Associations of Sex Hormones and Hormonal Status With Arterial Stiffness in a Female Sample From Reproductive Years to Menopause

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Associations of Sex Hormones and Hormonal Status With Arterial Stiffness in a Female Sample From Reproductive Years to Menopause © 2021 the Authors Published version Laakkonen, Eija K.; Karppinen, Jari E.; Lehti, Satu; Lee, Earric; Pesonen, Emilia; Juppi, Hanna-Kaarina; Kujala, Urho M.; Haapala, Eero A.; Aukee, Pauliina; Laukkanen, Jari A.; Ihalainen, Johanna K. Laakkonen, E. K., Karppinen, J. E., Lehti, S., Lee, E., Pesonen, E., Juppi, H.-K., Kujala, U. M., Haapala, E. A., Aukee, P., Laukkanen, J. A., & Ihalainen, J. K. (2021). Associations of Sex Hormones and Hormonal Status With Arterial Stiffness in a Female Sample From Reproductive Years to Menopause. Frontiers in Endocrinology, 12, Article 765916. https://doi.org/10.3389/fendo.2021.765916 2021 Associations of Sex Hormones and Hormonal Status With Arterial Stiffness in a Female Sample From Reproductive Years to Menopause Eija K. Laakkonen 1,2 *, Jari E. Karppinen 2 , Satu Lehti 1,2 , Earric Lee 2 , Emilia Pesonen 2 , Hanna-Kaarina Juppi 1,2 , Urho M. Kujala 2 , Eero A. Haapala 2,3 , Pauliina Aukee 4 , Jari A. Laukkanen 2,5 and Johanna K. Ihalainen 2 1 Gerontology Research Center, University of Jyväskylä, Jyväskylä, Finland, 2 Faculty of Sport and Health Sciences, University of Jyväskylä, Jyväskylä, Finland, 3 Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland, 4 Department Obstetrics and Gynecology and Pelvic Floor Research and Therapy Unit, Central Finland Health Care District, Jyväskylä, Finland, 5 Institute of Clinical Medicine, School of Medicine, University of Eastern Finland, Kuopio, Finland Objective: Loss of sex hormones has been suggested to underlie menopauseassociated increment in cardiovascular risk. We investigated associations of sex hormones with arterial stiffness in 19–58-years-old women. We also studied associations of specific hormonal stages, including natural menstrual cycle, cycle with combined oral contraceptives (COC) and menopausal status with or without hormone therapy (HT), with arterial stiffness. Methods: This study includes repeated measurements of 65 healthy women representing reproductive (n=16 natural, n=10 COC-users) and menopause (n=5 perimenopausal, n=26 postmenopausal, n=8 HT-users) stages. Arterial stiffness outcomes were aortic pulse wave velocity (PWVao) and augmentation index (AIx%) assessed using Arteriograph-device. Generalized estimating equation models were constructed to investigate associations of each hormone (wide age-range models) or hormonal stage (age-group focused models) with arterial stiffness. PWVao models with cross-sectional approach, were adjusted for age, relative fitness, fat mass and mean arterial pressure, while models with longitudinal approach were adjusted for mean arterial pressure. AIx% models used the same approach for adjustments and were also adjusted for heart rate. Results: Negative and positive associations with arterial stiffness variables were observed for estradiol and follicle-stimulating hormone, respectively, until adjustment for confounding effect of age. In naturally menstruating women, AIx% was higher at ovulation (B=3.63, p<0.001) compared to the early follicular phase. In COC-users, PWVao was lower during active (B=-0.33 - -0.57, p<0.05) than inactive pills. In menopausal women, HT-users had higher PWVao (B=1.43, p=0.03) than postmenopausal non-HT-users. Frontiers in Endocrinology | www.frontiersin.org November 2021 | Volume 12 | Article 7659161 Edited by: James Harper, Sam Houston State University, United States Reviewed by: Peter Mikael Nilsson, Lund University, Sweden Maureen Jane MacDonald, McMaster University, Canada *Correspondence: Eija K. Laakkonen eija.k.laakkonen@jyu.fi Specialty section: This article was submitted to Endocrinology of Aging, a section of the journal Frontiers in Endocrinology Received: 31 August 2021 Accepted: 12 November 2021 Published: 30 November 2021 Citation: Laakkonen EK, Karppinen JE, Lehti S, Lee E, Pesonen E, Juppi H-K, Kujala UM, Haapala EA, Aukee P, Laukkanen JA and Ihalainen JK (2021) Associations of Sex Hormones and Hormonal Status With Arterial Stiffness in a Female Sample From Reproductive Years to Menopause. Front. Endocrinol. 12:765916. doi: 10.3389/fendo.2021.765916 ORIGINAL RESEARCH published: 30 November 2021 doi: 10.3389/fendo.2021.765916 Conclusions: When using wide age-range assessments covering reproductive to menopausal lifespan it is difficult to differentiate ageand hormone-mediated associations, because age-mediated influence on arterial stiffness seemed to overrule potential hormone-mediated influences. However, hormonal status associated differentially with arterial stiffness in age-group focused analyses. Thus, the role of sex hormones cannot be excluded. Further research is warranted to resolve potential hormone-mediated mechanisms affecting arterial elasticity. Keywords: vascular aging, vascular stiffness, pulse wave analysis, reproductive hormones, menstruation, hormonal contraception, hormone replacement therapy, women’s health INTRODUCTION Cardiovascular (CV) disease is the leading cause of death in postmenopausal women (1). Lack of sex steroid estradiol (E2), either due to natural or surgical menopause, has been found to associate with an increased risk of CV diseases (2–4). Potential mechanisms include both indirect and direct hormone-mediated effects affecting endothelial and smooth muscle cell signaling as well as the extracellular matrix properties of the vasculature (5, 6). Increased arterial stiffness is a clinically important early sign of vascular aging as it predicts future CV events and associates with CV and all-cause mortality (7–9). Increased arterial stiffness refers to a reduced ability of arteries to respond to the pulse wave energy, which can be detected as changes in aortic pulse wave velocity (PWV) and augmentation index (AIx). PWV describes the speed of a pulse wave in the vasculature, while AIx expresses the resistance of the peripheral vascular sites as a reflection of a pulse wave from the peripheral arterial tree (10). The higher the PWV and the AIx are, the stiffer are the arteries. Although PWV increases with age in both men and women, doubling between ages 18 and 80, it is considered to be an indicator of aging-related change in arterial compliance rather than concomitant atherosclerosis (11). Notably, PWV has been found to be lower in women than in aged-matched men throughout reproductive years since puberty until early postmenopause (12), indicating that PWV may be a valuable marker for the arterial stiffening affected by the availability of the sex hormones. AIx, on the other hand, has been shown to be