P.K. Zhang J. Rainczuk A. et al.Proteomic characterization of midproliferative and midsecretory human endometrium.J Proteome Res. 2009; 8: 2032-2044Crossref PubMed Scopus (82) Google Scholar) correlated with published gene array data; the other 50% showed no or only minor changes in mRNA expression. Immunohistochemistry validated a number of the identified proteins as present in endometrial epithelial cells and at significantly different levels between proliferative and secretory endometrium (Fig. 2). Two-dimensional DiGE has also proven useful in identifying substrates of proprotein convertase (PC)6—a protease essential for implantation in mice—in human secretory phase endometrium (36Heng S. Cervero A. Simon C. Stephens A.N. Li Y. Zhang J. et al.Proprotein convertase 5/6 is critical for embryo implantation in women: regulating receptivity by cleaving EBP50, modulating ezrin binding, and membrane-cytoskeletal interactions.Endocrinology. 2011; 152: 5041-5052Crossref PubMed Scopus (27) Google Scholar, 37Kilpatrick L.M. Stephens A.N. Hardman B.M. Salamonsen L.A. Li Y. Stanton P.G. et al.Proteomic identification of caldesmon as a physiological substrate of proprotein convertase 6 in human uterine decidual cells essential for pregnancy establishment.J Proteome Res. 2009; 8: 4983-4992Crossref PubMed Scopus (16) Google Scholar, 38Nie G. Stephens A.N. A proteomic protocol to identify physiological substrates of pro-protein convertases.Methods Mol Biol. 2011; 768: 325-341Crossref PubMed Scopus (1) Google Scholar, 39Paule S.G. Airey L.M. Li Y. Stephens A.N. Nie G. Proteomic approach identifies alterations in cytoskeletal remodelling proteins during decidualization of human endometrial stromal cells.J Proteome Res. 2010; 9: 5739-5747Crossref PubMed Scopus (22) Google Scholar). Although analysis of tissue biopsies has some merits, it has many disadvantages. Endometrial tissue displays a remarkable diversity in its morphology, with substantial changes both in its architecture and cellular composition (including leukocyte content) between the cycle phases. For example, we identified one protein, coronin 1, as differentially regulated between proliferative and secretory phases (unpublished data): the immunohistochemical analysis revealed that it was wholly contained in leukocyte populations and not in the endometrial stromal, epithelial, or vascular compartments. Laser capture can be used to reduce the complexity and heterogeneity of tissue samples for 'omics analyses, and both stromal and epithelial compartments captured in this way have been used in gene arrays after mRNA amplification (40Evans G.E. Martinez-Conejero J.A. Phillipson G.T. Simon C. McNoe L.A. Sykes P.H. et al.Gene and protein expression signature of endometrial glandular and stromal compartments during the window of implantation.Fertil Steril. 2012; 97 (1365–73.e1–2)Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, the low yield of protein provided by this approach makes the proteomic analysis of these samples challenging. Furthermore, many proteins identified in tissue biopsy samples are either the abundant structural proteins or proteins associated with basic cell functions such as proliferation. Substantial prefractionation of the samples before analysis and the application of the newer "gel-free" techniques (41Meehan K.L. Rainczuk A. Salamonsen L. Stephens A. Proteomics and the search for biomarkers of reproductive disease.Reproduction. 2010; 140: 505-519Crossref PubMed Scopus (21) Google Scholar) may enable detection of the lower abundance proteins that are functionally important for receptivity. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is also applicable to tissue sections. This allows the spatial localization of hundreds of masses simultaneously, can provide molecular signatures related to morphology, and has also identified protein sequences of biomarkers for prognosis and disease severity in cancer-related biopsies (42Balluff B. Schone C. Hofler H. Walch A. MALDI imaging mass spectrometry for direct tissue analysis: technological advancements and recent applications.Histochem Cell Biol. 2011; 136: 227-244Crossref PubMed Scopus (101) Google Scholar, 43Cazares L.H. Troyer D.A. Wang B. Drake R.R. Semmes O.J. MALDI tissue imaging: from biomarker discovery to clinical applications.Anal Bioanal Chem. 2011; 401: 17-27Crossref PubMed Scopus (81) Google Scholar, 44Gemoll T. Habermann J.K. Lahmann J. Szymczak S. Lundgren C. Bundgen N.K. et al.Protein profiling of genomic instability in endometrial cancer.Cell Mol Life Sci. 2012; 69: 325-333Crossref PubMed Scopus (8) Google Scholar). Application of MALDI-IMS may provide receptivity-specific signatures for endometrial luminal and glandular epithelium or for the stromal compartment (45Burnum K.E. Cornett D.S. Puolitaival S.M. Milne S.B. Myers D.S. Tranguch S. et al.Spatial and temporal alterations of phospholipids determined by mass spectrometry during mouse embryo