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Proteomics of the human endometrium and uterine fluid: a pathway to biomarker discovery
Proteomics of the human endometrium and uterine fluid: a pathway to biomarker discovery Lois A. Salamonsen, Ph.D.,a Tracey Edgell, Ph.D.,a Luk J. F. Rombauts, M.D., Ph.D.,b,c Andrew N. Stephens, Ph.D.,a David M. Robertson, Ph.D.,a Adam Rainczuk, Ph.D.,a Guiying Nie, Ph.D.,a and Natalie J. Hannan, Ph.D.a a Prince Henry’s Institute of Medical Research, Monash Medical Centre; b Monash University, Department of Obstetrics and Gynaecology; and c Monash IVF, Clayton, Victoria, Australia
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 twodimensional 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 Use your smartphone clearly defining the molecular mechanisms underlying successful implantation. (Fertil SterilÒ to scan this QR code 2013;99:1086–92. Ó2013 by American Society for Reproductive Medicine.) and connect to the Key Words: 2D-DIGE, biomarkers, multiplex analysis, uterine fluid, uterine receptivity Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/salamonsenl-proteomics-human-endometrium-uterine-fluidbiomarker/
ENDOMETRIAL CYCLICITY AND RECEPTIVITY 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
Received August 14, 2012; revised September 4, 2012; accepted September 7, 2012; published online October 6, 2012. L.A.S. has received grants from NHMRC (Australia), Monash IVF, and Merck Serono GFI (unrelated to this work). T.E. received a Merck Serono Grant for Fertility Innovation Award after completion of the work reported in this review, an NHMRC project grant pending application for further work to identify uterine receptivity markers, and a grant awarded by Monash IVF Education and Research Foundation to undertake research into uterine receptivity markers (2011–2012 and 2012–2013), and also plans to patent in the field of uterine receptivity biomarkers. L.J.F.R. has consulted with MSD (not related to this work) and received a travel grant (not related to this work). A.N.S. has nothing to disclose. D.M.R. has nothing to disclose. A.R. has nothing to disclose. G.N. has nothing to disclose. N.J.H. has nothing to disclose. Supported by the National Health and Medical Research Council of Australia: program grant 494802, project grant (611804 to G.N. and L.J.F.R.), fellowship grants (1002028 to L.A.S.; 494803 to D.M.R. and 494808 to G.N.), and postdoctoral training award (628927 to N.J.H.); the Monash IVF Research and Education Foundation, and the Victorian Government’s Operational Infrastructure Program. Reprint requests: Lois A. Salamonsen, Ph.D., Prince Henry’s Institute of Medical Research, Uterine Biology, P.O. Box 5152, Clayton, Victoria 3168, Australia (E-mail: [email protected]). Fertility and Sterility® Vol. 99, No. 4, March 15, 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.09.013 1086
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restored. After ovulation, and driven by rising progesterone levels, the various cell types differentiate in preparation for implantation should conception occur (1). 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 (2). Embryo transfer studies in animals during the 1970s and 1980s (3, 4) 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) (5). VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility® 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 (6). 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.
FIGURE 1
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 (7, 8); 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 (9) and within the epithelial cells from which the secretions arise (e.g., changes in junctional complexes, Caþþ regulators, growth factors, cytokines, chemokines, and prostanoids) (10–13).
THE NEED FOR MARKERS OF RECEPTIVITY 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 (14). While infertility clinics have focused on providing highquality 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 (15, 16; 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) (17–19), 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 (20, 21). 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.
THE CASE FOR A PROTEOMIC APPROACH
The intrauterine environment for implantation. (A) In the uterine cavity, glandular secretions including nutrients, enzymes, cytokines, antiproteases, and transport proteins provide the milieu for final blastocyst development and attachment. (B) At the luminal epithelial surface, changes include those in the glycocalyx (e.g., mucins), along with adhesion molecules such as integrins. Within the luminal and glandular epithelial cells, changes occur in junctional complexes, Caþþ regulators, and secreted molecules as above. Salamonsen. Proteomics of the human endometrium. Fertil Steril 2013.
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Genomewide analyses applied to the endometrium have generated distinct molecular profiles in terms of cycle stage (22–24); disease status, such as endometriosis (25); fertility status; and after various ovarian stimulation protocols (26–28). 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 (29, 30). Comparison of proteomic data with published gene expression data in similar cohorts of women (25, 29, 31) 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 1087
THE ENDOMETRIUM endometrial disorders (32, 33). 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.
