Human Reproduction, Vol.29, No.6 pp.

1271 1278, 2014

Advanced Access publication on April 4, 2014 doi:10.1093/humrep/deu065

ORIGINAL ARTICLE Reproductive biology

The impact of using the combined oral

contraceptive pill for cycle scheduling on
gene expression related to endometrial
receptivity
Alfonso Bermejo 1,*, Carlos Iglesias 1, Mara Ruiz-Alonso 2, David Blesa 3,
Carlos Simon 3, Antonio Pellicer 4, and Juan Garca-Velasco1
Instituto Valenciano de Infertilidad, Av. Del Talgo 68 (28023), Madrid, Spain 2IVIOMICS, Paterna, Valencia, Spain 3Fundacion IVI, Universidad
de Valencia, Valencia, Spain 4Instituto Valenciano de Infertilidad, Universidad de Valencia, Valencia, Spain

*Correspondence address. Tel: +34-91-180-29-00; Fax: +34-91-180-29-10; E-mail: juan.garcia.velasco@ivi.es

Submitted on November 13, 2013; resubmitted on March 4, 2014; accepted on March 7, 2014

study question: Does the combined oral contraceptive pill (COCP) change endometrial gene expression when used for cycle
programming?

summary answer: COCP used for scheduling purposes does not have a signicant impact on endometrial gene expression related to
endometrial receptivity.

what is known already: Controversy exists around COCP pretreatment for IVF cycle programming, as some authors claim that it
might be detrimental to the live birth rate. Microarray technology applied to the study of tissue gene expression has previously revealed the behavior of genes related to endometrial receptivity under different conditions.
study design, size, and duration: Proof-of-concept study of 10 young healthy oocyte donors undergoing controlled ovarian
stimulation (COS) recruited between June 2012 and February 2013.
participants/materials, setting, and methods: Microarray data were obtained from endometrial biopsies from 10
young healthy oocyte donors undergoing COS with GnRH antagonists and recombinant FSH. In group A (n 5), COCP pretreatment was
used for 12 16 days, and stimulation began after a 5-day pill-free interval. Stimulation in group B (n 5) was initiated on cycle day 3 after a spontaneous menses. Endometrial biopsies were collected 7 days after triggering with hCG.

main results and the role of chance: No individual genes exhibited increased or decreased expression (fold change (FC) .2)
in patients with prior COCP treatment (group A) compared with controls (group B). However, the results of the functional analysis showed a total
of 11 biological processes that were signicantly enriched in group A compared with group B (non-COCP).
limitations, reasons for caution: The Endometrial Receptivity Array (ERA) has only been validated on endometrial samples
obtained in natural cycles and after hormonal replacement treatment (HRT). Therefore, it was not possible in this study to classify the endometrial
samples as receptive or non-receptive. We used the ERA to focus on 238 genes that are intimately related to endometrial receptivity, thus simplifying the analysis and understanding of the data.
wider implications of the findings: Cycle scheduling is common in IVF units and is used to avoid weekend retrievals and/or to
distribute evenly the workload for better efciency. Our failure to detect any relevant changes in the genes related to the window of implantation
when cycles were programmed with COCP pretreatment suggests that, despite controversial clinical results in previous studies, the use of COCPs
in this way does not affect uterine receptivity adversely.
study funding/competing interest(s): Funding for this study was provided by an unrestricted grant from Merck Sharp &
Dohme. C.S. and A.P. are co-inventors (with Patricia Diaz-Gimeno) of the Endometrial Receptivity Array and hold the patent. The other
authors have no conicts of interest to declare.

& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: journals.permissions@oup.com

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1272

Bermejo et al.

trial registration number: EudraCT registration number is 2011-003250-34.

