# Hormonal and metabolic responses across phases of combined oral contraceptive use and menstrual cycle in young elite female athletes

Katia Collomp, Agnès Olivier, Caroline Teulier, Juliette Bonnigal, Nathalie Crépin, Corinne Buisson, Magnus Ericsson, Emmanuelle Duron, Eric Favory, Mathieu Zimmermann, et al.

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## ► To cite this version:

Katia Collomp, Agnès Olivier, Caroline Teulier, Juliette Bonnigal, Nathalie Crépin, et al. *Hormonal and metabolic responses across phases of combined oral contraceptive use and menstrual cycle in young elite female athletes*. European Journal of Applied Physiology, 2025, 125, pp.1923-1932. 10.1007/s00421-025-05745-x. hal-04985565

HAL Id: hal-04985565

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Submitted on 18 Nov 2025

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# Hormonal and metabolic responses across phases of combined oral contraceptive use and menstrual cycle in young elite female athletes

Katia Collomp<sup>1,2,3,4</sup> · Agnès Olivier<sup>5</sup> · Caroline Teulier<sup>1,2</sup> · Juliette Bonnigal<sup>5</sup> · Nathalie Crépin<sup>4</sup> · Corinne Buisson<sup>4</sup> · Magnus Ericsson<sup>4</sup> · Emmanuelle Duron<sup>6</sup> · Eric Favory<sup>5</sup> · Mathieu Zimmermann<sup>7</sup> · Virgile Amiot<sup>7</sup> · Carole Castanier<sup>1,2</sup>

Received: 26 September 2024 / Accepted: 18 February 2025 / Published online: 9 March 2025
© The Author(s) 2025

## Abstract

**Purpose** Despite the significant number of female athletes using combined oral contraceptives (COCs), there is scant literature on their hormonal and metabolic effects across different phases.

**Methods** In order to contribute to a wider knowledge of COC-action mechanisms involved in athletes' performance and health, we therefore examined the effects of low-dose monophasic COC (ethinylestradiol/levonorgestrel) intake on sex hormones (estradiol, progesterone, sex hormone binding protein (SHBG)) as well as on a large number of pituitary (LH, TSH, prolactin) and peripheral (triiodothyronine, cortisol, DHEA, DHEA-S, aldosterone, osteocalcin, 25(OH)D) basal hormone levels in nine young elite female athletes, across COC administration (first and second half of active hormone intake, washout phases), compared to eleven female athletes without hormonal contraception across their normal menstrual cycle (NMC, i.e., early follicular, end follicular/peri-ovulatory, mid-luteal phases).

**Results** COC vs. NMC increased SHBG (p < 0.01), TSH, cortisol and 25(OH)D (p < 0.05), and decreased DHEA and DHEA-S (p < 0.05) concentrations. Across COC and NMC phases, higher estradiol and aldosterone concentrations (p < 0.05) were observed during the washout and mid-luteal phases, respectively.

**Conclusion** In highly trained female athletes, COC vs. NMC induced several hormonal alterations, irrespective of the phases, leading to potential ergogenic and clinical repercussions that merit clarification. In NMC athletes, the impact of endogenous sex hormone fluctuations on the parameters studied appeared limited, perhaps mitigated by intense physical training, with only aldosterone change. Given the high prevalence of vitamin D insufficiency, it seems warranted to monitor this parameter, not yet routinely considered in female athletes, taking into account COC intake.

**Trial registration** : ID-RCB:2020-A02965-34, France

Communicated by Fabio Fischetti.

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Katia Collomp
katia.collomp@univ-orleans.fr

<footnote n="1">1 CIAMS, Université d'Orléans, Pôle STAPS, Orléans, France</footnote>

<footnote n="2">2 CIAMS, Université Paris-Saclay, Orsay, France</footnote>

<footnote n="3">3 Fédération SAPREM, Université d'Orléans, Orléans, France</footnote>

<footnote n="4">4 LADF, Université Paris-Saclay, Orsay, France</footnote>

<footnote n="5">5 IFCE, Cadre Noir, Saint-Hilaire-Saint-Florent, Saumur, France</footnote>

<footnote n="6">6 Hôpital Brousse, AP-HP, Equipe INSERM MOODS-CESP, Villejuif, Kremlin-Bicêtre, France</footnote>

<footnote n="7">7 Médecine du Sport, CHU Orléans, Orléans, France</footnote>
## Graphical Abstract

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**Keywords** Ethinylestradiol/levonorgestrel · Sex-hormone status · Highly trained · TSH · Aldosterone · 25(OH)D

