Human Reproduction, pp.

1–10, 2020
doi:10.1093/humrep/deaa322

ORIGINAL ARTICLE Infertility

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Impact of low serum progesterone
levels on the day of embryo transfer
on pregnancy outcome: a prospective
cohort study in artificial cycles with
vaginal progesterone
Elena Labarta1,2,*, Giulia Mariani1,†, Stefania Paolelli1,2,
Cristina Rodriguez-Varela2, Carmina Vidal1,2, Juan Giles1,2,
José Bellver1,2, Fabio Cruz1,2, Alicia Marzal1,2, Paula Celada1,2,
Ines Olmo1,2, Pilar Alamá1,2, José Remohi1,2, and Ernesto Bosch1,2
1
Human Reproduction Department, IVI RMA Valencia, Valencia 46015, Spain 2IVI Foundation, Health Research Institute La Fe, Valencia
46026, Spain

*Correspondence address. Human Reproduction Department, IVI RMA Valencia, Plaza Policı́a Local, 3, Valencia 46015, Spain.
E-mail: elena.labarta@ivirma.com

Submitted on March 9, 2020; resubmitted on October 14, 2020; editorial decision on October 29, 2020

STUDY QUESTION: Is there a serum progesterone (P) threshold on the day of embryo transfer (ET) in artificial endometrium
preparation cycles below which the chances of ongoing pregnancy are reduced?
SUMMARY ANSWER: Serum P levels <8.8 ng/ml on the day of ET lower ongoing pregnancy rate (OPR) in both own or donated
oocyte cycles.
WHAT IS KNOWN ALREADY: We previously found that serum P levels <9.2 ng/ml on the day of ET significantly decrease OPR in a
sample of 211 oocyte donation recipients. Here, we assessed whether these results are applicable to all infertile patients under an artificial
endometrial preparation cycle, regardless of the oocyte origin.
STUDY DESIGN, SIZE, DURATION: This prospective cohort study was performed between September 2017 and November 2018
and enrolled 1205 patients scheduled for ET after an artificial endometrial preparation cycle with estradiol valerate and micronized vaginal
P (MVP, 400 mg twice daily).
PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients 50 years old with a triple-layer endometrium 6.5 mm underwent
transfer of one or two blastocysts. A total of 1150 patients treated with own oocytes without preimplantation genetic testing for aneuploi-
dies (PGT-A) (n ¼ 184), own oocytes with PGT-A (n ¼ 308) or donated oocytes (n ¼ 658) were analyzed. The primary endpoint was the
OPR beyond pregnancy week 12 based on serum P levels measured immediately before ET.
MAIN RESULTS AND THE ROLE OF CHANCE: Women with serum P levels <8.8 ng/ml (30th percentile) had a significantly lower
OPR (36.6% vs 54.4%) and live birth rate (35.5% vs 52.0%) than the rest of the patients. Multivariate logistic regression showed that serum
P < 8.8 ng/ml was an independent factor influencing OPR in the overall population and in the three treatment groups. A significant negative
correlation was observed between serum P levels and BMI, weight and time between the last P dose and blood tests and a positive
correlation was found with age, height and number of days on HRT. Multivariate logistic regression showed that only body weight was an
independent factor for presenting serum P levels <8.8 ng/ml. Obstetrical and perinatal outcomes did not differ in patients with ongoing
pregnancy regardless of serum P levels being above/below 8.8 ng/ml.
LIMITATIONS, REASONS FOR CAUTION: Only women with MVP were included. Extrapolation to other P administration forms
needs to be validated.

†
Present address: IVI RMA Roma, Largo Ildebrando Pizzetti 1, Rome 00197, Italy.
C The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved.
V
For permissions, please email: journals.permissions@oup.com
2 Labarta et al.

WIDER IMPLICATIONS OF THE FINDINGS: This study identified the threshold of serum P as 8.8 ng/ml on the day of ET for artificial
endometrial preparation cycles necessary to optimize outcomes, in cycles with own or donated oocytes. One-third of patients receiving

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MVP show inadequate levels of serum P that, in turn, impact the success of the ART cycle. Monitoring P levels in the mid-luteal phase is
recommended when using MVP to adjust the doses according to the needs of the patient.
STUDY FUNDING/COMPETING INTEREST(S): None.
TRIAL REGISTRATION NUMBER: NCT03272412.