higher in women compared to men throughout adulthood and to adopt a steeper increasing curve at middle-age in both sexes (13). During a woman’s monthly hormonal cycle, blood concentrations of E2 and other hormones fluctuate considerably according to the menstrual cycle phase. In normal menstrual cycle, E2 levels are the lowest during menstrual bleeding at the early follicular phase and rise thereafter until ovulation. The second E2 peak occurs at the mid luteal phase. A surge of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) occurs just before ovulation, while progesterone (P4) levels rise after ovulation and are at their highest at the mid luteal phase. In contrast, women with oral contraception (OC) have reduced endogenous hormone production, leading to substantially or completely attenuated peaking of E2, P4, FSH and LH during the menstrual cycle (14). In a study by Hayashi and co-authors, carotid arterial compliance was shown to follow menstrual cycle; compliance increased from early follicular to the ovulatory phase until a sharp decline in the luteal phase (15). However, the study did not find menstrual cycle to associate with peripheral arterial stiffness. In two studies, AIx was shown to follow a similar pattern reaching its peak value during days corresponding to ovulation and dropping thereafter being lowest at the luteal phase (16,17). The studies did not find AIx to correlate with E2 or P4 (no other hormones were investigated) across menstrual cycle, but the latter study found E2 to correlate negatively with AIx at luteal phase and P4 to correlate negatively with AIx at ovulatory and luteal phase. Other literature reports indicate that the observed changes in AIx or PWV over the natural menstrual cycle are subtle or non-existent (17–21). Only a few studies have investigated differences in arterial stiffness between women with natural menstrual cycle and women using OC and the results have remained inconclusive. One study found PWV to be slightly higher among OC-users than non-users (22), while others found no group differences (23–26). The recent study by Enea et al. (26) compared OC-users at active pill phase to the non-users at bleeding phase and found no difference in PVW but lower AIx in OC-users compared to non-users. This contrasts with findings by Seeland et al., who reported AIx to be higher in OC-users than non-users, accompanied by a negative association with E2 level (25). PVW across OC cycle phases has only been studied by Priest et al. (23) and Yu et al. (24). Neither of the studies found differences in PVW between different pill phases. After the reproductive stage with cyclic monthly fluctuations in sex hormones, their production in the ovaries gradually declines through perimenopause to postmenopause. This leads to a permanent reduction of circulating E2 and P4 concomitant to heightened FSH levels, which is why menopause is considered to end the protective effects of the hormones towards vasculature (27). Hormone replacement therapy (HT), if started in early menopause, has been suggested to be beneficial for CV health by decreasing both carotid artery intima-media thickness and blood pressure (28), but evidence regarding PWV and AIx have remained inconclusive. The pioneering finding in the field showed PWV to be higher in postmenopausal non-HT-users compared to premenopausal women, and lower in HT-users compared to non-using postmenopausal women (29). However, the later studies have shown mixed results regarding associations Laakkonen et al. Sex Hormones and Arterial Stiffness Frontiers in Endocrinology | www.frontiersin.org November 2021 | Volume 12 | Article 7659162 of menopausal stage or HT-use with differences in PWV or AIx (30–33). Furthermore, to our knowledge, the association of serum concentrations of sex hormones with both PWV and AIx has not been investigated in menopausal women. A limited number of studies have investigated arterial stiffness combining both reproductive and menopausal women having either natural hormonal status or taking hormonal preparations. Including hormone assessments into the vascular study models is even rarer. The aim of this study was to investigate the associations of sex hormones with arterial stiffness in a female sample spanning from reproductive years to postmenopause. Furthermore, we investigated the associations of the menstrual cycle, contraception pill cycle and menopausal status with PWV and AIx. We hypothesized that sex hormones influence arterial stiffness, which in our study could be seen as significant negative associations for E2 and P4 values and significant positive associations for FSH and testosterone values with arterial stiffness measurements or between different hormonal states and arterial stiffness. METHODS Participants and Study Design This study uses data and samples from two studies: the Endogenous and exogenous hormones and performance in women (MEndEx) study and the Estrogen, MicroRNAs and the Risk of Metabolic Dysfunction (EsmiRs) study. MEndEx study protocol has been published in (34). Briefly, healthy women aged 18–40 years were recruited by advertisements in the local newspaper and via social media. Each prospective participant was asked to complete a health questionnaire and the Low energy availability in females -questionnaire (LEAF-Q) (35) before the study onset. Inclusion criteria required a participant to be recreationally physically active with a body mass index (BMI) of 18–25 kg/m 2 and LEAF-Q score < 8. Participants were excluded if they were pregnant or lactating, if they had conditions affecting ovarian function, amenorrhea, endocrine disorders, or chronic diseases, or if they were taking medication that may affect exercise responses. Women using OCs were included. A total of 33 women enrolled for the MEndEx study, of which 28 participated in the measurements. Participated women of reproductive age (REPRO) formed two sub-groups, natural without any type of hormonal contraception (REPRO-NAT group) and combined OC users (REPRO-COC group). Two COC users were excluded from the current study due to lack of arterial stiffness measurement at cycle days of the low estradiol state, i.e., days with withdrawal bleeding during the placebo pill. Finally, 26 young women (16 REPRO-NAT and 10 REPRO-COC) were included in this study. Four experimental testing sessions were completed by each participant over an individual menstrual or contraceptive cycle. The phase of the menstrualorpillcycleinwhichfirst experimental testing commenced was randomized. A priori familiarization session was scheduled for each participant during which they filled questionnaires and were familiarized to the protocols of the experimental testing sessions. Blood samples were taken, and body composition and vascular measurements were performed in the