Circadian serum progesterone variations on the day of frozen embryo transfer in artificially prepared cycles

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ARTICLE
Circadian serum progesterone variations on
the day of frozen embryo transfer in
artificially prepared cycles
BIOGRAPHY
Sara Loreti is currently attending the last year of residency in Obstetrics and Gynecology at
Ospedale Maggiore Policlinico, Clinica Mangiagalli, Milan, Italy. During her residency, she worked for
18 months in Brussels IVF, Vrije Universiteit Brussel, Belgium, where she specialized in her field of
interest, reproductive medicine.
Sara Loreti
1,2,
*, Caroline Roelens
1
, Panagiotis Drakopoulos
1,3
, Neelke De Munck
1
,
Herman Tournaye
1
, Shari Mackens
1
, Christophe Blockeel
1
KEY MESSAGE
A statistically significant intra-day variation of serum progesterone was observed on the day of artificially prepared frozen
embryo transfer. This highlights the importance of a standardized procedure when measuring progesterone on the day of
the transfer, especially when clinical actions are taken when low or high progesterone measurements are encountered.
ABSTRACT
Research question: What is the intra-day variation of serum progesterone related to vaginal progesterone administration on the
day of frozen embryo transfer (FET) in an artificial cycle?
Design: A prospective cohort study was conducted including 22 patients undergoing a single blastocyst artificial cycle (AC)FET
from August to December 2022. Endometrial preparation was achieved by administering oestradiol valerate (2 mg three times
daily) and consecutively micronized vaginal progesterone (MVP; 400 mg twice daily). A blastocyst FET was performed on the 6th
day of MVP administration. Serum progesterone concentrations were measured on the day of transfer at 08:00, 12:00, 16:00
and 20:00 hours. The first and last blood samples were collected just before MVP was administered.
Results: The mean age and body mass index of the study population were 33.95 §3.98 years and 23.10 §1.95 kg/m
2
. The mean
P-values at 08:00, 12:00, 16:00 and 20:00 hours were 11.72 §4.99, 13.59 §6.33, 10.23 §3.81 and 9.28 §3.09 ng/ml,
respectively. A significant decline, of 2.41 ng/ml (95% confidence interval 0.814.00), was found between the first and last
progesterone measurements.
Conclusion: A statistically significant intra-day variation of serum progesterone concentrations on the day of FET in artificially
prepared cycles was observed. This highlights the importance of a standardized procedure for the timing of progesterone
measurement on the day of ACFET. Of note, the study results are applicable only to women using MVP for luteal phase
support; therefore it is necessary to confirm its validity in comparison with the different existing administration routes of
progesterone.
KEYWORDS
Artificial cycle
Circadian variation
Frozen embryo transfer
Serum progesterone
1
Brussels IVF, Universitair Ziekenhuis Brussel, Brussels, Belgium
2
Infertility Unit, Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico, Milan, Italy
3
IVF Greece, Athens, Greece
© 2023 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. E-mail address: saraloreti92@hotmail.it (S. Loreti). https://doi.org/10.1016/j.rbmo.2023.103601 1472-
6483/© 2023 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Declaration: The authors report no financial or commercial conflicts of interest.
1RBMO VOLUME 48 ISSUE 1 2024 103601

INTRODUCTION
In the context of assisted reproductive
technology, the use of frozen embryo
transfer (FET) has increased over
recent years, with an increase of 86%
between 2014 and 2019 (Fertility treatment
2019: trends and figures HFEA). FET
cycles have gained popularity due to the
increasing use of preimplantation genetic
testing for aneuploidies and the advantage
of scheduling the day of embryo transfer
(Labarta and Rodríguez, 2020). In
addition, a freeze-all strategy reduces the
incidence of ovarian hyperstimulation
syndrome, one of the most frequent and
severe complications of ovarian
stimulation, and may improve outcomes in
women who develop an elevated
progesterone concentration at the time of
ovulation triggering (Groenewoud et al.,
2018;Melo et al., 2021). FET can be
performed in both natural and artificial
cycles (Mackens et al., 2017) but none of
the endometrial preparations has been
proven superior to the others with regard
to reproductive outcomes (Ghobara et al.,
2017;Labarta and Rodríguez, 2020;Yarali
et al., 2016).
