Proteomics of the human

endometrium and uterine uid:

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 Henrys 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 dened 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 modications 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 denition of cellular location, timing of production of identied 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 proles necessary to inform clinical prac-
tice and serve as an end point for optimizing stimulation cycles in IVF clinics as well as more
clearly dening the molecular mechanisms underlying successful implantation. (Fertil Steril Use your smartphone
2013;99:108692. 2013 by American Society for Reproductive Medicine.) to scan this QR code
Key Words: 2D-DIGE, biomarkers, multiplex analysis, uterine uid, uterine receptivity and connect to the
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ENDOMETRIAL CYCLICITY shed during menstruation while, in par- restored. After ovulation, and driven
AND RECEPTIVITY allel, the denuded surface is rapidly re- by rising progesterone levels, the
epithelialized. During the proliferative various cell types differentiate in prep-
The endometrium is unique among
phase, under the inuence of estrogen, aration for implantation should con-
adult tissues in the extent of remodeling
all the cellular compartments and their ception occur (1).
that it undergoes during each menstrual
supporting extracellular matrices are For most of the menstrual cycle, the
cycle. The outer functionalis layer is
endometrium is not receptive for an
Received August 14, 2012; revised September 4, 2012; accepted September 7, 2012; published online embryo to implant; indeed, for at least
October 6, 2012. some of the time, specically in the
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 proliferative phase, it appears to be
the work reported in this review, an NHMRC project grant pending application for further work actively hostile (2). Embryo transfer
to identify uterine receptivity markers, and a grant awarded by Monash IVF Education and Re-
search Foundation to undertake research into uterine receptivity markers (20112012 and
studies in animals during the 1970s
20122013), and also plans to patent in the eld of uterine receptivity biomarkers. L.J.F.R. has and 1980s (3, 4) demonstrated that
consulted with MSD (not related to this work) and received a travel grant (not related to this developmental synchrony between the
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. embryo and endometrium is essential
Supported by the National Health and Medical Research Council of Australia: program grant 494802, for successful implantation and hence
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 for establishment of pregnancy.
and Education Foundation, and the Victorian Governments Operational Infrastructure Program. In women, this receptive phase is
Reprint requests: Lois A. Salamonsen, Ph.D., Prince Henrys Institute of Medical Research, Uterine Biol-
ogy, P.O. Box 5152, Clayton, Victoria 3168, Australia (E-mail: lois.salamonsen@princehenrys.org). of only approximately 4 days in
duration, occurring at about 5 to 10
Fertility and Sterility Vol. 99, No. 4, March 15, 2013 0015-0282/$36.00 days after the luteinizing hormone
Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.fertnstert.2012.09.013 (LH) surge (the midsecretory phase) (5).