Key words: endometrial receptivity / contraceptive pill / GnRH antagonist / gene expression / cycle programming

Materials and Methods

For IVF applications, cycle planning provides improved organization and

distribution of laboratory work at assisted reproduction centers. Cycle
planning evenly distributes the number of oocyte retrievals throughout
the week, which leads to cost savings by reducing the requirement for
weekend personnel (Zorn et al., 1987).
Protocols that include GnRH antagonists (GnRHant) have been associated with a reduction in ovarian hyperstimulation syndrome, shorter
stimulation times and lower gonadotrophin dosages. The GnRHant
protocols have achieved pregnancy rates similar to those achieved
with longer protocols (Devroey et al., 2009; Al-Inany et al., 2011).
However, GnRHant protocols have the drawback that the stimulation
cycle must begin with spontaneous menstruation. In contrast, the long
protocol allows one to delay the onset of stimulation once pituitary desensitization has been achieved. To plan the start of a GnRHant protocol, different approaches have been described: a exible start on Days 2
or 3, a delay or advance of the hCG trigger or steroid pretreatment
(Barmat et al., 2005; Tremellen and Lane, 2010; Cedrin-Durnerin
et al., 2012).
Over the last few years, controversy has arisen regarding whether
pretreatment with a combined oral contraceptive pill (COCP) may
have an impact on the IVF cycle outcome (Kolibianakis et al., 2006;
Rombauts et al., 2006; Bellver et al., 2007). A recent meta-analysis
suggested that COCPs had a negative impact on IVF results (Griesinger
et al., 2010); however, not all authors agree (Garca-Velasco et al.,
2011). This discrepancy prompted us to further evaluate endometrial
receptivity in IVF cycles where COCPs were administered as a
pretreatment.
Over the last decade, with the arrival of microarray technology, investigations have focused on the genes related to endometrial receptivity during
natural and stimulated cycles. The Endometrial Receptivity Array (ERA) is a
diagnostic tool that has been validated for analyzing the expression of 238
genes that are intimately related to endometrial receptivity (GarridoGomez et al., 2013). Microarray technology has provided the ability to
study, over a reduced period of time, the behavior of thousands of genes
in a specic tissue under various conditions. Thus, this technology has
been applied to the evaluation of broids (Horcajadas et al., 2008a,b)
and polyps (Liu et al., 2012), the premature elevation of progesterone
(Labarta et al., 2011) and the impact of ovarian stimulation on endometrial
receptivity (Horcajadas et al., 2008a,b).
In the case of cycle planning with GnRHant, a microarray analysis can
allow the assessment of endometrial receptivity from a molecular expression standpoint. This information can be linked to whether steroid
pretreatment applied prior to the start of the cycle might have a molecular effect on endometrial receptivity and whether steroid pretreatment
might inuence the clinical results of IVF. In the present study, we
compared endometrial gene expression between women treated
with COCP and those allowed to undergo spontaneous menstruation
without COCP.

Study design
This was a single-center, proof-of-concept study conducted in a
university-afliated private infertility clinic between June 2012 and February
2013. The study was designed to compare the gene expression prole in
the endometrium between two groups: A, the study group (ve women)
who received a COCP during the cycle prior to stimulation to schedule
the cycle starting on Day 1 (D1) of menses, and B, the control group (ve
women) who began stimulation directly with a spontaneous menses
without prior COCP priming.

Study population and randomization

A total of 10 volunteers from our egg donor program were included in the
study. The inclusion criteria were as follows: Caucasian; age 18 35 years;
a regular menstrual cycle (25 35 days); normal basal serum levels of follicle
stimulating hormone (FSH), luteinizing hormone (LH; both ,10 UI/ml) and
estradiol (E2; ,60 pg/ml); normal karyotype; body mass index 1825 kg/m2;
normal cervical smear from the past year; and a normal vaginal ultrasound.
The exclusion criteria were the presence of the following: endometriosis
(AFS classication stage .2); polycystic ovary syndrome (according to the
revised Rotterdam criteria); or an intrauterine device within the last
3 months.
All patients agreed to participate in the study and signed the informed
consent form on the hCG triggering day. The Institutional Review Board
and the Institutional Ethics Committee approved the study. The EudraCT
registration number is 2011-003250-34.