morning measurements, whereas the cardiorespiratory fitness was assessed later on that same day. EsmiRs study performed a four-year follow up for the Estrogenic Regulation of Muscle Apoptosis (ERMA) study participants (36). Of the ERMA women, 811 participants who had provided consent to be contacted with new study invitations were approached by postal participation invitation. The EsmiRs study proceeded in three waves; wave-1: postal questionnaire survey, wave-2: laboratory visit repeating ERMA baseline sampling and physiological measurements and wave-3: laboratory visits for metabolic measurements. EsmiRs wave-3 concerned only those women who had participated in wave-2 and did not fulfill additional exclusion criteria. Included participants needed to be either pre-, perior postmenopausal, having BMI between 18.5 and 30 kg/m 2 , and being metabolically healthy. E2and/or P4-containing medication was allowed only for women as menopausal HT. Exclusion criteria were regular use of medication for metabolic disorders (e.g., diabetes, thyroid dysfunction or dyslipidemia), alphaor beta-blockers, regular use of sedatives or analgesics, and current regular smoking. Occasional smoking was allowed if the participant did not smoke within two days before laboratory visits and not more than a few cigarettes in a month. Wave-3 contained two laboratory visits. The first visit included indirect calorimetry at rest, medical examination and oral glucose tolerance test, and the second visit contained indirect calorimetry during bicycle ergometer test. Vascular measurements were performed at the beginning of both laboratory visits. The flow of the EsmiRs women to the current study was briefly as follows. Of the 811 invited women, 522 responded (response rate 64.4%), but 28 were not willing to participate. Wave-1 questionnaires were returned by 494 women. Of them, 304 participated in wave-2 laboratory measurements and 42 in wave-3 laboratory measurements. Arterial stiffness measurements were not done for two participants due to the unavailability of the measuring device. One participant turned out to be voluntarily on strict low energyand carbohydrate diet resulting in ketosis, hence she was excluded from the study. Thus, data was available for the current study from 39 menopausal women (MENO group). Of them, five were perimenopausal without external hormonal preparations (MENO-PERI group), 26 were naturally postmenopausal without the use of HT (MENO-POST group) and eight were postmenopausal women who were currently using HT (MENOHT group). The MEndEx and EsmiRs studies adhered to the Declaration of Helsinki. The MEndEx study was approved by the Ethical Committee of the University of Jyväskylä (22 October 2018) and the EsmiRs study by the Ethics committee of the Central Finland Health Care District (9U/2018). All participants received detailed information about the study design, measurements, and procedures and provided signed informed consent before the onset of the study measurements. All laboratory measurements Laakkonen et al. Sex Hormones and Arterial Stiffness Frontiers in Endocrinology | www.frontiersin.org November 2021 | Volume 12 | Article 7659163 were performed at the Health and Sports Laboratory of the University of Jyväskylä, Finland, during 2019 and 2020. Figure 1 presents a diagram illustrating the timepoints in which data was collected in the MEndEx and EsmiRs. In both studies, the blood samples were drawn on the same morning as the arterial stiffness was assessed. Reproductive and Menopausal Stage of the Participants The REPRO-NAT group included reproductive-age women who did not use any sex hormone containing medications or contraceptives, while the REPRO-COC group included reproductive-age women who were currently using combined estrogenand progestogen-based OCs. The used products were either third generation COC-pills with ethinyl estradiol coupled with cyproterone acetate (n=2, brand name Vreya) or fourth generation COC-pills with ethinyl estradiol coupled with drospirenone (n=7, brand names Yaz, Yasmin, Stefaminelle and Tasminetta) or estradiol hemihydrate coupled with nomegestrol acetate (n=1, brand name Zoely) as effective hormone components. Active pills of these specimens contain similar amounts of estrogenic (0.02–0.035 mg ethinyl estradiol) and progestogenic (2–3 mg) compounds except that Zoely has 1.5 mg of a bioidentical E2 called estradiol hemihydrate. Taken together, COC-pills used by the MEndEx study participants provide a relatively stable hormonal condition for 21–24 active pill days followed by 4–7 hormone-free (inactive pill) days, although endogenous hormonal profiles may vary between individuals even when OCs employ the same mechanism of action (37). Menopausal status of the MENO group women was defined according to the Stages of Reproductive Aging Workshop + 10 guidelines (38) using menstrual cycle and serum FSH data. Women who self-reported not have had menstrual bleeding for over 12 months and had at the EsmiRs wave-2 a measured FSH level over 30 IU/L were considered postmenopausal, while women who reported still having occasional menstrual bleedings were considered perimenopausal. Of the postmenopausal women, eight were current HT-users. All MENO-HT group women used bioidentical E2 combined with a synthetic progestogen. Of them, six used an oral product with 1 mg estradiol hemihydrate and 5 mg dydrogesterone (n=5, brand name Femoston-conti) or with 1 mg estradiol valerate and 0.5 mg norethisterone acetate (n=1, brand name Cliovelle), while two used transdermal patches containing either 3.2 mg estradiol hemihydrate and 11.2 mg norethisterone acetate (n = 1, brand name Evorel Conti) or 0.585 mg estradiol hemihydrate with 5 mg oral dydrogesterone (n = 1, brand names Estradot and Terolut). Blood Collection and Biochemical Analysis Fasting venous blood samples were drawn from the antecubital vein at a supine position between 7:00 and 10:00 a.m. From A B C FIGURE 1 | Data collection timepoints in the MEndEx (A, B) and EsmiRs (C) studies. Laakkonen et al. Sex Hormones and Arterial Stiffness Frontiers in Endocrinology | www.frontiersin.org November 2021 | Volume 12 | Article 7659164 REPRO-NAT women, the samples were drawn at four different data collection weeks representing different menstrual cycle phases. Counting from the onset of menstrual bleeding, samples were drawn on days 2–4 (bleeding), on days 7–11 (follicular phase), on days 11–16 (ovulation), and on days 18– 25 (luteal phase) of the menstrual cycle. Ovulation was identified using a combination of counting the days from the commencement of bleeding and daily urine tests starting mid follicular phase (from day 8–9 onwards) to identify the LH surge. Urine was tested by using an at-home ovulation test (Dipro, LH Ovulation Strip, Aidian