Recent clomiphene citrate exposure does not impact subsequent clinical outcomes in single euploid frozen embryo transfer cycles

Abstract

Study question:
Do infertile couples who recently utilized clomiphene citrate (CC) for ovulation induction or ovarian stimulation (<90 days previously) followed by a single euploid embryo transfer (SEET) have lower implantation potential compared with patients who were not exposed to CC within 90 days before embryo transfer (ET)?

Summary answer:
There does not appear to be an association between recent CC exposure and lower implantation potential in patients who undergo a frozen embryo transfer (FET) of euploid embryos.

What is known already:
Clomiphene has been found to be associated with lower pregnancy rates when compared against other ovarian stimulation medications. The majority of published research about the effects of CC on implantation potential suggest an anti-estrogenic effect on the endometrium. Quality evidence and information about utilization of CC and its effect on implantation potential after euploid ETs is lacking in the literature.

Study design, size, duration:
A retrospective cohort study with propensity score matching was carried out. We included all patients that underwent an autologous SEET from September 2016 to September 2022 at a single academic-private ART center.

Participants/materials, setting, methods:
The study group included patients that had utilized CC during either ovulation induction cycles and/or controlled ovarian stimulation at least 90 days before FET. A propensity score-matched control group of patients that were unexposed to CC within 90 days prior to SEET was used for comparisons. The primary outcome was positive pregnancy test (defined as a positive serum β-hCG measured 9 days after ET), with other outcomes including clinical pregnancy, ongoing pregnancy, biochemical pregnancy loss, and clinical pregnancy loss rates per SEET. Multivariate regression analyses fitted with generalized estimating equations were utilized to analyze if there was an association between CC utilization and IVF outcomes. Furthermore, the study evaluated the cumulative effect of CC and endometrial receptivity in vivo and subsequent IVF outcomes.

Main results and the role of chance:
A total of 593 patients with utilization of CC in <90 days before ET were compared with 1779 matched controls. Positive pregnancy test rates were comparable among the control group and the CC exposed groups, respectively (74.3% versus 75.7%, P = 0.79), as were clinical pregnancy (64.0% versus 65.0%, P = 0.60), ongoing pregnancy (51.8% versus 53.2%, P = 0.74), biochemical pregnancy loss (15.7% versus 14.03%, P = 0.45), and clinical pregnancy loss rates were also comparable among cohorts (17.1% versus 18.1%, P = 0.71). No association was found between utilization of clomiphene and lower implantation rates (adjusted odds ratio 0.95, 95% CI 0.76-1.18). Also, no differences were observed in sub-analyses based on multiple CC utilization periods. Finally, no association was found between the number of consecutive cumulative clomiphene cycles and sub-optimal IVF outcomes.

Limitations, reasons for caution:
The study has inherent bias that originated from its retrospective design. Serum levels of CC were not measured and sample size for the sub-analyses was small.

Wider implications of the findings:
There does not appear to be an association between recent CC exposure and lower implantation potential in patients who undergo a FET of euploid embryos. This finding remains consistent, even in patients who undergo multiple, consecutive clomiphene cycles prior to ET. There were no long-term effects of CC on endometrial development and clinical characteristics examined in this study. Patients that utilized CC medication prior to a SEET cycle for either ovarian stimulation or ovulation induction, can be assured that there is no evidence of a residual effect of recent CC administration that could jeopardize their pregnancy probability.

Study funding/competing interest(s):
No funding was received for the realization of this study. A.C. is advisor and/or board member of Sema4 (stakeholder in data) and Progyny. The other authors have no conflicts of interest to declare.

Trial registration number:
N/A.

Full text

Recent clomiphene citrate exposure
does not impact subsequent clinical
outcomes in single euploid frozen
embryo transfer cycles
Carlos Hernandez-Nieto
1,
*, Joseph Lee
1
, Tamar Alkon-Meadows
1
,
Luz Soto-Cossio
1
, Benjamin Sandler
1,2
, Tanmoy Mukherjee
1,2
,
and Alan Copperman
1,2
1
Reproductive Medicine Associates of New York, New York, NY, USA
2
Department of Obstetrics, Gynecology and Reproductive
Science, Icahn School of Medicine at Mount Sinai, New York, NY, USA
*Correspondence address. Reproductive Medicine Associates of New York, New York, NY, USA and Department of Obstetrics,
Gynecology and Reproductive Science, Icahn School of Medicine at Mount Sinai, 635 Madison Ave, Floor 10th, New York, NY 10022, USA.
Tel: þ1-212-756-5777; E-mail: chernandez@rmany.com https://orcid.org/0000-0002-6703-1341
Submitted on October 18, 2022; resubmitted on March 27, 2023; editorial decision on April 03, 2023
STUDY QUESTION: Do infertile couples who recently utilized clomiphene citrate (CC) for ovulation induction or ovarian stimulation
(<90 days previously) followed by a single euploid embryo transfer (SEET) have lower implantation potential compared with patients who
were not exposed to CC within 90 days before embryo transfer (ET)?
SUMMARY ANSWER: There does not appear to be an association between recent CC exposure and lower implantation potential in
patients who undergo a frozen embryo transfer (FET) of euploid embryos.