Key words: artificial cycle / serum progesterone / hormonal replacement therapy / pregnancy outcome / luteal phase support

.. Study population
Introduction ..
.. The study enrolled 1205 infertile patients scheduled for ET under
Artificial endometrial preparation with HRT is frequently used for fro- ..
.. an artificial endometrial preparation treatment with HRT, of which
zen embryo transfer (FET) and egg donation cycles (Groenewoud .. 1150 were ultimately included. Among these, 658 cases (57.2%) were
et al., 2018). However, there is insufficient evidence to recommend ..
.. oocyte donation cycles, whereas 492 (42.8%) were own oocyte cycles
any one protocol for endometrial preparation (Ghobara et al., 2017) .. with 308 preimplantation genetic testing for aneuploidies (PGT-A)
and there are no clear guidelines for HRT. While estrogen administra- ..
.. treatments and 184 regular FET. Participating women were 50 years
tion is tailored to patient need (Labarta et al., 2017), the same proges- .. old with adequate endometrial pattern (triple layer) and thickness
terone (P) dose is given to all patients without individualization of ..
.. (6.5 mm) after estrogen treatment in the proliferative phase and LPS
luteal phase support (LPS) since an optimal P exposure before embryo .. with only MVP (400 mg twice daily for 5 days) before ET. One or two
transfer (ET) has not been well established (van de Vijver et al., 2016).
..
.. blastocysts were transferred. We wanted to reflect the broad range of
P doses and administration forms (i.e. vaginal or subcutaneous) are .. patients encountered in clinical practice, so all patients meeting eligibil-
instead chosen based on patient and doctor preferences (Vaisbuch
..
.. ity criteria for ET were included in the study. Patients with uterine or
et al., 2014). .. adnexal anomalies did not undergo ET according to our protocol, so
There is likely a minimum serum P concentration below which
..
.. these patients were not recruited for the study.
ET success rates are significantly lower when using natural micron- ...
ized vaginal P (MVP) in artificial endometrial preparation cycles. .. Endpoints
..
Most studies on the relation between serum P and pregnancy out- .. The primary endpoint was the OPR in patients who underwent ET af-
come are retrospective (Yovich et al., 2015; Alsbjerg et al., 2018; .. ter endometrial preparation artificial cycles based on serum P levels
..
Cédrin-Durnerin et al., 2019; Gaggiotti-Marre et al., 2019) except .. measured on the day of ET. Secondary endpoints were (i) the critical
one published by our group (Labarta et al., 2017), which analyzed .. threshold of serum P on the day of ET below which pregnancy rates
..
the relationship between serum P levels on the day of ET and on- .. were significantly lower, (ii) pregnancy outcomes according to
going pregnancy rate (OPR). Patients with serum P < 9.2 ng/ml, .. the threshold, (iii) factors that determine serum P levels and (iv) the
..
which corresponded to the 25th percentile, had a 20% lower OPR .. relationship between serum P levels and obstetric outcomes.
than those with higher values (P < 0.05) in an oocyte donation set- .. Pregnancy outcome was determined by a positive b-hCG test
..
ting. Despite all patients having received natural MVP at 400 mg .. (serum levels of b-hCG > 10 IU/ml 11 days after ET); clinical preg-
twice daily, one in every four patients had inadequate serum
.. nancy was defined as the presence of at least one gestational sac on
..
P levels. .. ultrasound; implantation was defined as the presence of a gestational
We sought to determine whether these findings could be extrap-
.. sac per embryo transferred; miscarriage rate was defined as any preg-
..
olated to the infertile population undergoing artificial cycles for .. nancy loss before Week 12, including biochemical miscarriage with a
ET, including treatments with own or donated eggs. Moreover, we
.. positive b-hCG test without evidence of a gestational sac and clinical
..
utilized a larger sample size to define the critical threshold of serum .. miscarriage after confirmation of an intrauterine gestational sac; ectopic
.. pregnancy was defined as a gestational sac located outside the uterine
P on the day of ET in HRT cycles that significantly alters the OPR ..
and live birth rates (LBRs). We also identified intrinsic factors that .. cavity; OPR was defined as the presence of at least one viable fetus
.. beyond Week 12; and LBR was defined as the number of deliveries
may be predictive of having low serum P levels; finally, we deter- ..
mined whether these low levels influence obstetric or neonatal .. that resulted in at least one live born neonate.
..
outcomes. ..
.. Sample size
.. Based on our previously published results, it was estimated that 25%
..
.. of the population could show inadequate serum P levels on the day of
Materials and methods .. ET (Labarta et al., 2017). The study population was calculated to de-
..
.. tect a 10% difference in OPR between two groups according to serum
Design and setting .. P levels (expected to be 40% in the suboptimal serum P group and
..
This prospective cohort study is registered in clinicaltrials.gov .. 50% in the optimal serum P group). By accepting an alpha risk of 0.05
(NCT03272412) and was approved by the Institutional Review Board .. (95% CI) and a beta risk of 0.2 (80% statistical power) in a two-sided
..
of IVI RMA Valencia, Spain. The study was conducted at IVI RMA .. test, and a ratio of 3:1 between groups (serum P levels >p25 vs
Valencia between September 2017 and November 2018.
.. p25), 1050 patients were needed to determine a statistically
Low serum progesterone impairs pregnancy outcome 3

..
significant difference. A drop-out rate of 15% was anticipated, so 1205 .. All ETs were performed by senior gynecologists under transabdominal
patients were needed. The ARCSINUS approximation was used for .. ultrasound guidance.
..
..