Oy, Finland). Ovulation occurs within 1–2 days of positive urine test, i.e., LH surge. Thus, ovulation phase laboratory visit was scheduled for 1–2 days after obtaining positive test result and luteal phase laboratory visit was scheduled approximately a week after ovulation (mean 6.3 +/- 0.7 days duration between visits). Women in the REPRO-COC group were also tested at four different timepoints, which are throughout the text presented in the order corresponding to the natural menstrual cycle, i.e., withdrawal bleeding is presented first although it actually occurs at the end of pill cycle after few days of the onset of inactive pill phase. Thus, sampling and measurements which took place at pill days 22–28 were referred as bleeding, at pill day 8 as active pill 1, at pill day 15 as active pill 2 and at pill days 21–24 as inactive pill. Both bleeding and inactive measurement points took place during inactive pill phase. The overlap on days between these phases is due to individual differences on how rapidly withdrawal bleeding starts after changing to the inactive pills. From all MENO women, the blood samples were drawn at wave-2 laboratory visit and at both wave-3 laboratory visits. Wave-2 FSH measurements were used only for the initial group assignments. E2, FSH, P4 and testosterone measurements from both wave-3 sampling points were used in the statistical analyses as week-1 and week-2 timepoints. Lipid and lipoprotein measurements were done only using wave-2 blood samples for the MENO groups while they were available from all sampling weeks for the REPRO-NAT and REPRO-COC groups. For serum separation, whole blood was left to clot for 30 min at room temperature and centrifuged at 2,200 ×g before aliquoting and storing the sera at −80°C until analysis. Serum LH (only for REPRO groups), E2, P4, FSH and testosterone concentrations were determined using IMMULITE 2000 XPi (Siemens Healthcare Diagnostics, UK) and serum total cholesterol, low-density lipoprotein (LDL)-cholesterol, highdensity lipoprotein (HDL)-cholesterol and triglycerides were measured using KONELAB 20 XTi or Indico analyzer (Thermo Fischer Scientific, Finland) according to manufacturer’s instructions. Assessment of Aortic Pulse Wave Velocity and Augmentation Index Participants were instructed to avoid moderate-to-vigorous physical activity 36–48 h before vascular measurements. All measurements were done between 7:00 and 10:00 a.m. Participants rested in a supine position for ten minutes before measurements. Oscillometric pulse wave analysis was then performed from the right upper arm using Arteriograph-device (Arteriograph; TensioMed Ltd., Budapest, Hungary) in the supine position (39).Thedeviceprovidesanautomatic assessment of resting heart rate (HR), systolic (SBP) and diastolic blood pressure (DBP), pulse pressure, aortic pulse wave velocity (PWVao), and augmentation index (Aix%). First, the device measures actual systolic blood pressure and subsequently inflates the cuff 35 mmHg above measured SBP, and then measures the fluctuations in the brachial artery. The signals are passed on to a tablet computer, recorded, and analysed as pulse waves. Mean arterial pressure (MAP) was computed by the formula MAP = DBP + 1/3(SBP –DBP). PWVao (m/s) was calculated from the time difference between the first systolic wave (direct) and the second systolic wave (reflected) and was related to the distance from the jugulum to the pubic symphysis. AIx% was computed from the pressure difference between the first (P1) and second (P2) wave in relation to the pulse pressure by the formula AIx% = [(P2-P1)/pulse pressure] x 100. We have previously reported a good short-term reproducibility for PWVao (intraclass correlation coefficient=0.90, coefficient of variation=3.7%) and moderate reproducibility for AIx% (intraclass correlation coefficient=0.88, coefficient of variation=29.1%) in adolescents (40). Within the EsmiRs study sample, using the two repeated measurements that were obtained during data collection wave-3 with a median duration between the measurements being two weeks, moderate reproducibility for PWVao (intraclass correlation coefficient=0.76, coefficient of variation=8.4%) and for AIx% (intraclass correlation coefficient=0.68, coefficient of variation=12.3%) were obtained. Furthermore, Arteriograph-derived PWVao has an acceptable agreement with invasively measured PWVao in adults (41,42) and with non-invasive tonometric SphymoCor-device (43). Anthropometrics and Body Composition Body weight was measured with a beam scale and height by a stadiometer, with the participant wearing only undergarments. BMI was calculated as weight (kg)/height squared (m 2 ). Body composition was assessed with a multifrequency bioelectrical impedance analyzer (InBody™720; Biospace, Seoul, Korea) at the beginning of each laboratory visit in both studies. The InBody-device provides estimates of fat mass and fat-free mass (FFM) of the body and calculates body fat percentage (fat mass (kg)/body weight (kg) x100). It is known that bioimpedancebased methods tend to overestimate FFM, and underestimate fat mass and fat percentage compared to dual-energy x-ray absorptiometry (DXA), which is commonly considered as the golden standard method. However, the use of DXA contains a health risk because each DXA-scan exposes participant to a small amount of radiation. Therefore, its use needs to be carefully justified for repeated measurements. Furthermore, a recent study showed multifrequency bioimpedance to provide similar change estimates of FFM and fat mass compared to the DXA (44). Therefore, we considered InBody ™720 to provide adequate enough estimates of body composition for the purposes of the current study. Laakkonen et al. Sex Hormones and Arterial Stiffness Frontiers in Endocrinology | www.frontiersin.org November 2021 | Volume 12 | Article 7659165 Cardiorespiratory Fitness In the MEndEx study, peak oxygen uptake (V O 2PEAK )was determined using a standard treadmill test with an incremental protocol at the same time of day +/- 2h to avoid the possible confounding effects of circadian rhythms. An exercise test started with a 5-min warm-up performed at a self-selected pace. The treadmill incline remained constant at 0.5° throughout the entire test. Treadmill velocity was 6 km·h −1 for the first 3-min stage of the test and was increased by 1 km·h −1 every third minute until volitional exhaustion. HR was recorded continuously using a HR monitor (Polar 800, Polar Electro, Kempele, Finland). The V O 2 was measured breath-by-breath using a portable gas analyzer (Oxycon Mobile®, Jaeger, Hoechberg, Germany), which was calibrated before each test according to the manufacturer’s instructions. V O 2PEAK was defined as the highest average 30 s V O 2 value. In the EsmiRs study, V O 2PEAK was determined during an