Artificially prepared FET, i.e. a hormonal
replacement therapy (HRT) cycle, has
been the most commonly used
endometrial preparation method due to
the minimal cycle monitoring needed,
including fewer hormonal analyses and
ultrasound examinations, and the possible
use in women without regular menses
(Wallach et al., 1996). However, there are
significant drawbacks related to this
strategy, including costs, discomfort,
medication intake and the possible adverse
consequences of oestrogen
supplementation, such as an increase in
thrombotic risk. Additionally, a higher risk
of pre-eclampsia due to the absence of a
corpus luteum has been observed in HRT
cycles (Conrad et al., 2022;Roelens et al.,
2022;von Versen-H€
oynck et al., 2019).
In HRT cycles, oestrogens are
administered to induce proliferative
changes in the endometrium. When an
adequate endometrial thickness is
reached, progestogens are initiated to
mimic the mid-cycle transition to the
secretory phase (Mackens et al., 2017).
Progesterone is the key factor for inducing
endometrial receptivity and can be
administered via different routes;
consequently, the different
pharmacodynamics of serum
progesterone related to the various routes
of administration needs to be taken into
consideration. There is an ongoing debate
on the best progesterone protocol in
terms of dose, type, route and duration of
administration (Connell et al., 2015).
However, there is no proof that one
strategy is superior to another, and
therefore luteal phase support approaches
vary among regions, physicians, clinics and
patient preferences (van de Vijver et al.,
2016;van der Linden et al., 2015).
Low serum progesterone concentrations
around the day of embryo transfer in
patients undergoing HRTFET have been
shown to have a negative impact on
pregnancy outcomes (Brady et al., 2014;
Gaggiotti-Marre et al., 2019;Labarta et
al., 2017). In the first study published by
Labarta and colleagues a critical
concentration of serum progesterone
(9.2 ng/ml) was identified below which the
ongoing pregnancy rate (OPR) dropped by
almost 20% (32.7% versus 52.8%) (Labarta
et al., 2017). To validate these findings in
the general population, a large prospective
study with a diverse group of patients was
developed (Labarta, 2020). A lower cut-off
was found (8.8 ng/ml), and below this value
the OPR declined by 18% (Labarta et al.,
2021). In addition, it seems that one-third
of the population in this study had a lower
progesterone concentration than the cut-
off.
Further research has shown that additional
progesterone supplementation in cases of
low progesterone values on the day of FET
could rescue pregnancy outcomes
(Labarta et al., 2022). However, intra-day
variations in serum progesterone
concentrations have been observed in
both the late follicular and mid-luteal
phases of gonadotrophin-stimulated IVF
cycles (Gonz
alez-Foruria et al., 2019;
Thomsen et al., 2018), in addition to the
spontaneous cycle of healthy women
(Filicori et al., 1984). Nonetheless, no
previous studies have evaluated the
progesterone variation on the day of
transfer in an HRTFET cycle.
Another aspect to take into consideration
is the factors that have an impact on serum
progesterone concentrations on the day of
HRTFET. Gonzalez-Foruria and
collaborators discovered a positive
correlation between increasing age and
progesterone concentration the day
before HRTFET. On the other hand, it
was found that increased body weight, the
time of blood collection during the day and
a history of serum progesterone
concentrations less than 10 ng/ml had
significant adverse correlations with
progesterone concentrations (Gonz
alez-
Foruria et al., 2020). Furthermore,
research by Maignien and colleagues
showed that body mass index (BMI), parity
and non-European geographical origin
were all independent factors linked to
serum progesterone concentrations below
9.8 ng/ml on the day of HRTFET
(Maignien et al., 2022).
Given the clinical implications of serum
progesterone measurement on the day of
FET and the lack of a standardized timing
for its assessment, the current study aimed
to evaluate the existence of an intra-day
variation of progesterone on the day of
FET when micronized vaginal
progesterone (MVP) was administered.
Understanding daily progesterone
variations and pharmacodynamics during
HRTFET is especially important when
clinical actions, such as additional
progesterone supplementation, are
considered when encountering low or high
progesterone values.
MATERIAL AND METHODS
Design and setting
This prospective cohort study was
conducted at Brussels IVF, a tertiary
university-based hospital in Brussels,
Belgium, between August and December
2022 (NCT05511272). The study was
approved by the ethics committee of
Universitair Ziekenhuis Brussel (EC-2022-
166, approval date 29 June 2022).