WHAT IS KNOWN ALREADY: Clomiphene has been found to be associated with lower pregnancy rates when compared against
other ovarian stimulation medications. The majority of published research about the effects of CC on implantation potential suggest an
anti-estrogenic effect on the endometrium. Quality evidence and information about utilization of CC and its effect on implantation potential
after euploid ETs is lacking in the literature.
STUDY DESIGN, SIZE, DURATION: A retrospective cohort study with propensity score matching was carried out. We included all
patients that underwent an autologous SEET from September 2016 to September 2022 at a single academic-private ART center.
PARTICIPANTS/MATERIALS, SETTING, METHODS: The study group included patients that had utilized CC during either
ovulation induction cycles and/or controlled ovarian stimulation at least 90 days before FET. A propensity score-matched control group of
patients that were unexposed to CC within 90 days prior to SEET was used for comparisons. The primary outcome was positive
pregnancy test (defined as a positive serum b-hCG measured 9 days after ET), with other outcomes including clinical pregnancy, ongoing
pregnancy, biochemical pregnancy loss, and clinical pregnancy loss rates per SEET. Multivariate regression analyses fitted with generalized
estimating equations were utilized to analyze if there was an association between CC utilization and IVF outcomes. Furthermore, the study
evaluated the cumulative effect of CC and endometrial receptivity in vivo and subsequent IVF outcomes.
MAIN RESULTS AND THE ROLE OF CHANCE: A total of 593 patients with utilization of CC in <90 days before ET were compared
with 1779 matched controls. Positive pregnancy test rates were comparable among the control group and the CC exposed groups, re-
spectively (74.3% versus 75.7%, P¼0.79), as were clinical pregnancy (64.0% versus 65.0%, P¼0.60), ongoing pregnancy (51.8% versus
53.2%, P¼0.74), biochemical pregnancy loss (15.7% versus 14.03%, P¼0.45), and clinical pregnancy loss rates were also comparable
among cohorts (17.1% versus 18.1%, P¼0.71). No association was found between utilization of clomiphene and lower implantation rates
(adjusted odds ratio 0.95, 95% CI 0.76–1.18). Also, no differences were observed in sub-analyses based on multiple CC utilization periods.
Finally, no association was found between the number of consecutive cumulative clomiphene cycles and sub-optimal IVF outcomes.
LIMITATIONS, REASONS FOR CAUTION: The study has inherent bias that originated from its retrospective design. Serum levels of
CC were not measured and sample size for the sub-analyses was small.
V
CThe Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved.
For permissions, please email: journals.permissions@oup.com
Human Reproduction, Vol.38, No.6, pp. 1151–1161, 2023
Advance Access Publication on April 19, 2023 https://doi.org/10.1093/humrep/dead072
ORIGINAL ARTICLE Reproductive endocrinology
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WIDER IMPLICATIONS OF THE FINDINGS: There does not appear to be an association between recent CC exposure and lower
implantation potential in patients who undergo a FET of euploid embryos. This finding remains consistent, even in patients who undergo
multiple, consecutive clomiphene cycles prior to ET. There were no long-term effects of CC on endometrial development and clinical
characteristics examined in this study. Patients that utilized CC medication prior to a SEET cycle for either ovarian stimulation or ovulation
induction, can be assured that there is no evidence of a residual effect of recent CC administration that could jeopardize their pregnancy
probability.
STUDY FUNDING/COMPETING INTEREST(S): No funding was received for the realization of this study. A.C. is advisor and/or
board member of Sema4 (stakeholder in data) and Progyny. The other authors have no conflicts of interest to declare.
TRIAL REGISTRATION NUMBER: N/A.
Key words: clomiphene citrate / frozen embryo transfer / IVF / preimplantation genetic testing / controlled ovarian hyperstimulation /
ovulation induction
Introduction
Clomiphene citrate (CC) is a non-steroidal synthetic ovulatory stimu-
lant categorized as a selective estrogen receptor modulator (SERM)
that is used in the treatment of female infertility of endocrine origin
and can be used as a coadjutant during ovarian stimulation for IVF
(Kamath et al., 2017). CC is a triphenyl-ethylene derivative that yields
both estrogenic agonist and estrogenic antagonist properties. Its mech-
anism of action is by competitive binding to estrogen receptors in the
hypothalamus and pituitary, followed by a reduced signaling of estro-
gen via its receptors. In this way, CC is interfering with the feedback
mechanism of endogenous estrogen resulting in an increase in FSH
and LH secretion that subsequently will stimulate ovarian follicular
recruitment and development (Kerin et al.,1985;Maruncic and
Casper, 1987).
Despite its effectiveness in achieving ovulation since its development
in the late 1960s, CC has been found to be associated with lower
pregnancy rates when compared against other ovarian stimulation
medications (Bonhoff et al.,1996;Kamath et al.,2017;Zhang et al.,
2020). The majority of published research about the effects of CC on
implantation potential suggest an anti-estrogenic effect on the cervical
mucus (Marchini et al.,1989;Randall and Templeton, 1991), endome-
trial vascular resistance alterations (Kupesic and Kurjak, 1993), and,
more importantly, direct effects on endometrial structural develop-
ment and thinning of the endometrial lining during stimulation as po-
tential causes of altered endometrial receptivity (Eden et al., 1989).