incremental bicycle ergometer test, which consisted of submaximal and maximal phases. All tests were performed between 7:00 and 9:00 a.m. The submaximal phase started at 20 W, and the workload increased for 20 W every four minutes until respiratory exchange ratio (RER) ~1.0 was reached. After that, participants continued directly to the maximal phase. The maximal phase started at 100 W, and the workload was increased 1 W/3 s (20 W/min) until voluntary exhaustion. Participants were instructed to cycle the ergometer (Ergoselect 200, Ergoline GmbH,Germany)at70±5rpm.HRandworkloadwere recorded continuously with a 12-lead ECG -system (CardioSoft v.5.02, GE Medical System Corina, GE Medical System Inc., USA). Gas exchange was measured with Vmax Encore 92 metabolic cart (Sensormedics, Yorba Linda, CA, USA), which was calibrated before each test according to the manufacturer’s instructions. Gas exchange data were averaged for every 10 s, and V O 2PEAK was determined as the highest rolling 30 s of V O 2. We used cutoffs RER MAX ≥1.10 or age-predicted maximal heart rate (210 - age) ≥99% to assess whether exercise test was maximal. One participant did not quite meet the set limits, but her data was still considered reliable. Respiratory gas exchange data was unreliable in two participants due to equipment failure or difficulties wearing the mask. For these two participants, V O 2PEAK was determined from W MAX using the equation by Storer et al. (45). For all study participants, the relative V O 2PEAK was computed as V O 2PEAK relative to FFM (V O 2PEAK /FMM) obtained by bioimpedance measurements and expressed as ml/kg FFM/min. Statistics Means, standard deviations and frequencies were used for descriptive purposes. When available, data points which are commonly considered to represent the lowest E2 phase of the menstrual or pill cycle, i.e., menstrual bleeding at early follicular stage and withdrawal bleeding during placebo pill, were used to describe REPRO-NAT and REPRO-COC groups and as references in generalized estimating equation (GEE) models. Correspondingly, for the MENO group, MENO-POST group was chosen as a reference in the GEE models. In descriptive tables, the MENO group week-1 data point was used except for one participant whose week-2 data was used due to missing data at week-1. Group comparisons between REPRO and MENO groups and between REPRO-NAT and REPRO-COC groups were performed using Student’s t-test or, if Shapiro-Wilk test indicated variable not to follow a normal distribution, MannWhitney U test. Group comparisons between MENO-PERI, -POST and -HT groups were correspondingly done using ANOVA or Kruskall-Wallis test. Linear GEE models with an unstructured working correlation matrix were constructed for the complete dataset to investigate potential associations of hormones (wide age-range models) with PWVao and AIx%. Age-group focused GEE models were also constructed for each group to investigate associations of menstrual cycle phases (REPRO-NAT group, four datapoints), COC-pill cycle phases (REPRO-COC group, four datapoints) and menopausal stages (two datapoints for each MENO group) with PWVao and AIx%. The models with cross-sectional nature (wide age-range models and of the age-group focused models, the MENO group model) were adjusted for age, relative cardiorespiratory fitness level (V O 2PEAK /FMM), body fat mass, MAP and AIx% models also for heart rate to avoid differences in these metrics to influence the interpretation of the results. The models with longitudinal nature (of the age-group focused models, REPRO-NAT and REPROCOC group models) were adjusted only for MAP, and in the case of AIx%, also for heart rate. Regression coefficients (B), standard errors (SE), P-values and 95% confidence intervals (95% CI) are reported for each model, and both unadjusted and adjusted models are presented. The benefitofGEEmodelover repeated-measures ANOVA is that participants are not lost due to incomplete observations (e.g., from participants who have information available from only some of the datapoints) (46). In the REPRO-NAT group, there was missing data for PWVao and AIx% for one woman at follicular phase, three women at ovulation phase and two women at luteal phase, for V O 2PEAK /FMM for two women at bleeding phase, two women at follicular phase, four women at ovulation phase and for two women at luteal phase and for E 2 , FSH, P4 and testosterone for two women at ovulation phase. In the REPRO-COC group, there was missing data for PWVao and AIx% for one woman at inactive pill phase, and for V O 2PEAK /FMM for two women at bleeding phase and one woman at active pill 1 phase. In the MENO group, there was missing data for PWVao and AIx% for one woman at week-1 and two women at week-2. P≤0.05 was considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics 24.0. RESULTS Descriptive Characteristics and Hormonal Status of the Study Participants Table 1 presents descriptive characteristics of the study participants. Participated women were 26 adult 19 to 37 years Laakkonen et al. Sex Hormones and Arterial Stiffness Frontiers in Endocrinology | www.frontiersin.org November 2021 | Volume 12 | Article 7659166 old women representing the reproductive-age period and 39 adult 52 to 58 years old women representing the menopause-age period. All women in the REPRO group and 74% of the MENO group women were never smokers. Of the MENO women, five were perimenopausal and 34 were postmenopausal. Of the REPRO group, 62% and of the MENO group, 80% had natural status without the current use of external hormone preparations. All participants combined, ten were current COC-users, nine had previously used progestogen-releasing coils while eight were current and one was a former HT-user. Figure 2 presents the variation of hormone levels between different menstrual and COC-pill cycle phases and menopausal groups. The typical menstrual cycle dependent variation is evident for E2, P4, LH, and FSH, while testosterone did not change at group mean level among the naturally menstruating women (REPRO-NAT group, Figure 2A). In the REPRO-COC group, there were only modest differences in LH and FSH levels across the pill cycle phases with active and inactive pills (Figure 2B). For the menopausal women, statistics were performed as means of the two data collection weeks and compared between MENO-PERI, -POST and -HT groups (Figure 2C). As expected, E2 levels were lowest and FSH levels were highest among MENO-POST women. Considerable variation was observed in P4 among MENO-PERI women indicating that participants still had an active menstrual cycle. Indicators of Cardiovascular Health of the Study Participants Most of the measured parameters indicated REPRO group to have better CV health than menopausal women (Table 2). REPRO group had higher absolute and relative