Proteomic Analysis of Tissue Biopsies A number of studies (31, 34, 35) 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 twodimensional gel electrophoresis (2D DiGE) and MALDI-TOFMS/MS has enabled the identification of 196 differentially expressed spots between cycle phases (31). 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 (31). It is interesting that only 50% of the proteins identified in our proteomic analysis (31)
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 (36–39). 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 (40). However, the low yield of protein provided by this
FIGURE 2
Molecular changes in endometrial tissue between the proliferative and secretory phases. Differentially regulated proteins, initially identified by differential two-dimensional gel electrophoresis, were validated by immunohistochemistry. Upper panel: CLIC1 is detected in epithelium predominantly during the midsecretory phase. Lower panel: PGRMC1 is present in the stromal compartment maximally during the proliferative phase. Brown coloration represents positive staining. Salamonsen. Proteomics of the human endometrium. Fertil Steril 2013.
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Fertility and Sterility® 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 (41) 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 (42–44). Application of MALDI-IMS may provide receptivity-specific signatures for endometrial luminal and glandular epithelium or for the stromal compartment (45). 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 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 (46). This finding has been replicated in mice (47, 48), 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 (49, 50). 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 mL 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 (51). 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 periimplantation environment. VOL. 99 NO. 4 / MARCH 15, 2013
Discovery Studies: Uterine Lavage 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 (52). Thus, their secretion during the midsecretory phase could be anticipated. Measurement of factors known as critical for implantation such as leukemia inhibitory factor (LIF) (53) and PC6 (54, 55) in uterine fluid confirms this supposition. Analysis of uterine lavage by 2D-DiGE is confounded by the abundance of certain serum proteins, particularly albumin and gamma globulins, along with hemoglobin if there is blood contamination during sampling. These comprise 90% of the total protein in the samples, hence masking the less abundant proteins and making their detection and analysis difficult (56). These same serum proteins are also present in uterine aspirate (57). Much earlier studies in the ewe support the selective transudation of serum proteins rather than their direct derivation from blood, as the proteome of uterine fluid is very different from that of serum (58). The amino acid content is likewise different and requires active transport from the blood (8). Depletion of the highly abundant serum proteins or prefractionation of the samples can largely overcome this issue and has enabled identification of many proteins of lower abundance in uterine fluid (56). The combined data from our and other studies applying 2D-DiGE to uterine lavage (7, 59) provide a number of common proteins that are differentially regulated between the proliferative and secretory phases, between midsecretory samples from fertile and infertile women, and between proreceptive and receptive endometrium in fertile women. All these are worthy of further study. Uterine cavity aspirate has also proven applicable to analysis of the secretory phase endometrial secretome, using a range of gel-based and chromatographic techniques combined with mass spectrometry. One such study (57) identified >800 proteins in uterine fluid, including a number of proteins of relevance to the intrauterine environment such as mucins, defence proteins, S100 proteins, MMP9, and TIMP1. One potential problem with the use of aspirates is that the very small volume precludes removal of any floating cells before freezing and hence some structural proteins may be detected. Multiplex analysis such as Luminex, an alternative approach to blinded proteomic analysis, enables the simultaneous measurement of multiple analytes; these are limited by antibody availability and also compatibility with the multiplex format (i.e., they do not cross-react when multiplexed). Prefractionation of the samples is not required, but dilution requirements for individual analytes need to be carefully determined. A substantial body of work by Boomsma et al. (10, 60) measured 17 cytokines simultaneously in aspirates of the uterine cavity. These demonstrated marked quantitative differences in cytokine abundance between artificially stimulated cycles and the gold standard of natural cycles in fertile women, supporting gene array data (29, 30) that suggested disturbance of the endometrium by ovarian stimulation protocols. Furthermore, a profile of interleukin1b (IL-1b) and tumor necrosis factor-a (TNF-a) levels 1089
THE ENDOMETRIUM conducive to clinical pregnancy and applicable to the prediction of clinical pregnancy were identified. This profile was additive to the predictive value of embryo quality (60). In a larger multiplex analysis that included 42 cytokines, chemokines, and growth factors, 31 different analytes were detectable in uterine lavage at concentrations in the pg/mg of protein range (2). However, absolute levels were highly variable between women, even those at the same phase of the cycle. Validation studies using immunohistochemistry determined that endometrial epithelial cells were the source of the analytes tested; these included factors such as vascular endothelial growth factor A (VEGFA), which is generally an endothelial cell product but is also highly expressed and secreted by the endometrial epithelium. Final confirmation of local glandular production was that a similar secretory profile with similar rank order was detected in culture medium from primary endometrial epithelial cells (61).