## Abbreviations

<table><tr><td>ALD</td><td>Aldosterone</td></tr><tr><td>ANOVA</td><td>Analyse of variance</td></tr><tr><td>CBP</td><td>Cortisol-binding protein</td></tr><tr><td>COC</td><td>Combined oral contraceptive</td></tr><tr><td>COC0</td><td>Washout period (inactive hormone intake)</td></tr><tr><td>COC1</td><td>First half of active hormone intake</td></tr><tr><td>COC2</td><td>Second half of active hormone intake</td></tr><tr><td>COR</td><td>Cortisol</td></tr><tr><td>DHEA</td><td>Dehydroepiandrosterone</td></tr><tr><td>DHEA-S</td><td>Dehydroepiandrosterone-sulphate</td></tr><tr><td>EE</td><td>Ethinylestradiol</td></tr><tr><td>E2</td><td>Estradiol</td></tr><tr><td>FEI</td><td>Free estradiol index</td></tr><tr><td>FSH</td><td>Follicle-stimulating hormone</td></tr><tr><td>FT3</td><td>Free triiodothyronine</td></tr><tr><td>HPG</td><td>Hypothalamo-pituitary-gonadal</td></tr><tr><td>LH</td><td>Luteinizing hormone</td></tr><tr><td>LVN</td><td>Levonorgestrel</td></tr><tr><td>NMC</td><td>Normal menstrual cycle</td></tr><tr><td>NMP1</td><td>Early follicular phase</td></tr><tr><td>NMP2</td><td>End follicular/peri-ovulatory phase</td></tr><tr><td>NMP3</td><td>Mid-luteal phase</td></tr><tr><td>OST</td><td>Osteocalcin</td></tr><tr><td>PG</td><td>Progesterone</td></tr><tr><td>PRL</td><td>Prolactin</td></tr></table>

<table><tr><td>SEM</td><td>Standard error of the mean</td></tr><tr><td>SHBG</td><td>Sex hormone binding protein</td></tr><tr><td>TSH</td><td>Thyroid-stimulating hormone</td></tr><tr><td>25(OH) D</td><td>Calcidiol (25-hydroxyvitamin D)</td></tr></table>

## Introduction

Between 20 and 70% of elite female athletes use hormonal contraception (Larsen et al. 2020; Martin et al. 2018; Oxfeldt et al. 2020), with a large majority opting for 2nd-generation combined oral contraceptives (COCs). These COCs, whether mono-, bi- or triphasic, contain synthetic estrogens and progestins, typically ethinylestradiol (EE) as the estrogen at concentrations between 20 and 50 µg per pill and levonorgestrel (LVN) as the progestin at concentrations of 50 to 150 µg (Castanier et al. 2021). They are administered for 21 days followed by a 7 day break (i.e., washout phase), with or without inactive non-hormonal pills, to mimic the menstrual cycle but without the peaks of estradiol (E2) and progesterone (PG), thus stabilising hormone levels through inhibition of the hypothalamo-pituitary-gonadal (HPG) axis and suppression of gonadotropin secretion produced by the pituitary gland, follicle-stimulating hormone (FSH) and luteinizing hormone (LH).

<figure><img src="image_5.png" /></figure>
While COCs are known to significantly decrease E2 and PG, free and total blood testosterone, as well as dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulphate (DHEA-S), with a parallel increase in sex hormone-binding protein (SHBG) concentrations in healthy women (Coelingh Bennink et al. 2017; Enea et al. 2009; Knutsson et al. 2023), the literature on a female athlete population or on the other COC hormonal effects is sparse and lacking consensus. Indeed, few studies, most of which were carried out on non-physically trained women and/or with higher COC doses, reported during the active hormonal pill phase an increase or not in prolactin (PRL), thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), cortisol (COR), aldosterone (ALD), osteocalcin (OST) and calcidiol (25(OH)D) (Aden et al. 1998; Kuhl et al. 1985a, 1985b; Özcan et al. 2023; Raps et al. 2014; Sawin et al. 1978; Weeke and Hansen 1975; Wiegratz et al. 2003). As these hormones directly or indirectly regulate metabolism, COC-induced changes may play a role in athletes' performance and health. Moreover, even fewer studies have explored potential modifications in hormonal and metabolic status during the COC washout phase (Aden et al. 1998; Ihalainen et al. 2021; Kuhl et al. 1985a; Martin et al. 2021; Rechichi et al. 2008; Weeke and Hansen 1975) and across the intake period, despite LVN concentrations only reaching a steady state in the second half of each active hormone intake treatment cycle (Kuhnz et al. 1992).

In order to contribute to a wider knowledge of COC-action mechanisms in highly trained female athletes, we therefore aimed to examine the effects of low-dose monophasic COC (i.e. EE/LVN) intake on various pituitary and peripheral hormone secretions and metabolic responses in young elite female athletes, taking into account the phase of COC administration (first and second half of active hormone intake, and washout phases), compared to female athletes with a normal menstrual cycle (NMC), during the early follicular phase (low E2), end follicular/peri-ovulatory phase (high E2 & low PG) and mid-luteal phase (high E2 & high PG). We hypothesised that COC vs. NMC will induce several marked changes in these pituitary and peripheral hormones in an elite female population, with modulation across the different phases of active/inactive pill-taking. In parallel, we will assess in NMC athletes the impact of endogenous sex hormone fluctuations across the menstrual cycle on the parameters studied.