V O 2PEAK than MENO group. They also had healthier body composition as indicated by lower BMI and fat mass and higher FFM. Total cholesterol and LDL-cholesterol levels were lower in the REPRO than in the MENO group. REPRO group also had lower SBP, DBP, PWVao and AIx% values indicating less rigid arteries compared to the MENO group. For the sub-group mean values and comparisons between REPRO-NAT to REPRO-COC and between MENO-PERI, -POST and -HT groups see Table S1.Of the women of reproductive age, REPRO-COC women had an average higher triglyceride, BMI, SBP, DBP and PWVao values than REPRO-NAT women. No within-group differences among menopausal women were observed. The variations in PWVao and AIx% within the menstrual cycle and COC-pill cycle phases are presented in Figure 3. Association of Sex Hormones With Arterial Stiffness Age is known to associate with arterial stiffness, which was also the case in our sample; age association with PWVao was B= 0.09, 95% CI 0.07 to 0.11 (p < 0.001) and with AIx% B= 0.95, 95% CI 0.84 to TABLE 1 | Descriptive characteristics of the study participants. REPRO group MENO group n=26 n=39 Age (years) 24.9 ± 4.2 55.0 ± 1.7 Smoking, n(%) Never 26 (100) 29 (74.4) Former 8 (20.5) Occasional 2 (5.1) Current reproductive status, n(%) Reproductive, regularly menstruating, natural 16 (61.5) – Reproductive, regularly menstruating, COC-user 10 (38.5) – Perimenopausal, irregular cycle –5 (12.8) Postmenopausal, none menstruating, natural –26 (66.7) Postmenopausal, none menstruating, HT-user –8 (20.5) Use of hormonal contraceptives, n(%) Never 16 (61.5) 30 (77.9) Former per type IUD, hormonal coil –9 (23.1) Current per type Oral combined 10 (38.5) – Use of menopausal hormonal therapy, n(%) Never 26 (100) 31 (77.5) Former 1 (2.5) Current per type Oral combined 6 (15.0) Transdermal combined 2 (5.0) Estradiol (pmol/l) 249.8 ± 147.8 181.8 ± 204.1 Follicle-stimulating hormone (IU/l) 4.9 ± 2.5 71.4 ± 36.0 Progesterone (nmol/) 1.6 ± 1.4 1.1 ± 4.5 Luteinizing hormone (IU/l) 4.8 ± 3.4 – Testosterone (nmol/l) 0.8 ± 0.6 0.5 ± 0.3 Values are mean ± standard deviation unless otherwise stated. COC, combined oral contraception; HT, hormone therapy; IUD, intrauterine device. Laakkonen et al. Sex Hormones and Arterial Stiffness Frontiers in Endocrinology | www.frontiersin.org November 2021 | Volume 12 | Article 7659167 1.06 (p < 0.001). The associations of the measured hormones with PWVao and AIx% were investigated with GEE-models presented in Tables 3,4, respectively. Associations were first inspected using univariable models, which were then adjusted for age (multivariable model 1) and finally for age and relative fitness level, body fat mass, MAP, and for AIx% also for heart rate (multivariable model 2). E2 was not associated with AIx% in any of the models but it was negatively associated with PWVao in the univariable model (B=-0.55, p=0.05). However, adjustment for age attenuated the association between E2 and PWVao. Statistically significant association between age and PWVao remained even after further adjustments (multivariable model 2). FSH was positively associated with PWVao and AIx% in unadjusted models (B=0.04, p=0.038 and B=0.26, p<0.001, respectively). Further adjustment for age attenuated these associations. Age remained to be significantly associated with AIx% but not with A B C FIGURE 2 | Hormone levels across data collection time points and study groups. Hormone concentrations during the menstrual cycle of the natural reproductive women (A), during pill cycle of the combined oral contraceptive users (B) and among menopausal women (C). In all figures, bold lines in graphs represent means with standard deviations. For menopausal women, the means were calculated as average concentrations of the two measurement points (the first measurement point is presented with lighter and the second is with darker colors). In C, progesterone was log-transformed for better visualization purposes only. P for trend is provided by Wald Chi-Squared Test of the generalized estimating equation models. FSH, follicle-stimulating hormone. Laakkonen et al. Sex Hormones and Arterial Stiffness Frontiers in Endocrinology | www.frontiersin.org November 2021 | Volume 12 | Article 7659168 ACKNOWLEDGMENTS Statistician Markku Kauppinen is thanked for the statistical consultancy and the personnel of the Laboratories of the Faculty of Sport and Health Sciences, University of Jyväskylä for their work during data collection and measurements. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2021. 765916/full#supplementary-material REFERENCES 1. De Kleijn MJJ, van der Schouw YT, Verbeek ALM, Peeters PHM, Banga JD, van der Graaf Y. Endogenous Estrogen Exposure and Cardiovascular Mortality Risk in Postmenopausal Women. Am J Epidemiol (2002) 155 (4):339–45. doi: 10.1093/aje/155.4.339 2. Rosano GMC, Vitale C, Marazzi G, Volterrani M. Menopause and Cardiovascular Disease: The Evidence. Climacteric (2007) 10(Suppl 1):19– 24. doi: 10.1080/13697130601114917 3. Lobo RA. Surgical Menopause and Cardiovascular Risks. Menopause (2007) 14(3):562–6. doi: 10.1097/gme.0b013e318038d333 4. Honigberg MC, Zekavat SM, Aragam K, Finneran P, Klarin D, Bhatt DL, et al. Association of Premature Natural and Surgical Menopause With Incident Cardiovascular Disease. JAMA (2019) 322(24):2411–21. doi: 10.1001/ jama.2019.19191 5. Akishita M, Yu J. Hormonal Effects on Blood Vessels. Hypertens Res (2012) 35:363–9. doi: 10.1038/hr.2012.4 6. Dupont JJ, Kenney RM, Patel AR, Jaffe IZ. Sex Differences in Mechanisms of Arterial Stiffness. Br J Pharmacol (2019) 176:4208–25. doi: 10.1111/bph.14624 7. Vlachopoulos C, Aznaouridis K, Stefanadis C. Prediction of Cardiovascular Events and All-Cause Mortality With Arterial Stiffness. JACC (2010) 55 (13):1318–27. doi: 10.1016/j.jacc.2009.10.061 8. van Popele NM, Grobbee DE, Bots ML, Asmar R, Topouchian J, Reneman RS, et al. Association Between Arterial Stiffness and Atherosclerosis: The Rotterdam Study. Stroke (2001) 32(2):454–60. doi: 10.1161/01.STR.32.2.454 9. London GM, Marchais SJ, Guerin AP, Pannier B. Arterial Stiffness: Pathophysiology and Clinical Impact. Clin Exp Hypertens (2004) 26(7– 8):689–99. doi: 10.1081/CEH-200031982 10. Obara S, Hayashi S, Hazama A, Murakawa M, Katsuda S. Correlation Between Augmentation Index and Pulse Wave Velocity in Rabbits. J Hypertens (2009) 27:332–40. doi: 10.1097/HJH.0b013e32831ac951 11. Avolio AP, Chen S-G, Wang R-P, Zhang C-L, Li M-L, O’Rourke MF. Effects of Aging on Changing Arterial Compliance and Left Ventricular Load in a Northern Chinese Urban Community. Circulation (1983) 68:50–8. doi: 10.1161/01.CIR.68.1.50 12. Tomiyama H, Yamashina A, Arai T, Hirose K, Koji Y, Chikamori T, et al. Influences of Age and Gender on Results of Noninvasive Brachial-Ankle Pulse Wave Velocity Measurement –A Survey of 12 517 Subjects. Atherosclerosis (2003) 166:303–9. doi: 10.1016/S0021-9150(02)00332-5 13. Paiva AMG, Mota-Gomes MA, Brandão AA, Silveira FS, Silveira MS, Okawa RTP, et al. Reference Values of Office Central Blood Pressure, Pulse Wave Velocity, and Augmentation Index Recorded by Means of the Mobil-O-Graph PWA Monitor. Hypertens Res (2020) 43(11):1239–48. doi: 10.1038/s41440020-0490-5 14. Rivera R, Yacobson I, Grimeset D. The Mechanism of Action of Hormonal Contraceptives and Intrauterine Contraceptive Devices. Am J Obstet Gynecol (1999) 181(5 Pt 1):1263–9. doi: 10.1016/S0002-9378(99)70120-1 15. Hayashi K, Miyachi M, Seno N, Takahashi K, Yamazaki K, Sugawara J, et al. Variations in Carotid Arterial Compliance During the Menstrual Cycle in Young Women. Exp Physiol (2006) 91(2):465–72. doi: 10.1113/expphysiol. 