Commercially available CC is a racemic mixture of two stereoisom-
ers with different pharmacodynamics and pharmacokinetics:
Enclomiphene, which is completely anti-estrogenic and the isomer
Zuclomiphene, which is mildly estrogenic, as well as anti-estrogenic
(Mikkelson et al.,1986). In general, CC has a half-life of 5–7days but
metabolites have been detected in humans up to 6 weeks after admin-
istration. This extended bioavailability is possible because of extensive
plasma protein binding, enterohepatic cycling, and accumulation in fatty
tissues. This aspect of CC is thought to be clinically important, as the
effect after therapy could continue for several months (Young et al.,
1999;Ghobadi et al., 2009). Based on these observations, the CC
pharmacologic profile and its undesirable adverse effect on pregnancy
rates during ART, clinicians have pointed toward the necessity for
patients to utilize a freeze-all strategy after the utilization of CC for
ovarian stimulation by way of avoiding fresh embryo transfers (ETs)
and optimizing pregnancy rates (Reed et al.,2018).
Quality evidence and information about utilization of CC and its ef-
fect on implantation potential after ET is lacking in the literature.
Evidence-based recommendations of the optimal wash out time for
transferring an embryo after utilization of CC remain controversial. A
study by Nakagawa et al. (2014) demonstrated patients who utilized
CC within 90 days of frozen/thawed ET experienced significantly lower
implantation rates than those who were unexposed to CC.
Conversely, a more recent study by Kato et al. (2018) showed that
CC administration <60 days from last dose exhibited a detrimental ef-
fect on fresh ET although subsequent vitrified/thawed ETs were not
associated with impaired implantation regardless of the timing of em-
bryo replacement and the CC exposure.
Currently published studies about CC exposure and implantation
potential have some methodological shortcomings that could limit their
generalizability. Moreover, no study has analyzed CC exposure and
subsequent transfer of a chromosomally screened euploid embryo,
which is of the utmost importance when analyzing the characteristics
of early implantation in vivo. Therefore, the objective of our study was
to analyze infertile couples who utilized CC for ovulation induction
and/or ovarian stimulation followed by a single euploid blastocyst
transfer and assess the association between CC utilization and sus-
tained implantation rates. Furthermore, the study evaluated the cumu-
lative effect of CC and endometrial receptivity in vivo and subsequent
IVF outcomes.
Material and methods
Study design and patient populations
The retrospective propensity score-matched cohort analysis was per-
formed at a single academic-private ART center and included infertility
patients who underwent an IVF cycle with preimplantation genetic
testing for aneuploidy (PGT-A) with a subsequent single euploid em-
bryo transfer (SEET) from September 2016 to September 2022. All
patients underwent controlled ovarian stimulation (COS), ICSI, ex-
tended embryo culture, and trophectoderm (TE) biopsy. All PGT-A
analyses were performed with next-generation sequencing technology.
All ovarian stimulation protocols and laboratory methods used in the
study had been previously described (Hernandez-Nieto et al., 2020).
As a study group, all patients who underwent a SEET and had uti-
lized CC before their ET preparation cycle either during ovulation
1152 Hernandez-Nieto et al.
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induction cycles and/or COS including CC were included. Only em-
bryos generated during the COS cycle that utilized CC were included
in this group. Dosage, duration of CC, and number of consecutive
prior CC cycles were recorded. Time from exposure to CC was de-
fined from the date of last ingestion of pill until the day of the ET pro-
cedure. Control patients were not exposed to CC before the ET nor
had exposure to CC within 90 days of treatment. All control cases
were selected from a matched 3:1 cohort of subjects that were identi-
fied using a propensity score matching algorithm based on clinical
parameters including: oocyte age at retrieval, BMI, and serum anti-
Mu¨ llerian hormone (AMH) levels. Cases with incomplete information
were excluded from the analysis, also cases involving multiple TE biop-
sies, multiple thaw/freeze procedures, and cases with patients utilizing
donor oocytes or testicular sperm extraction. Similarly, patients with
recurrent implantation failure, recurrent pregnancy losses, or patients
with known chromosomal rearrangements were excluded from
analysis.
For the ETs, all cases underwent synthetic endometrial preparation,
as previously described (Hernandez-Nieto et al., 2019). For all cases,
thawing and transfer of the embryos were carried out on the sixth day
of progesterone supplementation regardless of the day of embryo de-
velopment at time of vitrification. Euploid embryos with the top mor-
phology grade were selected for transfer. In gender selection for family
balancing cases, the highest graded embryo of the preferred genetic
sex was transferred. When patients have multiple embryos frozen that
share the same morphological grades, Day 5 embryos were preferen-
tially selected over biopsied Day 6 or 7 embryos. Among embryos
biopsied on the same day of development, inner cell mass grade was
prioritized in embryo selection, followed by expansion grade, and then
TE grade, as described previously (Nazem et al.,2019).