# Methods

## Participants

Twenty elite female athletes (judokas and riders), recruited from centers and technical platforms hosting top-level

athletes, volunteered for the study (age: 19.6±0.4 years; height: 164.4±2.5 cm; weight: 62.0±2.6 kg; BMI: 22.9±0.5 kg/m2; % fat mass: 19.8±2.0). After being informed of the study's objectives and risks, participants signed a written consent form. All procedures performed in the study were approved by the ethics committee (ID-RCB:2020-A02965-34) and were in accordance with the Declaration of Helsinki.

Prior to the study, subjects were screened with a medical history and physical examination. Inclusion criteria required participants to be highly physically active (6 to 11 training sessions per week, 3 to 6 h per day, for at least 3 years). Exclusion criteria included cardiovascular, liver, biliary or renal disease; hyperlipidaemia; uncontrolled high blood pressure; endocrinological disorder; oligomenorrhoea or amenorrhoea; pregnancy; history of thromboembolic disorder. Subjects were divided into 2 groups: Group 1 (9 subjects) had used monophasic COC: 20 µg/100 µg (7); 30 µg/150 µg (2) of ethinylestradiol and levonorgestrel per pill, respectively, for at least six months, with pills during the washout phase for all participants; Group 2 (11 subjects) had NMC, without hormonal contraception for at least 1 year. Subjects reported regular menstrual cycle between 25 and 29 days during the last three cycles.

## Experimental procedure

Each participant visited the laboratory 3 times after the inclusion visit, at 5 week intervals, in order to systematically shift the subjects' cycle phase and reach the 3 target statuses, in the winter and spring seasons, at mid-latitude. For the COC group, visits were scheduled during the first (Day3–Day10, i.e. COC1) and second (Day11–Day18, i.e. COC2) periods of pill intake as well as during the washout without hormone intake (W2–W7, i.e. COC0). For the NMC group, visits occurred during the early follicular phase (Day1–Day7, i.e. NMP1) with Day1 being the first day of bleeding, the end follicular/peri-ovulatory phase (Day8–Day14, i.e. NMP2) and during the mid-luteal phase (Day18–Day24, i.e. NMP3). Ovulation was detected through PG measurement in all NMC participants, and menstrual phases were retrospectively confirmed by analyzing serum sex female hormones.

## Blood collection and analysis

Blood samples were collected in the morning (7:00–8:00 a.m.) after an overnight fast. Participants were asked to maintain their normal dietary intake during the experimental period, and to refrain from consuming caffeine or alcohol and from making strenuous efforts for at least 24 h prior to the trial sessions. Venous blood samples were collected from an antecubital vein using standard
procedures, and the blood was transferred into serum, EDTA and heparin tubes. The serum and plasma were obtained by centrifuging the tubes at 3500 rpm for 10 min before being stored at –20 °C for later analysis. Vitamin D (25(OH)D) was determined by electrochemiluminescence, and calcium by a classical photometric test. The concentrations of E2, PG, SHBG, LH, PRL, TSH, FT3, DHEA, DHEA-S, COR, ALD and OST were determined by ELISA (kits from DRG Diagnostic®, Germany). The free estradiol index (FEI) was determined by [E2/SHBG]. The analytical sensitivities for the assays were: Vitamin D: 3.0 ng/ml; calcium: 8 mg/L; E2: 10.6 pg/ml; PG: 0.045 ng/ml; SHBG: 0.41 nmol/L; LH: 1.27 mIU/ml; PRL: 0.35 ng/ml; TSH: 0.06 mIU/ml; FT3: 0.38 pg/ml; DHEA-S: 0.04 mg/L; DHEA: 0.63 ng/ml, COR: 1.3 ng/ml, ALD: 10.65 pg/ml and OST: 0.31 ng/ml, respectively. Assays were made in duplicate, and coefficients of variation for all parameters were always < 10%.

## Statistical analyses

Results are presented as mean values ± standard error of the mean (SEM). After evaluating the normality of the samples, differences in blood parameters between the trials were analyzed with a two-way (treatment and phase) analysis of variance (ANOVA) with repeated measurements. A post hoc Fisher test was performed to locate the differences, in the event of an ANOVA revealing a significant main effect. Size of treatment was estimated with Cohen's d scores and 95% CI for d. Correlations were calculated using Pearson's product moment correlation test. The null hypothesis was rejected at p < 0.05.