Study groups and stimulation protocol

Patients from group A received a COCP (Microgynon30, Bayer, Berlin,
Germany) during the cycle prior to stimulation. The dosing consisted of 1
tablet per day for 12 16 days to schedule the cycle starting on D1 of
menses. In group A, stimulation began after a 5-day pill-free interval. Patients
from group B began stimulation directly on D2/D3 of the spontaneous menstrual cycle without prior COCP priming.
The stimulation started with a xed dose of 150 IU recombinant FSH
administered subcutaneously (sc; Puregon, MSD, Madrid, Spain) for 4
days. After that, doses were adjusted according to the follicular response,
as visualized on ultrasound. To avoid a premature increase in LH, a daily
dose of 0.25 mg GnRHant (Orgalutran, MSD, Madrid, Spain) was administered sc on xed D5 until the day of triggering.
Final oocyte maturation was induced as soon as three follicles with a mean
diameter .17 mm were observed on ultrasound. This was designated day
LH+0. For triggering, patients received 250 mg recombinant HCG sc (Ovitrelle, Merck-Serono, Geneva, Switzerland). Follicle aspiration was conducted 36 h after triggering.
For luteal phase support, patients in both groups applied 200 mg
vaginal micronized progesterone (Progefk, Efk, Madrid, Spain) every
12 h. This medication was continued until the day of the biopsies in all patients
(LH+7).

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Introduction

Oral contraception and endometrial gene expression

1273

Hormonal and statistical analysis

reproducible in a second ERA test that was applied 29 40 months after

the rst ERA test (Daz-Gimeno et al., 2013).

Donors were tested for progesterone (P4), LH and estradiol (E2) concentrations on the day of recombinant HCG administration (LH+0) and on days
LH+2, LH+4 and LH+6. Serum E2, P4 and LH were evaluated using a commercially available, microparticle enzyme immunoassay kit (Abbott Laboratories, Abbott Park, IL, USA). Plasma samples from all donors were centrifuged at
200 g for 5 min, and the supernatant was stored at 2808C until assayed.
The mean hormonal concentrations were compared with a one-way analysis of variance or Kruskal Wallis test based on the normality of the distribution. Signicance was set at P , 0.05. The Mann Whitney U-test was
used for pairwise comparisons of values that were not normally distributed,
which yields results identical to the Kruskal Wallis test for two independent
samples. If multiple comparisons were performed, Bonferroni correction for
multiple comparisons was used to analyze the differences between the protocols. All statistical analyses were performed with the SPSS (Statistical
Package for the Social Sciences) 18.0 software package (SPSS, Inc.).

At 7 days after triggering (LH+7), an endometrial biopsy was obtained under

sterile conditions with a Pipelle catheter (Pipelle de Cornier, Prodimed,
Neuilly-en-Thelle, France) from the uterine fundus of each woman.
Samples were immediately placed in cryovials, homogenized in 1.5 ml
RNA Later solution (QIAGEN, Barcelona, Spain), vigorously shaken for a
few seconds and stored at room temperature for subsequent ERA analysis.
All samples were processed within 48 72 h.

Sample labeling and microarray hybridization

Total RNA was extracted with the QIAGEN RNeasy mini Kit (QIAGEN,
Chatsworth, CA, USA). Approximately 1 2 mg of total RNA per mg of
endometrial tissue was obtained. The RNA quality was assessed by loading
300 ng of total RNA onto an RNA LabChip and running the chip on an
Agilent A2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA,
USA). Good-quality RNA samples, with an RNA Integrity Number equal
to or greater than seven, were a prerequisite for ERA analysis.
A total of 10 separate hybridizations were performed. Sample preparation
and hybridization were adapted from the Agilent technical manual (onecolor). In short, rst-strand cDNA was reverse transcribed from 200 ng of
total RNA with T7-Oligo(dT) Promoter Primers. Samples were transcribed
in vitro and labeled with Cy-3 with the Low Input Quick Amp Labeling kit
(Agilent Technologies, Inc., Santa Clara, CA, USA). The labeling reaction typically yielded 4 5 mg of complementary RNA (cRNA) with a specic activity
greater than six. Fragmented cRNA samples were hybridized onto the customized ERA array by incubating at 658C for 17 h with constant rotation. The
microarray was then washed in two 1-min steps and two washing buffers
(Agilent Technologies, Inc., Santa Clara, CA, USA). The microarrays with
hybridized cRNAs were scanned with an Axon 4100A scanner (Molecular
Devices, Sunnyvale, CA, USA), and the data were extracted using the
GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA).

