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blastocyst (as cited in [6]). This dialogue is the result of a series of
complex autocrine, paracrine and/or juxtacrine interactions of differ-ent
molecules and their receptors, which are expressed in a coordi-nated
manner in the endometrium and/or the embryo [7,8]. The
embryo implantation process can be summarised as follows: the em-bryo,
in the morula stage, enters the uterine cavity where it continues
to divide to form the blastocyst. This happens in a defined time period
of between 72 and 96 h after fertilisation. Once inside the uterine cav-ity,
a dialogue is established between the blastocyst and the receptive
endometrium, mediated by hormones and growth factors (reviewed
in [9]), and the blastocyst is oriented correctly (apposition). The blas-tocyst
then comes into contact with the endometrial epithelium and
adheres to its surface (adhesion). Following this, penetration of the
endometrium by the blastocyst occurs (invasion) (reviewed in
[2,10]). This process is mediated by immune cells, cytokines, growth
factors, chemokines and adhesion molecules (reviewed in [9]). To
reach this point, the endometrium needs to display a proper, coordi-nated
gene expression regulation during the whole endometrial
cycle.
1.1. Transcriptomics of the human endometrium
During the endometrial cycle, there are morphological and func-tional
changes that are specific to each menstrual cycle phase and
which are caused by the response of the different endometrial cell
types to steroid hormones and paracrine molecules. Since 1950, the
histological point of view has been used for endometrial dating [11].
Afterwards, the need to understand the genetic mechanism underly-ing
the histological changes emerged. Before the genomic era, re-searchers
were limited to studying “gene by gene” to determine the
molecular changes responsible for the alterations observed. However
in the “genomic” era, the general trend is a global screening of all the
genes transcribed and their interactions [12]. So in the last decade,
the transcriptional mechanisms underlying endometrial biology
have been broadly investigated.
For global transcriptomic analyses, the preferred technique has
been microarray-based gene expression (for a review see: [12–21]).
Table 1 summarises some of the most representative reviews on en-dometrial
transcriptomics in natural cycles analysed by microarray
technology. Nevertheless despite its obvious advantages, this tech-nology
is not without its limitations [12,14]. Ponnampalam et al.
[22] were the first authors to propose the transcriptomic characteri-sation
of the human endometrium throughout the menstrual cycle
by using this technology as an alternative to histological dating. How-ever,
other groups had previously used this tool to search for a molec-ular
signature of receptivity [23–26].
Endometrial transcriptomics studies could be classified according
to their main objective: firstly, identification of the transcriptomic
physiological profile in each endometrial cycle phase (with emphasis
placed on endometrial receptivity) [22–38]; secondly, a comparison
between the endometrial profiles of fertile patients and those with
recurrent implantation failure [39–41]; thirdly, comparisons of pro-files
between healthy women and women with endometrial pathol-ogies
[42,43]; and finally, studies on the altered transcriptomic
profiles following different ovarian stimulation protocols [44,45].
Table 2 lists some of the most representative original papers on en-dometrial
transcriptomics in natural cycles analysed by microarray
technology.
There is some disagreement among transcriptomic studies, which
may be attributed to differences in experimental designs, in the type
of array used (distinct probes in different arrays), sampling conditions,
sample selection criteria, sample size, day of the cycle when the sample
Table 1
Reviews on endometrial transcriptomics.
Authors Date Reference
Giudice, L.C. 2003 [19]
Horcajadas et al. 2004 [13]
White and Salamonsen 2005 [14]
Sherwin et al. 2006 [12]
Giudice, L.C. 2006 [16]
Simmen and Simmen 2006 [15]
Horcajadas et al. 2007 [20]
Aghajanova et al. 2008 [17]
Aghajanova et al. 2008 [21]
Haouzi et al. 2011 [18]
Table 2
Original papers on endometrial transcriptomics.
Authors Date Time of biopsya Comparativeb Array ID Ref
Kao et al. 2002 CD 8–10 vs. LH+(8–10) LP vs. MS HG U95A (Affymetrix) [23]
Borthwick et al. 2003 CD 9–11 vs. LH+(6–8) LP vs. MS HG U95A–E (Affymetrix) [25]
Kuokkanen et al. 2010 CD 11–13 vs. CD 19-23 LP vs. MS with RNA and miRNA
expression
HG U133 Plus 2.0 (Affymetrix) and
miRCHIP V1 Array
[36]
Carson et al. 2002 LH+(2–4) vs. LH+(7–9) ES vs. MS HG U95A (Affymetrix) [24]
Riesewijck et al. 2003 LH+2 vs. LH+7 ES vs. MS HG U95A (Affymetrix) [26]
Mirkin et al. 2005 LH+3 vs. LH+8 ES vs. MS HG U95Av2 (Affymetrix) [33]
Haouzi et al. 2009 LH+2 vs. LH+7 ES vs. MS HG U133 Plus 2.0 (Affymetrix) [27]
Critchley et al. 2006 Dating by Noyes MS vs. LS HG U133A (Affymetrix) [35]
Tseng et al. 2010 Dating by Noyes ES vs. MS vs. LS HG U133 Plus 2.0 (Affymetrix) [37]
Punyadeera et al. 2005 CD 2–5 vs. CD 11–14 M vs. LP HG U133A (Affymetrix) [28]
Ponnampalam et al. 2004 Complete cycle, dating by Noyes EP vs. MP vs. LP vs. ES vs.
MS vs. LS vs. M
Home made (Peter MacCallum Cancer Institute) [22]
Talbi et al. 2006 Complete cycle, dating by Noyes EP vs. MP vs. LP vs. ES vs.
MS vs. LS
HG U133 Plus 2.0 (Affymetrix) [34]
Diaz-Gimeno et al. 2011 (LH+1 vs. +3 vs. +5 vs. +7)
(LH+(1–5) vs. LH+7 vs. CD 8–12)
LP vs. ES vs. MS vs. LS HG U133A (Affymetrix) and
Homemade “ERA”
[38]
Koler et al. 2009 CD 21 Fertility vs. infertility Array-Ready Oligo Set for the Human
Genome Version 3.0 (Operon)
[40]
Altmae et al. 2010 LH+7 Fertility vs. infertility Whole Human Genome Oligo Microarray
(Agilent Technologies)
[39]
Revel et al. 2011 CD 20–24 Fertility vs. infertility with miRNA Taqman Human MiRNA Array Card A
(Applied Biosystems)
[29]
Yanahaira et al. 2005 CD 9–11 Epithelial vs. stromal cells in
proliferative phase
BD Atlas Nylon cDNA Expression Array;
BD Biosciences (Clontech)
[31]
Van Vaerenbergh et al. 2010 LH+(5–7) MS vs. pregnant HG U133 Plus 2.0 (Affymetrix) [30]
a CD: Cycle day; LH+: LH surge+days.
b EP: Early-proliferative; MP: mid-proliferative; LP: late-proliferative; ES: early-secretory; MS: mid-secretory; LS: late-secretory; M: menstrual.
3. M. Ruiz-Alonso et al. / Biochimica et Biophysica Acta 1822 (2012) 1931–1942 1933
is collected, the statistical analysis applied to the results, separation or
not of tissue compartments, among others [13,15,27,34].
To our knowledge, there are very few human studies that aim to
define the transcriptomic profile throughout all the menstrual cycle
phases [22,34]. Most research has focused on finding a receptive
transcriptomic signature and on comparing the expression profile
during the WOI to other phases [23–26,32,33,35–38,46] for the pur-pose
of developing a tool that dates endometrial receptivity [38], or
to identify abnormalities in the endometrium [35,37,38].
1.2. Discovery of specific endometrial transcriptomic profiles
The most widely used analysis of gene expression data is hierar-chical
clustering and principal component analyses (PCA) with visual
heatmap representations. With these analyses, the grouping of the
various types of samples based on their transcriptomic profiles can
be detected. In all the studies conducted [22,26,34,35,37,38], samples
are clearly grouped according to the stage to which they belong. Gen-erally
in these studies, phase assignment is completed based on pre-vious
histological dating, and is usually performed by at least two
independent pathologists.
Grouping samples in accordance with the endometrial cycle is
clearly observed with the hierarchical clustering method, especially
in the case of those studies that analyse the entire menstrual cycle,
which is the case of Ponnampalam et al. [22] and of Talbi et al. [34].
In these two studies, samples are grouped into two main branches
which are divided into other sub-branches. In both studies, a major
branch contains the proliferative and early-secretory phases (and
the menstrual phase in the case of Ponnampalam et al. [22]), while
the other main branch groups include the samples in the mid-secretory
and the late-secretory phases. The PCA analysis reported
by Talbi et al. [34] detects four clusters of samples corresponding to
the predominant proliferative, early-secretory, mid-secretory or
late-secretory phases. Despite the sets of genes they used in this
study for PCA and hierarchical clustering being different, the same
clusters were found by both methods. In addition, although six sam-ples
were dated histologically as “ambiguous”, both PCA and hierar-chical
clustering categorise them in the same phase. These facts
favour the existence of well-defined transcriptomic profiles for each
menstrual phase. In addition, Ponnampalam et al. [22] found differ-ences
between the clustering based on the expression profiles of
some samples and the group they were classified as by the histologi-cal
criteria. The authors suggested for the first time that trans-criptome
profiling can detect those abnormalities, which are not
identified by histology and, even more importantly, the expression
of the specific gene clusters were found to be delayed by 2 days
with specific induction ovulation treatments [44].