<figure><img src="image_6.png" /><figcaption>**Fig. 1** Concentrations of E2 (pg/ml) and PG (ng/ml) in COC and NMC subjects. For COC: COC0 (washout); COC1 (1st half of active pill); COC2 (2nd half of active pill); For NMC: NMP1 (early follicular); NMP2 (end follicular/peri-ovulatory); NMP3 (mid-</figcaption></figure>

## Results

### Sex hormones, SHBG and FEI (Figs. 1 and 2)

E2 and PG concentrations were lower in COC vs. NMC (p < 0.01, Cohen's d: –1.57, 95% CI for d: –2.15,–0.99; p < 0.01, Cohen's d: –2.34, 95% CI for d: –3.00,–1.69, respectively). There was also a treatment effect for SHBG and FEI, with higher values in COC vs. NMC for SHBG (p < 0.01, Cohen's d: 1.35, 95% CI for d: 0.79, 1.91), and lower values for FEI (p < 0.01, Cohen's d: –1.23, 95% CI for d: –1.79,–0.78). In COC subjects, higher E2 was obtained in COC0 vs. COC1 and COC2 (p < 0.05). In NMC subjects, higher E2 was obtained in NMP2 and NMP3 vs. NMP1, and higher PG in NMP3 vs. NMP2 and NMP1 (p < 0.01). No difference in E2 and FEI between COC0 and NMP1 was found.

### Pituitary hormones and FT3 (Fig. 3)

There were decreased LH and increased TSH (p < 0.05, Cohen's d: –0.91, 95% CI for d: –1.44,–0.38; Cohen's d: 1.09, 95% CI for d: 0.55, 1.64 respectively) concentrations in COC vs. NMC. No treatment effect occurred for either PRL or FT3 concentrations. There was an increased LH in NMP2 vs. other conditions (p < 0.01).

### Adrenal and mixed hormones (Fig. 4)

DHEA (p < 0.05, Cohen's d: –1.44, 95% CI for d: –2.02,–0.87) and DHEA-S (p < 0.05, Cohen's d: –1.04, 95% CI for d: –1.59,–0.51) concentrations were lower and COR levels higher in COC vs. NMC (p < 0.05, Cohen's d: 1.06, 95% CI for d: 0.55, 1.53). There was a significant increase in ALD in NMP3 vs. the other conditions (p < 0.05).

<figure><img src="image_7.png" /></figure>

luteal)⁺, p < 0.05,⁺⁺, p < 0.01, difference COC vs. NMC \*, p < 0.05, \*, p < 0.01, difference across NMC phases, \*p < 0.05, difference across COC phases
<figure><img src="image_8.png" /><figcaption>**Fig. 2** Concentrations of SHBG (nmol/L) and free estradiol index (FEI) in COC and NMC subjects For COC: COC0 (washout); COC1 (1st half of active pill); COC2 (2nd half of active pill); For NMC: NMP1 (early follicular); NMP2 (end follicular/peri-ovulatory);</figcaption></figure>

<figure><img src="image_9.png" /></figure>

NMP3 (mid-luteal)⁺, p < 0.05, ++, p < 0.01, difference COC vs. NMC
*, p < 0.05, **, p < 0.01, difference across NMC phases, ⚮p < 0.05,
difference across COC phases

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 3** Concentrations of LH (mIU/ml), PRL (ng/ml), TSH (mIU/ml) and FT3 (pg/ml) in COC and NMC subjects For COC: COC0 (wash-out); COC1 (1st half of active pill); COC2 (2nd half of active pill);</figcaption></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

For NMC: NMP1 (early follicular); NMP2 (end follicular/peri-ovu-
latory); NMP3 (mid-luteal)⁺, p < 0.05, difference COC vs. NMC *,
p < 0.05, **, p < 0.01, difference across NMC phases

**Osteocalcin, vitamin D, and calcium (Fig. 5)**

No change in COC vs. NMC was found for osteocalcin

or calcium, whereas a treatment difference was found for
25(OH)D, with higher values in COC vs. NMC (p < 0.05,
<figure><img src="image_14.png" /><figcaption>**Fig. 4** Concentrations of DHEA-S (mg/L), DHEA (ng/ml), COR (ng/ml) and ALD (pg/ml) in COC and NMC subjects For COC: COC0 (washout); COC1 (1st half of active pill); COC2 (2nd half of active</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>pill); For NMC: NMP1 (early follicular); NMP2 (end follicular/peri-ovulatory); NMP3 (mid-luteal)⁺, p < 0.05, difference COC vs. NMC *; p < 0.05, difference across NMC phases</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>**Fig. 5** Concentrations of OST (ng/ml), 25(OH)D (ng/ml) and calcium (mg/L) in COC and NMC subjects For COC: COC0 (washout); COC1 (1st half of active pill); COC2 (2nd half of active pill); For</figcaption></figure>

NMC: NMP1 (early follicular); NMP2 (end follicular/peri-ovulatory); NMP3 (mid-luteal)⁺, p < 0.05, difference COC vs. NMC

Cohen's d: 1.27, 95% CI for d: 0.71,1.82). No change in either phase was observed for these parameters.

correlations (p < 0.05) between PG and ALD (r = 0.56) as well as between FT3 and 25(OH)D (r = 0.47).