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this calculation (Casagrande et al., 1978).
... Statistical analysis
Study protocol ..
.. Statistical analysis was performed using IBM SPSS Statistics v25 soft-
Endometrial preparation
.. ware (SPSS Inc., Chicago, IL, USA). Continuous variables were
..
Only patients who underwent egg donation cycles using fresh embryos .. expressed as mean and SD, whereas categorical variables were
R
.. expressed as percentages. Patients were classified into 10 groups
were given a GnRH agonist (DecapeptylV 3.75 mg IM, single dose, ..
Ipsen Pharma, Barcelona, Spain) administered in the mid-luteal phase .. according to the deciles of serum P. OPR was calculated in each group
..
of the previous menstrual cycle, or a GnRH antagonist (0.25 mg/day) .. to detect a critical threshold related to the probability of OPR.
R
for 5 days from the first day of menstruation (OrgalutranV 0.25 mg/
.. Categorical variables were compared with a v2 test, and a Student’s t-
..
0.5 ml SC, single dose, Merck Sharp & Dohme, Madrid, Spain) (Vidal .. test was used to compare the continuous variables between the two
.. groups defined by the threshold.
et al., 2018). After transvaginal ultrasound to confirm ovarian quies- ..
cence, estrogen treatment commenced on Days 2–3 of menstruation. .. To analyze the influence of patient characteristics on serum P on
.. the day of ET, multivariate logistic regression analysis was performed.
Estradiol was administered orally at either 6 mg/day of estradiol valer- ..
R
ate (ProgynovaV, Bayer Hispania, Barcelona, Spain; MeriestraV,
R .. Variables that were correlated in a univariable analysis with serum
.. P levels were included (age, height, weight, BMI, time frame between
Novartis, Barcelona, Spain) or transdermally with two patches of 75 lg ..
R
estradiol hemihydrate (EvopadV, Janssen Cilag, Madrid, Spain) every
.. the last dose of MVP and blood tests and days on HRT before ET).
.. Similarly, to analyze the net impact of low serum P on OPR, a multi-
48 h. After 10–14 days on estrogens, a vaginal two-dimensional (2D) ..
.. variate logistic regression analysis was conducted and adjusted for the
ultrasound was performed to measure endometrial thickness (EMT) ..
.. following variables: age, weight, height, serum E2 in the proliferative
and to confirm a triple-layer pattern, and a blood sample was drawn
.. phase, EMT, number of embryos transferred in previous cycles, num-
for estradiol (E2) and P determinations to ensure that no spontaneous .. ber of embryos transferred in the current cycle, quality of the trans-
ovulation had occurred. If EMT was 6.5 mm, the endometrial pattern ..
.. ferred embryo, day of blastocyst development (Day 5 or 6) and donor
was trilaminar, and serum P < 1.0 ng/ml, ET was scheduled. LPS began ..
5 days before ET with MVP at a dose of 400 mg twice daily
.. age in oocyte donation cycles. All variables were considered potential
.. confounding when the P-value was <0.2 in univariate analysis.
R R
(UtrogestanV, SEID, Barcelona, Spain or ProgeffikV, Effik, Madrid, ..
Spain). The last dose before ET (10th dose) was administered on the
.. Multivariate logistic regression analysis was performed both in the gen-
.. eral population and in each of the three groups according to the type
morning of ET. If pregnancy occurred, hormone treatment was main- ..
.. of treatment (own oocytes without PGT-A, own oocytes with PGT-A
tained until pregnancy Week 12 in accordance with routine practice. .. and donated oocytes).
..
Selecting patients .. To evaluate the predictive capability of serum P on OPR, the receiv-
.. ing operating characteristic (ROC) curve was described and the AUC
All eligible patients at our clinic were offered participation in the study ..
and those interested provided written informed consent. A blood test
.. was calculated. The optimal threshold was defined according to sensi-
.. tivity and specificity to predict OPR.
was performed 1–2 h before ET to determine serum E2 and P levels. ..
..
Hormone measurement
..
..
..
Hormone measurements taken the day of ET (E2 and P) were blinded
.. Results
to the doctor, embryologist, and patient and the results were not ..
available until the end of the study. Blood samples were analyzed by .. Descriptive analysis
..
R
an electrochemiluminescence immunoassay (CobasV e411 analyzer, .. Of the 1205 eligible patients, 1150 were analyzed. The reasons for ex-
Roche diagnostics GmbH, Germany). Intra- and inter-assay coefficients
..
.. clusion were withdrawal (n ¼ 20), change in the exogenous P dose
of variation for the P determinations were 1.2–11.8% and 3.6–23.1%, ..
.. (n ¼ 25) and failure to undergo blood testing (n ¼ 10). This meant that
respectively, for P-values between 0.22 and 51.6 ng/ml. Sensitivity was .. 55 patients (4.6%) could not be included in the analysis.
0.03 ng/ml. The intra- and inter-assay coefficients of variation for E2 ..
.. The mean overall population age was 39.6 § 4.6 years, mean BMI
determinations were 2.4–9.5% and 2.5–11.9%, with a measurement .. was 23.7 § 4.2 kg/m2 and mean EMT was 8.8 § 1.5 mm when receiv-
range of 25.4–2932 pg/ml. Sensitivity was 5 pg/ml. ..
.. ing estrogen therapy. A mean of 1.1 § 0.3 blastocysts were transferred
.. (88% were single ETs). Ongoing pregnancy outcome was reported in
IVF laboratory ..
Intracytoplasmic sperm injection was used in all cases. Either fresh or
.. 1148 patients while live birth outcome was available for 1125 patients.
.. The overall OPR was 49.0% (95% CI: 46.2–51.9) and the LBR was
vitrified oocytes were used in oocyte donation cycles since there are ..
.. 47.0% (95% CI: 44.1–49.9).
no differences in pregnancy rates between them (Cobo et al., 2008; ..
Rienzi et al., 2010). Likewise, ET was performed with fresh or thawed ..
.. Clinical outcome according to serum P on
blastocysts. ..
Embryo quality was classified according to the Spanish ASEBIR .. the day of embryo transfer
..
(Asociación para el estudio de la biologı́a de la reproducción) classifica- .. The mean serum P level on the day of ET was 12.1 § 7.0 ng/ml.
tion (Pons, 2015) and only embryos graded A to C were transferred.
.. Patients were divided in 10 groups by deciles of serum P and OPR
4 Labarta et al.