Endometrial receptivity array analysis

ERA gene expression values were pre-processed and normalized, and the
endometrial receptivity status was diagnosed with the ERA computational
predictor (Daz-Gimeno et al., 2011). The ERA test indicated whether the
endometrial samples were Receptive (R) or Non-receptive (NR), with an
associated diagnostic probability. However, the ERA test has only been validated on endometrial samples obtained in natural cycles and after hormonal
replacement treatment (HRT). Therefore, in this study, we used the ERA to
focus on 238 genes that are intimately related to endometrial receptivity. The
accuracy and consistency of the ERA diagnostic tool has been demonstrated
to be superior to endometrial histology. The results were completely

All analyses were performed using the Babelomics web-based suite (Medina
et al., 2010). The Agilent array background was corrected with robust multiarray averaging (RMA) methodology. The intensity signal was standardized
across arrays with the quantile normalization algorithm.
The data obtained from group A were compared with the data from group B
to evaluate the effects of COCP pretreatment. For all comparisons, we
assessed the differential gene expression with limma moderated t-statistics.
Standard microarray analysis techniques were used to perform one test for
each gene (or a probe-set) in the microarray and for comparisons. Thus, for
each gene, a t-test statistic is reported together with its corresponding P-value.
The raw P-values had to be corrected for multiple testing to minimize the
amount of false positives in the study. In this analysis, we used the conventional multiple testing P-value correction procedures proposed by Benjamini
Hochberg (Benjamini et al., 2001) to derive adjusted P-values.
The gene Set Analysis was performed for each of the comparisons
explored in the study. We used the logistic regression models described in
previous studies (Montaner and Dopazo, 2010; Sartor et al., 2010) to identify
functional blocks that were enriched in either of the conditions. In this study,
we used the functional blocks described in the GO Biological Process database (Dennis et al., 2003). The conventional multiple testing P-value correction procedure proposed by Benjamini Yekutieli (Benjamini and Yekutieli,
2001) was used to derive the adjusted P-values.
When performing the array analysis, we opted for a two-way approach to
overcome type II error: (a) instead of using triplicates as usual, we chose ve
samples per group to minimize inter-patient variation and obtain a homogenous group, and (b) to obtain as much information as possible from each
sample, we decided that they should be tested individually.

Results
Epidemiological characteristics and the baseline ovarian stimulation parameters for each group are summarized in Table I. There were no signicant differences in age, body mass index or the number of prior
donations. With respect to ovarian stimulation, there were no signicant
differences in the gonadotrophin doses received, the number of mature
Table I Baseline characteristics and parameters of
ovarian stimulation.
A (COCP,
n 5 5)

B (non-COCP,
n 5 5)

P-value

........................................................................................
Age (years)

23.0 + 1.4

23.8 + 1.6

ns

BMI (Kg/m2)

20.9 + 0.9

20.7 + 1.2

ns

Total dose FSH (IU)

1485 + 314

1580 + 218

ns

Follicles .15 mm
triggering day

12.6 + 2.4

14.4 + 2.7

ns

No. of oocytes
retrieved

20.8 + 4.6

19 + 2.7

ns

Days of stimulation

10.4 + 0.7

9.6 + 0.6

ns

E2 on day of
triggering (pg/ml)

2110 + 350

1975 + 280

ns

Values represent means + SD.

COCP, combined oral contraceptive pill; BMI, body mass index; FSH, follicle
stimulating hormone; IU, international units; E2, plasma estradiol; ns, not signicant.

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Sample attainment

Data processing and data analysis

1274
oocytes retrieved, the pre-puncture estradiol levels or the number of
days of stimulation.
The serum levels of estradiol, LH and progesterone are summarized in
Fig. 1 for each of the three donor sampling points: the day of oocyte retrieval (LH + 2), 48 h later (LH + 4) and prior to endometrial biopsy
(LH+7). No signicant differences were found between group A and
the non-COCP group (B).