## Correlation

Considering all subjects, there was a significant correlation (p < 0.05) between E2 and LH (r = 0.59), E2 and DHEA (r = 0.62), DHEA and DHEA-S (r = 0.59), SHBG and cortisol (r = 0.56), SHBG and TSH (r = 0.46), SHBG and 25(OH)D (r = 0.46). In NMC, there were significant

## Discussion

Given the scarcity of studies carried out on female athletes, we examined in this population the impact of COC (EE/LVN), across the different phases of active/inactive pill-taking, on various pituitary and peripheral hormones that play an important role in athlete performance and health. In

<figure><img src="image_17.png" /></figure>
parallel, we assessed in NMC athletes the effects of endoge-
nous sex hormone fluctuations across the menstrual cycle on
the parameters studied. Intake of COC vs. NMC, irrespective
of the phase, increased TSH, SHBG, cortisol and 25(OH)
D, and decreased DHEA and DHEA-S concentrations. Few
parameters were influenced by the COC or NMC phases,
with only E2 and aldosterone concentrations being higher
during the washout and mid-luteal phases, respectively.

As anticipated (Castanier et al. 2021; Martin et al. 2018;
Oxfeldt et al. 2020), in NMC subjects, we observed higher
concentrations of LH during NMP2, with significantly
elevated estradiol concentrations during NMP2 and NMP3
compared to NMP1. Progesterone, significantly higher dur-
ing NMP3, was utilized to verify ovulation and thus the nor-
mality of the menstrual cycle in our NMC subjects. COCs
logically inhibited ovulation, resulting in no LH peak nor
PG increase, with significantly lower E2 concentrations
in COC versus NMC subjects, except during the washout
phase. Interestingly, there was a significant increase in E2
secretion during this hormone-free period, compared to the
period of hormone intake, with similar values versus the
early follicular phase. There is no consensus on this increase,
or lack thereof, in E2 during COC0. This may be explained
by the type of COC used and/or the timing of sampling.
Indeed, our subjects were tested after an average washout
of four days, following low doses of EE/LNG, unlike stud-
ies conducted on female athletes at the start of the inactive
pill or with unspecified progestin nature (Ihalainen et al.
2021). In line with the literature (Coelingh Bennink et al.
2017; Knutsson et al. 2023; Özcan et al. 2023), we observed
an increase in SHBG concentrations by more than 100%
in our study with COCs, irrespective of the phase of COC
administration. However, free estradiol index (E2/SHBG),
considered to reflect estradiol activity (Gilberg et al. 2002),
was not significantly different during the washout phase
compared to the early follicular phase, in contrast to the
first and second half of active hormone intake, where it was
strongly decreased compared to NMC subjects. This sug-
gests a relative increase in endogenous estrogenic activity
during the COC-washout vs. COC-active hormone phases,
which could partially compensate for the drop in exogenous
hormone levels.

Prolactin and thyroid hormones each have pleotropic
and independent roles allowing the body to accommodate
to physical activity and exercise but they also share an inter-
relation in their responses, since hypothalamic thyrotropin-
releasing hormone (TRH) release stimulates the release of
TSH and thus the thyroids as well as the release of PRL,
estrogen serving as an interconnective regulatory link by
stimulating the release of both the thyroids and prolactin
(Hackney and Saeidi 2019). Despite the variations of endog-
enous/exogenous estrogen concentrations across NMC and
COC phases, no change in PRL levels was observed in the

present study, either as a function of the menstrual cycle or
hormonal contraception, and similarly, TSH values remained
consistent irrespective of the menstrual cycle period. These
results align with previous studies reporting little or no
effect of the endogenous hormonal status in women (Aden
et al. 1998; Kuhl et al. 1985a; Sawin et al. 1978; Weeke and
Hansen 1975). However, we found increased TSH but not
FT3 concentrations in our COC subjects, maintaining this
increase during all COC periods. Higher TSH concentra-
tions with COCs were initially described with concomitant
higher total and free T3 and T4 concentrations (Kuhl et al.
1985b; Weeke and Hansen 1975), but most recent studies
have reported an increase in TSH but not in free thyroid
hormones, due to the parallel increase in thyroxine-binding
globulin (TBG) protein (Özcan et al. 2023; Raps et al. 2014;
Wiegratz et al. 2003). It may therefore be suggested that the
discrepancy may be related to the lower doses of synthetic
estrogens and progestins used in COCs today, limiting their
thyroid impact.

With COC administration, we found a significant decrease
in both DHEA and DHEA-S compared to NMC. This find-
ing agreed with experiments performed on sedentary or
recreationally active female subjects, with COCs including
different progestins (Coelingh Bennink et al. 2017; Knut-
son et al. 2023). COCs lower androgen status may drive
mood and sexual function alterations in women although
studies of these effects have yielded conflicting results (Bur-
rows et al. 2012; Coelingh Bennink et al. 2017; Oinonen
and Mazmanian 2002). Similarly, the higher total serum
COR obtained here in the COC versus the NMC group was
consistent with most previous works (Özcan et al. 2023).
Unfortunately, we did not analyze cortisol-binding protein
(CBP), but studies showed that COCs stimulate the liver
production of binding proteins, including CBP, with, conse-
quently, a probable lack of change in free cortisol concentra-
tions and/or effects (Özcan et al. 2023; Vibarel-Rebot et al.
2015). Regarding NMC phases, according to the literature,
we found no modification in the androgenic adrenal/gonadal
DHEA and DHEA-S concentrations nor in COR throughout
the menstrual cycle (Kanaley et al. 1992; Kuhl et al. 1985a;
Knutsson et al. 2023; Vibarel-Rebot et al. 2015), whereas
aldosterone levels were significantly increased in NMP3
compared with other conditions. There is very little data on
aldosterone variations during the menstrual cycle, but one
study (De Souza et al. 1989) previously noticed an increase
during the mid-luteal phase. It may be hypothesized that
this increase, whose involvement during endurance exercise
needs to be determined, was mediated by the increase in its
PG precursor, in view of the significant correlation obtained
(r = 0.56).