..
was calculated in each group (Fig. 1). Additional results of positive .. Exploratory analysis of factors related to
..
beta-hCG, LBRs and miscarriage rates according to the deciles of se- .. serum P levels on the day of embryo
rum P are shown in Supplementary Fig. S1. ..

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.. transfer
Considering the lower limit of the 95% CI of the overall OPR ..
(46.2%) as a fair rate, a critical cutoff of 8.8 ng/ml was observed. This .. Correlation analysis identified which variables could impact serum
.. P levels. Pearson’s coefficient (r) showed a significant association
cutoff corresponded to the 30th percentile. Table I shows the baseline ..
clinical data and results in patients whose serum P levels fell below or .. (P < 0.05) of serum P levels with BMI (r ¼ 0.16), weight (r ¼
.. 0.13), time between last dose of MVP and blood tests (r ¼ 0.07),
above 8.8 ng/ml, according to the type of treatment. Overall, patients ..
with serum P levels <8.8 ng/ml yielded a significantly lower OPR of
.. age (r ¼ 0.07), height (r ¼ 0.07) and days on HRT until ET (r ¼ 0.08).
.. Among them, multivariate logistic regression showed that only body
36.6% vs 54.4%; crude odds ratio (OR), 95% CI: 0.49 (0.35–0.63); ..
P < 0.001; lower LBR of 35.5% vs 52.0%; OR (95% CI): 0.51 (0.39–
.. weight remained statistically significant for presenting serum P levels
..
0.66); P < 0.001 and higher clinical miscarriage rate at 13.5% vs 23%; .. below the critical threshold (P ¼ 0.02). Patients with a serum P level
.. of <8.8 ng/ml had a significantly higher body weight than the rest
OR (95% CI): 1.9 (1.2–2.9); P ¼ 0.006) (Fig. 2). Crude OR for com- ..
parison of OPRs reached statistical significance in the group with own .. (65.9 vs 63.4 kg, P ¼ 0.001). The mean time between the last dose of
.. P and blood sample was 341 § 140 min. Mean serum P was
oocytes with PGT-A and in the group with donated oocytes, as shown ..
in Fig. 3. .. 12.9 § 8.6 ng/ml vs 11.5 § 5.4 ng/ml when the last dose of P was
..
Multivariate logistic regression showed that serum P below 8.8 ng/ml .. within below or above 6 h, respectively (P ¼ 0.007). This difference did
remained an independent factor for decreasing OPRs in the three dif- .. not affect OPR; 50.3% vs 44.3%, P ¼ 0.08.
..
ferent populations after adjusting for all confounding variables (Table II). .. The ROC curve showed a significant predictive value of serum
The resulting adjusted OR (95% CI) for ongoing pregnancy was 0.49 .. P levels on the day of ET for both OPR and LBR, with an AUC (95%
..
(0.37–0.64; P < 0.001) and for live birth 0.52 (0.40–0.69; P < 0.001) for .. CI) of 0.58 (0.55–0.62) for both outcomes (P < 0.001). The serum
patients with serum P levels <8.8 ng/ml on the day of ET.
.. P threshold of 8.8 ng/ml offered a sensitivity of 77.6% for OPR,

Figure 1. Ongoing pregnancy rate according to the deciles of serum progesterone (P) on the day of embryo transfer. Data are
expressed as mean, 95% CI.
Low serum progesterone impairs pregnancy outcome 5

Table I Comparison of baseline clinical characteristics based on serum P level on the day of embryo transfer below or above
8.8 ng/ml, denoted as the critical threshold.