1086 VOL. 99 NO. 4 / MARCH 15, 2013


During the past decade, many molecular changes associ-
ated with endometrial receptivity in women have been iden-
tied. These occur in the luminal and glandular epithelium, in
the stromal compartment during the initiation of decidualiza-
tion 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 rst apposition
of the blastocyst to the luminal epithelial surface, its attach-
ment and invasion of trophoblast cells between the epithelial
cells, and syncytialization and invasion of the extracellular
trophoblast through the developing decidua until some tro-
phoblast cells invade and remodel the spiral arterioles (6). Pre-
paredness 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 com-
partment 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
The intrauterine environment for implantation. (A) In the uterine
cavity, glandular secretions including nutrients, enzymes, cytokines,
antiproteases, and transport proteins provide the milieu for nal
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, Ca regulators, and secreted molecules as
above.
Salamonsen. Proteomics of the human endometrium. Fertil Steril 2013.
VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility
Molecular changes in the glands and luminal epithelium
result in changes in their secretions into the uterine cavity.
Typically uterine uid 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, Ca regulators, growth factors,
cytokines, chemokines, and prostanoids) (1013).
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 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 re-
sult 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) (1719), 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
Genomewide analyses applied to the endometrium have gen-
erated distinct molecular proles in terms of cycle stage
(2224); disease status, such as endometriosis (25); fertility
status; and after various ovarian stimulation protocols
(2628). However, changes in gene expression are not
necessarily reected 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 identied 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 two-
dimensional gel electrophoresis (2D DiGE) and MALDI-TOF-
MS/MS has enabled the identication of 196 differentially
expressed spots between cycle phases (31). Not unexpectedly,
the vast majority of increases in protein abundance were ob-
served in secretory phase endometrium (196 vs. 39 in the se-
cretory vs. proliferative phase, respectively). Of these, 42
sequences identied separate gene products, and 34 se-
quences were isoforms of either different pI (suggesting dif-
ferential phosphorylation or glycosylation and presenting
as protein spot trains) or different size and charge, indicat-
ing differences in processing (31). It is interesting that only
50% of the proteins identied in our proteomic analysis (31)
FIGURE 2
Molecular changes in endometrial tissue between the proliferative and secretory phases. Differentially regulated proteins, initially identied 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.
1088
correlated with published gene array data; the other 50%
showed no or only minor changes in mRNA expression. Im-
munohistochemistry validated a number of the identied pro-
teins as present in endometrial epithelial cells and at
signicantly different levels between proliferative and secre-
tory endometrium (Fig. 2). Two-dimensional DiGE has also
proven useful in identifying substrates of proprotein conver-
tase (PC)6a protease essential for implantation in micein
human secretory phase endometrium (3639).
Although analysis of tissue biopsies has some merits, it
has many disadvantages. Endometrial tissue displays a re-
markable diversity in its morphology, with substantial
changes both in its architecture and cellular composition (in-
cluding leukocyte content) between the cycle phases. For ex-
ample, we identied one protein, coronin 1, as differentially
regulated between proliferative and secretory phases (unpub-
lished data): the immunohistochemical analysis revealed that
it was wholly contained in leukocyte populations and not in
the endometrial stromal, epithelial, or vascular compart-
ments. 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 amplication
(40). However, the low yield of protein provided by this
VOL. 99 NO. 4 / MARCH 15, 2013