VALIDATION AND FUNCTIONAL STUDIES The utility of increasingly sensitive mass-spectrometric analyses provides progressively more potential markers of endometrial receptivity; however, these proteomic evaluations are invariably performed on only a small number of individuals (7, 10, 59). Thus, we can assume that many identified markers will prove to be false positives arising from individual variation. It is thus essential that any proposed biomarkers are subject to analysis on sufficiently large cohorts of individuals. Strong validation usually relies also on additional sample test sets. A further complication is the apparent multifactorial nature of endometrial infertility, and it is unlikely that a single biomarker of receptivity will emerge. In recent times, we have seen the acceptance of multivariate diagnostics (62), and this would seem the likely future of endometrial receptivity testing. This methodology combines multiple complementary markers into a single value index by identifying complex patterns in data sets. However, it must be remembered that it may also result in the inadvertent incorporation of artifactual patterns associated with unforeseen biases in sample collection. Thus, it remains essential to define clear inclusion/exclusion criteria and use of sufficiently large sample cohorts from multiple sites to ensure a robust biomarker validation. Another important consideration is the lack of consensus between laboratories as to which cohorts of tissue are most relevant. Is it better to compare proliferative phase tissue or fluid with those from the midsecretory phase or the midsecretory phase samples from fertile versus infertile women? Markers thus identified may not be relevant to individuals undergoing ovarian stimulation, upon which many genomic studies have focused. Although functional studies are not essential for validation, demonstration of actions related to the physiologic roles of each potential marker add strength and provide mechanistic insight. Knowledge of function gained through genetically modified mice has been useful in some instances. For example, mice null for the leukemia inhibitory factor (LIF) gene are infertile due to implantation failure (63). In women, LIF expression in the endometrial epithelium suggests secretion 1090
into the uterine lumen, and soluble LIF has been measured in luminal secretions (53). Despite this, the analysis of LIF levels in uterine fluid from infertile and fertile women has not been consistent, and its value as a single marker of receptivity is still not clear (64, 65). Endometrial PC6 is likewise essential for implantation in mice (66) and is appropriately expressed in human endometrial epithelium and secreted into the uterine cavity. The levels of PC6 are reduced in the midsecretory phase of a cohort of infertile women but not in all, again indicating the need for multiplexing (54). More detailed functional studies have been made possible with the development of in vitro human models for implantation. Uterine fluid stimulates the adhesive capacity of endometrial epithelium, along with the outgrowth of mouse blastocysts; these actions can be replicated by certain individual factors present in the fluid including VEGF (51). The chemokine CX3CL1, which is secreted into the uterine cavity, can regulate trophoblast adhesion, at least in part via differential actions on adhesion and extracellular matrix molecules including integrins and osteopontin (SPP1) (67), which are important at the implantation site. Trophoblast spheroid attachment to endometrial epithelial cell layers is reduced in stably transfected cells with reduced PC6 secretion; this occurs, at least in part, via PC6 cleavage of integrins on the endometrial epithelial surface into their functional forms (68).
NEXT STEPS The application of proteomics has the potential to provide biomarkers for endometrial receptivity. Our experience has demonstrated that endometrial tissue itself is not ideal for biomarker discovery, given its cellular complexity and variability both among individuals and among cycle stages. Analysis of uterine fluid, collected by lavage or aspiration, has the major advantage of containing higher levels of locally secreted proteins than could be detected in other sample types (such as blood or urine). Changes in this fluid represent the actual microenvironment for implantation. Uterine fluid also contains relatively low levels of cellular proteins, making it much less complex than tissue and consequently more amenable to proteomic investigations. A combination of prefractionation techniques, including protein enrichment using hydrogel nanoparticles (69, 70) along with improving proteomic technologies, will enable the identification of lower abundance proteins and posttranslational modifications, several of which may be biomarkers of fertility status. It is unlikely that any single marker will enable identification of disturbed receptivity in all affected women; identification of a fingerprint representative of endometrium with impaired receptivity and its application in a multiplex format is a more likely scenario. A major limitation still remains in terms of uterine fluid collection, which provides considerable variability and requires standardization. Testing for receptivity is likely to provide the next leap forward for infertility clinics. Such a test would provide clear information on whether inadequate uterine receptivity is the primary cause of infertility, and will guide changes in protocols for ovarian stimulation and luteal phase support. This knowledge will provide for prediction of the likely outcome VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility® of embryo transfer in any given treatment or natural cycle. Such receptivity markers may also prove to be attractive targets for contraceptive development.