The cited studies conclude that it is possible to accurately cata-logue
endometria at different stages based on their transcriptomic
profiles, thus facilitating the transition from the anatomical to the
molecular medicine of the human endometrium [16].
1.3. Identification of common temporal gene expression patterns
A classic gene expression data analysis is the detection of clusters of
genes with similar expression patterns throughout the endometrial
cycle. Based on the data from samples taken at different cycle phases
(early-proliferative, mid-proliferative, late-proliferative, early-secretory,
mid-secretory, late-secretory and menstrual), Ponnampalam et al. [22]
found for those genes with a fold change greater than or equal to 2,
sevenmain groups of genes with the same expression pattern through-out
the menstrual cycle, and with a peak of expression in all seven de-fined
cycle phases (menstrual, early-proliferative, mid-proliferative,
late-proliferative, early-secretory, mid-secretory and late-secretory).
This finding agrees with those obtained by Talbi et al. [34], who found
groups of genes with a peak of expression in all their studied phases
(proliferative, early-secretory, mid-secretory, late-secretory). Diaz-
Gimeno et al. [38] also detected clusters of different genes between
prereceptive and receptive samples.
Based on these results, the existence of groups of genes that be-have
similarly in terms of the expression level in all the cycle phases
can be established. This should have implications for the physiological
processes that characterise each phase. However, it is difficult to find
a functional association with the phase in which they are over- or
underexpressed.
1.4. Specific gene expression profiles in endometrial tissue compartments
The endometrium is a complex tissue with different cell compartments
and, in general, the endometrial samples analysed for transcriptomic pro-filing
consist in a combination of the different compartments or tissues.
Therefore, the reported expression measured for each gene is a weighting
of the expression of the given gene in each cell compartment analysed.
Thisweighting task can mask large differences in expression between dif-ferent
compartments, and it has been proposed to be a main cause of the
resulting differences reported in apparently similar studies [24]. To solve
this issue, laser capture microdissection to separate different tissue com-partments
has been tested in mice [47] and the human endometrium
[31,46,48,49].
Yanaihara et al. [31] conducted a study in the proliferative cycle
phase (days 9–11) by separating stroma and the epithelium. By com-paring
the various compartments, they found differentially expressed
up-regulated genes (including enzymes, growth-related proteins, and
intra/extracellular matrix proteins), most of which were present in
the stroma with functions relating to cell growth and remodelling.
Another study, conducted in the menstrual phase, found different,
specific profiles for stromal cells or glands, and for different depths
of the endometrium [49]. Nevertheless, this technique is not without
its disadvantages as it requires prior manipulation using frozen sec-tions
that extend the processing time and, in some cases, may intro-duce
errors given the need for an RNA amplification of the epithelial
compartment [20].
2. Transcriptomics of the different phases of menstrual cycle
2.1. Menstruation
Onset of menstruation corresponds to day 1 of the cycle, with an
approximate duration of 3–5 days, and includes loss of the functional
layer of the endometrium due to a drop in oestrogen levels [4].
This is one of the stages where major changes occur at the trans-criptomic
level. The main processes involved are apoptosis and tissue
breakdown. In different studies, the analysis of temporal gene expres-sion
patterns has revealed a peak of expression of those genes related
to apoptosis, inflammation, signal transduction, transcription and
DNA repair [22,28,35]. These include natural cytotoxicity-triggering
receptor 3 (NCR3) [22,28], Wnt family members (Wnt5a and
Wnt7a) [28] and the family of matrix metalloproteinases (MMPs)
[28,35,49].
NCR3 was reported for the first time by Ponnampalam et al. [22],
which increased towards the late-secretory phase and was sustained
in the menstrual phase, then its levels lowered in the proliferative
phase [22,28]. This gene is involved in the inflammatory response in
recognition of no-HLA ligands by natural killer cells [50]. NCR3 is ac-tivated
by the presence of NK cells and mimics the expression profile
of NK cells in the endometrium [22]. Wnt5a and Wnt7a are other im-portant
genes involved in this stage which belong to the Wnt path;
their high expression level during the menstrual phase lowers in
the proliferative phase, probably due to an increase in the expression
of the specific inhibitors of Wnt action. In mice, they have proven
most important in uterine glandular development [51]; thus, perhaps,
they may be implicated in endometrial gland development [28].
4. 1934 M. Ruiz-Alonso et al. / Biochimica et Biophysica Acta 1822 (2012) 1931–1942
In general, the expression of MMPs begins to increase towards the
late-secretory phase [35] before dropping back to the late-proliferative
phase [28]. These metalloproteinases are involved in tis-sue
desquamation [52]. This pattern has been observed for MMP1, -3
and ‐10, up-regulated in the menstrual phase vs. the proliferative
phase. In contrast, MMP26 (endometase) shows increased expression
in the proliferative phase; this is possibly because it is involved in tis-sue
remodelling, which occurs during this phase [28].
Other genes detected in the premenstrual phase with maximum ex-pression
levels are protease-activated receptor type 1 (F2R (PAR-1)) and
lysyl oxidase (LOX) [35]. The thrombin receptor (PAR-1) is responsible
for mediating many thrombin functions in the cardiovascular system,
and is located on the surface of endothelial cells and platelets. Activation
of this molecule may lead to platelet aggregation, vascular smooth mus-cle
mitogenesis and vasoconstriction. It is likely that PAR-1 plays an im-portant
role in the events preceding and is, at least partly, responsible for
menstrual bleeding [35]. On the other hand, the LOX gene encodes an
amino oxidase, which is crucial in the collagen deposition process and
various related wound-healing functions [53]; therefore, it seems likely
that it plays an important role in endometrial repair [35].
Despite all these studies, the menstrual phase is the least studied
from the global gene expression perspective.
2.2. Proliferative phase
The proliferative phase lasts from the end of the menstrual phase
(days 3–5) until ovulation (days 12–14). During this phase, oestrogen
levels increase, leading to stromal and glandular proliferation, and to
the elongation of spiral arteries to create a new functional layer [4].
The proliferative phase involves several processes, including cellu-lar
proliferation and differentiation, extracellular matrix remodelling,
angiogenesis and vasculogenesis [15,34,36,54]. Moreover, there is
also a high expression of the genes related to adhesion and ion chan-nels
[34]. The cellular proliferation in this stage is evidenced by mitot-ic
activity, DNA synthesis and increased tissue weight (as cited in
[34]).
It is noteworthy that there is a global decrease in gene expression
in the transition from the menstrual to the proliferative phase be-cause
up to 64% of the genes that are differentially expressed between
these two phases are down-regulated in the proliferative phase [28].
The processes that increase in this transition (with implicate up-regulated
genes) are tissue remodelling (MMP26, TFF3) [25,28], cell
differentiation (Hoxa10, Hoxa11) and vasculogenesis (Connexin-37,
Hoxb7, sFRP) [28].
MMPs are repressed in this stage, except for MMP26, whose ex-pression
is activated and maintained until the WOI. This expression
pattern suggests that MMP26 could be implicated in both tissue
remodelling and blastocyst invasion [28]. In addition, TFF3 is another
candidate gene that falls in the endometrial remodelling factor cate-gory
[25]. The trefoil family of peptides (TFF) are mucin-associated
peptides and their high expression in the proliferative vs. secretory
phases [23,25] makes them candidates as participants in endometri-um
regeneration after the menstrual phase [25].
Hox family genes are up-regulated in this stage if compared to the
menstrual phase. This overexpression appears to be caused by in-creased
oestrogen levels that inhibit the Wnt5a and Wnt7a genes to
allow the expression of Hoxa10 and Hoxa11. The Hox gene expres-sion
completes glandular development and differentiation, which
were initiated by Wnt5a and Wnt7a [28]. This context explains the
up-regulation of numerousWnt inhibitors (like sFRP and WIF). More-over
one of these inhibitors, sFRP, has been associated with vascular
remodelling and maturation, while Hoxb7 has been related with vas-cular
development induction (cited in [28]).
Towards the end of the proliferative phase and during the transi-tion
to the secretory phase, there is a change in the predominant pro-cesses
within the endometrium in relation to the early-secretory
phase, which include: cell motility, communication and cell adhesion,
Wnt receptor signalling, the I-κB kinase/nuclear factor (NF)-κB cas-cade,
cellular signalling, cellular cycle regulation, cellular division,
cellular proliferation, collagen metabolism, extracellular matrix regu-lation,
signal transduction and regulation of enzymes and ion chan-nels.