Estrogens are considered as the “key regulator” of bone
metabolism in women (Khosla and Monroe 2018) and
changes in vitamin D and osteocalcin concentrations have
been previously reported either across the menstrual cycle or
with COC intake in healthy women or female athletes (Har-
mon et al. 2016, 2020; Jürimäe et al. 2011). Vitamin D is
a fat-soluble steroid pro-hormone that occurs in two forms,
vitamin D2 and D3, which may be obtained from sunlight
exposure of the skin as well as from the diet. Both vitamin
D2 and D3 undergo hydroxylation in the liver, where they
are converted into 25(OH)D, further hydroxylated in the
kidney to form 1,25-hydroxyvitamin D (calcitriol), a bio-
logically active metabolite which then binds to vitamin D
receptors (VDBP) at target tissues (Farrokhyar et al. 2015;
Harju et al. 2022; Sist et al. 2023). Despite its well-recogn-
nized importance for both skeletal and non-skeletal systems,
including cardiovascular, immune, inflammatory and muscle
functions, a large proportion of the sporting population is
deficient in vitamin D, with the risk increasing significantly
in high latitudes, as well as in winter and spring, although
specific prevalence in female athletes remains unknown
(Farrokhyar et al. 2015; Harju et al. 2022; Sist et al. 2023).
In our NMC group, 25(OH)D concentrations were below
the recommended value of 30 ng/ml in most of our subjects
(9/11), with two of them even showing vitamin D deficiency
(< 20 ng/ml). These percentages (80%, 45% and 20% below
30, 25 and 20 ng/ml, respectively) appeared greater than
those observed in sedentary or moderately active women
(Harmon et al. 2020) and may reflect the increased needs
resulting from intensive physical training. The same pre-
vious study only reported minimal change in calciotropic
hormones during the menstrual cycle (Harmon et al. 2020)
but, in women with 25(OH)D values below 30 ng/ml, lower
mean E2 across the menstrual cycle. Partially in line with
this study, we found similar 25(OH)D values across the
menstrual cycle, probably because of its long half-life,
but without any correlation with E2 levels. Interestingly,
we observed in parallel relatively low calcium concentra-
tions in our population and significant correlation between
25(OH)D and FT3 concentrations. There was surprisingly
little information in female athletes (Nikolaidis et al. 2003)
but it may be suggested that low blood calcium and FT3
may be partially resulted from low 25(OH)D values. Lastly,
we found similar osteocalcin levels across the menstrual
cycle or between our groups of subjects. This finding was
partially in accordance with previous studies (Jürimäe
et al. 2011; Martin et al. 2021) reporting no change across
NMC but lower osteocalcin with COC administration (i.e.,
20 mg of EE and 75 mg of gestodene) compared to NMC
(Jürimäe et al. 2011). One hypothesis to explain the discrep-
ancy with our COC data is the use of different progestins,
whose specific impact on osteocalcin remains to be con-
firmed. However, our COC subjects had significantly higher
25(OH)D versus NMC subjects, with only one subject with
vitamin D deficiency. This increase in 25(OH)D by COC
intake was previously reported (Harmon et al. 2016) but

as COC induced in parallel a 30% VDPB increase (Özcan
et al. 2023), vitamin D status may be overestimated in COC
subjects and the biological pathways involved remain to be
determined (Harmon et al. 2016).

Conclusion

In highly trained female athletes, low-dose monophasic COC induced, compared to NMC, several hormonal alterations, irrespective of the active/inactive hormone intake phases, leading to potential ergogenic and clinical repercussions that merit clarification. In NMC female athletes, the impact of endogenous sex hormone fluctuations on the parameters studied appears to be very limited, perhaps mitigated by intense physical training, with only a change in aldosterone concentrations. Finally, given the high prevalence of vitamin D insufficiency, it seems warranted to monitor this parameter, not yet routinely considered in female athletes, taking into account COC intake as well as training, competition, season and environmental conditions (Farrokhyar et al. 2015; Kraus et al. 2020).

**Acknowledgements** The authors wish to express their gratitude to all the study participants for their dedicated effort. We also extend our thanks to Dr Alexandre Marchand and Dr Morgane Duquenoy for their expert assistance.