approach makes the proteomic analysis of these samples chal-
lenging. Furthermore, many proteins identied in tissue bi-
opsy samples are either the abundant structural proteins or
proteins associated with basic cell functions such as prolifer-
ation. Substantial prefractionation of the samples before
analysis and the application of the newer gel-free tech- (52). Thus, their secretion during the midsecretory phase could
niques (41) may enable detection of the lower abundance pro-
teins that are functionally important for receptivity.
Matrix-assisted laser desorption/ionization (MALDI) im-
aging mass spectrometry (IMS) is also applicable to tissue sec-
tions. This allows the spatial localization of hundreds of
masses simultaneously, can provide molecular signatures re-
lated to morphology, and has also identied protein se-
quences of biomarkers for prognosis and disease severity in
cancer-related biopsies (4244). Application of MALDI-IMS
may provide receptivity-specic signatures for endometrial
luminal and glandular epithelium or for the stromal compart-
ment (45). Finally, several studies have suggested that post-
translational modication of proteins is an important
feature of endometrial biology; these modications will con-
tinue to challenge researchers in this eld.
UTERINE FLUID
Uterine uid (a protein-rich histotroph), and in particular the
glandular secretions that provide many of its components, is
critical for implantation. This was rst denitively estab-
lished in ewes in which endometrial glandular development
had been inhibited during early postnatal life. In the resultant
adults, conceptus development was retarded, and implanta-
tion failed to occur (46). This nding 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 rst trimester (49, 50).
The components of uterine uid are derived from a number
of sources: secretions from the luminal epithelium and
glands, proteins selectively transudated from blood, and
likely contributions from tubal uid; 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 uid is small: it is difcult
to retrieve more than 10 mL from a woman by aspiration.
Uterine uid has a much less complex proteome than that
of endometrial tissue because it lacks high abundance cellular
proteins. Furthermore, collection of uterine uid by either
lavage or aspiration is less invasive than tissue biopsy; both
have been used for proteomic analyses. Given that the endo-
metrial 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 quanti-
tatively 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. Impor-
tantly, because the blastocyst rst enters the uterine cavity
approximately 4 days after ovulation and then completes its
preimplantation development, early midsecretory phase uid
provides a highly representative sampling of the peri-
implantation environment.
VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility
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
be anticipated. Measurement of factors known as critical for
implantation such as leukemia inhibitory factor (LIF) (53)
and PC6 (54, 55) in uterine uid conrms 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 difcult
(56). These same serum proteins are also present in uterine as-
pirate (57). Much earlier studies in the ewe support the selec-
tive transudation of serum proteins rather than their direct
derivation from blood, as the proteome of uterine uid 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 identication of many proteins of lower
abundance in uterine uid (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 com-
bined with mass spectrometry. One such study (57) identied
>800 proteins in uterine uid, including a number of proteins
of relevance to the intrauterine environment such as mucins,
defence proteins, S100 proteins, MMP9, and TIMP1. One po-
tential problem with the use of aspirates is that the very small
volume precludes removal of any oating cells before freez-
ing and hence some structural proteins may be detected.
Multiplex analysis such as Luminex, an alternative ap-
proach to blinded proteomic analysis, enables the simulta-
neous measurement of multiple analytes; these are limited
by antibody availability and also compatibility with the mul-
tiplex 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 de-
termined. 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 articially
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 prole of interleukin-
1b (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 identied. This prole
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 var-
iable between women, even those at the same phase of the
cycle. Validation studies using immunohistochemistry deter-
mined that endometrial epithelial cells were the source of the
analytes tested; these included factors such as vascular endo-
thelial growth factor A (VEGFA), which is generally an endo-
thelial cell product but is also highly expressed and secreted
by the endometrial epithelium. Final conrmation of local
glandular production was that a similar secretory prole
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 anal-
yses provides progressively more potential markers of endo-
metrial receptivity; however, these proteomic evaluations
are invariably performed on only a small number of individ-
uals (7, 10, 59). Thus, we can assume that many identied
markers will prove to be false positives arising from
individual variation. It is thus essential that any proposed
biomarkers are subject to analysis on sufciently 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 dene clear inclusion/exclusion
criteria and use of sufciently 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
uid with those from the midsecretory phase or the midsecre-
tory phase samples from fertile versus infertile women?
Markers thus identied may not be relevant to individuals un-
dergoing ovarian stimulation, upon which many genomic
studies have focused.
Although functional studies are not essential for valida-
tion, demonstration of actions related to the physiologic roles
of each potential marker add strength and provide mechanis-
tic insight. Knowledge of function gained through genetically
modied mice has been useful in some instances. For exam-
ple, 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 uid from infertile and fertile women has
not been consistent, and its value as a single marker of recep-
tivity 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 implanta-
tion. Uterine uid stimulates the adhesive capacity of endo-
metrial epithelium, along with the outgrowth of mouse
blastocysts; these actions can be replicated by certain individ-
ual factors present in the uid including VEGF (51). The che-
mokine 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 in-
cluding integrins and osteopontin (SPP1) (67), which are im-
portant at the implantation site. Trophoblast spheroid
attachment to endometrial epithelial cell layers is reduced in
stably transfected cells with reduced PC6 secretion; this oc-
curs, at least in part, via PC6 cleavage of integrins on the en-
dometrial epithelial surface into their functional forms (68).
NEXT STEPS
The application of proteomics has the potential to provide bio-
markers for endometrial receptivity. Our experience has dem-
onstrated that endometrial tissue itself is not ideal for
biomarker discovery, given its cellular complexity and vari-
ability both among individuals and among cycle stages. Anal-
ysis of uterine uid, 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 uid represent the actual mi-
croenvironment for implantation. Uterine uid also contains
relatively low levels of cellular proteins, making it much less
complex than tissue and consequently more amenable to pro-
teomic investigations. A combination of prefractionation
techniques, including protein enrichment using hydrogel
nanoparticles (69, 70) along with improving proteomic
technologies, will enable the identication of lower
abundance proteins and posttranslational modications,
several of which may be biomarkers of fertility status. It is
unlikely that any single marker will enable identication of
disturbed receptivity in all affected women; identication of
a ngerprint 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 uid 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 proto-
cols for ovarian stimulation and luteal phase support. This
knowledge will provide for prediction of the likely outcome
VOL. 99 NO. 4 / MARCH 15, 2013

of embryo transfer in any given treatment or natural cycle.
Such receptivity markers may also prove to be attractive tar-
gets for contraceptive development.
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