Some highly represented molecular functions are those related
to cell cycle regulation, DNA synthesis and steroid binding, and the
most prominent cellular components involved include chromosomes,
replication fork, DNA polymerase, microtubule skeleton, extracellular
matrix, organelles not bound to membranes and collagen [34].
The genes described as being overexpressed are consistent with
mitotic activity and tissue remodelling, which occur in this phase
[34]: Olfactomedin 1 [25,34], SOX4, MMP11, tPA (PLAT), ADAM12,
retinoic acid, members of the IGF and TGF family, FGF1, HGF, FGFR 3
and 1.2 and specific activator protein-kinase activities [34].
Olfactomedin1 (OLFM1) is suppressed when comparing the mid-secretory
phase with the early-secretory one [38], suggesting that
this gene is regulated by progesterone, which would inhibit its ex-pression
[34]. In contrast, SOX4 shows not only a down-regulation
in the proliferative vs. the menstrual phase [28], but a down-regulation
in the early secretory vs. the proliferative phase, implying
that it is inhibited by oestrogen [34]. The aforementioned cytokines
are the factors involved in immune reactions, but are also involved
in cell communication processes. Therefore during the proliferative
phase, these molecules act as paracrine, or even autocrine, molecules
by participating in oestrogen modulation and progesterone control of
endometrial cell differentiation. IL1, TNF alpha, TGF alpha, EGF or Csf1
(M-CSF) are involved in establishing a specific regional distribution of
cell populations and their differential proliferative activity [1].
On the other hand, high levels of DNA replication licensing mem-bers
(such as MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, and origin
recognition complex proteins) have been found. These data suggest
that, in addition to cell cycle regulation, DNA replication licensing is
likely to be another key regulatory point in the hormonal regulation
of epithelial cell proliferation in the human endometrium [36].
2.3. Secretory phase
The secretory phase takes place between ovulation (days 12–14)
and onset of menstruation in a new cycle (days 28–30). It is sub-divided
into early-, mid- and late-secretory phases, and corresponds
to the luteal phase of the ovary [4]. Traditionally, this phase has been
the most studied because the endometrium becomes receptive during
the mid-secretory subphase, allowing for embryo implantation.
During the secretory phase, the endometrium is subjected to in-creasing
levels of oestrogen and progesterone. A rise in progesterone
involves glycogen and mucus secretion. In the late-secretory phase,
oestrogen and progesterone levels drop in the absence of implanta-tion,
implying that the menstrual phase undergoes preparation [4].
The endometrium's state of receptivity includes a limited time pe-riod
in which the endometrial epithelium acquires a transient state
that is dependent on steroid hormones during blastocyst adhesion,
and penetration occurs [9,55]. It is the result of the synchronised, in-tegrated
interaction of ovarian hormones, endometrial factors and
signals from the embryo [9]. Moreover to achieve this state of recep-tivity,
the endometrium responds to a process mediated by growth
factors, lipid mediators, transcription factors and cytokines with para-crine
action (reviewed in [8]). This process is tightly controlled in
terms of space and time. This period of receptivity, or the WOI, was
suggested in the 1970's [10]. The WOI opens on either day 19 or 20
of the menstrual cycle [5] or on days 4 or 5 after progesterone pro-duction
or administration [56]. This window expands for 4–5 days,
corresponding to days 19–24 of the menstrual cycle [5,56]. This oc-curs
for most women with regular cycles, with implantation on days
7–10 after the LH surge. However in a small percentage of women,
implantation can occur from days 6 to 12 after the LH surge [57]. In
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assisted reproductive treatments, it is possible to induce a clinical
WOI by supplying progesterone and oestradiol to synchronise with
the embryo transfer [58]. WOI duration has been proven by several
studies which followed the embryo implantation process (cited in
[23]), onset of structural changes and the emergence of pinopodes
[55], or were based on implantation success rates on different days
during or after the WOI [57].
Given the interest shown in this phase, several studies have inves-tigated
its transcriptomic profile. The main objective has been to find
a molecular signature that is characteristic of a receptive endometri-um
to gain insight into the implantation phenomenon Some of
these studies were designed by comparing samples taken in the re-ceptive
phase vs. samples taken in the prereceptive (early-secretory)
phase [24,26,33,37,38,46,59]. Other studies were designed to use
proliferative phase [23,25,36] or late-secretory phase samples
[35,37] (reviewed in [20]). Different studies also vary in terms of
the many other parameters used, such as number of patients, taking
samples from the same patient or not, using pools of samples, days
of the cycle when samples are taken, or types of data analysis
[13,15,16,60]. Despite these differences, they all agree on the exis-tence
of a transcriptomic profile that is typical of the WOI. However,
and perhaps due to differences in design, no consensus has been
reached on the genes making up this transcriptomic signature.
According to the review by Giudice et al. [16] if compared with
some studies like that of Talbi et al. [34], we see that of the 75 up-regulated
genes and the 56 down-regulated ones in Riesewijck et al.
[26], only 41 and 11 respectively match. Of the 74 up-regulated and
the 76 down-regulated genes in the study of Carson et al. [24], only
11 and 1 respectively agree with the study of Talbi et al. [34]. Of the
49 up-regulated and the 58 down-regulated genes in the study of
Mirkin et al. [33], only 14 and 3 respectively agree. However, these
studies are complementary to each other and shed light on the com-plexity
of endometrial receptivity and the molecules (known and
new) involved in the implantation process [13].
Despite the variability in transcriptomic studies, potentially im-portant
factors in endometrial receptivity have been found: growth
factors, cytokines, lipoproteins, transcription factors and other mole-cules
regulated by steroid hormones [9,19,24]. Furthermore, the over-all
set of results suggests that transcriptional activation has to occur
to achieve a state of receptivity. This is because most genes are up-regulated
if compared to their expression in the prereceptive phase
[25–27,38,44,59]. For this reason, the term “the transcriptional awak-ening
process” was coined [44]. However, this activation process is
detected when samples were taken on LH +7 because, in those stud-ies
in which samples were taken on LH+8–10 [23,24,33], more genes
were found to be down-regulated than up-regulated.
2.3.1. Early-secretory
The early-secretory phase is characterised by the predominance of
the processes related to cell metabolism (fatty acids, lipids, eicosa-noids,
and amino alcohols), transport (with a large representation
of transporters for the biological molecules involved in these meta-bolic
processes), germ cell migration (which could facilitate the
transport of sperm and ensure an aseptic environment) and negative
cell proliferation regulation. An increase in metabolism is consistent
with the fact that this phase is biosynthetically highly active, probably
in preparation for embryo implantation, and mitosis inhibition is
supported by the down-regulation of numerous growth factors [34].
Some of the prominent genes up-regulated in the early secretory
phase are MSX1 [26,33], PIP5K1B [33], Keratin 8, 17βHSD, Osteopontin
(SSP1), secretoglobins, metallothionein, arginase 2, phospholipase C,
DKK1, Aquaporin 3 and MUC1 [34]. Of the genes listed, the following
are highlighted; MSX1, which belongs to the family of HOX genes that
correlate temporally and spatially with critical morphogenesis events
[33]; 17βHSD, which regulates oestradiol availability in the endometri-um
[34]; PIP5K1B, which regulates a wide range of cellular processes,
including proliferation, survival and cytoskeleton organisation [33];
and MUC1, which maintains cell surface hydration by lubricating and
protecting cells against microorganisms and enzymes [34,61].
Wnt pathway regulation is striking. Some ligands, but not others,
are down-regulated. Furthermore some Wnt inhibitors, such as
sFRP1, are repressed, but DKK1 (another inhibitor of Wnt action) is
highly up-regulated in this phase vs. the proliferative phase [34] to
further increase in the mid-secretory phase [24].
2.3.2. Mid-secretory
This phase is characterised by being highly active metabolically,
secretory, non-proliferative, with great importance being placed on
the innate immune response, stress response and response to
wounding [15,16,34].
The secretory phase is when more gene expression changes are
detected due to the presence of the progesterone peak. These changes
coincide with the time that the endometrium becomes receptive. The
genes whose expression changes during the transition between the
early- and the mid-secretory phases, and between the mid- and the
late-secretory phases, are potential candidate genes to be regulated
by progesterone [23,25,34]. In fact, Ponnampalam et al. [22] detected
a cluster of genes that follows a temporal regulation pattern during
the endometrial cycle which mimics the increase in circulating pro-gesterone.
These genes have been identified in some of the major bio-logical
processes taking place during implantation, such as signalling,
growth, differentiation and cell adhesion. However, there are no sig-nificant
changes associated with the peak of oestrogen.
The genes whose expression alters during the transition from the
early- to the mid-secretory phases are mainly involved in cell cycle
regulation, ion binding, transport of signalling proteins, and members
of the family of immunomodulators [33]. The genes that are repressed
in this subphase transition are mainly sFRP, PR, PR membrane compo-nent
1, ER-α, MUC1, 17βHSD-2, MMP11 [34], Endothelin 3 (EDN3)
[23,24,26] and, especially, Olfactomedin-1 [23,24,26,34], which is
present in most studies.