Study population
The study included 22 non-smoking,
subfertile women scheduled for FET in an
artificial cycle. The patients were between
18 and 40 years old, had a normal BMI
(18.5 and 25 kg/m
2
) and were not taking
any other medication during the study
period. To be included, patients had to
reach an adequate endometrial pattern
(triple layer) and thickness (6.5 mm) after
oestrogen treatment in the proliferative
phase. After this, MVP (400 mg twice daily)
was administered, and a single-blastocyst
FET was performed on the 6th day of MVP
administration.
Patients who underwent a transfer of an
embryo using in-vitro maturation or
oocyte/embryo donation were excluded.
In addition, women suffering from uterine
pathologies (fibroids, polyps, chronic
2RBMO VOLUME 48 ISSUE 1 2024

endometritis), uterine malformations or
hydrosalpinx were excluded.
All eligible patients were offered
participation in the study and those
interested provided written informed
consent. No medical treatments in
addition to the ones used for the standard
HRTFET cycle were administered in this
study.
End-points
The primary objective of the study was to
evaluate the existence of a statistically
significant variability of serum
progesterone concentration on the day of
transfer in an ACFET cycle, considering
the moment of progesterone
administration, and thus the
pharmacokinetics of MVP.
The pregnancy outcomes of the enrolled
participants were also described. The
clinical and ongoing pregnancy rates (CPR,
OPR) and the miscarriage rate were
examined. Pregnancy was determined by a
positive b-human chorionic gonadotrophin
(HCG) test result (serum concentrations
of b-HCG over 10 IU/l at 12 days after
embryo transfer). Clinical pregnancy was
defined as the presence of at least one
gestational sac on transvaginal
ultrasonography, miscarriage was defined
as any pregnancy loss before 12 weeks
(including biochemical miscarriage with a
positive b-HCG test without evidence of a
gestational sac, and clinical miscarriage
after confirmation of a gestational sac) and
ongoing pregnancy was defined as the
presence of at least one viable fetus
around 12 weeks of gestation.
Sample size
The sample size, i.e. 22 patients, was
calculated using a paired t-test to detect a
difference of 15% between the first and last
progesterone measurements with a 5%
false-positive rate in a two-sided test with
80% statistical power and a 95%
confidence interval (CI).
Study protocol
The blastocysts involved in this study were
vitrified on day 5 or 6 of embryo culture if
they reached at least the full blastocyst
stage with a good-quality inner cell mass
and trophectoderm, i.e. at least type Bl3BB
according to the Gardner and Schoolcraft
scoring system (Gardner and Schoolcraft,
1999). The vitrification protocol used has
previously been described in detail
elsewhere (Van Landuyt et al., 2011).
Endometrial preparation
Endometrial preparation was achieved by
starting oestradiol valerate (6 mg/day) on
the second or third day of the menstrual
cycle until an adequate endometrial
pattern (triple line) and thickness
(6.5 mm) were demonstrated by bi-
dimensional (2D) transvaginal
ultrasonography. MVP (800 mg/day) was
started 5 days prior to blastocyst FET.
During the treatment, blood samples were
taken to assess FSH, LH, oestradiol and
progesterone. HRT cycles in which there
was an ‘escape’follicular growth were
excluded from the study.
Hormone measurement
On the day of FET, the participants had
four blood samples taken (at 08:00, 12:00,
16:00 and 20:00 hours). In the first blood
sample, at 08:00 hours, serum oestradiol
and progesterone were both measured. At
12:00, 16:00 and 20:00 hours only
progesterone was evaluated. If the serum
progesterone measurement at
08:00 hours was below 8.8 ng/ml, oral
dydrogesterone (Duphaston 10 mg three
times daily) was added alongside the
vaginal dose, starting on the evening of the
transfer, after the last blood sample had
been taken. According to the study
protocol, MVP was given just after the
collection of the first and last blood
samples of the day, at 08:00 and
20:00 hours, to avoid the interference that
the recent administration of the drug
could have on serum progesterone
concentrations.