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Own oocytes no PGT-A (n 5 184) Own oocytes with PGT-A (n 5 308) Oocyte donation (n 5 658)

P < 8.8 ng/ml P  8.8 ng/ml P P < 8.8 ng/ml P  8.8 ng/ml P P < 8.8 ng/ml P  8.8 ng/ml P
............................................................................................................................................................................................................................
Number (%) of patients 57 (31%) 127 (69%) 99 (32.1%) 209 (67.9%) 188 (28.6%) 470 (71.4%)
Age 35.6 § 3.3 35.1 § 3.3 0.290 37.4 § 3.6 37.9 § 3.4 0.268 41.5 § 4.5 41.7 § 3.9 0.603
BMI 24.2 § 3.4 23.0 § 3.5 0.034 24.5 § 5.0 23.1 § 3.7 0.014 24.5 § 4.8 23.6 § 4.2 0.019
Weight (kg) 65.5 § 9.9 62.8 § 11.1 0.114 66.7 § 13.7 62.8 § 11.4 0.018 65.7 § 13.1 63.8 § 11.4 0.096
Height (m) 1.64 § 0.1 1.64 § 0.1 0.543 1.64 § 0.1 1.64 § 0.1 0.844 1.63 § 0.1 1.64 § 0.1 0.133
Proliferative phase
Serum E2 (pg/ml) 236 § 156 230 § 168 0.809 220 § 149 273 § 315 0.115 271 § 185 291 § 283 0.371
Serum P (ng/ml) 0.12 § 0.17 0.13 § 0.17 0.828 0.09 § 0.11 0.15 § 0.17 <0.001 0.21 § 0.23 0.19 § 0.20 0.317
Endometrial thickness (mm) 9.0 § 1.8 9.0 § 1.5 0.996 8.9 § 1.5 8.8 § 1.6 0.324 8.6 § 1.5 8.8 § 1.6 0.117
Luteal phase
Serum E2 (pg/ml) 257 § 134 232 § 145 0.279 252 § 215 237 § 173 0.521 224 § 118 250 § 181 0.034
Serum P (ng/ml) 6.5 § 1.5 13.8 § 5.4 <0.001 6.5 § 1.9 14.3 § 10.0 <0.001 6.4 § 1.9 14.8 § 5.7 <0.001
Days on HRT until ET 16.7 § 3.4 16.6 § 3.1 0.908 16.6 § 3.5 16.9 § 3.2 0.531 18.4 § 4.2 18.9 § 4.4 0.124
Time between last P dose and 334 § 160 346 § 120 0.624 346 § 115 336 § 158 0.732 349 § 158 336 § 132 0.354
blood test (min)
No. embryos transferred 1.3 § 0.4 1.2 § 0.4 0.315 1.1 § 0.3 1.1 § 0.3 0.761 1.1 § 0.3 1.1 § 0.3 0.665
Proportion of single embryo 75.4% 81.9% 0.325 91.8% 92.8% 0.817 86.6% 87.9% 0.695
transfer (% SET)
No. previous embryos 1.6 § 2.1 1.3 § 1.7 0.299 0.9 § 1.5 0.6 § 1.3 0.064 2.2 § 3.4 1.4 § 2.2 0.004
transferred
Embryo quality of the best embryo transferred
A (%) 9.8 10.7 0.709 3.9 3.7 0.943 13.8 23.1 0.008
B (%) 56.1 62.1 68.8 71.0 63.1 62.3
C (%) 34.1 27.2 27.3 25.3 22.5 14.6
Gestational outcome (%)
Positive beta-hCG test 52.6 70.1 0.030 52.5 68.4 0.008 56.4 68.2 0.005
(>10 IU/l)
Implantation 44.7 59.4 0.076 42.9 60.0 0.005 43.0 53.7 0.001
Clinical pregnancy 43.9 61.4 0.036 42.4 61.2 0.002 50.0 62.0 0.007
Ongoing pregnancy 38.6 53.5 0.079 34.3 54.1 0.001 37.2 54.7 <0.001
Live birth 36.8 51.2 0.080 34.3 53.1 0.002 35.7 51.8 <0.001
Biochemical miscarriage 16.7 12.4 0.546 19.2 10.5 0.144 11.3 8.5 0.437
Clinical miscarriage 16.0 15.4 1.000 19.0 11.7 0.297 26.6 13.8 0.007

Data are expressed as mean § SD or n (%). Bold entries mean that the P-value was < 0.05, which is statistically significant.
PGT-A, preimplantation genetic testing for aneuploidies; E2, estradiol; P, progesterone; ET, embryo transfer.

..
whereas specificity was 37.1%. The optimal serum P threshold at .. 23. Serum P levels below or above 8.8 ng/ml on the day of ET did not
which sensitivity and specificity for OPR were both >50% was .. influence obstetrical or perinatal outcomes in the overall population;
10.4 ng/ml (63.6% sensitivity, 50.1% specificity).
... e.g. percentages of term deliveries (86.1% vs 85.4%), normal
..
.. birth weight (83.6% vs 91.3%), pregnancy-associated hypertension
.. (12.9% vs 9%), gestational diabetes (8.1% vs 7.4%), risk of preterm
Obstetrical and perinatal outcomes ..
.. labor (4.8% vs 4.9%) and bleeding during the first (32.3% vs 24.6%) or
according to serum P below or above ..
.. second/third trimester (2.4% vs 2.8%). Notably, serum P levels
8.8 ng/ml on the day of embryo transfer .. <8.8 ng/ml trended toward higher risk of pregnancy-associated
..
A total of 555 pregnant women remained pregnant beyond Week 20. .. hypertension in oocyte donation cycles but did not reach statistical
Of them, 529 had a confirmed live birth; there were two stillbirths .. significance (15.7% vs 8.2%, P ¼ 0.07). This was not seen in treatments
..
and one perinatal death; and no birth was confirmed for the remaining . with own oocytes (8.9% vs 9.9%, P ¼ 1.00).
6 Labarta et al.