On the other hand, there are those genes that are overexpressed in
the mid-secretory vs. the early-secretory. Here there are some genes
whose known function seems to be involved in implantation prepara-tion,
and they are involved in processes related to cell adhesion, me-tabolism,
response to external stimuli, signalling, immune response,
cell communication and negative regulation of proliferation and de-velopment
[34,38]. The processes related to the immune system in-clude
stress response, defence response, humoral immune response,
innate immune response and response to wounding [38]. The trans-criptomic
detection of activation of the immune response in mid-stage
secretory is consistent with previous studies which have al-ready
linked it to implantation [62,63]. The overexpressed molecular
functions in this stage include three terms: oxidoreductase activity,
carbohydrate binding and receptor binding, and cellular components
and spindle microtubules [38]. It is worth noting some of the genes
involved in these processes: cysteine-rich secretory stress protein 3
(CRISP3) [34,38], glutathione peroxidase 3 (GPX3) [22,26,34,38],
metallothionein 1G, 1H, 1E, 1F, 1L, 1X and 2A [23,25,33,34,38], leukae-mia
inhibitory factor (LIF) (in contrast to other studies: [24,26,33], but
supported by others: [34,38,64]), granulysin, Glycodelin (PAEP, PP-14)
[23,25,26,34,38], IGFBP-3 [26,34], Apolipoprotein D [23,24,26] and
Dickkopf 1 (DKK1) [23,24,26,36].
A highly represented process during the mid-secretory phase is
cell adhesion. Entrance to the blastocyst in the receptive endometrium
triggers the production of cytokines by endometrial epithelial cells.
These cytokines modulate receptivity by regulating the expression of
the adhesion molecules in mammals [65]. Deregulation of cytokines
and their signals leads to implantation failure or abnormalities in pla-centa
formation [66]. Although activation of LIF in this stage is disput-ed
in some studies (possibly due to the use of different microarray
versions, according to [21]), it is a cytokine that induces proliferation
6. 1936 M. Ruiz-Alonso et al. / Biochimica et Biophysica Acta 1822 (2012) 1931–1942
and differentiation of many tissue types. Some women with infertility
or recurrent abortions present low levels of LIF or mutations in the
coding region of this gene [67–69]. Recently, LIF has been shown to
play an important role in the adhesion and invasion stages during im-plantation
due to an anchor effect in the trophoblast [70]. These find-ings
suggest that LIF plays an important role in implantation and many
studies have focused on this (reviewed in [21]). According to the re-view
by Rashid et al. [9], optimal levels of LIF are necessary for im-plantation
since the neutralisation of LIF in vitro models inhibits
implantation of the blastocyst. Homogeneous LIF production in preg-nant
women seems to be consistent with its importance in the re-productive
process [71]. Another important molecule involved in
adhesion is Osteopontin (SPP1), a structural protein of the extracellu-lar
matrix [33]. SPP1 has been found to be up-regulated in most of the
studies presented. This glycoprotein is a ligand for αvβ3 integrin, it
mediates cellular adhesion and migration during implantation, and
is regulated by progesterone [72]. In addition, SPP1 shows its peak of
expression in endometrial epithelial cells and has also proven its pres-ence
on the surface of pinopodes [73].
The cellular metabolism is a well represented process in the secre-tory
phase. Apolipoprotein D is a multifunctional transporter involved
in both lipid metabolism and the transport of cholesterols. The activa-tion
of its expression detected in most studies is consistent with the
activation of lipid metabolism in the mid secretory phase [17].
The immune response also plays an important role throughout the
secretory phase. In the mid-secretory phase, an up-regulation of the
genes involved in the activation of the innate immune response
takes place (complement, antimicrobial peptides, Toll-like receptor
expression), as well as increased chemotaxis of monocytes, T cells
and NK cells (CXCL14, granulysin, IL15, carbohydrate sulfotransferase
2, and suppression of NK and T-cell activation) [34]. CXCL14 is a che-mokine
that is thought to be the major recruiter of monocytes during
the WOI due to its strong overexpression in this stage [34]. In addi-tion,
a role for this chemokine has been recently proposed in that it
stimulates the chemotaxis of natural killer cells for clustering around
epithelial glands [74]. IL15 is thought to play important roles in uNK
cell proliferation and differentiation [75], and has been suggested to
be involved in the recruitment of peripheral blood cells CD16-NK
[76]. Glycodelin has been broadly studied in relation to implantation
and it also participates in immune response regulation [23] by an im-munosuppressive
effect promoting the opening of the WOI by
lowering maternal immunological responses to the implanting em-bryo
(as cited in [21]). The complement gene family is also regulated
in this stage, with some genes being overexpressed and others
underexpressed in relation to the early-secretory phase [26,33,34].
Some overexpressed genes are involved in protecting the
endometrium and/or the embryo in this phase. Metallothioneins
and GPXs (antioxidants) protect the cell from free radicals and
heavy metals; this function can prove important in protecting the
embryo against these molecules [34]. GPX3 is a selenium-dependent
protein and, interestingly, it has been associated with infertility in
selenium-deficient women [77]. It protects the cell from oxidative
damage by catalysing the reduction of hydrogen peroxide, lipid per-oxides
and organic hydroxyperoxide by glutathione [26]. On the
other hand, the decay accelerating factor (DAF) is a complement reg-ulatory
protein for which two possible functions are postulated: pro-tection
of the embryo from the maternal complement-mediated
attack and prevention of epithelium destruction by an increased ex-pression
of the complement at the time of implantation [46]. In the
study into expression by Franchi et al. [46], which separately analysed
different tissues, it is noted that although DAF is expressed in both
compartments, the expression is greater in the epithelium. In addi-tion,
this protein was found to decrease in the endometrium of pa-tients
with recurrent abortions associated with the antiphospholipid
syndrome [78]. Another protective mechanism activated in this
stage is response to stress, which is also the case with GADD45 (a
member of a group of stress-inducible genes). Its up-regulation
shows an increase in the protective mechanisms to anticipate implan-tation
and blastocyst invasion [17].
Embryo implantation in the endometrium and its apposition have
been compared to what happens between leukocytes and the endo-thelium
[79], where L-selectin is important in this process. The ex-pression
of this molecule occurs in the blastocyst [80] while that of
its ligand occurs in endometrial epithelial cells [81]. Although no dif-ferences
were found between the early‐ and the mid-secretory sub-phases,
L-selectin ligand levels have been seen to vary between
groups of women with and without successful implantation (with
higher levels in the first group) [81]. IVF patients with progesterone
and oestradiol supplementation present an increased expression of
the L-selectin ligands in the endothelium of the endometrium [82].
In the transition between the mid-secretory and the late-secretory
phases, the genes that mostly change their expression are related not
only to the immune response of the innate immune system, but also
to the humoral and cellular immune response [34]. The study by
Tseng et al. [37] identified 126 up-regulated genes in the mid-secretory
and the late-secretory phases. Overexpressed processes in-clude
coagulation cascades and complex metabolism, including car-bohydrates,
glucose, lipids, cofactors, vitamins, xenobiotics and
amino acids, all of which suggest extracellular remodelling in the
mid-secretory phase [37].
Of the overexpressed genes in this subphase, some peak in themid-secretory
expression to then gradually decrease in the late-secretory.
This is the case of S100 calcium-binding protein P (S100P), aquaporin
3 (AQP3), chemokine (CXC motif) ligand 14 (CXCL14) [22,34,38],
Claudin-4 [24,26], Annexin4 (ANXA4) [22,23,26,33,34,37], Osteopontin
(SPP1) [23–26,33,34,46], FoxO1 [33,36], the DAF for complement CD55,
monoamine oxidase A (MAOA), GADD45 [23,25,26,33,34], MAPKKK5
[23,25,33,34], IL15 [23,24,26,33,46], ID4 [23,26,33], SERPING 1 and CIR
[33]. Other genes maintain a high expression in the late-secretory
phase, such as Apo E and Stanniocalcin 1 [34].
2.3.3. Late-secretory
During the late secretory phase, oestrogen and progesterone
levels decrease and the main processes that take place include extra-cellular
matrix degradation, inflammatory response and apoptosis
[15,16].
In the transition from the mid-secretory to the late-secretory
phases, the changes taking place in the extracellular matrix and cyto-skeleton
undergo preparation with favoured processes such as vaso-constriction,
smooth muscle contraction, haemostasis, and the
transition of the immune response to an inflammatory response
[37,83]. The genes that are regulated in this transition mostly relate
to the immune response of the innate immune system, the humoral
and cellular immune response [34], haemostasis, blood coagulation,
steroid biosynthesis and the metabolism of prostaglandins [35],
which is consistent with previous contributions. The processes repre-sented
in this late-secretory stage, such as matrix degradation, in-flammatory
response and cell apoptosis, do not favour implantation.
Thus, the transition from the mid- to the late-secretory phase define
the WOI closure and a return to the non-receptive endometrial phe-notype
[34].