Progesterone values were measured at the
Centre for Reproductive Medicine,
Universitair Ziekenhuis Brussel, using a
validated electrochemiluminescence
immunoassay (Cobas 8000 e801, Roche
Diagnostics, Roche Diagnostics, France)
with a measured sensitivity and total
imprecision (coefficient of variation) of
0.03 mg/l and less than 7%, respectively
(Racca et al., 2021). In a previously
published paper (Lim et al., 2019), this
analyser demonstrated an acceptable
performance concerning precision,
linearity, reference range validation and
correlation.
All participants underwent embryo transfer
under ultrasound guidance in the morning
(between 10:00 and 12:00 hours). The
blastocysts were warmed on the morning
of the day of transfer and immediately
evaluated for morphological survival. They
were eligible for transfer if at least 50% of
the cells survived. The morphological
quality of the blastocyst was classified
according to the Gardner and Schoolcraft
score (Gardner and Schoolcraft, 1999)as
previously described (De Munck et al.,
2015); only good-quality embryos were
transferred, to avoid a confounding effect
of embryo quality on pregnancy outcome.
After the embryo transfer, the patient
continued her medication for at least
12 days. On the 12th day, if the b-HCG
result was negative, the medications were
all stopped. If the b-HCG result was
positive, the medications were continued,
and the patient was followed up with blood
samples and transvaginal ultrasound
examinations until the proper evolution
and viability of the pregnancy were
demonstrated. The medications were all
discontinued at around 8 weeks of
gestation by a specific step-down protocol
according to the standard clinical practice
of the centre.
Statistical analysis
Progesterone concentrations were
measured over the day of HRTFET, and,
pairwise percentage differences were
computed for each patient. Categorical
variables are shown as percentages, and
continuous variables as mean (§standard
deviation [SD]) values. To analyse the
variation in progesterone concentrations
between 08:00 and 20:00 hours, the 95%
CI for the difference in paired means using
the Wilcoxon signed-rank test was
calculated. Furthermore, comparisons were
made for patients with low 8.8 ng/ml
versus normal (>8.8 ng/ml) progesterone
concentrations, and a possible correlation
between women’sageandBMIandthe
progesterone concentration on the day of
transfer was assessed using univariate and
multivariate linear regression analysis. All
statistical tests used a two-tailed aof 0.05. All
analyses were performed using STATA
(StataCorp LLC, United States) 13.0.
RESULTS
TABLE 1 shows in detail the characteristics of
the patients and the HRTFET cycles.
Most of the patients were of caucasian
origin, three had African American origin
and three were of Asiatic origin. None of
the women had a particular medical or
surgical history or a positive report of
smoking or alcohol/drug abuse. Seven
patients (31.8 %) in the cohort had already
achieved a pregnancy after previous FET,
of whom six had already delivered a live
RBMO VOLUME 48 ISSUE 1 2024 3

baby and one pregnancy had resulted in a
miscarriage.
All 22 participants underwent the four
blood samples as per the study protocol
on the day of HRTFET, and therefore no
drop-outs were registered. The mean
values of progesterone, their variability
indexes and the coefficient of variations
measured at four different time points on
the day of FET are displayed in TABLE 2.A
significant decline, of 2.41 ng/ml (95% CI
0.814.00), was found between the first
and last progesterone measurements of
the day, and a decline of 4.31 ng/ml
(31.71%) between 12:00 and 20:00 hours.
Statistically significant differences in
progesterone values were observed
between the measurements performed at
08:00 and 20:00 hours (P= 0.007), 08:00
and 12:00 hours (P<0.001), 12:00 and
16:00 hours (P<0.001), and 16:00 and
20:00 hours (P= 0.004). An overview of
different progesterone concentrations and
profiles on the day of HRTFET is shown in
Figures 1 and 2.
The proportion of patients with low
progesterone values, defined as a
progesterone concentration 8.8 ng/ml,
varied during the day. The percentage of
participants with a lower progesterone
than the cut-off was 27.3% at 08:00 hours,
13.6% at 12:00 hours, 40.9% at
16:00 hours and 36.4% at 20:00 hours.
Moreover, a statistically significant
difference in the percentage of patients
with progesterone concentrations over
8.8 ng/ml was found between 08:00 and
12:00 hours and between 12:00 and
16:00 hours (P= 0.009 and P= 0.01,
respectively), with the highest number of
patients above the cut-off at 12:00 hours.
No statistically significant difference was
observed in the percentage of patients
with progesterone values over 8.8 ng/ml
between 08:00 and 20:00 hours (P= 0.07)
and between 16:00 and 20:00 hours
(P= 0.3).