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Positive beta-hCG test

Clinical Pregnancy
Pregnancy Outcome

Ongoing Pregnancy

Live Birth

BiochemicalMiscarriage

Clinical Miscarriage

,0 ,5 1,0 1,5 2,0 2,5 3,0

Odds Ratio (95% CI)

Figure 2. Crude odds ratios for the clinical outcomes in patients with serum P levels <8.8 ng/ml on the day of embryo transfer
compared to patients with higher levels. All differences were statistically significant except for biochemical miscarriage.

..
Discussion .. subcutaneous or intramuscular) is lacking. Serum P can be measured
.. only when using natural-like progestogens, such as MVP. Indeed, syn-
This prospective study aimed to analyze the relationship between se- .. thetic progestogens like dydrogesterone make P measurements futile
..
rum P levels on the day of ET in artificial cycles and pregnancy out- .. because the molecule completely differs and will not be detectable by
comes. Our results confirm that low serum P levels on the day of ET ..
.. this test (Griesinger et al., 2019).
lead to worse pregnancy outcomes. In fact, regardless of the type of .. Serum P levels vastly differ when using MVP compared to subcuta-
treatment (own oocytes with or without PGT-A and oocyte dona- .. neous or intramuscular P, due to different pharmacokinetics (PK) and
..
tion), the impact of serum P on the day of ET is present after adjusting .. pharmacodynamics (PD) of the distinct compounds (Miles et al.,
for all possible confounding factors. Two important messages can be ..
.. 1994). Compared to injected P, MVP leads to lower serum P levels
obtained from this study. First, all patients who receive MVP in HRT .. and higher intrauterine P levels due to the first uterine pass effect
cycles need to reach a minimum of 8.8 ng/ml circulating P to maintain
..
.. (Bulletti et al., 1997). Moreover, steady levels are more stable when
pregnancy, regardless of the origin of the oocytes. Second, 30% of .. using vaginal P (Duijkers et al., 2018), which facilitates its measurement
patients receiving MVP at a dose of 400 mg twice daily are below the
..
.. and interpretation. Although no direct correlation between uterine
optimal P level. .. and serum P levels have been found in PK and PD studies (Paulson
This study allowed us to better define the critical threshold of serum
..
.. et al., 2014), current evidence showing an association between serum
P values needed in artificial endometrial preparation cycles to maintain .. P levels and OPR or LBR demonstrates the relevance of serum P lev-
..
OPRs above 45%. The threshold hereby defined (8.8 ng/ml) is slightly
... els in determining pregnancy success (Yovich et al., 2015, Labarta
different to the one described in our previous study (9.2 ng/ml) .. et al., 2017, Alsbjerg et al., 2018; Cédrin-Durnerin et al., 2019;
(Labarta et al., 2017). The goal of the present study was to better de- .. Gaggiotti-Marre et al., 2019). We hypothesize that one of the reasons
..
fine this threshold; for that purpose, a much larger population (1150 in .. for this relationship is the immunomodulatory role of P in early preg-
the current study vs 211 in the previous one) of every-day patients .. nancy stages (Shah et al., 2018), which favors embryo tolerance and
..
was included. .. prevent miscarriage. This is not a direct effect on the uterus, but
Our research focused on women using natural MVP for LPS. Thus, .. rather a systemic effect that requires adequate P levels in the blood-
..
applicability to other forms of P administration needs to be validated. .. stream to have positive effects on maintaining pregnancy.
In fact, information about the percentage of patients with inadequate
.. Since the first published study that demonstrated a relation
..
serum P levels using other administration routes (e.g. rectal, oral, . between serum P levels and pregnancy outcome in artificial cycles
Low serum progesterone impairs pregnancy outcome 7

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Figure 3. Mean ongoing pregnancy rate (95% CI) and crude odds ratios in the three treatment groups based on serum P level
on the day of ET below or above 8.8 ng/ml. ET, embryo transfer.

Table II Multivariate logistic regression evaluating the effect of variables on ongoing pregnancy rate.