The prominent genes repressed in the transition to the late-secretory
phase are GABARAPL1, GABARAPL3, DKK1 [37]. GABARAPL1
and GABARAPL3 are involved in autophagy, thus suggesting that
autophagic degradation may be involved in the mechanism responsi-ble
for the formation of new blood vessels [37].
In contrast, the genes exhibiting a peak expression at the end of
the secretory phase include SOX4, ADAMTS5, GNG4, Integrin α2, pro-lactin
receptor, MMPs, ADAMS, PLAU, PLAT, EBAF [34], endothelin re-ceptor
B (EDNRB) and MMP10 [35]. It is known that progesterone
inhibits stromal cell-derived proteases (uPA, MMP3) (as cited in
[34]). So a drop in progesterone levels is important for extracellular
7. M. Ruiz-Alonso et al. / Biochimica et Biophysica Acta 1822 (2012) 1931–1942 1937
matrix degradation. Accordingly, the up-regulation in late-secretory
metalloproteinases (MMPs and Adams), serine proteases (PLAU and
PLAT) and associated inhibitors is consistent with the progressive
drop in progesterone levels. The overexpression of PLAU allows the
activation of TGF [84] and converts plasminogen into plasmin, a po-tential
activator of MMPs [49]. Hence, PLAU plays a key role in prepar-ing
tissue for desquamation [34]. TGF family members are also
involved in tissue breakdown, and EBAF stimulates pro-MMP3 and
MMP7 production in the absence of progesterone (as this inhibits
the expression of EBAF and MMPs) (as cited in [34]). Furthermore,
the drop in progesterone allows the overexpression of inflammatory
mediators [83], as well as local mediators of function endometrial re-ceptor
endothelial B (EDNRB) and MMP10 [35].
This stage witnesses intense immune system activity [34], which
is consistent with the histological observations of leukocyte extrava-sation
[85]. Regarding the immune activation, to highlight the over-expression
of Fc receptors, MHC molecules, NK molecules and T-cell-
secreted molecules, it corresponds to the preparation of an innate
and adaptive immunity response. Through the up-regulation of the Fc
receptors, monocytes and granulocytes are prepared to respond to
antibodies, and by expressing MHC-II molecules, antigen presenting
cells are more effective [34]. TNF alpha and IL beta are secreted by
the white blood cells in the stromal compartment at the end of the
cycle by stimulating the release of matrix-degrading enzymes, thus
contributing to the scaling of the vascular basal membrane and con-nective
tissue [86]. All that is described above reveals that the pre-dominant
activities in the late-secretory phase correspond to an
endometrium preparing for scaling in the next menstrual phase,
which is when the process starts again.
The most significant biological processes regulated, and the genes
highlighted in this manuscript which change among the different men-strual
cycle phases, are summarised in Fig. 1 and Table S1, respectively.
2.4. Transcriptomics in pregnancy
There is a unique in vivo study of the comparative transcriptomic
profile during pregnancy in humans [30]. Verenbergh et al. [30] stud-ied
a case in which a biopsy was taken in the mid-secretory phase
from a patient who was subsequently found to be pregnant. This valu-able
sample was compared with non-pregnant patient samples from
the same phase and a total of 394 differentially expressed genes
were found. The major networks represented by these genes include
those of post-translational modification, cell signalling, cell move-ment,
cell development and haematologic system functioning. The
significant canonical routes involved in the endometrium of the preg-nant
patient were oxidative phosphorylation, ephrin receptor signal-ling,
neurotrophin/tyrosine kinase receptor (TRK) signalling, the
purine metabolism and peroxisome proliferator-activated receptor
(PPAR) signalling. These networks and canonical pathways form
part of the molecular mechanisms known to be involved in both the
embryo–endometrial dialogue and implantation in mice and humans.
However, this study does not indicate the distance between sampling
and the implantation site location, so the question remains as to
whether the observed changes are due to paracrine and/or to endo-crine
effects.
The aforementioned study is the only in vivo study conducted in
humans to date. Nevertheless, there are other in vitro studies with
cells in co-culture with blastocysts that help gain a better under-standing
of the embryo–endometrium relationship [21].
2.5. miRNA expression in the endometrium
MicroRNAs (miRNAs) are RNA molecules of 21–23 nucleotides
that regulate the expression of other genes through the complemen-tary
binding sites in their mRNA targets. MiRNAs can lead to the
degradation or repression of their targets [87]. Recently, the number
of publications whose aim is to study the expression of miRNAs in
the endometrium has increased, particularly during the WOI [36],
but also by comparing the natural cycle vs. the ovarian stimulation
cycles [88]. Furthermore, studies have also been done to profile the
miRNA expression in women with implantation failure vs. women
with proven fertility, and different profiles were found in both groups
[29]. As result of all these studies, the existence of an miRNA expres-sion
profile of each cycle phase has been proposed [36,88,89].
Kuokkannen et al. [36] compared the miRNA expression profile
between the late-proliferative and the mid-secretory phases to find
that the miRNAs up-regulated in the mid-secretory phase were
miRNAs whose target genes are involved in DNA replication licensing
and cell cycle regulators. These results are consistent with the sup-pression
of cell proliferation during the secretory phase. Among
these up-regulated miRNA, we find MIR31, MIR29B, MIR29C,
MIR30B, MIR30D and MIR210, whose targets are key regulators of
the cell cycle, cyclins and their cyclin-dependent kinase (CDK) part-ners;
for example, replication-licensing factors MCM2 and MCM3,
and E2F transcript factor 3 (a transcription factor that induces the ex-pression
of those genes regulating the cell cycle and promoting cell
cycle progression). The overexpression of these miRNAs in the mid-secretory
phase is consistent with the repression detected in some
of its target genes, but not in all of them. Kuokkannnen et al. [36]
suggested that these targets could be regulated at the translation
level without, therefore, any effect on the gene expression.
Shah et al. [88] also found a different profile between the different
sub-phases, in this case between the early-secretory and the mid-secretory
phases (8 up-regulated miRNAs and 12 down-regulated
miRNAs on LH +7 vs. LH +2). The bioinformatics analysis revealed
that these miRNAs regulate a large number of genes, some of which
are known to be differentially expressed during the WOI. For exam-ple,
SPP1 is a target of MIR424 and, during the receptive phase, the
expression of SPP1 increases [23–26,33,34,46], while that of MIR424
decreases [88]. Therefore, as expected, the differential expression of
miRNAs may play an important role in establishing and maintaining
endometrial receptivity [88].
3. Gene expression in ovarian stimulation regimes
Ovarian stimulation is a usual process in assisted reproductive
treatments which is becoming increasingly common in societies of
developed countries. However, some researchers argue that ovarian
stimulation may affect endometrial receptivity if compared to a natu-ral
cycle [44,45,59,64,90–93], suggesting that it may be due to high
concentrations of steroids [92]. In fact, clinical data have associated
stimulated cycles with a lower implantation rate than in natural
cycles [91], and different studies have addressed the fact that
supraphysiological concentrations of oestradiol on the day of human
chorionic gonadotrophin (HCG) administration are deleterious to em-bryonic
implantation [94–97].
Several studies have investigated the effect of stimulated cycles on
endometrial receptivity transcriptomics [98]. Some studies have
compared stimulated (agonist or antagonist) vs. natural cycles
[44,59,64,92,93], and others have compared the effect between ago-nists
and antagonists [45,99,100]. In those studies that focus on the
secretory phase, it is generally observed that while the expression
patterns in the prereceptive phase are similar between natural and
stimulated cycles, notable differences are found when compared in
the WOI [44,45,59,64,92,93]. The principal differentially expressed
genes are involved in processes of angiogenesis, negative regulation
of proliferation and up-regulation of apoptosis, among others [44].
Horcajadas' group [44] emphasised the fact that the transition be-tween
the prereceptive and the receptive phase is not as obvious in
stimulated cycles (hCG +5 vs. hCG +7) as in natural cycles (LH +5
versus LH +7). This group crossed the secretory phase by sampling
8. 1938 M. Ruiz-Alonso et al. / Biochimica et Biophysica Acta 1822 (2012) 1931–1942
Fig. 1. Biological processes along the endometrial cycle. Cartoon depicting the endometrium tissue evolution along the menstrual cycle. Underneath are represented the relative
levels of the main regulatory hormones affecting the endometrium. Principal biological processes are indicated at the cycle phase when they are more significantly regulated.
on days LH+1,+3,+5,+7,+9 and hCG+1,+3,+5,+7,+9, and
noted that the hCG +9 profile is more similar to the LH +7 than the
hCG +7 profile, thus concluding that the endometrium in stimulated
cycles does not reach the state of receptivity in the same way as in
natural cycles [44]. Moreover, Haouzi et al. [59] analysed samples in
both the prereceptive and receptive phases in stimulated (hCG +2
and hCG +5) and natural cycles (LH +2 and LH +7) with the
same patient. They observed moderate to severe expression changes
at the time of endometrial receptivity in stimulated vs. natural cycles.