For the main analysis, the cohorts were created using a cutoff value
of 90 days since last CC utilization and compared with control cases
that includes patients with more than 90 days since last CC utilization
and/or patients unexposed to the medication. This criterion was
based on the reports from Nakagawa et al. (2014) of lower pregnancy
rates when ETs were performed in <90 days from the last administra-
tion of CC. Consequently, a sensitivity analysis considering multiple
periods of CC exposure was performed, with cohorts for this analysis
separated in periods of <30 days of CC utilization (Group A);
31–60 days (Group B); 61–90 days (Group C); >90 days (Group D);
and a different group of patients unexposed to CC (Group E). Finally,
another sub-analysis was performed observing the total number of
consecutive cumulative CC cycles prior to the ET cycle. Cohorts were
categorized as: <3 cumulative cycles (Group 1); 4–6 cycles (Group 2);
>6 cycles (Group 3); and unexposed patients (Group 4).
Outcome measures
The primary outcome of the study was implantation rate, determined
here by the positive pregnancy test rate and defined as a positive se-
rum b-hCG measured 9 days after ET. Secondary outcomes included
implantation rate in the setting of a SEET, defined as the number of
gestational sacs observed at vaginal ultrasound 3–5weeks after ET di-
vided by the number of embryos transferred; clinical pregnancy rate
(CPR): the proportion of patients with ultrasonographically detectable
fetal cardiac activity; biochemical pregnancy loss rate (BPL): pregnancy
loss occurring after the presence of a positive pregnancy test followed
by a decrease or lack of increase of b-hCG serum levels in serial
measurements 48h after the first measurement and/or without detec-
tion of a gestational sac visualized by vaginal ultrasound at the fifth
week of pregnancy; clinical pregnancy loss rate (CPL): pregnancy loss
occurring after the presence of a confirmed gestational sac; and ongo-
ing pregnancy rate (OPR), a sustained pregnancy after detected fetal
heart beat on a vaginal ultrasound and/or complete delivery of a prod-
uct of fertilization after 22 completed weeks of gestational age,
which breathes or shows evidence of life (Zegers-Hochschild et al.,
2017). Secondary analysis objectives are to assess if multiple time peri-
ods between last dose of CC and ET are associated with differences
in IVF outcomes previously described. Last, we analyzed if there was a
cumulative effect of CC on IVF outcomes based on the total cumula-
tive number of cycles that utilized CC before the ET cycle.
Statistical methods
Statistical analyses were performed using SAS version 9.4 (SAS
Institute Inc., Cary, NC, USA). Demographic and embryological data
were registered for all participants. Medians, means, SD, interquartile
ranges (IQRs), and frequencies were calculated for all variables.
Descriptive and comparative unadjusted analysis was performed by
Student’s t-test, Mann–Whitney Utest, Fisher’s exact, and v
2
tests, as
appropriate. A multivariate logistic regression fitted with generalized
estimating equations (GEE) with an exchangeable correlation structure
to account for patients who underwent multiple frozen embryo trans-
fer (FET) cycles was utilized. Adjusted odds ratio (aOR) with 95% CI
were calculated. All variables that showed significance on the unad-
justed analysis and/or variables that were thought to be clinically rele-
vant were encompassed and adjusted for as covariates in the models.
All P-values were two-sided with a clinical significance level determined
at P<0.05. For the primary outcome, on a power analysis calculation,
a sample size of 329 patients per group was required to detect a dif-
ference of 10% in positive pregnancy test rates and to have an 80%
power with an alpha of 0.05.
Regulatory approval
This retrospective analysis was approved by an academic Institutional
Review Board (HS #: STUDY-18-00441). Patient information was de-
identified before data analysis.
Results
A total of 2372 SEET cycles from 2162 infertile couples were included
in the analysis. A total of 593 patients who utilized CC in <90 days
prior to ET day were compared against 1779 propensity score-
matched controls. Demographic and baseline characteristics of
cohorts, including embryological and IVF outcomes, were compared
among cohorts (Table I).
In the unadjusted analysis, significant differences were found in the
days from last CC utilization to the ET date in controls versus patients
that used CC <90days from ET (median days 302.5 IQR 613, versus
64 IQR 31, P0.0001), and differences in the total number of prior
CC cycles (mean 0.7 SD §1.4, versus 2.5 SD §1.7, P0.0001) and
cumulative dose of CC (median 0 IQR 500 mg, versus 1000 IQR
1500 mg, P0.0001) were found when comparing controls against
Clomiphene and euploid embryo transfers 1153
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exposed patients, respectively. Female patients age at the moment of
ET was younger in the CC exposed group compared with controls
(mean 37.4 §3.9, versus 36.6 §3.7, P0.001) and moreover, a dif-
ference was found in the total number of prior euploid ETs among the
exposed group compared with controls (mean 0.7 SD §1.0, versus
0.2 SD §0.7, P0.0001, respectively). No differences were found in
age at oocyte retrieval, baseline AMH, BMI, baseline serum estradiol,
progesterone, FSH, and LH levels among cohorts. Additionally, maxi-
mum estradiol and progesterone levels measured at conversion (start
of exogenous progesterone) were similar among groups. Notably, en-
dometrial thickness was similar among groups (9.2 mm §3.0 in con-
trols versus 9.4 mm §3.0, P¼0.15). Regarding IVF outcomes: no
differences were found in the total number of good quality embryos
transferred. Positive pregnancy test rates were comparable among the
control group and the CC exposed group, respectively (74.3% versus
75.7%, P¼0.79), also clinical pregnancy (64.0% versus 65.0%,
P¼0.60), ongoing pregnancy (51.8% versus 53.2%, P¼0.74), bio-
chemical pregnancy loss (15.7% versus 14.03%, P¼0.45), and clinical
pregnancy loss rates were comparable among cohorts (17.1% ver-
sus 18.1%, P¼0.71) (Table I and Fig. 1). In a sensitivity analysis, af-
ter assessing IVF outcomes exclusively on patients that underwent
their first ET after the oocyte extraction cycle, we observed no sig-
nificant differences between the control group (n ¼1507) and
<90 days of matched CC exposed patients (n ¼532), and the posi-
tive pregnancy test, clinical pregnancy, ongoing pregnancy, and preg-
nancy loss rates were comparable among cohorts (Supplementary
Table SI).