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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 twodimensional 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 Use your smartphone clearly defining the molecular mechanisms underlying successful implantation. (Fertil SterilÒ to scan this QR code 2013;99:1086–92. Ó2013 by American Society for Reproductive Medicine.) and connect to the Key Words: 2D-DIGE, biomarkers, multiplex analysis, uterine fluid, uterine receptivity Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/salamonsenl-proteomics-human-endometrium-uterine-fluidbiomarker/
ENDOMETRIAL CYCLICITY AND RECEPTIVITY 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
Received August 14, 2012; revised September 4, 2012; accepted September 7, 2012; published online October 6, 2012. L.A.S. has received grants from NHMRC (Australia), Monash IVF, and Merck Serono GFI (unrelated to this work). T.E. received a Merck Serono Grant for Fertility Innovation Award after completion of the work reported in this review, an NHMRC project grant pending application for further work to identify uterine receptivity markers, and a grant awarded by Monash IVF Education and Research Foundation to undertake research into uterine receptivity markers (2011–2012 and 2012–2013), and also plans to patent in the field of uterine receptivity biomarkers. L.J.F.R. has consulted with MSD (not related to this work) and received a travel grant (not related to this work). A.N.S. has nothing to disclose. D.M.R. has nothing to disclose. A.R. has nothing to disclose. G.N. has nothing to disclose. N.J.H. has nothing to disclose. Supported by the National Health and Medical Research Council of Australia: program grant 494802, project grant (611804 to G.N. and L.J.F.R.), fellowship grants (1002028 to L.A.S.; 494803 to D.M.R. and 494808 to G.N.), and postdoctoral training award (628927 to N.J.H.); the Monash IVF Research and Education Foundation, and the Victorian Government’s Operational Infrastructure Program. Reprint requests: Lois A. Salamonsen, Ph.D., Prince Henry’s Institute of Medical Research, Uterine Biology, P.O. Box 5152, Clayton, Victoria 3168, Australia (E-mail: [email protected]). Fertility and Sterility® Vol. 99, No. 4, March 15, 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.09.013 1086
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restored. After ovulation, and driven by rising progesterone levels, the various cell types differentiate in preparation for implantation should conception occur (1). 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 (2). Embryo transfer studies in animals during the 1970s and 1980s (3, 4) 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) (5). VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility® 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 (6). 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.
FIGURE 1
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 (7, 8); 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 (9) and within the epithelial cells from which the secretions arise (e.g., changes in junctional complexes, Caþþ regulators, growth factors, cytokines, chemokines, and prostanoids) (10–13).
THE NEED FOR MARKERS OF RECEPTIVITY 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 (14). While infertility clinics have focused on providing highquality 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 (15, 16; 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) (17–19), 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 (20, 21). 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.
THE CASE FOR A PROTEOMIC APPROACH
The intrauterine environment for implantation. (A) In the uterine cavity, glandular secretions including nutrients, enzymes, cytokines, antiproteases, and transport proteins provide the milieu for final blastocyst development and attachment. (B) At the luminal epithelial surface, changes include those in the glycocalyx (e.g., mucins), along with adhesion molecules such as integrins. Within the luminal and glandular epithelial cells, changes occur in junctional complexes, Caþþ regulators, and secreted molecules as above. Salamonsen. Proteomics of the human endometrium. Fertil Steril 2013.
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Genomewide analyses applied to the endometrium have generated distinct molecular profiles in terms of cycle stage (22–24); disease status, such as endometriosis (25); fertility status; and after various ovarian stimulation protocols (26–28). 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 (29, 30). Comparison of proteomic data with published gene expression data in similar cohorts of women (25, 29, 31) 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 1087
THE ENDOMETRIUM endometrial disorders (32, 33). 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.