Differential gene expression

All samples were adequately hybridized and pre-analyzed. Data normalization reduced the technical artifacts and boosted detection of the biological signal.

Bermejo et al.

We analyzed 238 genes in the ERA and compared genetic expression

levels between the two groups. Only one gene, tyrosine-3 monooxygenase (TH), exhibited increased differential expression (fold change (FC)
.2) in patients with prior COCP treatment (group A) compared with
the patients without prior cycle planning (group B). However, the difference was not statistically signicant (adjusted P-value .0.05). The heat
map plot (Fig. 2) showed a similar gene prole for samples within
groups A (COCP) and B (non-COCP). The principal component analysis
(PCA) plot is shown in Fig. 3.
The results obtained from the ERA bioinformatic predictor do not
have clinical value in our study because the results and predictions
have only been validated in natural or hormone replacement therapy
cycles. Assuming this limitation, all analyzed samples were classied as receptive by the ERA predictor.

Functional analysis of gene expression

Figure 1 Mean (n 5) serum levels of luteinizing hormone (LH, A),

estradiol (E2, B) and progesterone (P4, C) at each of the three sampling
points (LH+2, LH+4 and LH+7, considering day of triggering as day
LH+0). Blue and red lines represent group A (combined oral contraceptive pill, COCP) and B (controls, non-COCP) respectively.

Figure 2 Gene clustering by Pearsons correlation. A1 A5 and B1

B5 represent samples from group A (combined oral contraceptive pill,
COCP) and group B (controls, non-COCP), respectively. On the left
of the heat map is the color-coding of gene transcripts: red signies
up-regulation, blue down-regulation and white unchanged.

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Functional blocks from the GO Biological Process database were used in

this analysis (Dennis et al., 2003). The GO database is unique in that the
functions or blocks of genes are organized into a Directed Acyclic Graph
(DAG) structure. However, this procedure makes many of the signicantly enriched functions redundant. We displayed only the signicant,
non-redundant terms (functions); that is, among the signicant terms,
we selected the more specic terms.
The results showed that 11 biological processes were signicantly
enriched samples from group A (COCP) compared with those from
group B (non-COCP). These processes were dened as follows: response to metal ion (regulation of changes or cellular activities in response to stimulation with a metal ion); organ morphogenesis (the
development of different organs upon stimulation from specic

1275

Oral contraception and endometrial gene expression

Table II GO terms related to differentially expressed

genes.
GO term

Name

Genes

P-value

........................................................................................
Response to metal ion

11

0.033

GO:0009887

Organ morphogenesis

23

0.023

GO:1901700

Response to oxygen-containing
compound

29

0.024

GO:0009888

Tissue development

37

0.011

GO:0071310

Cellular response to organic

substance

40

0.019

GO:0044700

Single organism signaling

110

0.024

GO:0007154

Cell communication

114

0.032

GO:0044238

Primary metabolic process

131

0.023

GO:0044237

Cellular metabolic process

127

0.033

GO:0071704

Organic substance metabolic

process

137

0.023

GO:0050789

Regulation of biological process

158

0.023

Figure 3 Normalized data from Principal Component Analysis (PCA)

plots. PCA was done on the 10 samples from the 2 groups of this study.
The samples are represented spatially according to the gene expression
value for the 238 Endometrial Receptivity Array (ERA) genes. The
groups are represented by different colors: Group A (combined oral
contraceptive pill, COCP), red; Group B (non-COCP), blue. PC1 and
PC2 are rst and second principal components that explain the
highest variability that separates samples in the space.

activities); response to oxygen-containing compounds (regulation of

cellular activity in response to an oxygenated compound); tissue development (regulation of tissue development until the denitive structure is
achieved); cellular response to organic substance (regulation of the cellular response to certain molecular stimuli); single organism signaling
(cell signaling processes); cell communication (mediation of intercellular
or extracellular matrix communications); primary metabolic process
(normal cellular catabolic or anabolic processes); cellular metabolic
process (chemical processes by which a cell transforms substances);
organic substance metabolic process (chemical reactions for processing
molecules with a carbon backbone); and regulation of biological processes (control of genetic expression, protein changes and molecular
interactions). The affected biological processes, the implicated genes
and their respective P-values are summarized in Table II.