A subsequent analysis was performed of IVF outcomes of patients
with exposure to CC in <90 days prior to ET according to the type of
stimulation in which CC was utilized prior to the FET preparation cy-
cle. CC was used for COS in 334 patients and CC was utilized for
ovulation induction (OI) in 259 cases. When compared with patients
unexposed or patients that utilized CC >90 days from the ET, no dif-
ferences were observed in positive pregnancy test, clinical pregnancy,
ongoing pregnancy, biochemical pregnancy loss, and clinical pregnancy
loss rates among groups (Supplementary Table SII).
.............................................................................................................................................................................................................................
Table I Demographic characteristics, IVF outcomes, and data comparison between groups categorized by clomiphene
citrate exposure.
Control group Clomiphene citrate (<90 days)
n51779 n 5593
Mean/median SD/IQR Mean/median SD/IQR P-value
Time between CC and embryo transfer (days) 302.5 613.0 64.0 31.0 <0.0001*
Prior CC cycles 0.7 1.4 2.5 1.7 <0.0001*
Cumulative dose of CC (mg) 0.0 500.0 1000.0 1500.0 <0.0001*
Age at oocyte retrieval (years) 36.7 3.9 36.5 3.8 0.73**
Age at ET (years) 37.5 4.0 36.6 3.8 <0.001**
Anti-Mu¨ llerian hormone (ng/ml) 2.9 3.2 2.8 3.7 0.72**
BMI (kg/m
2
) 23.7 4.2 23.7 4.1 0.83**
Baseline serum estradiol (pg/ml) 37.7 26.4 37.3 26.0 0.08*
Baseline serum progesterone (ng/ml) 0.4 0.2 0.4 0.2 0.31*
Baseline LH (IU/l) 4.8 3.0 4.8 2.9 0.96*
Baseline FSH (IU/l) 6.5 3.7 6.5 3.6 0.96*
Estradiol at conversion (pg/ml) 271.6 204.4 264.1 183.4 0.62*
Serum progesterone at exogenous progesterone start (ng/ml) 0.3 0.2 0.3 0.2 0.34*
Endometrial thickness at transfer (mm) 9.2 3.0 9.4 3.0 0.15*
Previous euploid ETs 0.7 1.0 0.2 0.7 <0.0001*
Top embryo quality ET 1259/1779 70.77% 446/593 75% 0.40
v
Positive pregnancy test (N/%) 1322/1779 74.3% 449/593 75.7% 0.79
v
Clinical pregnancy (N/%) 1114/1779 64.0% 386/593 65.0% 0.60
v
Ongoing pregnancy (N/%) 923/1779 51.8% 316/593 53.2% 0.74
v
Biochemical pregnancy loss (N/%) 208/1322 15.7% 63/449 14.03% 0.45
v
Clinical pregnancy loss (N/%) 191/1114 17.1% 70/386 18.1% 0.71
v
Data presented as mean and SD, or median and interquartile range (IQR) and frequencies unless stated otherwise. Statistical significance, P<0.05.
N: sample size; CC: clomiphene citrate; ET: embryo transfer.
*Student’s t-test.
**Mann–Whitney U-test.
v
Chi-squared test.
1154 Hernandez-Nieto et al.
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In a multivariate regression analysis fitted with a GEE, after adjusting
for patient age at ET, embryonic grade, prior number of CC cycles,
and prior number of euploid ETs per patient, no association was found
between utilization of CC in <90 days from the ET and lower positive
pregnancy test rates (aOR 0.95, CI 95% 0.76–1.18). Similarly, no asso-
ciation was found with lower CPR (aOR 0.99, CI 95% 0.80–1.21) and
OPR (aOR 0.96, CI 95% 0.79–1.16). Furthermore, no association was
found between CC exposure and higher odds of biochemical preg-
nancy loss (aOR 1.09, CI 95% 0.81–1.48) or clinical pregnancy loss
(aOR 1.10, CI 95% 0.82–1.49).
Another sub-analysis was performed analyzing multiple periods of
days since the last CC utilization and ET of a single euploid blastocyst.