Proteomic Analysis of Tissue Biopsies A number of studies (31, 34, 35) 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 twodimensional gel electrophoresis (2D DiGE) and MALDI-TOFMS/MS has enabled the identification of 196 differentially expressed spots between cycle phases (31). 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 (31). It is interesting that only 50% of the proteins identified in our proteomic analysis (31)
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 (36–39). 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 (40). However, the low yield of protein provided by this
FIGURE 2
Molecular changes in endometrial tissue between the proliferative and secretory phases. Differentially regulated proteins, initially identified by differential two-dimensional gel electrophoresis, were validated by immunohistochemistry. Upper panel: CLIC1 is detected in epithelium predominantly during the midsecretory phase. Lower panel: PGRMC1 is present in the stromal compartment maximally during the proliferative phase. Brown coloration represents positive staining. Salamonsen. Proteomics of the human endometrium. Fertil Steril 2013.
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Fertility and Sterility® 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 (41) 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 (42–44). Application of MALDI-IMS may provide receptivity-specific signatures for endometrial luminal and glandular epithelium or for the stromal compartment (45). 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 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 (46). This finding has been replicated in mice (47, 48), 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 (49, 50). 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 mL 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 (51). 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 periimplantation environment. VOL. 99 NO. 4 / MARCH 15, 2013
Discovery Studies: Uterine Lavage 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 (52). Thus, their secretion during the midsecretory phase could be anticipated. Measurement of factors known as critical for implantation such as leukemia inhibitory factor (LIF) (53) and PC6 (54, 55) in uterine fluid confirms this supposition. Analysis of uterine lavage by 2D-DiGE is confounded by the abundance of certain serum proteins, particularly albumin and gamma globulins, along with hemoglobin if there is blood contamination during sampling. These comprise 90% of the total protein in the samples, hence masking the less abundant proteins and making their detection and analysis difficult (56). These same serum proteins are also present in uterine aspirate (57). Much earlier studies in the ewe support the selective transudation of serum proteins rather than their direct derivation from blood, as the proteome of uterine fluid is very different from that of serum (58). The amino acid content is likewise different and requires active transport from the blood (8). Depletion of the highly abundant serum proteins or prefractionation of the samples can largely overcome this issue and has enabled identification of many proteins of lower abundance in uterine fluid (56). The combined data from our and other studies applying 2D-DiGE to uterine lavage (7, 59) provide a number of common proteins that are differentially regulated between the proliferative and secretory phases, between midsecretory samples from fertile and infertile women, and between proreceptive and receptive endometrium in fertile women. All these are worthy of further study. Uterine cavity aspirate has also proven applicable to analysis of the secretory phase endometrial secretome, using a range of gel-based and chromatographic techniques combined with mass spectrometry. One such study (57) identified >800 proteins in uterine fluid, including a number of proteins of relevance to the intrauterine environment such as mucins, defence proteins, S100 proteins, MMP9, and TIMP1. One potential problem with the use of aspirates is that the very small volume precludes removal of any floating cells before freezing and hence some structural proteins may be detected. Multiplex analysis such as Luminex, an alternative approach to blinded proteomic analysis, enables the simultaneous measurement of multiple analytes; these are limited by antibody availability and also compatibility with the multiplex format (i.e., they do not cross-react when multiplexed). Prefractionation of the samples is not required, but dilution requirements for individual analytes need to be carefully determined. A substantial body of work by Boomsma et al. (10, 60) measured 17 cytokines simultaneously in aspirates of the uterine cavity. These demonstrated marked quantitative differences in cytokine abundance between artificially stimulated cycles and the gold standard of natural cycles in fertile women, supporting gene array data (29, 30) that suggested disturbance of the endometrium by ovarian stimulation protocols. Furthermore, a profile of interleukin1b (IL-1b) and tumor necrosis factor-a (TNF-a) levels 1089
THE ENDOMETRIUM conducive to clinical pregnancy and applicable to the prediction of clinical pregnancy were identified. This profile was additive to the predictive value of embryo quality (60). In a larger multiplex analysis that included 42 cytokines, chemokines, and growth factors, 31 different analytes were detectable in uterine lavage at concentrations in the pg/mg of protein range (2). However, absolute levels were highly variable between women, even those at the same phase of the cycle. Validation studies using immunohistochemistry determined that endometrial epithelial cells were the source of the analytes tested; these included factors such as vascular endothelial growth factor A (VEGFA), which is generally an endothelial cell product but is also highly expressed and secreted by the endometrial epithelium. Final confirmation of local glandular production was that a similar secretory profile with similar rank order was detected in culture medium from primary endometrial epithelial cells (61).