**Author contributions** KC and CC: study conception, data analyses, interpretation and manuscript writing. AO, CT: study conception and interpretation. JB: data analyses. EF, MZ, VA, NC: participation in the experiment. CB, ME, ED: critical revision of the manuscript for critical intellectual content. All authors read and approved the final version of the manuscript.

**Funding** Open access funding provided by Université d'Orleans. This project was supported by the World Anti-Doping Agency (WADA) and the Institut Français du Cheval et de l'Equitation (IFCE).

**Data availability** The data that support the findings are available from the corresponding author upon reasonable request.

Declarations

**Conflict of interest** The authors declare no conflict of interest.

**Institutional review board statement** The study was conducted in accordance with the 1964 Declaration of Helsinki, and approved by the Ethics Committee (ID-RCB:2020-A02965-34).

**Informed consent** Informed consent was obtained from all subjects involved in the study.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated
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## References

Aden U, Jung-Hoffmann C, Kuhl H (1998) A randomized cross-over study on various hormonal parameters of two triphasic oral contraceptives. Contraception 58(2):75-81. https://doi.org/10.1016/S0010-7824(98)00071-7

Burrows LJ, Basha M, Goldstein AT (2012) The effects of hormonal contraceptives on female sexuality: a review. J Sex Med 9(9):2213-2223. https://doi.org/10.1111/j.1743-6109.2012.02848.x

Castanier C, Bougault V, Teulier C et al (2021) The specificities of elite female athletes: a multidisciplinary approach. Life (Basel) 11(7):622. https://doi.org/10.3389/fphys.2023.1185343

Coelingh Bennink HJT, Zimmerman Y, Laan E et al (2017) Maintaining physiological testosterone levels by adding dehydroepan-drosterone to combined oral contraceptives: I Endocrine Effects. Contraception 96(5):322-329. https://doi.org/10.1016/j.contracept-ion.2016.06.022

De Souza MJ, Maresh CM, Maguire MS et al (1989) Menstrual status and plasma vasopressin-renin activity, and aldosterone exercise responses. J Appl Physiol 67:736-743. https://doi.org/10.1152/jappl.1989.67.2.736

Enea C, Boisseau N, Ottavy M et al (2009) Effects of menstrual cycle, oral contraception, and training on exercise-induced changes in circulating DHEA-sulphate and testosterone in young women. Eur J Appl Physiol 106(3):365-373. https://doi.org/10.1007/S00421-009-1017-6

Farrokhyar F, Tabasinejad R, Dao D et al (2015) Prevalence of vita-min D inadequacy in athletes: a systematic-review and meta-analysis. Sports Med 45(3):365-378. https://doi.org/10.1007/S40279-014-0267-6

Gillberg P, Olofsson H, Mallmin H et al (2002) Bone mineral density in femoral neck is positively correlated to circulating insulin-like growth factor (IGF)-I and IGF-binding protein (IGFBP)-3 in Swedish men. Calcif Tissue Int 70(1):22-29. https://doi.org/10.1007/s002230020048

Hackney AC, Saeidi A (2019) The thyroid axis, prolactin, and exercise in humans. Curr Opin Endocr Metab Res 9:45-50. https://doi.org/10.1016/j.coemr.2019.06.012

Harju T, Gray B, Mavroedi A et al (2022) Prevalence and novel risk factors for vitamin D insufficiency in elite athletes: systematic review and meta-analysis. Eur J Nutr 61(8):3857-3871. https://doi.org/10.1007/s00394-022-02967-z

Harmon QE, Umbach DM, Baird DD (2016) Use of estrogen-contain-ing contraception is associated with increased concentrations of 25-hydroxy vitamin D. J Clin Endocrinol Metab 101(9):3370-3377. https://doi.org/10.1210/jc.2016-1658

Harmon QE, Kissell K, Jukic AMZ et al (2020) Vitamin D and reproductive hormones across the menstrual cycle. Hum Reprod 35(2):413-423. https://doi.org/10.1007/s00394-022-02967-z

Ihalainen JK, Löfberg I, Kotkajuuri A et al (2021) Influence of menstrual cycle or hormonal contraceptive phase on energy intake and metabolic hormones-a pilot study. RS Endocrines 2(2):79-90. https://doi.org/10.3390/endocrines2020008

Jürimäe J, Vaiksaar S, Mäestu J et al (2011) Adiponectin and bone metabolism markers in female rowers: eumenorrheic and oral contraceptive users. J Endocrinol Investig 34:835-839. https://doi.org/10.3275/7415

Kanaley JA, Boileau RA, Bahr JM et al (1992) Cortisol levels during prolonged exercise: the influence of menstrual phase and menstrual status. Int J Sports Med 13(4):332-336. https://doi.org/10.1055/s-2007-1021276

Khosla S, Monroe DG (2018) Regulation of bone metabolism by sex steroids. Cold Spring Harb Perspect Med 8(1):a031211. https://doi.org/10.1101/cshperspect.a031211

Knutsson JE, Ekström L, Hirschberg AL (2023) Disposition of serum steroids in response to combined oral contraceptives and menstrual cycle phases: a double-blind, randomized, placebo-controlled study. Drug Test Anal 15(1):134-138. https://doi.org/10.1002/dta.3373