Cohorts for this analysis were separated into periods of <30days of
CC utilization (Grou p A, n ¼6); 31–60 days (Group B, n ¼223);
61–90 days (Group C, n ¼365); >90 days (Group D, n ¼534); and
patients unexposed to CC (Group E, n ¼1244). IVF outcomes are
depicted in Fig. 2. No significant differences were found in positive
pregnancy test, CPR, OPR, BPL, and CPL rates among all groups. No
differences were found in endometrial thickness at the time of ET
among cohorts analyzed (P¼0.14).
A final sub-analysis was performed to assess the impact of cumu-
lative and consecutive cycles of CC and CC dosages on implanta-
tion rates after a SEET. Cohorts were categorized as: <3
cumulative cycles (Group 1), n ¼856; 4–6 cycles (Group 2),
n¼240; >6cycles(Group3),n¼31; and unexposed patients
(Group 4), n ¼1245. No difference was found in endometrial thick-
ness at ET among cohorts (Group 1: mean 9.7 SD 2.3 mm; Group
2: 9.7 §2.2; Group 3: 10.2 §2.7; and Group 4: 9.5 §2.1,
P¼0.09). A significant difference was found among the median and
IQR dosage of cumulative CC utilized (Group 1: 1000 mg IQR
1000; Group 2: 2000 mg IQR 1000; Group 3: 4000 mg IQR 1500
and Group 4: 0 mg IQR 0, P¼<0.0001). No differences were
found in positive pregnancy test rates, CPR, OPR, BPL, and CPL
rates among the cohorts (Fig. 3).
In a multivariate analysis fitted with a GEE and using as a reference
Group 4 (patients unexposed to CC) after adjusting for age, BMI, em-
bryo grading, endometrial thickness at ET, and prior euploid ETs per
patient, no association was found between higher number of cumula-
tive CC cycles and lower odds of implantation, clinical pregnancy, and
ongoing pregnancy rates. Finally, no association was found with
Figure 1. IVF outcomes comparison between groups categorized by clomiphene citrate exposure. N: sample size; CC: clomiphene
citrate. *Chi-squared test.
Clomiphene and euploid embryo transfers 1155
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increased odds of biochemical pregnancy loss and clinical pregnancy
loss rates (Table II).
Discussion
CC has been utilized in different ART treatment regimens for many
decades. Despite its effectiveness and safety profile, some concerns
surrounding its anti-estrogenic effects have kept clinicians cautious to-
ward CC use and its potentially adverse effects on pregnancy out-
comes. Our study findings suggest that patients who were exposed to
CC are not at risk of impaired implantation, clinical pregnancy, and on-
going pregnancy rates. Even after adjusting for some subtle clinical dif-
ferences found among both cohorts, such as age at ET, and other
important covariates, such as embryo grading and prior euploid ETs,
we found that there is no association of CC utilization with lower im-
plantation, clinical pregnancy, and ongoing pregnancy rates. Also, there
was no association with increased biochemical and clinical pregnancy
loss rates. Furthermore, while looking only at the first ET in this same
populations, we observed no significant differences in positive preg-
nancy test and pregnancy loss rates among cohorts, and finally, no dif-
ferences were found regardless of whether CC was used for COS or
as a ovulation induction agent.
Clinical scrutiny of the pharmacological properties of CC has been
highly controversial. Prior studies have suggested that CC may cause
lower pregnancy rates in distinct protocols of ART treatment (Dickey
et al., 1965;Garcia et al.,1977;Franks et al.,1985;Gadalla et al.,
2018) and increased pregnancy loss rates (Goldfarb et al.,1968).
These undesirable outcomes have been attributed to a direct anti-
estrogenic effect of clomiphene on the endometrium. Moreover, some
authors had hypothesized that the utilization of CC is associated with
altered structural development of the endometrium, clinically observed
as a thinning of the endometrial lining (Randall and Templeton, 1991;
Practice Committee of the American Society for Reproductive
Medicine, 2003;Zollner et al., 2003;Chaube et al.,2005;Cha et al.,
2012;Mahajan and Sharma, 2016;Weiss et al.,2017). This clinical ef-
fect was likely caused by an inhibition of epithelial cell proliferation and
estrogen response element transactivation in the endometrial tissue
(Amita et al.,2010), as well as decreased pinopode cell formation
(Creus et al.,2003). Besides the endometrial lining thinning effect,
other researchers had proposed that CC could alter endometrial re-
ceptivity by different mechanisms such inhibition of prostaglandin syn-
thesis (Lerner et al., 1975). Furthermore, CC also diminishes the
expression of estrogen and progesterone receptors in the endome-
trium, and downregulates several proteins, growth factors, and cyto-
kines related to epithelial function, adhesion, cell invasion, and
Figure 2. IVF outcome comparisons of different clomiphene citrate exposure time categories and unexposed patients. N: sample
size; CC: clomiphene citrate. *Chi-squared test.
1156 Hernandez-Nieto et al.
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decidualization during the implantation window (Palomino et al.,2005;
Bao et al.,2009;Wallace et al.,2011;Valdez-Morales et al.,2015;
Chen et al.,2016;Mehdinejadiani et al.,2019). Moreover, CC has
been associated with suboptimal uterine vascular flows (Ng et al.,
2006;Thessaloniki ESHRE/ASRM-Sponsored PCOS Consensus
Workshop Group, 2008;Omran et al., 2018), abnormal endometrial
tissue maturation (Yeko et al., 1992;Reed et al.,2018;Montenegro
et al.,2021), and detrimental effects on cervical mucus (Marchini et al.,
1989;Massai et al., 1993).