Own oocytes no Own oocytes with Oocyte donation Total

PGT-A (n 5 184) PGT-A (n 5 308) (n 5 658) (1150)

Adjusted OR P Adjusted OR P Adjusted OR P Adjusted OR P

(95% CI) (95% CI) (95% CI) (95% CI)
............................................................................................................................................................................................................................
Age 0.95 (0.85–1.05) 0.325 0.98 (0.91–1.10) 0.593 0.97 (0.93–1.02) 0.217 0.98 (0.95–1.01) 0.099
Weight 1.03 (0.99–1.06) 0.114 1.02 (1.00–1.04) 0.061 1.00 (1.00–1.01) 0.680 1.01 (1.00–1.02) 0.124
Height 0.21 (0.00–51.7) 0.578 1.08 (0.03–46.4) 0.970 2.95 (0.19–45.2) 0.437 1.77 (0.24–13.06) 0.577
E2 proliferative phase 1.00 (1.00–1.00) 0.695 1.00 (1.00–1.00) 0.403 1.00 (1.00–1.00) 0.045 1.0 (1.00–1.00) 0.039
Endometrial thickness 1.13 (0.91–1.39) 0.276 0.99 (0.86–1.15) 0.920 1.12 (1.00–1.26) 0.045 1.07 (0.99–1.16) 0.098
No. embryos previously transferred 0.98 (0.82–1.17) 0.832 0.96 (0.81–1.15) 0.686 0.83 (0.76–0.91) <0.001 0.88 (0.83–0.94) <0.001
No. embryos transferred 1.05 (0.45–2.44) 0.917 2.57 (0.94–6.99) 0.065 2.09 (1.23–3.52) 0.006 1.80 (1.22–2.65) 0.003
Quality of transferred embryo 2.32 (1.31–4.12) 0.004 0.73 (0.45–1.19) 0.210 1.83 (1.37–2.44) <0.001 1.59 (1.28–1.97) <0.001
Day of blastocyst development 0.56 (0.27–1.17) 0.122 0.80 (0.45–1.41) 0.434 0.99 (0.63–1.56) 0.977 0.79 (0.58–1.08) 0.140
(d5 vs d6)
Donor’s age 0.95 (0.92–0.99) 0.009
P < 8.8 ng/ml the day of ET 0.49 (0.24–0.99) 0.048 0.38 (0.23–0.65) <0.001 0.54 (0.37–0.78) 0.001 0.49 (0.37–0.64) <0.001

Results were calculated in each type of treatment and in the overall population. Data are presented as odds ratio (OR) and 95% confidence interval. Bold entries mean that the P-value
was < 0.05, which is statistically significant. Impact of low serum progesterone levels on the day of embryo transfer on pregnancy outcome. A prospective cohort study in artificial
cycles with vaginal progesterone.
8 Labarta et al.

..
(Yovich et al., 2015), several studies have found similar results. .. threshold level (9.2 ng/ml) between our first study (Labarta et al.,
However, they were all retrospective analyses that reached the .. 2017) and the current one (8.8 ng/ml) is not due to the timing of
..
same conclusion: a minimum threshold of serum P levels needs to .. measurement (mean time 5.7 h in the current study). Regarding ob-