implantation.J Lipid Res. 2009; 50: 2290-2298Crossref PubMed Scopus (125) Google Scholar). Finally, several studies have suggested that posttranslational modification of proteins is an important feature of endometrial biology; these modifications will continue to challenge researchers in this field. Uterine fluid (a protein-rich histotroph), and in particular the glandular secretions that provide many of its components, is critical for implantation. This was first definitively established in ewes in which endometrial glandular development had been inhibited during early postnatal life. In the resultant adults, conceptus development was retarded, and implantation failed to occur (46Gray C.A. Taylor K.M. Ramsey W.S. Hill J.R. Bazer F.W. Bartol F.F. et al.Endometrial glands are required for preimplantation conceptus elongation and survival.Biol Reprod. 2001; 64: 1608-1613Crossref PubMed Scopus (265) Google Scholar). This finding has been replicated in mice (47Dunlap K.A. Filant J. Hayashi K. Rucker 3rd, E.B. Song G. Deng J.M. et al.Postnatal deletion of Wnt7a inhibits uterine gland morphogenesis and compromises adult fertility in mice.Biol Reprod. 2011; 85: 386-396Crossref PubMed Scopus (115) Google Scholar, 48Jeong J.W. Kwak I. Lee K.Y. Kim T.H. Large M.J. Stewart C.L. et al.Foxa2 is essential for mouse endometrial gland development and fertility.Biol Reprod. 2010; 83: 396-403Crossref PubMed Scopus (136) Google Scholar), which use hemochorial placentation and are more similar to humans than sheep (epitheliochorial placentation). In women, histotroph derived from uterine glands is important throughout the first trimester (49Burton G.J. Watson A.L. Hempstock J. Skepper J.N. Jauniaux E. Uterine glands provide histotrophic nutrition for the human fetus during the first trimester of pregnancy.J Clin Endocrinol Metab. 2002; 87: 2954-2959Crossref PubMed Scopus (247) Google Scholar, 50Hempstock J. Cindrova-Davies T. Jauniaux E. Burton G.J. Endometrial glands as a source of nutrients, growth factors and cytokines during the first trimester of human pregnancy: a morphological and immunohistochemical study.Reprod Biol Endocrinol. 2004; 2: 58Crossref PubMed Scopus (148) Google Scholar). The components of uterine fluid are derived from a number of sources: secretions from the luminal epithelium and glands, proteins selectively transudated from blood, and likely contributions from tubal fluid; in a conception cycle, secretions from the developing blastocyst will also be present. Because the uterine luminal surfaces are closely apposed, the volume of uterine fluid is small: it is difficult to retrieve more than 10 μL from a woman by aspiration. Uterine fluid has a much less complex proteome than that of endometrial tissue because it lacks high abundance cellular proteins. Furthermore, collection of uterine fluid by either lavage or aspiration is less invasive than tissue biopsy; both have been used for proteomic analyses. Given that the endometrial surface is covered by a substantial glycocalyx that almost certainly binds a number of secreted factors, these two methods of retrieval would not be expected to be quantitatively or even qualitatively identical, though most of the proteins retrieved are common (51Hannan N.J. Nie G. Rainzcuk A. Rombauts L.J. Salamonsen L.A. Uterine lavage or aspirate: which view of the intrauterine environment?.Reprod Sci. 2012; 19: 1125-1132Crossref PubMed Scopus (28) Google Scholar). However, it is reasonable to assume that both should contain useful biomarkers. Importantly, because the blastocyst first enters the uterine cavity approximately 4 days after ovulation and then completes its preimplantation development, early midsecretory phase fluid provides a highly representative sampling of the peri-implantation environment. Many proteins that are maximally expressed by endometrial luminal and/or glandular epithelium during the midsecretory phase are localized toward the apical compartment of the cells, whereas earlier in the cycle they are more basally located (52Jones R.L. Hannan N.J. Kaitu'u T.J. Zhang J. Salamonsen L.A. Identification of chemokines important for leukocyte recruitment to the human endometrium at the times of embryo implantation and menstruation.J Clin Endocrinol Metab. 2004; 89: 6155-6167Crossref PubMed Scopus (238) Google Scholar). Thus, their secretion during the midsecretory phase could be anticipated. Measurement of factors known as critical for implantation such as leukemia inhibitory factor (LIF) (53Ledee-Bataille N. Lapree-Delage G. Taupin J.L. Dubanchet S. Frydman R. Chaouat G. Concentration of leukaemia inhibitory factor (LIF) in uterine flushing fluid is highly predictive of embryo implantation.Hum Reprod. 2002; 17: 213-218Crossref PubMed Scopus (173) Google Scholar) and PC6 (54Heng S. Hannan N.J. Rombauts L.J. Salamonsen L.A. Nie G. PC6 levels in uterine lavage are closely associated with uterine receptivity and significantly lower in