Discussion
In the past few years, the use of COCPs for GnRHant cycle planning has
elicited substantial controversy (Kolibianakis et al., 2006; Rombauts et al.,
2006; Bellver et al., 2007). It has been proposed that COCPs could negatively affect clinical results due to the possible effect of progestogen on
endometrial receptivity (Griesinger et al., 2010). Additionally, it was suggested that COCP pretreatment may strongly suppress LH secretion and
impair folliculogenesis in subsequent cycles when only rFSH was used. In
the present study, we examined the expression of genes responsible for
endometrial receptivity in patients subjected to controlled ovarian stimulation (COS) under a GnRHant protocol in the presence or absence of

COCP treatment. The take home message of this study was that we
did not observe signicant differences in endometrial gene expression
related to endometrial receptivity in these patients, regardless of previous COCP treatment.
When analyzing our results, only one gene, TH, showed an increased
expression in the COCP group compared with patients without prior
cycle scheduling, but this increase was not signicant (adjusted P-value
.0.05). This enzyme catabolizes tyrosine conversion to phenylalanine,
an essential amino acid that acts as a dopamine precursor. TH gene
up-regulation within the window of implantation has been described previously by other authors (Daz-Gimeno et al., 2011) under natural conditions.
As expected, the secretory phase is marked by more gene expression
changes due to the presence of the progesterone peak. These changes
coincide with the time that the endometrium becomes receptive. The
genes that are overexpressed in the mid-secretory versus the early secretory phase have known functions that are involved in implantation preparation, and they are implicated in processes related to cell adhesion,
metabolism, response to external stimuli, signaling, immune response,
cell communication and negative regulation of proliferation and development (Talbi et al., 2006).
Eleven biological processes were signicantly up-regulated in women
pretreated with COCP compared with non-COCP. Three of them are
related to metabolic processes (involving primary, cellular and organic substances), and one (single organism signaling) was related to signaling functions. All of these processes are enriched, which occurs in a natural cycle
during the window of implantation (Ruiz-Alonso et al., 2012). Other key
processes for embryo implantation, such as cell adhesion, innate immune
response and the response to stress or wounding, were not modied in
the endometrial samples from the COCP group. Functional analysis of
the included gene in the ERA revealed that the most signicant annotation is the presence of 19 processes including ones relating to the
immune response, cytoskeleton proteins needed for remodeling the
endometrium, oxidoreductase activity, carbohydrate binding and receptor binding (Daz-Gimeno et al., 2011). None of these 19 biological

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GO:0010038

1276

(Fatemi et al., 2013). Interestingly, in addition to the deleterious effects of

progesterone at the beginning of the cycle, elevated progesterone levels
at the end of the stimulation may have a negative effect on endometrial
receptivity; this was recently studied using microarray technology. Important differences were observed in the genetic expression patterns
of patients with progesterone concentrations above or below 1.5 ng/ml
on the day of follicular maturation (Labarta et al., 2011).
In the present study, we used a 30-mg dose of COCP and implemented a 12- to 16-day treatment period with a 5-day washout period. We
did not observe differences in the gene expression patterns at the endometrial level that could affect endometrial receptivity. Thus, we concluded that the 10 samples were nearly identical in this respect
regarding the endometrial gene expression of the genes related to the
window of implantation.
All participants received the same protocol to avoid problems with reproducibility or cycle-to-cycle variations and to increase the consistency
of the gene expression patterns for assessing endometrial receptivity. A
recent report has demonstrated the safety and reproducibility of ERA
endometrial microarrays and compared the results with classical histological analyses. Duplicate biopsies were obtained under the same conditions from seven patients, with intervals between biopsies ranging from
29 to 40 months. They demonstrated 100% reproducibility between the
two samples (Daz-Gimeno et al., 2013).
We can conclude that the information we discovered regarding endometrial gene expression using microarray analysis suggests that the use of
COCP as a pretreatment did not negatively impact the prole of endometrial gene expression for women undergoing IVF with the GnRHant
protocol. When COCPs were used for a maximum of 16 days and a pillfree interval was allowed for 5 days, endometrial gene expression was
not modied.