Nevertheless, Mirkin et al. [99] found no significant differences in the
transcriptome of endometrial receptivity in stimulated cycles when
comparing LH +8 and hCG +9. The fact that Mirkin et al. [99]
found no significant changes when comparing LH +8 and hCG +9
if compared with other studies that compare LH +7 and hCG +7
[44,45,59,64,92,93] can be explained by the delay described in
Horcajadas' study [44] by which the hCG +9 profile presents a great-er
similarity to that of receptivity in natural cycles.
On the other hand, there have been comparisons made between
treatment with antagonists and treatment with agonists. Simon et
al. [45] compared samples in LH +2 and LH +7 (natural cycle) vs.
stimulated cycle samples with antagonists and agonists. On day +2,
the results were similar in all three groups, but on day +7, the
transcriptomic profiles of the groups in the stimulated cycles differed
from the natural cycle. Endometrial dating and the pinopode expres-sion
in the agonist-stimulated cycle suggested arrested endometrial
development if compared with the other regimes. These results are
consistent with those recently obtained by another group, Haouzi et
al. [100], which found more genes in common in the stimulated
cycle with antagonist vs. a natural cycle than in a stimulated cycle
with agonists vs. a natural cycle in the transition between the
prereceptive and the receptive phases (43% and 15%, respectively).
Regarding miRNA, Sha et al. [88] found that 22 miRNAs were sig-nificantly
deregulated between the natural and the stimulated cycle.
Notably, half of them had at least one putative ERE or PRE in the pro-moter
region, suggesting that the altered miRNA expression was
probably caused by suboptimal progesterone and oestrogen levels
during stimulation. In addition, the miRNA expression was more sim-ilar
between LH +7 and hCG +4 than with hCG +7, and they pro-posed
a change in endometrial maturation due to the stimulation
therapy [88]. Table 3 lists some of the most representative original
papers on endometrial transcriptomics in controlled ovarian stimula-tion
cycles analysed by microarray technology.
The presence of oestradiol and progesterone responsive elements
(ERE and PRE) was noteworthy in the promoter regions of differen-tially
expressed genes, suggesting that oestradiol and progesterone
directly modulate their expression and may affect the receptivity
state [92]. The alteration in the gene expression at the time of recep-tivity
with a stimulated cycle, which attempts to mimic the natural
cycle, suggests a shift in time in differentiation towards a receptive
endometrium caused by these treatments. This could have negative
9. M. Ruiz-Alonso et al. / Biochimica et Biophysica Acta 1822 (2012) 1931–1942 1939
Table 3
Original papers on endometrial transcriptomics in controlled ovarian stimulation regimes.
Authors Date Time of biopsya Comparativeb Study ID Ref
Horcajadas et al. 2008 LH+(1–9) vs. hCG+(1–9) NC vs. COS Gene expression [44]
Haouzi et al. 2009 LH (+2; +7) vs. hCG+(+2; +5) NC vs. COS Gene expression [59]
Liu et al. 2008 LH+7 vs. hCG+5 NC vs. COS Gene expression [92]
Macklon et al. 2008 LH+5 vs. hCG+2 NC vs. COS Gene expression [93]
Sha et al. 2011 LH (+2; +7) vs. hCG (+4; +7) NC vs. COS miRNA expression [88]
Horcajadas et al. 2005 LH (+2; +7) vs. hCG+7 NC vs. COH Gene expression [64]
Mirkin et al. 2004 LH+8 vs. hCG+9 Ag vs. Atg vs. NC Gene expression [99]
Simon et al. 2005 LH (+2; +7) vs. hCG (+2; +7) Ag vs. Atg vs. NC Gene expression [45]
Haouzi et al. 2010 LH (+2; +7) vs. hCG (+2; +5) Ag vs. Atg vs. NC Gene expression [100]
a hCG+: hCG administration+days; LH+: LH surge+days.
b NC: Natural cycle; COS: controlled ovarian stimulation; COH: controlled ovarian hyperstimulation; Ag: agonist; Atg: antagonist.
effects on the implantation process and could, therefore, be one of the
main causes with less success rates in ovarian stimulation using this
protocol [91].
4. Making the most of microarrays [115]
As we have noticed throughout the manuscript, the main objec-tives
of microarray studies are to: firstly, determine discrete homoge-neous
subsets of an entity based on gene expression profiles (class
discovery); secondly, identify the genes differentially expressed
among predefined classes of samples with different characteristics
(class comparison); thirdly, develop a mathematical function based
on the expression profiles that can accurately predict the biologic
group, diagnostic category or prognostic stage (class prediction)
[113,114].
The MicroArray Quality Control (MAQC) consortium is a
community-wide effort that sought to experimentally address the
key issues surrounding the reliability of DNA microarray data.
MAQC brought together more than a hundred researchers at 51 aca-demic,
government and commercial institutions to assess the perfor-mance
of seven microarray platforms in profiling the expression of
two commercially available RNA sample types. The results were com-pared
not only at different locations and between different micro-arrays
formats, but also in relation to three more traditional
quantitative gene expression assays. MAQC's main conclusions con-firmed
that, with careful experimental design and appropriate data
transformation and analysis, microarray data can indeed be reproduc-ible
and comparable among different formats and laboratories. The
data also demonstrated that the fold change results of microarray ex-periments
correlated closely with the results of assays like quantita-tive
reverse transcription PCR [115,116]. The MAQC effort ended
long-lasting controversy about the reliability of microarrays, but
their conclusions rely on “careful experimental design and appropri-ate
data transformation and analysis”.
Most of the transcriptomic studies reviewed in this manuscript
show, through unsupervised analyses, that discrete subsets of endo-metrial
phase-specific profiles can be identified (class discovery).
General agreement has been reached on this, and it shoulders the
standpoint that the endometrial cycle can be subclassified from a mo-lecular
point of view. However, class comparisons identify differen-tially
expressed genes in different studies, gene lists that do not
completely agree. As stated above, careful experimental design and
proper analysis are needed to compare transcriptome data. The
main reasons for these discrepancies are related to the experimental
design. Microarray platforms were a problem in the early stages of
this technology, but nowadays, commercial arrays have overcome
this, although the MAQC project detected that some laboratories pro-duced
lower quality data. Surely, the main factors for discrepancies
are those associated with samples; sampling conditions, selection
criteria, different criteria on the day of collection of samples, etc. Ulti-mately,
these criteria depend on the study objectives and how these
objectives were achieved. This introduces subtle differences, even
for apparently identical objectives that render different gene lists.
Sample sizes should vary depending on the number of truly differen-tially
expressed genes or when fold changes are smaller, which
should also be addressed in the experimental design. Given that thou-sands
of genes are tested in a single experiment, the proportion of
false positives among the genes declared significant can be very
high and the false discovery rate (FDR) must be controlled during
the data analysis. Likewise when identifying differentially expressed
gene lists, should there be many genes with more or less the same
correlation with classes, the reported restricted molecular signature
can strongly depend on the samples selection [114]. All these aspects
have contributed to the different gene lists shown in the papers
reviewed in this manuscript. Another fundamental aspect to maintain
the confidence in reported data and in class predictors is validation. A
basic way of performing validation is to divide the initial group of pa-tients
into two identical samples: one for the class comparison and
one to confirm the results using the same platform and prediction
rule if any has been developed to predict a clinical outcome. Cross-validation
methods, like leave-one-out, are an alternative to the split
sample methods [113]. Out of the 18 original papers listed in
Table 2, Riesewijk et al. [26], Punyadeera et al. [28], Revel et al. [29],
Yanaihara et al. [31], have validated the expression level of some of
their differentially expressed genes, with a different technique, in a
second set of samples from the same population. Haouzi et al. [27]
and Diaz-Gimeno et al. [38] validated their class predictors through
the leave-one-out method. These last two and the remaining authors
verified the level of expression of some reported genes, with a differ-ent
technique, in the same set of samples.
Finally, it is obvious that receptivity it is not only regulated by
gene expression for there are multiple downstream steps from gene
to function. Therefore, it can be assumed that even in carefully
designed, analysed and validated microarray experiments, part of
the receptivity characteristics will not be represented in a trans-criptomic
profile.
5. Transcriptomics as a diagnostic tool
Infertility is a major problem for many couples undergoing fertility
treatments to solve it. Despite the success of these treatments, em-bryo
implantation failures still occur. The main causes of implantation
failure include low endometrial receptivity, embryonic defects and
other multifactorial effects (diseases or disorders of the endometri-um)
[101]. It is currently assumed that two thirds of these implanta-tion
failures are caused by poor endometrial receptivity or defects in
the embryo–endometrial dialogue [59]. For many years, the histolog-ical
criteria established by Noyes [11] were considered the gold stan-dard
for endometrial dating to determine the receptive state [2,6,34].