On the day of FET, 6 out of 22 women
(27.3 %) had a progesterone value lower
than the ideal cut-off at the first
measurement of the day (08:00 hours).
They were therefore supplemented with
an extra dose of oral progesterone
(dydrogesterone 30 mg/day; Duphaston)
starting from the evening of the FET. Given
that this medication was added after the
last blood sample of the day had been
withdrawn, it did not interfere with the
progesterone measurement on the day of
the FET cycle.
After the study FET, seven women
obtained a positive pregnancy test. Four
reached an ongoing pregnancy at 12 weeks
of gestation and three pregnancies ended
in a miscarriage.
The participants’ages and BMI values were
correlated with the progesterone
concentration on the day of FET, using
univariate and multivariate regression
analysis (TABLES 3 and 4), but no significant
association was demonstrated between
these parameters and the first
progesterone evaluation of the day (08:00
hour), or with the difference in
progesterone concentration between 8:00
and 20:00 hours.
DISCUSSION
To the best of the authors’knowledge, this
is the first prospective study analysing the
intra-day variation of progesterone on the
day of HRTFET. The results highlight that
TABLE 1 CHARACTERISTICS OF THE PARTICIPANTS AND FROZEN EMBRYO
TRANSFERS
Characteristic Value
Baseline characteristics
Age (years) 33.95 §3.98
Body mass index (kg/m
2
) 23.10 §1.95
Basal FSH (IU/l) 7.84 §2.31
Previous FET (n) 1.81 §2.88
FET cycle characteristics
Day of planning of the embryo transfer/start of MVP administration
Serum FSH (IU/l) 6.94 §2.08
Serum oestradiol (ng/l) 208.51 §82.30
Serum progesterone (ng/ml) 0.19 §0.16
Endometrial thickness at ultrasound (mm) 8.44 §1.31
Number of days of oestradiol valerate intake
a
9.81 §2.78
Total oestradiol valerate dose (mg)
a
58.90 §16.73
Data are expressed as mean §standard deviation. n= 22.
a
From the first day of oestradiol valerate intake to the start of MVP.
FET, frozen embryo transfer; MVP, micronized vaginal progesterone.
TABLE 2 MEAN SERUM PROGESTERONE LEVELS AT FOUR DIFFERENT TIME POINTS DURING THE DAY OF FROZEN EMBRYO
TRANSFER IN AN ARTIFICIAL CYCLE
Time of blood sample (hours) Serum progesterone value (ng/ml) Variability Indexes Coefficient of variation P-value
08:00 11.72 §4.99 0.09 0.43 <0.001
a
12:00 13.59 §6.33 0.1 0.47 <0.001
a
16:00 10.23 §3.81 0.08 0.37 0.004
a
20:00 9.28 §3.09 0.07 0.33 0.007
b
Progesterone values are expressed as mean §standard deviation.
Comparisons were made using the Wilcoxon signed-rank test.
a
P-value describing the difference in progesterone measurement between that time point and the subsequent one along a 12-hour period.
b
P-value describing the difference in progesterone level concentration between 08:00 and 20:00 hours.
4RBMO VOLUME 48 ISSUE 1 2024

serum progesterone shows a clinically
relevant intra-day variation even in
exogenously supplemented FET cycles.
The current study showed lower
progesterone values in the morning, rising
at midday (after the absorption of the
morning dose of MVP), followed by a
descending curve in the afternoon and the
lowest values in the evening, just before the
second dose of MVP.
Progesterone concentrations
demonstrated a remarkable clinically and
statistically significant decline of 2.41 ng/ml
between 08:00 and 20:00 hours on the
day of HRTFET. Interestingly, the
percentage of participants considered to
have normal progesterone concentrations
(over 8.8 ng/ml) changed during the day:
normal progesterone values occurred in
72.7% of participants at baseline (08:00
hours), 86.3% at 12:00 hours, 59% at
16:00 hours and 63.6% at 20:00 hours.
The number of patients presenting lower
concentrations than the accepted cut-off
of 8.8 ng/ml at 20:00 hours was higher
than at 08:00 hours. Therefore, this study
demonstrates that measuring serum
progesterone in the afternoon and evening
differs significantly from assessing it in the
morning. The statistically significant
intra-day shift in progesterone values
and in the proportion of patients above
the critical progesterone cut-off
underlies the importance of measuring
serum progesterone at a standardized
time point, in order not to make a false
evaluation of its concentration, especially
when clinical actions are undertaken
accordingly.