Authors roles
A.B. designed the study, conducted the data analysis and interpretation
and wrote the manuscript. He is the corresponding author for the
reviewing procedure. M.R.-A. and D.B. processed the endometrial
samples and performed the microarray technology, data analysis and
manuscript writing. C.I. participated in donor recruitment and care
during the medical treatment and performed the endometrial biopsies
for sample collection. A.P. and C.S. contributed to the review of the
manuscript. J.G.-V. participated in the study design and reviewed the intellectual content of the manuscript.

Funding
Funding for this study was provided by an unrestricted grant from Merck
Sharp & Dohme.

Conict of interest
C.S. and A.P. are co-inventors (with Patricia Diaz-Gimeno) of the Endometrial Receptivity Array and hold the patent. None of the other authors
has any conicts of interest to declare.

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processes that characterize the receptive endometrium were affected in

the study group (Table II).
The estrogen and progestogen in COCP have been shown to affect
FSH and LH secretion, respectively (Tsai and Yen, 1971; Anderson
et al., 1990). However, the estrogen doses in the COCPs used in different studies have greatly varied from 15 to 50 mg. In addition, different
studies have varied the COCP-free interval prior to the start of stimulation between 2 and 5 days, with variation of prior treatment duration
between 10 and 21 days (Cedrin-Durnerin et al., 2007; Smulders et al,
2010). These variables could have an impact on clinical results. Previous
studies have demonstrated that after a 5-day pill-free interval, the endocrine prole of women pretreated with a COCP resembled that of
women allowed to undergo spontaneous menstruation (CedrinDurnerin et al., 2007).
The rst studies to assess the effects of the use of COCP in cycle planning with GnRHant suggested that COCP had a possible benecial effect
on clinical results (Biljan et al., 1998; Fukuda et al., 2000). This result contrasted with later studies, which suggested that COCP had a negative effect
on pregnancy rates (Pinkas et al., 2008; Meldrum et al., 2009). However, in
some studies, the washout period was only 2 days. A short washout interval has been linked to higher early pregnancy loss, due to the continued,
strong COCP-mediated suppression of endogenous gonadotrophin secretion during the early washout period (Griesinger et al., 2010). The variability in the type of COCP, the duration of treatment and the duration of
the pill-free interval has generated heterogeneous results and confusion
about the effects of COCP on IVF outcome.
A turning point occurred with the meta-analysis published in 2010
(Griesinger et al., 2010). That meta-analysis included six previous
studies and reported that the group that received prior COCP treatment
showed a signicant decrease in the rate of ongoing pregnancy, an
increased duration of stimulation, and greater consumption of gonadotrophins compared with those that did not receive COCP. Thus, it
was proposed that the progestogen in COCPs might have a negative
effect on endometrial receptivity.
A randomized controlled study from our group (Garca-Velasco et al.,
2011) evaluated the clinical results from 115 women who had received
prior treatment with COCP before starting a GnRHant protocol as
part of their IVF/ICSI cycle. These results were compared with the
results from 113 patients who received stimulation under a long protocol
without prior COCP treatment. We found no signicant differences in
the pregnancy, implantation or live birth rates between the groups.
The number of days of COCP administration is relevant because
COCP suppresses pituitary and ovarian activity. Prolonged COCP administration was associated with a greater suppression of activity (van
Heusden et al., 2002). Therefore, more stimulation days and higher
FSH doses would be required throughout the COS cycle, which would
have a negative economic impact on the cost of the cycle. In the
present study, we administered COCP for between 12 and 16 days,
which we considered sufcient to restrain FSH levels without excessive
inhibition. In this series and in our previous study (Garca-Velasco
et al., 2011), COCP pretreatment for 1216 days did not increase the
total dose of gonadotrophins required compared with non-COCP
patients.
High progesterone levels at the beginning of the cycle, which could
arise from the progestogen content of the COCP, have been linked to
a decreased probability of pregnancy; however, this effect remains controversial because recent publications have not conrmed those ndings

Bermejo et al.

Oral contraception and endometrial gene expression

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