2005.032011 16. RobbAO,MillsNL,DinJN,SmithIBJ,PatersonF,NewbyDE,etal.Influence of the Menstrual Cycle, Pregnancy, and Preeclampsia on Arterial Stiffness. Hypertension (2009) 53(6):952–8. doi: 10.1161/HYPERTENSIONAHA.109.130898 17. Umapathy E, Muthiraparampil ST, Namugowa A. Assessment of Variations in Arterial Tone During Different Phases of Menstrual Cycle. Int J Reprod Contracept Obstet Gynecol (2019) 8(5):1810–5. doi: 10.18203/2320-1770. ijrcog20191924 18. Williams MR, Westerman RA, Kingwell BA, Paige J, Blombery PA, Sudhir K, et al. Variations in Endothelial Function and Arterial Compliance During the Menstrual Cycle. J Clin Endocrinol Metab (2001) 86(11):5389–95. doi: 10.1210/jcem.86.11.8013 19. Ounis-Skali N, Mitchell GF, Solomon CG, Solomon SD, Seely EW. Changes in Central Arterial Pressure Waveforms During the Normal Menstrual Cycle. J Investig Med (2006) 54(6):321–6. doi: 10.2310/6650.2006.05055 20. Adkisson EJ, Casey DP, Beck DT, Gurovich AN, Martin JS, Braith RW. Central, Peripheral and Resistance Arterial Reactivity: Fluctuates During the Phases of the Menstrual Cycle. Exp Biol Med (Maywood) (2010) 235(1):111–8. doi: 10.1258/ebm.2009.009186 21. Spaczynski RZ, Mitkowska A, Florczak M, Banaszewska B, Krauze T, Wykrętowicz A, et al. Decreased Large-Artery Stiffness in Midluteal Phase of the Menstrual Cycle in Healthy Women of Reproductive Age. Ginekol Pol (2014) 85(10):771–7. 22. Hickson SS, Miles KL, McDonnell BJ, Yasmin, Cockcroft JR, Wilkinson IB, et al. ENIGMA Study Investigators. Use of the Oral Contraceptive Pill Is Associated With Increased Large Artery Stiffness in Young Women: The ENIGMA Study. JHypertens(2011) 29(6):1155–9. doi: 10.1097/HJH. 0b013e328346a5af 23. Priest SE, Shenouda N, MacDonald MJ. Effect of Sex, Menstrual Cycle Phase, and Monophasic Oral Contraceptive Pill Use on Local and Central Arterial Stiffness in Young Adults. Am J Physiol Heart Circ Physiol (2018) 315(2): H357–65. doi: 10.1152/ajpheart.00039.2018 24. Yu A, Giannone T, Scheffler P, Doonan RJ, Egiziano G, Gomez Y-H, et al. The Effect of Oral Contraceptive Pills and the Natural Menstrual cYCLe on Arterial Stiffness and hemodynamICs (CYCLIC). J Hypertens (2014) 32(1):100–7. doi: 10.1097/HJH.0000000000000012 25. Seeland U, Demuth I, Regitz-Zagrosek V, Steinhagen-Thiessen E, König M. Sex Differences in Arterial Wave Reflection and the Role of Exogenous and Endogenous Sex Hormones: Results of the Berlin Aging Study Ii. J Hypertens (2020) 38(6):1040–6. doi: 10.1097/HJH.0000000000002386 26. Enea C, Laffetas P, Pichon A, Delpech N. Arterial Stiffness and Hemodynamics in Young Women: The Effects of Oral Contraceptive Intake and Physical Habits. Int J Environ Res Public Health (2021) 18:3393. doi: 10.3390/ijerph18073393 27. Ramesh SS, Christopher R, Devi BI, Bhat DI. The Vascular Protective Role of Oestradiol: A Focus on Postmenopausal Oestradiol Deficiency and Aneurysmal Subarachnoid Haemorrhage. Biol Rev Camb Philos Soc (2019) 94(6):1897–917. doi: 10.1111/brv.12541 28. Hodis HN, Mack WJ, Henderson VW, Shoupe D, Budoff MJ, Hwang-Levine J, et al. ELITE Research Group. Vascular Effects of Early Versus Late Postmenopausal Treatment With Estradiol. NEnglJMed(2016) 374 (13):1221–31. doi: 10.1056/NEJMoa1505241 29. Rajkumar C, Kingwell BA, Cameron JD, Waddell T, Mehra R, Christophidis N, et al. Hormonal Therapy Increases Arterial Compliance in Postmenopausal Women. J Am Coll Cardiol (1997) 30(2):350–6. doi: 10.1016/S0735-1097(97) 00191-5 30. Scuteri A, Lakatta EG, Bos AJ, Fleg JL. Effect of Estrogen and Progestin Replacement on Arterial Stiffness Indices in Postmenopausal Women. Aging (Milano) (2001) 13(2):122–30. doi: 10.1007/BF03351534 31. O’Neill SM, Liu J, O’Rourke MF, Khoo SK. The Menopausal Transition Does Not Appear to Accelerate Age-Related Increases in Arterial Stiffness. Climacteric (2013) 16(1):62–9. doi: 10.3109/13697137.2012.739220 32. Casiglia E, Tikhonoff V, Boschetti G, Giordano N, Mazza A, CaffiS, et al. Arterial Stiffness and Related Variables Across Menopausal Status: An Epidemiologic Study. J Women’s Health (Larchmt) (2013) 22(1):75–84. doi: 10.1089/jwh.2012.3666 Laakkonen et al. Sex Hormones and Arterial Stiffness Frontiers in Endocrinology | www.frontiersin.org November 2021 | Volume 12 | Article 76591615 33. Abbas SZ, Sangawan V, Das A, Pandey AK. Assessment of Cardiovascular Risk in Natural and Surgical Menopause. Indian J Endocrinol Metab (2018) 22 (2):223–8. doi: 10.4103/ijem.IJEM_620_17 34. Ihalainen JK, Löfberg I, Kotkajuuri A, Kyröläinen H, Hackney AC, TaipaleMikkonen RS. Influence of Menstrual Cycle or Hormonal Contraceptive Phase on Energy Intake and Metabolic Hormones –A Pilot Study. Endocrines (2021) 2:79–90. doi: 10.3390/endocrines2020008 35. Melin A, Tornberg ÅB, Skouby S, Faber J, Ritz C, Sjödin A, et al. The LEAF Questionnaire: A Screening Tool for the Identification of Female Athletes at Risk for the Female Athlete Triad. Br J Sports Med (2014) 48:540–5. doi: 10.1136/bjsports-2013-093240 36. Kovanen V, Aukee P, Kokko K, Finni T, Tarkka IM, Tammelin T, et al. Design and Protocol of Estrogenic Regulation of Muscle Apoptosis (ERMA) Study With 47 to 55 Years Old Women’s Cohort: Novel Results Show MenopauseRelated Differences in Blood Count. Menopause (2018) 25(9):1020–32. doi: 10.1097/GME.0000000000001117 37. Elliott-Sale KJ, Smith S, Bacon J, Clayton D, McPhilimey M, Goutianos G, et al. Examining the Role of Oral Contraceptive Users as an Experimental and/ or Control Group in Athletic Performance Studies. Contraception (2013) 88 (3):408–12. doi: 10.1016/j.contraception.2012.11.023 38. Harlow SD, Gass M, Hall JE, Lobo R, Maki P, Rebar RW, et al. STRAW 10 Collaborative Group. Executive Summary of the Stages of Reproductive Aging Workshop + 10: Addressing the Unfinished Agenda of Staging Reproductive Aging. Menopause (2012) 19:387–95. doi: 10.1097/gme.0b013e31824d8f40 39. Haapala EA, Laukkanen JA, Takken T, Kujala UM, Finni T. Peak Oxygen Uptake, Ventilatory Treshold, and Arterial Stiffness in