Nevertheless, this method has been criticised in recent years because
it is unable to discriminate between fertility and infertility because,
10. 1940 M. Ruiz-Alonso et al. / Biochimica et Biophysica Acta 1822 (2012) 1931–1942
although histologically endometrial morphology may submit a mid-secretory
phase, this does not imply that it is receptive [22,102,103].
For a long time now, endometrial receptivity foundations have
been studied from the morphological, biochemical, molecular and
cellular points of view to propose possible markers of receptivity.
However, none of the proposed markers have become a diagnostic
tool because they offer a low predictive value [5,17,18,56]. Some au-thors
have suggested that “bad receptivity” could be caused by an al-tered
gene expression [101,102,104], and that the search for markers
of receptivity should focus on analysing and understanding these
genes as a possible underlying cause of infertility [105]. With this ob-jective
in mind, several studies linking gene expression with implan-tation
failure have been conducted. This comparison is performed by
analysing endometrial samples in the prereceptive versus the recep-tive
phases in healthy women compared to women with implantation
failure. It was concluded that at least part of this implantation failure
is due to the endometrium being unable to enter the receptive phase
[102]. In this way, many studies that search for endometrial markers
have focused on a single molecule or on a family of specific molecules,
such as integrins [106,107], mucins [108], cytokines [109], mono-amine
oxidase A [110], C4BPA, CRABP2 and OLFM1 [111], among
others. Although the results of different research works relate a defi-cient
expression of a gene or of a reduced group of genes with im-plantation
failure, the molecules studied have not succeeded as
markers in the clinical practice, as demonstrated by LIF [112]. The
conclusion reached was that a single molecule does not suffice to de-scribe
such a complex phenomenon like receptivity. By acknowledg-ing
this fact, transcriptomic profiles can prove to be complex
enough to classify the different endometrial cycle phases, including
receptivity.
Ponannampalam's group [22] used the “GeneRaVe” algorithm to
identify a group of genes (Pyrophosphate (inorganic), outer dense
fibre of sperm tails 2, Ectonucleoside triphosphate diphosphohydrolase
3, Membrane cofactor protein and Zinc finger protein 549), which are
capable of dating the correct cycle phase for 36 of the 37 samples they
analysed. In their search for marker genes involved in implantation,
Allegra et al. [71] conducted a study with samples from women who
had become pregnant after one IVF/ICSI cycle. Heavily regulated genes
(FC>23 or b−11) selected from the studies of Carson et al. [24], Kao
et al. [23], Borthwick et al. [25], Riesewijck et al. [26] and Mirkin et al.
[33], plus 13 genes involved in the apoptosis pathway, were analysed.
Of the total of 43 genes, only six showed a homogeneous expression
among all the samples (VEGF, LIF, PLA2G2A, ALPL, NNMT and STC1);
of these, only the first two showed any involvementwith the implanta-tion
process. In order to establish a list of possibly relevant genes for en-dometrial
receptivity, Horcajadas et al. [20] compared the results
obtained from three different situations: a natural cycle in fertile
women, a stimulated cycle, and refractory conditions (IUD). The result
of this comparison was that the three works only shared 25 WOI
genes. The 25 genes were regulated in one sense in the natural cycle,
and conversely in COS and IUD (dysregulated). Among them, some
genes are classically involved in endometrial receptivity, such as
glycodelin, LIF or α-catenin.
Given the need for reliable, objective molecular endometrium dat-ing,
our institution developed a specific tool based on the trans-criptomic
signature of different menstrual cycle phases. We named
this tool Endometrial Receptivity Array (ERA), which has been de-scribed
in the work of Diaz-Gimeno et al. [38]. The ERA tool consists
in a custom array containing 238 genes that are differentially
expressed in different endometrial cycle phases, as well as a compu-tational
predictor capable of cataloguing samples depending on
whether the expression profile of these 238 genes is similar, or not,
to the profiles of a set of training samples obtained from normal en-dometria.
These 238 genes were selected by means of an exhaustive
study, which compared the expressions of the samples obtained in
the prereceptive and the receptive phases of women with proven
fertility, and genes were selected with a fold change greater than 3.
Of the 238 genes, 134 were a specific transcriptomic signature of
the receptivity stage. This signature was determined by selecting
the common genes between comparisons made in the early vs. mid-secretory
and mid-secretory vs. proliferative phases (74 genes were
up-regulated and 60 were down-regulated). The group of selected
genes included those genes independently proposed in other publica-tions
(18 of the 25 proposed by Horcajadas et al. [20], 61 of the 126
proposed by Tseng et al. [37], and laminin beta 3 and microfibril-associated
protein 5 reported as being potential markers of receptiv-ity
[27]).
The endometrial dating results using the molecular tool proposed
by Diaz-Gimeno et al. [38] indicated an ACC of 95% and an AUC of
0.94, with a specificity and sensitivity of 0.8857 and 0.99758, respec-tively,
which shows the analytic power of this molecular tool for en-dometrial
dating. This is the first time that a molecular tool based
on microarray technology can be used clinically in reproductive med-icine
to assess the endometrium. Specifically, it has been proven to
discriminate among patients with an implantation failure of endome-trial
origin those that are non-receptive, which accounts for approxi-mately
23% of cases. We view the possibility that this test will be
expanded to be used routinely as an endometrium diagnostic tool in
the basic infertility work-up. Moreover, ERA is also a new molecular
research tool for endometrial research as it contains a finite number
of genes involved in endometrial receptivity, thus avoiding the use
of whole genome microarrays to cut costs and to simplify data mining
[38].
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.bbadis.2012.05.004.
Acknowledgements
Thanks to Francisco Domínguez and to José A. Martínez-Conejero
for their useful comments, and to Tamara Garrido for the Fig. 1
cartoon.
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![Biochimica et Biophysica Acta 1822 (2012) 1931–1942
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier .com/locate/bbadis
The genomics of the human endometrium☆,☆☆
Maria Ruiz-Alonso a, David Blesa b, Carlos Simón c,⁎
a IVIOMICS, Parc Cientific Universitat de Valencia, Calle Catedrático Agustín Escardino, 9, 46980 Paterna, Valencia, Spain
b IVI Foundation, Parc Cientific Universitat de Valencia, Calle Catedrático Agustín Escardino, 9, 46980 Paterna, Valencia Spain
c University of Valencia, Department of Paediatrics, Obstetrics and Gynaecology, Faculty of Medicine, Avenida Blasco Ibañez, 15, 46010, Valencia, Spain
a r t i c l e i n f o a b s t r a c t
Article history:
Received 14 December 2011
Received in revised form 4 April 2012
Accepted 6 May 2012
Available online 24 May 2012
Keywords:
Endometrium
Transcriptomic
Menstrual cycle
Receptivity
Infertility
The endometrium is a complex tissue that lines the inside of the endometrial cavity. The gene expression of
the different endometrial cell types is regulated by ovarian steroids and paracrine-secreted molecules from
neighbouring cells. Due to this regulation, the endometrium goes through cyclic modifications which can
be divided simply into the proliferative phase, the secretory phase and the menstrual phase. Successful em-bryo
implantation depends on three factors: embryo quality, the endometrium's state of receptivity, and a
synchronised dialogue between the maternal tissue and the blastocyst. There is a need to characterise the
endometrium's state of receptivity in order to prevent reproductive failure. No single molecular or histolog-ical
marker for this status has yet been found. Here, we review the global transcriptomic analyses performed
in the last decade on a normal human endometrium. These studies provide us with a clue about what global
gene expression can be expected for a non-pathological endometrium. These studies have shown endometri-al
phase-specific transcriptomic profiles and common temporal gene expression patterns. We summarise the
biological processes and genes regulated in the different phases of natural cycles and present other works on
different conditions as well as a receptivity diagnostic tool based on a specific gene set profile. This article is
part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
The endometrium is a complex tissue that lines the inside of the en-dometrial
cavity. It is morphologically divided into functional and basal
layers. The functional layer occupies two thirds of the endometrial
thickness and it presents different cellular compartments: the luminal
epithelium, the glandular epithelium, stroma and the vascular compart-ments.
The epitheliumis composed of epithelial cells that may be on the
surface or coat epithelial glands, and the stroma consists of an extracel-lular
matrix (ECM) and fibroblasts that differentiate during the
decidualization process. In the stroma, blood vessels (spiral arteries)
are also present, alongwith immune resident cells such asmacrophages
and NK cells [1–3]. Regarding their function, the functional layer is re-sponsible
for proliferation, secretion and tissue degeneration, while
the regenerative capacity of this organ lies in the basal layer [2–4].
The gene expression of the different endometrial cell types is reg-ulated
by ovarian steroids and paracrine-secreted molecules from
neighbouring cells. Due to this endocrine/paracrine regulation, the
endometrium goes through cyclic modifications which compose the
endometrial cycle. An endometrial cycle can be divided simply into
the proliferative phase, corresponding to the follicular phase in the
ovary, the secretory phase, corresponding to the luteal phase in the
ovary, and the menstrual phase. The first day of the endometrial
cycle corresponds to the beginning of the menstrual phase (the first
day of menstrual bleeding) and desquamation of the functional
layer takes place during this phase. From the menstrual phase to ovu-lation,
the proliferative phase takes place and, at this time, oestrogen
levels begin to rise, leading to not only a proliferation of stromal cells
and glands, but to the elongation of spiral arteries. Finally, the secre-tory
phase is defined as the time between ovulation and menstrua-tion.