Our main findings demonstrate no detrimental effect of recent CC
exposure on IVF outcomes. Our findings contrast with the results pub-
lished by Nakagawa et al., in which they observed that patients who
were exposed to CC within 90 days of ET experienced significantly
lower pregnancy rates than those who were not exposed to CC.
Those authors recommended a 90-day wash-out window before
attempting the transfer of a frozen embryo in patients utilizing CC
medication for ovarian stimulation (Nakagawa et al., 2014); it is impor-
tant to mention that the study findings were based on analysis of a
Figure 3. IVF outcomes based on different clomiphene citrate cumulative cycles before embryo transfer. N: sample size; CC: clomi-
phene citrate. *Chi-squared test.
.............................................................................................................................................................................................................................
Table II Multivariate regression analysis for IVF outcomes based on different clomiphene citrate cumulative cycles before
embryo transfer.
Group 1 (CC <3 cycles) Group 2 (CC 4–6 cycles) Group 3 (CC >6 cycles) Group 4 (unexposed)
aOR (95% CI) aOR (95% CI) aOR (95% CI)
Positive pregnancy test rate 0.90 (0.7–1.1) 0.80 (0.5–1.1) 0.70 (0.2–1.8) Reference
Clinical pregnancy rate 1.003 (0.8–1.2) 0.88 (0.6–1.2) 0.73 (0.3–1.60) Reference
Ongoing pregnancy rate 0.94 (0.7–1.15) 0.8 (0.6–1.1) 0.71 (0.2–1.8) Reference
Biochemical loss rate 0.80 (0.5–1.09) 0.83 (0.5–1.38) 1.008 (0.3–3.3) Reference
Clinical pregnancy loss rate 1.18 (0.8–1.7) 1.25 (0.7–2.14) 1.15 (0.3–3.9) Reference
Data presented as adjusted odds ratios (aORs) with 95% CI.
CC: clomiphene citrate.
Clomiphene and euploid embryo transfers 1157
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relatively small sample size (219 CC exposure patients (<90 days prior
to ET); 159 control patients). Also, the study included multiple cleav-
age stage ETs that were performed in fresh endometrial stimulated
cycles. Those factors that could explain the relatively low pregnancy
rates found (control group 36.7% and exposed patient cohort 25.1%,
(P<0.05)). As described previously, cleavage stage transfers and fresh
ETs could yield lower implantation rates than blastocyst transfers and
FETs (Zaat et al.,2021;Glujovsky et al.,2022). In contrast, the results
of our study showed comparable positive pregnancy test rates after
the transfer of single euploid FETs over synthetically prepared endo-
metrium (74.3% control group versus 75.7% in patients exposed to
<90 days of CC, P¼0.49). Our IVF outcomes and findings correlate
with another study by Kato et al. (2018) in which the authors found
no differences in pregnancy rates and neonatal outcomes in patients
exposed to <60 days of CC after a single vitrified-warmed blastocyst
transfer. Furthermore, no association was found for the duration be-
tween the last day of CC administration and the day of the blastocyst
transfer (Kato et al., 2018). Those findings agree with our adjusted
analysis in which we observed no associations with decreased positive
pregnancy test, clinical and ongoing pregnancy rates, or increased rates
of pregnancy losses after adjusting for potential cofounders and
covariates.
Another important circumstance to consider is the possible detri-
mental effect of cumulative CC exposure. This question is paramount
as it is common for patients to undergo multiple consecutive cycles of
CC prior to high complexity ART. To date, no study has analyzed the
impact of consecutive CC cycles on implantation rates in vitrified-
warmed euploid blastocyst transfer cycles. This is relevant for any re-
productive endocrinologist and clinical data are lacking. Commercially
available CC is a mixture of two diastereoisomers: enclomiphene
(62%) and zuclomiphene (38%) (Ernst et al., 1976). The trans-isomer,
enclomiphene, is described as having a higher response on receptors
and is primarily responsible for the ovulation-inducing actions of CC
(Clark and Markaverich, 1981). In pharmacokinetic studies, enclomi-
phene levels are observed to rapidly rise after administration and fall
to undetectable concentrations soon thereafter, with a half-life: 10.5h
(Mikkelson et al.,1986). The other cis-isomer, zuclomiphene, is elimi-
nated in a slower period of time. The serum levels of zuclomiphene
remain detectable for more than 30 days after administration and may
accumulate over consecutive cycles of treatment (Young et al., 1999).
Based on these properties, some authors had hypothesized that long-
lasting estrogen receptor occupancy by CC might alter endometrial
cell function, thus disturbing the implantation window (Palomino et al.,
2005). In our study, we performed a sub-analysis including multiple
cohorts of patients exposed to CC over consecutive cycles before ET.
We observed that the positive pregnancy test, clinical pregnancy, and
ongoing pregnancy rates were similar among all the cohorts analyzed.