It is well known that, in the natural
menstrual cycle of healthy women, given
the rapidly fluctuating concentrations of
progesterone in the mid-late luteal phase,
the value of a single estimation of this
hormone in the assessment of luteal phase
function is questionable. On the other
hand, in artificial cycles, progesterone
rapidly enters the systemic circulation and
reaches a steady state 6 h after vaginal
progesterone administration (Labarta and
Rodríguez, 2020), making its measurement
more reliable. The pharmacokinetics of
progesterone in artificially induced cycles
demonstrates rapid absorption when using
vaginal tablets, reaching mean peak plasma
concentrations after 36 h, with a quick
mean elimination half-life of 13 h following
administration (Archer et al., 1995;Levy,
1999;von Eye Corleta et al., 2004).
Nonetheless, the results of the present
study demonstrate that progesterone
concentrations vary during the day, even
when exogenous hormones are given, due
to the metabolism and pharmacokinetics
of the drug. These results reflect earlier
research on vaginal progesterone tablets,
which showed that mean serum
progesterone values decreased with time
after the dose of vaginal progesterone
(Gonz
alez-Foruria et al., 2020).
FIGURE 1 Representation of progesterone concentrations at 08:00, 12:00, 16:00 and 20:00 hours
on the day of frozen embryo transfer in an artificial cycle. The boxes represent the median value and
interquartile range of the different progesterone measurements at four different time points over a
12 h interval. Micronized vaginal progesterone was administered to the participants just after the
blood samples had been taken in the morning (08:00 hours) and in the evening (20:00 hours).
FIGURE 2 Individual serum progesterone concentrations measured at four different time points over a 12 h interval on the day of frozen embryo
transfer in an artificial cycle.
RBMO VOLUME 48 ISSUE 1 2024 5

Given the widely acknowledged negative
impact of low serum progesterone
concentrations on the day of HRTFET on
pregnancy outcomes, the clinical
implications of the current study are of the
utmost importance. Progesterone
concentrations prior to FET have been
shown to be an independent factor
associated with low live birth rates (LBRs)
(Brady et al., 2014;Gaggiotti-Marre et al.,
2019;Labarta et al., 2017). Thanks to the
first study by Yovich and colleagues (Yovich
et al., 2015) and then later ones by Labarta
and co-workers (Labarta, 2020;Labarta et
al., 2022,2021,2017), serum progesterone
concentrations were shown to be related
to pregnancy rate in artificially prepared
FET cycles.
Nonetheless, there is still disagreement
about the optimum progesterone
concentrations for maximizing pregnancy
outcomes. Interestingly, the concentration
does not appear to be constant across all
female patients, as observed in earlier
research, revealing significant histological
endometrial variances in individuals
receiving the same regimen (Sudoma et
al., 2011). It is unknown why certain women
have decreased progesterone
concentrations after receiving the same
MVP dose but this may be due to poor
vaginal progesterone absorption in some
cases or even an impact of the frequency
of sexual intercourse (Merriam et al.,
2015).
According to this study’sfindings, age and
BMI were not correlated with the first
progesterone measurement of the day or
with the difference between the
progesterone concentrations at 08:00 and
20:00 hours, yet the study’s strict inclusion
criteria could have created a bias in the
construction of the linear regression
analysis. Therefore, further studies
assessing a wider range of ages and BMI
values are needed. In fact, previously
published larger studies (Gonz
alez-Foruria
et al., 2020;Maignien et al., 2022) found
that age above 40 years, increasing body
weight, parity, geographical origin, a history
of serum progesterone concentrations
below 10 ng/ml on the day of a previous
HRTFET and the time of blood extraction
were either positively or negatively
correlated with the first progesterone
measurement around the day of FET.
One of the study’s key strengths is its
prospective longitudinal design, which
aimed to remove confounding bias and
increase the power. Serum progesterone
was measured in the same laboratory, at
the same four predetermined intervals
throughout the day of HRTFET, and
consistently under the same
circumstances.