Adolescnents. Eur J Appl Physiol (2018) 118(11):2367–76. doi: 10.1007/s00421-018-3963-3 40. Haapala EA, Veijalainen A, Kujala UM, Finni T. Reproducibility of Pulse Wave Velocity and Augmentation Index Derived From non-Invasive Occlusive Oscillometric Tonometry Analysis in Adolescents. Clin Physiol Funct Imaging (2019) 39:22–8. doi: 10.1111/cpf.12528 41. Horvath IG, Nemeth A, Lenkey Z, Alessandri N, Tufano F, Kis P, et al. Invasive Validation of a New Oscillometric Device (Arteriograph) for Measuring Augmentation Index, Central Blood Pressure and Aortic Pulse Wave Velocity. J Hypertens (2010) 28(10):2068–75. doi: 10.1097/HJH. 0b013e32833c8a1a 42. Rossen NB, Laugesen E, Peters CD, Ebbehøj E, Knudsen ST, Poulsen PL, et al. Invasive Validation of Arteriograph Estimates of Central Blood Pressure in Patients With Type 2 Diabetes. Am J Hypertens (2014) 27:674–9. doi: 10.1093/ ajh/hpt162 43. Rajzer MW, Wojciechowska W, Klocek M, Palka I, Brzozowska-Kiszka M, Kawecka-Jaszcz K. Comparison of Aortic Pulse Wave Velocity Measured by Three Techniques: Complior, SphygmoCor and Arteriograph. J Hypertens (2008) 26(10):2001–7. doi: 10.1097/HJH.0b013e32830a4a25 44. Antonio J, Kenyon M, Ellerbroek A, Carson C, Burgess V, Tyler-Palmer D, et al. Comparison of Dual-Energy X-Ray Absorptiometry (DXA) Versus a Multi-Frequency Bioelectrical Impedance (InBody 770) Device for Body Composition Assessment After a 4-Week Hypoenergetic Diet. JFunct Morphol Kinesiol (2019) 4(2):23. doi: 10.3390/jfmk4020023 45. Storer TW, Davis JA, Caiozzo VJ. Accurate Prediction of VO2max in Cycle Ergometry. Med Sci Sports Exerc (1990) 22(5):704–12. doi: 10.1249/00005768199010000-00024 46. Ma Y, Mazumdar M, Memtsoudis SG. Beyond Repeated-Measures Analysis of Variance: Advanced Statistical Methods for the Analysis of Longitudinal Data in Anesthesia Research. Reg Anesth Pain Med (2012) 37:99–105. doi: 10.1097/ AAP.0b013e31823ebc74 47. Tsao CW, Lyass A, Larson MG, Levy D, Hamburg NM, Vita JA, et al. Relation of Central Arterial Stiffness to Incident Heart Failure in the Community. JAm Heart Assoc (2015) 4(11):e002189. doi: 10.1161/JAHA.115.002189 48. Mitchell GF, Hwang SJ, Vasan RS, Larson MG, Pencina MJ, Hamburg NM, et al. Arterial Stiffness and Cardiovascular Events: The Framingham Heart Study. Circulation (2010) 121:505–11. doi: 10.1161/CIRCULATIONAHA.109.886655 49. Chirinos JA, Kips JG, Jacobs DR Jr, Brumback L, Duprez DA, Kronmal R, et al. Arterial Wave Reflections and Incident Cardiovascular Events and Heart Failure: MESA (Multiethnic Study of Atherosclerosis). J Am Coll Cardiol (2012) 60:2170–7. doi: 10.1016/j.jacc.2012.07.054 50. Williams JS, MacDonald MJ. Influence of Hormonal Contraceptives on Peripheral Vascular Function and Structure in Premenopausal Females: A Review. Am J Heart Circ Physiol (2021) 320:H77–89. doi: 10.1152/ ajpheart.00614.2020 51. Meendering JR, Torgrimson BN, Miller NP, Kaplan PF, Minson CT. Ethinyl Estradiol-to-Desogestrel Ratio Impacts Endothelial Function in Young Women. Contraception (2009) 79(1):41–9. doi: 10.1016/j.contraception. 2008.07.025 52. Thompson AK, Przemska A, Vasilopoulou D, Newens KJ, Williams CM. Combined Oral Contraceptive Pills Containing Desogestrel or Drospirenone Enhance Large Vessel and Microvasculature Vasodilation in Healthy Premenopausal Women. Microcirculation (2011) 18(5):339–46. doi: 10.1111/j.1549-8719.2011.00094.x 53. Angerer P, Kothny W, Störk S, von Schacky C. Hormone Replacement Therapy and Distensibility of Carotid Arteries in Postmenopausal Women: A Randomized, Controlled Trial. J Am Coll Cardiol (2000) 36(6):1789–96. doi: 10.1016/S0735-1097(00)00969-4 54. Kawecka-Jaszcz K, Czarnecka D, Olszanecka A, Rajzer M, Jankowski P. The Effect of Hormone Replacement Therapy on Arterial Blood Pressure and Vascular Compliance in Postmenopausal Women With Arterial Hypertension. J Hum Hypertens (2002) 16(7):509–16. doi: 10.1038/sj.jhh. 1001431 55. Hildreth K, Kohrt WM, Moreau KL. Oxidative Stress Contributes to Large Elastic Arterial Stiffening Across the Stages of the Menopausal Transition. Menopause (2014) 21(6):624–32. doi: 10.1097/GME.0000000000000116 56. Nagai Y, Earley CJ, Kemper MK, Bacal CS, Metter EJ. Influence of Age and Postmenopausal Estrogen Replacement Therapy on Carotid Arterial Stiffness in Women. Cardiovasc Res (1999) 41(1):307–11. doi: 10.1016/S0008-6363(98) 00219-3 57. Sriprasert I, Hodis HN, Bernick B, Mirkin S, Mack WJ. Effects of Estradiol Dose and Serum Estradiol Levels on Metabolic Measures in Early and Late Postmenopausal Women in the REPLENISH Trial. JWomen’sHealth (Larchmt) (2020) 29(8):1052–8. doi: 10.1089/jwh.2019.8238 58. Karvinen S, Jergenson MJ, Hyvärinen M, Aukee P, Tammelin T, Sipilä S, et al. Menopausal Status and Physical Activity are Independently Associated With Cardiovascular Risk Factors of Healthy Middle-Aged Women: CrossSectional and Longitudinal Evidence. Front Endocrinol (Lausanne) (2019) 10:589. doi: 10.3389/fendo.2019.00589 59. Ding M, Manson JE. HDL-C and Arterial Calcification in Midlife Women: Does an HDL Paradox Exist? Menopause (2021) 28(3):231–3. doi: 10.1097/ GME.0000000000001734 60. The Reference Values for Arterial Stiffness’Collaboration. Determinants of Pulse Wave Velocity in Healthy People and in the Presence of Cardiovascular Risk Factors: ‘Establishing Normal and Reference Values’.Eur Heart J (2010) 31(19):2338–50. doi: 10.1093/eurheartj/ehq165 61. Benas D, Kornelakis M, Triantafyllidi H, Kostelli G, Pavlidis G, Varoudi M, et al. Pulse Wave Analysis Using the Mobil-O-Graph, Arteriograph and Complior Device: A Comparative Study. Blood Press (2019) 28(2):107–13. doi: 10.1080/08037051.2018.1564236 62. Papaioannou TG, Thymis J, Benas D, Triantafyllidi H, Kostelli G, Pavlidis G, et al. Measurement of Central Augmentation Index by Three Different Methods and Techniques: Agreement Among Arteriograph, Complior, and Mobil-O-Graph Devices. J Clin Hypertens (Greenwich) (2019) 21(9):1386–92. doi: 10.1111/jch.13654 63. El Khoudary SR, Aggarwal B, Beckie TM, Hodis HN, Johnson AE, Langer RD, et al. American Heart Association Prevention Science Committee of the Council on Epidemiology and Prevention; and Council on Cardiovascular and Stroke Nursing. Menopause Transition and Cardiovascular Disease Risk: Implications for Timing of Early Prevention: A Scientific Statement From the American Heart Association. Circulation (2020) 142(25):e506–32. doi: 10.1161/CIR.0000000000000912 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in Laakkonen et al. Sex Hormones and Arterial Stiffness Frontiers in Endocrinology | www.frontiersin.org November 2021 | Volume 12 | Article 76591616 this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2021 Laakkonen, Karppinen, Lehti, Lee, Pesonen, Juppi, Kujala, Haapala, Aukee, Laukkanen and Ihalainen. 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