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be reached to optimize outcomes in artificial cycles when using MVP ... stetrical outcomes, we found no differences between the two
(Alsbjerg et al., 2018; Cédrin-Durnerin et al., 2019; Gaggiotti-Marre .. groups of P levels. Interestingly, the prevalence of hypertensive dis-
..
et al., 2019). Only one large retrospective study does not agree .. orders during pregnancy was not statistically different between
with this statement, which reported that patients with serum P lev- .. patients with low and good P levels. This suggests that the higher
..
els <10 ng/ml obtained similar results to those with good levels .. prevalence of hypertensive disorders in artificial cycles vs natural
(Volovsky et al., 2020). Nevertheless, this study presented a major .. cycles might be related to something else, perhaps relaxin levels,
..
flaw because patients with lower P levels (<8 ng/ml) were supple- .. rather than to serum P levels as suggested (Conrad et al., 2019; von
mented with more exogenous P (no details of route and added .. Versen-Höynck et al., 2019).
..
doses were provided) from the day of ET onward. Therefore, the .. One limitation of our study is that no cleavage-stage ETs were in-
net effect of low P levels could not be fully analyzed because it
..
.. cluded, so we cannot ensure that the threshold is the same for this ap-
would have biased their conclusions, as later suggested (Alsbjerg .. proach. Yet, PK studies demonstrate that steady levels of P are
et al., 2020).
..
.. reached after 6 h of progesterone exposure and can be maintained by
Our prospective design has the advantage of performing blinded P .. continued dosing. Thus, we hypothesize that the same threshold can
determinations and avoiding the risk of modifying LPS according to
..
.. be used for Days 2–3 ETs. Indeed, measurement of serum P on Day
hormonal measurements. This is often the case with other retrospec- .. 4, the day before blastocyst transfer, is related to pregnancy outcome
tive studies, which makes it difficult to interpret the results (Volovsky
..
.. and shows a similar serum P cutoff (Gaggiotti-Marre et al., 2019).
et al., 2020). In our study, patients with later modified LPS (according .. Another limitation could be the heterogeneity in the population in-
..
to the doctor’s criteria, and mainly due to bleeding) were excluded .. cluded (own oocytes þ PGT-A, own oocytes without PGT-A, oocyte
from analysis. .. donation), and for this reason we have shown the results separately in
..
Although serum P levels are associated with pregnancy outcome, .. these three groups.
they do not show a high predictive value for ongoing pregnancy as .. In summary, we confirm the relevance of serum P levels when using
..
seen on the ROC curve analysis. This implies that P is not a single pre- .. natural MVP in HRT cycles, not only in oocyte donation cycles as pre-
dictor of treatment success because other factors, such as embryo ..
.. viously published (Labarta et al., 2017) but also in cycles with own
quality, determine the cycle’s fate. After adjusting for all the confirmed .. oocytes. Research into individualized luteal phase supplementation
confounding factors, including embryo quality, serum P below the opti- ..
.. (iLPS) (Labarta, 2020) should be prioritized as not all patients may
mal threshold was still associated with outcome in the three groups .. benefit from a one-size-fits all protocol. Questions about the best
according to the type of treatment. According to our recent experi- ..
.. dose and route of administration of exogenous P according to individ-
ence, in almost 80% of patients (unpublished data), levels of P are sim- .. ual characteristics and type of cycle are still remaining. Future studies
ilar in subsequent cycles if doses of exogenous P are not modified. ..
.. are needed to demonstrate if iLPS is clearly effective in this particular
This could be a plausible explanation of the findings in oocyte donation .. set of patients with inadequate levels of serum P.
recipients with low serum P. In fact, they showed a higher number of
..
..
embryos previously transferred, which could have failed due to this ..
reason; whereas patients with a good level of serum P had less previ-
..
.. Supplementary data
ous failed attempts and had the possibility of doing an elective transfer ..
of a grade A embryo. We do not know if this is the cause or the con-
.. Supplementary data are available at Human Reproduction online.
..
sequence, but it makes sense that these patients may have more im- ..
plantation failure due to insufficient progesterone exposure and are
..
..
conducting a second or third ET. .. Data availability
..
In this study, we identified variables related to serum P levels. ..
.. The data underlying this article will be shared on reasonable request
Although some variables were significantly correlated, the strength of
.. to the corresponding author.
the relation was very weak as all absolute Pearson’s r values were be- ..
low 0.2. The only variable to remain significant in the multivariate re- ..
..
gression analysis was body weight, which showed a negative ..
correlation. The difference in body weight between patients with low ..
..
Authors’ roles
or adequate levels of P was 2.5 kg, which is not clinically relevant, con- .. All authors made substantial contributions to data acquisition, re-
sidering that this did not impact pregnancy outcome. Indeed, weight ..
.. vised the article and approved the final version. The first and last
had no impact on PK when using MVP (Levy et al., 1999). A recent .. authors significantly contributed to the study conception and design,
retrospective analysis in 685 FET cycles confirmed our results, observ- ..
.. performed statistical analyses and data interpretation and drafted
ing a negative correlation between weight and serum P levels .. the article.
(González-Foruria et al., 2020). These interpersonal variations could ..
..
be due to a variable capacity of absorption, clearance and different dis- ..
tribution in fat tissue.
..
.. Funding
The decrease in mean serum P levels with increased time interval is ..
small and did not affect clinical outcomes. The difference in the
.. No external funding was either sought of obtained for this study.
Low serum progesterone impairs pregnancy outcome 9
..
Conflict of interest .. Ghobara T, Gelbaya TA, Ayeleke RO. Cycle regimens for frozen-
.. thawed embryo transfer. Cochrane Database Syst Rev 2017;7:
..
E.L. received a grant from Ferring in 2020, has provided consultancy .. CD003414.

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services for MSD and Ferring Pharmaceuticals and is part of the .. González-Foruria I, Gaggiotti-Marre S, Alvarez M, Martı́nez F, Garcı́a S,
..
Ferring Pharmaceuticals LIFE program and Merck Global program for .. Rodrı́guez I, Coroleu B, Polyzos N. Factors associated with serum
Fertility Innovation Leaders. During the past 12 months, she has re- .. progesterone concentrations the day before cryopreserved embryo
..
ceived honoraria from Angelini/IBSA, Merck, MSD and Ferring .. transfer in artificial cycles. Reprod Biomed Online 2020;40:797–804.
Pharmaceuticals for lecturing. E.B. received a grant from Finox in 2016,
.. Griesinger G, Tournaye H, Macklon N, Petraglia F, Arck P, Blockeel
..
has given lectures or provided consultancy services for MSD, Merck, .. C, van Amsterdam P, Pexman-Fieth C, Fauser BC.
IBSA and Ferring Pharmaceuticals. F.C. has received honoraria from
.. Dydrogesterone: pharmacological profile and mechanism of action
..
MSD and Ferring Pharmaceuticals for lecturing. Rest of authors have .. as luteal phase support in assisted reproduction. Reprod Biomed
..
nothing to declare. C.R.-V. received a grant from the Spanish Ministry .. Online 2019;38:249–259.
of Education, Culture and Sport in 2019 for the National Programme .. Groenewoud ER, Cohlen BJ, Macklon NS. Programming the endo-
..
for Training University Lecturers (FPU). .. metrium for deferred transfer of cryopreserved embryos: hormone
.. replacement versus modified natural cycles. Fertil Steril 2018;109:
..
.. 768–774.
.. Labarta E. Relationship between serum progesterone (P) levels and
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