Also, there were no differences in biochemical pregnancy and clinical
pregnancy losses. Even in an adjusted analysis controlling for important
covariates, we observed no association between cumulative CC cycles
and a detrimental effect on embryo implantation rates. So, besides a
potential cumulative effect of clomiphene isomers and their pharmaco-
kinetic/pharmacodynamic properties impacting on endometrial and
uterine tissues, no evidence surfaced to display impairing effects on
clinical IVF outcomes in this study.
Finally, the main analysis and sub-analysis showed that endometrial
thickness at the time of the ET was similar among all compared
cohorts, suggesting the effect of CC on impaired endometrial growth
or receptivity, if present, to be short-lasting and associated only with
the time throughout cycle when the medications are being utilized, es-
pecially during the course of fresh ET cycles or ovulation induction
cycles (Palomino et al., 2005;Nakagawa et al.,2014;Gadalla et al.,
2018;Hawkins Bressler et al.,2021). Therefore, our findings suggest
that there is no clinical long-lasting or cumulative effect of CC on en-
dometrial development and implantation rates during a vitrified/
thawed SEET cycle.
Above and beyond our best efforts to avoid biases, this study is not
without limitations. Primarily, the inherent bias originated from its ret-
rospective design. Also, CC serum and tissue levels were not mea-
sured and not incorporated into the study design since measuring drug
levels is not part of our standard clinical operating procedures. An im-
portant limitation to consider is that in the main analysis subtle popula-
tion differences can be found mostly owing to the difference in time
between CC exposure and the ET in the CC exposed group, and the
longer interval time to ET in the control group; differences that can be
attributed because some patients in the dataset had a failed ETs or dif-
ferent outcomes in prior cycles, although a sub-analysis showed that
the majority of the included cases was patients undergoing their first
ET after the COS cycle. In the sensitivity analysis, while exclusively ob-
serving patients that underwent the first ET after COS we observed
no significant differences in the analyzed outcomes, and, furthermore,
in the multivariate analysis, we adjusted for these differences between
the populations and for other important confounding variables. The fi-
nal analysis showed that these differences did not significantly affect
the outcomes analyzed. Another limitation is that the populations ana-
lyzed could be heterogeneous in nature because of their multiple infer-
tility diagnoses, although we excluded patients with a diagnosis of
recurrent implantation failure, recurrent pregnancy loss, patients with
uterine factor, patients with known chromosomal rearrangements and
cases involving multiple TE biopsies, and/or multiple thaw/freeze pro-
cedures. Likewise, we utilized a propensity score-matched control
group to improve the comparability of the controls and interest popu-
lations, and we utilized an adjusted multivariate analysis with a GEE
model trying to control for potential confounders and covariables,
including the same patient appearing multiple times in the same
dataset. Lastly, of all the studies analyzing CC and its pharmacologi-
cal consequences on FETs, our study includes the largest population
analyzed to date of chromosomally screened embryos via next-
generation sequencing that were transferred in a SEET protocol.
This approach helps us to aptly analyze the potential effect of the
CC on implantation rates by excluding the potential failure caused
by embryonic aneuploidy that exists in published studies and, thus,
potentially confounding the theoretical clinical consequences of CC
on implantation rates after IVF. Nonetheless, our sub-analysis find-
ings must be taken with caution owing to the increased risk of bias
caused by the limited sample sizes, the consequence of creating
multiple groups for data exploration.
Further clinical and pharmacogenomic research is needed and
should focus on prospectively analyzing serum and tissue CC concen-
trations along with multiomic profiles of endometrial cellular sub-types
or endometrial organoids in order to advance our understanding and
provide foundational information on the deep mechanisms of endome-
trial proliferation, receptivity, and the potential pharmacological anti-
estrogenic effects of CC.
1158 Hernandez-Nieto et al.
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In conclusion, there does not appear to be an association between
recent CC exposure and lower implantation potential in patients who
undergo FET of euploid embryos. This finding remains consistent even
when patients who undergo multiple, consecutive CC cycles prior to
ET are included. There were no long-term effects of CC on endome-
trial development and the clinical characteristics assessed in this study.
Patients that utilized CC medication prior a SEET cycle, either for
ovarian stimulation or ovulation induction, can be assured that there is
no evidence of a residual effect of recent CC administration that could
jeopardize their pregnancy probability.
Supplementary data
Supplementary data are available at Human Reproduction online.
Data availability
Some or all datasets generated during and/or analyzed during the cur-
rent study are not publicly available but are available from the corre-
sponding author upon reasonable request.
Acknowledgements
The authors thank all the physicians, fellows, embryologists, research,
and staff members for all the valuable work and help in realizing this
article.
Authors’ roles
All authors have made substantial contributions to the conception, de-
sign, performance, analysis, and writing of the study. C.H.-N. partici-
pated in the conception, design, acquisition of data, analysis of data,
drafting of article, and final approval. J.L. participated in the drafting of
article and final approval. T.A.-M. participated in acquisition of data
and the drafting of article and final approval. L.S.-C. participated in the
drafting of article and final approval. B.S. participated in the drafting of
article and final approval. A.C. participated in conception, drafting of
article, and final approval. T.M. participated in conception, design,
drafting of article, and final approval.
Funding
No funding was obtained for the realization of this article.
Conflict of interest
A.C. is advisor and/or board member of Sema4 and Progyny. The
remaining co-authors do not have any conflicts of interest.
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