The beginning of this phase is characterised by a rise in
progesterone levels [4]. The secretory phase has been the most stud-ied
because, in this phase, the endometrium acquires a receptive phe-notype
which allows the implantation of the blastocyst. This period of
receptivity is known as the “Window of implantation” (WOI). The
WOI opens on day 19 or 20 of the cycle, and lasts 4–5 days [5]. If im-plantation
does not occur, the progesterone and oestrogen levels de-crease,
accompanying a vasoconstriction of the spiral arteries and
leading to involution of the endometrium; then the cycle starts
again [4].
It is generally accepted that successful implantation depends on
three factors: embryo quality, the endometrium's state of receptivity,
and a synchronised dialogue between the maternal tissue and the
☆ This article is part of a Special Issue entitled: Molecular Genetics of Human
Reproductive Failure.
☆☆Disclosures: The authors declare that they have competing interests. CS is the
inventor of the patent application, AX090139WO, covering the Endometrial Recep-tivity
Array (ERA). All the authors work full- or part-time for IVIOMICS, which com-mercialises
the ERA.
⁎ Corresponding author. Tel.: +34 963903305; fax: +34 963902522.
E-mail addresses: maria.ruiz@iviomics.com (M. Ruiz-Alonso), David.Blesa@ivi.es
(D. Blesa), Carlos.Simon@ivi.es (C. Simón).
0925-4439/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2012.05.004](https://image.slidesharecdn.com/embryoimplantation7-141014224103-conversion-gate02/85/embryo-implantation-7-1-320.jpg)
![1932 M. Ruiz-Alonso et al. / Biochimica et Biophysica Acta 1822 (2012) 1931–1942
blastocyst (as cited in [6]). This dialogue is the result of a series of
complex autocrine, paracrine and/or juxtacrine interactions of differ-ent
molecules and their receptors, which are expressed in a coordi-nated
manner in the endometrium and/or the embryo [7,8]. The
embryo implantation process can be summarised as follows: the em-bryo,
in the morula stage, enters the uterine cavity where it continues
to divide to form the blastocyst. This happens in a defined time period
of between 72 and 96 h after fertilisation. Once inside the uterine cav-ity,
a dialogue is established between the blastocyst and the receptive
endometrium, mediated by hormones and growth factors (reviewed
in [9]), and the blastocyst is oriented correctly (apposition). The blas-tocyst
then comes into contact with the endometrial epithelium and
adheres to its surface (adhesion). Following this, penetration of the
endometrium by the blastocyst occurs (invasion) (reviewed in
[2,10]). This process is mediated by immune cells, cytokines, growth
factors, chemokines and adhesion molecules (reviewed in [9]). To
reach this point, the endometrium needs to display a proper, coordi-nated
gene expression regulation during the whole endometrial
cycle.
1.1. Transcriptomics of the human endometrium
During the endometrial cycle, there are morphological and func-tional
changes that are specific to each menstrual cycle phase and
which are caused by the response of the different endometrial cell
types to steroid hormones and paracrine molecules. Since 1950, the
histological point of view has been used for endometrial dating [11].
Afterwards, the need to understand the genetic mechanism underly-ing
the histological changes emerged. Before the genomic era, re-searchers
were limited to studying “gene by gene” to determine the
molecular changes responsible for the alterations observed. However
in the “genomic” era, the general trend is a global screening of all the
genes transcribed and their interactions [12]. So in the last decade,
the transcriptional mechanisms underlying endometrial biology
have been broadly investigated.
For global transcriptomic analyses, the preferred technique has
been microarray-based gene expression (for a review see: [12–21]).
Table 1 summarises some of the most representative reviews on en-dometrial
transcriptomics in natural cycles analysed by microarray
technology. Nevertheless despite its obvious advantages, this tech-nology
is not without its limitations [12,14]. Ponnampalam et al.
[22] were the first authors to propose the transcriptomic characteri-sation
of the human endometrium throughout the menstrual cycle
by using this technology as an alternative to histological dating. How-ever,
other groups had previously used this tool to search for a molec-ular
signature of receptivity [23–26].
Endometrial transcriptomics studies could be classified according
to their main objective: firstly, identification of the transcriptomic
physiological profile in each endometrial cycle phase (with emphasis
placed on endometrial receptivity) [22–38]; secondly, a comparison
between the endometrial profiles of fertile patients and those with
recurrent implantation failure [39–41]; thirdly, comparisons of pro-files
between healthy women and women with endometrial pathol-ogies
[42,43]; and finally, studies on the altered transcriptomic
profiles following different ovarian stimulation protocols [44,45].
Table 2 lists some of the most representative original papers on en-dometrial
transcriptomics in natural cycles analysed by microarray
technology.
There is some disagreement among transcriptomic studies, which
may be attributed to differences in experimental designs, in the type
of array used (distinct probes in different arrays), sampling conditions,
sample selection criteria, sample size, day of the cycle when the sample
Table 1
Reviews on endometrial transcriptomics.
Authors Date Reference
Giudice, L.C. 2003 [19]
Horcajadas et al. 2004 [13]
White and Salamonsen 2005 [14]
Sherwin et al. 2006 [12]
Giudice, L.C. 2006 [16]
Simmen and Simmen 2006 [15]
Horcajadas et al. 2007 [20]
Aghajanova et al. 2008 [17]
Aghajanova et al. 2008 [21]
Haouzi et al. 2011 [18]
Table 2
Original papers on endometrial transcriptomics.
Authors Date Time of biopsya Comparativeb Array ID Ref
Kao et al. 2002 CD 8–10 vs. LH+(8–10) LP vs. MS HG U95A (Affymetrix) [23]
Borthwick et al. 2003 CD 9–11 vs. LH+(6–8) LP vs. MS HG U95A–E (Affymetrix) [25]
Kuokkanen et al. 2010 CD 11–13 vs. CD 19-23 LP vs. MS with RNA and miRNA
expression
HG U133 Plus 2.0 (Affymetrix) and
miRCHIP V1 Array
[36]
Carson et al. 2002 LH+(2–4) vs. LH+(7–9) ES vs. MS HG U95A (Affymetrix) [24]
Riesewijck et al. 2003 LH+2 vs. LH+7 ES vs. MS HG U95A (Affymetrix) [26]
Mirkin et al. 2005 LH+3 vs. LH+8 ES vs. MS HG U95Av2 (Affymetrix) [33]
Haouzi et al. 2009 LH+2 vs. LH+7 ES vs. MS HG U133 Plus 2.0 (Affymetrix) [27]
Critchley et al. 2006 Dating by Noyes MS vs. LS HG U133A (Affymetrix) [35]
Tseng et al. 2010 Dating by Noyes ES vs. MS vs. LS HG U133 Plus 2.0 (Affymetrix) [37]
Punyadeera et al. 2005 CD 2–5 vs. CD 11–14 M vs. LP HG U133A (Affymetrix) [28]
Ponnampalam et al. 2004 Complete cycle, dating by Noyes EP vs. MP vs. LP vs. ES vs.
MS vs. LS vs. M
Home made (Peter MacCallum Cancer Institute) [22]
Talbi et al. 2006 Complete cycle, dating by Noyes EP vs. MP vs. LP vs. ES vs.
MS vs. LS
HG U133 Plus 2.0 (Affymetrix) [34]
Diaz-Gimeno et al. 2011 (LH+1 vs. +3 vs. +5 vs. +7)
(LH+(1–5) vs. LH+7 vs. CD 8–12)
LP vs. ES vs. MS vs. LS HG U133A (Affymetrix) and
Homemade “ERA”
[38]
Koler et al. 2009 CD 21 Fertility vs. infertility Array-Ready Oligo Set for the Human
Genome Version 3.0 (Operon)
[40]
Altmae et al. 2010 LH+7 Fertility vs. infertility Whole Human Genome Oligo Microarray
(Agilent Technologies)
[39]
Revel et al. 2011 CD 20–24 Fertility vs. infertility with miRNA Taqman Human MiRNA Array Card A
(Applied Biosystems)
[29]
Yanahaira et al. 2005 CD 9–11 Epithelial vs. stromal cells in
proliferative phase
BD Atlas Nylon cDNA Expression Array;
BD Biosciences (Clontech)
[31]
Van Vaerenbergh et al. 2010 LH+(5–7) MS vs. pregnant HG U133 Plus 2.0 (Affymetrix) [30]
a CD: Cycle day; LH+: LH surge+days.
b EP: Early-proliferative; MP: mid-proliferative; LP: late-proliferative; ES: early-secretory; MS: mid-secretory; LS: late-secretory; M: menstrual.](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)