VALIDATION AND FUNCTIONAL STUDIES The utility of increasingly sensitive mass-spectrometric analyses provides progressively more potential markers of endometrial receptivity; however, these proteomic evaluations are invariably performed on only a small number of individuals (7, 10, 59). Thus, we can assume that many identified markers will prove to be false positives arising from individual variation. It is thus essential that any proposed biomarkers are subject to analysis on sufficiently large cohorts of individuals. Strong validation usually relies also on additional sample test sets. A further complication is the apparent multifactorial nature of endometrial infertility, and it is unlikely that a single biomarker of receptivity will emerge. In recent times, we have seen the acceptance of multivariate diagnostics (62), and this would seem the likely future of endometrial receptivity testing. This methodology combines multiple complementary markers into a single value index by identifying complex patterns in data sets. However, it must be remembered that it may also result in the inadvertent incorporation of artifactual patterns associated with unforeseen biases in sample collection. Thus, it remains essential to define clear inclusion/exclusion criteria and use of sufficiently large sample cohorts from multiple sites to ensure a robust biomarker validation. Another important consideration is the lack of consensus between laboratories as to which cohorts of tissue are most relevant. Is it better to compare proliferative phase tissue or fluid with those from the midsecretory phase or the midsecretory phase samples from fertile versus infertile women? Markers thus identified may not be relevant to individuals undergoing ovarian stimulation, upon which many genomic studies have focused. Although functional studies are not essential for validation, demonstration of actions related to the physiologic roles of each potential marker add strength and provide mechanistic insight. Knowledge of function gained through genetically modified mice has been useful in some instances. For example, mice null for the leukemia inhibitory factor (LIF) gene are infertile due to implantation failure (63). In women, LIF expression in the endometrial epithelium suggests secretion 1090
into the uterine lumen, and soluble LIF has been measured in luminal secretions (53). Despite this, the analysis of LIF levels in uterine fluid from infertile and fertile women has not been consistent, and its value as a single marker of receptivity is still not clear (64, 65). Endometrial PC6 is likewise essential for implantation in mice (66) and is appropriately expressed in human endometrial epithelium and secreted into the uterine cavity. The levels of PC6 are reduced in the midsecretory phase of a cohort of infertile women but not in all, again indicating the need for multiplexing (54). More detailed functional studies have been made possible with the development of in vitro human models for implantation. Uterine fluid stimulates the adhesive capacity of endometrial epithelium, along with the outgrowth of mouse blastocysts; these actions can be replicated by certain individual factors present in the fluid including VEGF (51). The chemokine CX3CL1, which is secreted into the uterine cavity, can regulate trophoblast adhesion, at least in part via differential actions on adhesion and extracellular matrix molecules including integrins and osteopontin (SPP1) (67), which are important at the implantation site. Trophoblast spheroid attachment to endometrial epithelial cell layers is reduced in stably transfected cells with reduced PC6 secretion; this occurs, at least in part, via PC6 cleavage of integrins on the endometrial epithelial surface into their functional forms (68).
NEXT STEPS The application of proteomics has the potential to provide biomarkers for endometrial receptivity. Our experience has demonstrated that endometrial tissue itself is not ideal for biomarker discovery, given its cellular complexity and variability both among individuals and among cycle stages. Analysis of uterine fluid, collected by lavage or aspiration, has the major advantage of containing higher levels of locally secreted proteins than could be detected in other sample types (such as blood or urine). Changes in this fluid represent the actual microenvironment for implantation. Uterine fluid also contains relatively low levels of cellular proteins, making it much less complex than tissue and consequently more amenable to proteomic investigations. A combination of prefractionation techniques, including protein enrichment using hydrogel nanoparticles (69, 70) along with improving proteomic technologies, will enable the identification of lower abundance proteins and posttranslational modifications, several of which may be biomarkers of fertility status. It is unlikely that any single marker will enable identification of disturbed receptivity in all affected women; identification of a fingerprint representative of endometrium with impaired receptivity and its application in a multiplex format is a more likely scenario. A major limitation still remains in terms of uterine fluid collection, which provides considerable variability and requires standardization. Testing for receptivity is likely to provide the next leap forward for infertility clinics. Such a test would provide clear information on whether inadequate uterine receptivity is the primary cause of infertility, and will guide changes in protocols for ovarian stimulation and luteal phase support. This knowledge will provide for prediction of the likely outcome VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility® of embryo transfer in any given treatment or natural cycle. Such receptivity markers may also prove to be attractive targets for contraceptive development.
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