Kraus FB, Medenwald D, Ludwig-Kraus B (2020) Do extreme summers increase blood vitamin D (25-hydroxyvitamin D) levels? PLoS ONE 15(11):e0242230. https://doi.org/10.1371/journal.pone.0242230

Kuhl H, Gahn G, Romberg G et al (1985a) A randomized cross-over comparison of two low-dose oral contraceptives upon hormonal and metabolic parameters: I Effects upon Sexual Hormone Levels. Contraception 31(6):583-593. https://doi.org/10.1016/0010-7824(85)90058-7

Kuhl H, Gahn G, Romberg G et al (1985b) A randomized cross-over comparison of two low-dose oral contraceptives upon hormon- and metabolic serum parameters: II. Effects upon thyroid function, gastrin, STH, and glucose tolerance. Contraception 32(1):97-107. https://doi.org/10.1016/0010-7824(85)90119-2

Kuhnzal-YacoubFuhrmeister WGA (1992) Pharmacokinetics of lev-onorgestrel and ethinylestradiol in 9 women who received a low-dose oral contraceptive over a treatment period of 3 months and after a wash-out phase, a single oral administration of the same contraceptive formulation. Contraception 46(5):455-469. https://doi.org/10.1016/0010-7824(92)90149-n

Larsen B, Morris K, Quinn K et al (2020) Practice does not make perfect: a brief view of athletes' knowledge on the menstrual cycle and oral contraceptives. J Sci Med Sport 23:690-694. https://doi.org/10.1016/j.jsams.2020.02.003

Martin D, Sale C, Cooper SB et al (2018) Period prevalence and perceived side effects of hormonal contraceptive use and the menstrual cycle in elite athletes. Int J Sports Physiol Perform 13:926-932. https://doi.org/10.1123/ijspp.2017-0330

Martin D, Cooper SB, Tang JCY et al (2021) Bone metabolic marker concentrations across the menstrual cycle and phases of combined oral contraceptive use. Bone 145:115864. https://doi.org/10.1016/j.bone.2021.115864

Nikolaidis MG, Protosygellou MD, Petridou A et al (2003) Hematologic and biochemical profile of juvenile and adult athletes of both sexes: implications for clinical evaluation. Int J Sports Med 24(7):506-511. https://doi.org/10.1055/s-2003-42014

Oinonen KA, Mazmanian D (2002) To what extent do oral contraceptives influence mood and affect? J Affect Disord 70(3):229-240. https://doi.org/10.1016/S0165-0327(01)00356-1

Oxfeldt M, Dalgaard LB, Jørgensen AA et al (2020) Hormonal contraceptive use, menstrual dysfunctions, and self-reported side effects in elite athletes in Denmark. Int J Sports Physiol Perform 15:1377-1384. https://doi.org/10.1123/ijspp.2019-0636

Özcan Ö, den Elzen WPJ, Hillebrand JJ et al (2023) The effect of hormonal contraceptive therapy on clinical laboratory parameters: a literature review. Clin Chem Lab Med 62(1):18-40. https://doi.org/10.1515/cclm-2023-0384

Raps M, Curvers J, Helmerhorst FM et al (2014) Thyroid function, activated protein C resistance and the risk of venous thrombosis in users of hormonal contraceptives. Thromb Res 133(4):640-644. https://doi.org/10.1016/j.thromres.2013.12.041

Rechichi C, Dawson B, Goodman C (2008) Oral contraceptive phase has no effect on endurance test. Int J Sports Med 29(4):277-281. https://doi.org/10.1055/s-2007-965334
Sawin CT, Hershman JM, Boyd AE et al (1978) The relationship of changes in serum estradiol and progesterone during the menstrual cycle to the thyrotropin and prolactin responses to thyrotropin-releasing hormone. J Clin Endocrinol Metab 47:1296–1302. https://doi.org/10.1210/jcem-47-6-1296

Sist M, Zou L, Galloway SDR et al (2023) Effects of vitamin D supplementation on maximal strength and power in athletes: a systematic review and meta-analysis of randomized controlled trials. Front Nutr 10:1163313. https://doi.org/10.3389/fnut.2023.1163313

Vibarel-Rebot N, Rieth N, Lasne F et al (2015) Oral contraceptive use and saliva diurnal pattern of metabolic steroid hormones in young healthy women. Contraception 91(3):245–247. https://doi.org/10.1016/j.contraception.2014.11.010

Weeke J, Hansen AP (1975) Serum tsh and serum t3 levels during normal menstrual cycles and during cycles on oral contraceptives. Acta Endocrinol (Copenh) 79(3):431–438. https://doi.org/10.1530/acta.0.0790431

Wiegratz I, Kutschera E, Lee JH et al (2003) Effect of four different oral contraceptives on various sex hormones and serum-binding globulins. Contraception 67(1):25–32. https://doi.org/10.1016/j.contraception.2004.03.004

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