The limitations of the study include the
strict inclusion criteria, which could create
a bias when extrapolating the findings to a
larger subfertile population undergoing
HRTFET. Ideally, the results should be
confirmed in larger prospective trials with
a more diverse patient group. The design
of the study focused only on women using
MVP for luteal phase support. Therefore, it
is necessary to confirm the applicability to
various progesterone administration
methods, but there is currently a lack of
data on the proportion of individuals with
insufficient serum progesterone
concentrations who received the
medication via a different route (such as
intramuscular, subcutaneous or rectal).
In addition, it is only when employing
‘natural’progestogens, like MVP, that
serum progesterone can be detected;
‘synthetic’progestogens such as
dydrogesterone render progesterone tests
useless because the molecule is
fundamentally different and cannot be
detected by the current assays (Griesinger
et al., 2019).
Moreover, the pharmacokinetics and
pharmacodynamics of the various
substances result in different serum
progesterone concentrations when using
MVP in comparison to subcutaneous or
intramuscular progesterone (Miles et al.,
1994). Due to the uterine first-pass effect,
MVP causes lower blood progesterone
concentrations and higher intrauterine
progesterone concentrations compared
with injectable progesterone (Bulletti et al.,
1997). In addition, progesterone
concentrations are more consistent when
using vaginal progesterone (Duijkers et al.,
2018), making measurement and analysis
easier.
It is vital to emphasize that the findings of
this study have significant implications for
clinical practice. In the first place, it adds to
the growing body of evidence on the
importance of serum progesterone
concentrations on the day of FET. Given
that this method is gaining popularity in
clinical practice (De Geyter et al., 2018;
European IVF Monitoring Consortium
(EIM), for the European Society of Human
Reproduction and Embryology (ESHRE) et
al., 2022), the authors think that further
efforts should be made to increase the
success rates and pregnancy outcomes of
FET cycles. Second, this study shows the
existence of a statistically significant intra-
day variation of serum progesterone on the
day of HRTFET, which highlights the
need to find a standardized time point to
measure it.
In conclusion, the results of this study
clearly show a remarkable decrease in
progesterone values on the day of
HRTFET. Our findings emphasize the
importance of a systematic and
standardized process for timing
progesterone measurements on the day of
HRTFET, taking into account the most
recent administration of MVP. This is
TABLE 3 CORRELATION OF MATERNAL AGE AND BMI WITH SERUM
PROGESTERONE VALUE AT 08:00 HOURS ON THE DAY OF FROZEN EMBRYO
TRANSFER IN AN ARTIFICIAL CYCLE, USING REGRESSION ANALYSIS
Variables Correlation coefficient Standard error P-value 95% confidence interval
Age 0.04 0.02 0.07 0.005 to 0.09
BMI 0.03 0.05 0.50 0.14 to 0.07
BMI, body mass index.
TABLE 4 CORRELATION OF MATERNAL AGE AND BMI WITH THE DIFFERENCE IN
SERUM PROGESTERONE VALUES BETWEEN 08:00 AND 20:00 HOURS ON THE
DAY OF FROZEN EMBRYO TRANSFER IN AN ARTIFICIAL CYCLE, USING
REGRESSION ANALYSIS
Variables Correlation coefficient Standard error P-value 95% confidence interval
Age 0.06 0.21 0.75 0.38 to 0.51
BMI 0.87 0.44 0.84 -1.02 to 0.85
Multivariate linear regression analysis controlling for age and BMI.
BMI, body mass index.
6RBMO VOLUME 48 ISSUE 1 2024

crucial when clinical interventions, such as
extra progesterone supplementation,
are considered whenever low or high
progesterone values are observed; in fact,
this could lead to an under- or
overtreatment according to the moment
of progesterone measurement.
DATA AVAILABILITY
Data will be made available on request.
ACKNOWLEDGEMENTS
The authors thank the women who
voluntarily participated and the staff of
Brussels IVF, Brussels, Belgium.
AUTHOR CONTRIBUTIONS
C.B., S.M., C.R. and S.L. are responsible
for the concept and study design and for
manuscript writing. S.L. performed the
data collection and drafted the
manuscript. P.D. performed the statistical
and data analyses. H.T. and N.D.M. revised
the manuscript critically for important
intellectual content. All authors
contributed to the interpretation,
discussion and editing of the manuscript.
All the authors approved the final version.
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Received 18 June 2023; received in revised form 14
September 2023; accepted 5 October 2023.
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