In this study, we determined the resting cfDNA levels during the menstrual cycle and the changes in cfDNA levels during the menstrual cycle in subjects with a normal menstrual cycle. The results showed that the baseline values did not change significantly during the menstrual cycle, and exercise-induced changes in cfDNA levels and related blood markers may be associated with female hormone levels.
The lack of significant differences in the basic characteristics of the menstrual cycle suggests that these factors were unlikely to have influenced the results of this study. Oxygen uptake increased significantly at 8 to 10, 18 to 20, and 28 to 30 min of exercise compared with that Pre and was approximately 29–31 mL/min/kg over the three phases. The mean VO2max in this study was 45.3 ± 3.3 (mL/min/kg), 60% of which was 27.2 (mL/min/kg). This finding suggests that the exercise load was equivalent to 70% VO2max in this study.
In the present study, aromatase levels significantly increased at Post 0 immediately after exercise in all three phases. In addition, estradiol levels were significantly higher in the luteal than those in the menstrual phase, and progesterone levels were significantly higher in the luteal than those in the menstrual and ovulatory phases, indicating that fluctuations in female hormones during the normal menstrual cycle were observed. Previous studies have reported that the expression of aromatase increases immediately after exercise[26] and that aromatase is expressed in skeletal muscle and synthesizes estrogen via dehydroepiandrosterone (DHEA)[27, 28]. In addition, a previous study reported that exercise increased the expression of aromatase protein and estradiol in the skeletal muscles of ovariectomized rats[27]. Similarly, testosterone levels in skeletal muscles after transient exercise have been reported to significantly increase regardless of sex[28]. Furthermore, serum estradiol levels after exercise were significantly higher in ovariectomized rats that exercised for 3 months than those in the control, sham-operated, and ovariectomized groups[29]. Therefore, it is possible that estradiol level increases following exercise could be due to synthesis and secretion by organs other than the ovaries. Therefore, it is conceivable that, in the present study, the increase in aromatase associated with exercise stimulation promoted estrogen synthesis in the skeletal muscle, especially in the ovulatory and luteal phases, which significantly increased after exercise.
No significant differences in cfDNA levels were observed between the menstrual, ovulatory, and luteal phases in Pre, which is consistent with previous studies showing no significant variation in resting cfDNA levels during the menstrual cycle[20, 21]. Cyclic cell proliferation, differentiation, and endometrial shedding occur throughout the menstrual cycle. Menstruation, which occurs in the absence of fertilization and implantation of a fertilized egg[30], is associated with the apoptotic shedding of the outer endometrial epithelial layer, fragmentation of glands, and loss of adhesion molecules[31–33]. In addition, angiogenesis is involved in vascular bed repair[34], resulting in inflammation[35]. Therefore, although cfDNA derived from dead cells may increase with these events, it is unlikely that cfDNA leaks sufficiently to show significant changes from the perspective of shed blood and blood draining into the uterine cavity associated with menstruation[36].
The cfDNA levels were significantly higher at Post 0 immediately after exercise than at Pre in all three phases, indicating the influence of exercise load. It has been reported that cfDNA concentrations increased up to 5-fold from resting levels in an exercise stress test using all-out cycling[14, 15], suggesting that a certain level of exercise stress influenced cfDNA concentrations in the current study. The change in cfDNA levels from Pre showed a significant increase at Post 0 in all three phases, and the increase from Pre to Post 0 was significantly greater in the ovulatory than that in the luteal phase (p = 0.044). Among the cfDNA levels at Post 0 observed in this study, the comparison between the ovulatory and luteal phases had a p-value = 0.083, which was very close to significance. This suggests that the amount of cfDNA variation due to transient exercise may be related to the menstrual cycle phase (i.e., differences in female hormone concentrations). Generally, cfDNA appears in the blood owing to cell death via apoptosis, and it has been reported that cfDNA is also associated with inflammatory reactions[37, 38]. Recently, it has been shown that the appearance of cfDNA is not only due to apoptosis but also due to NETosis, in which neutrophils are stimulated by reactive oxygen species (ROS) and IL-8, an inflammatory cytokine, to release DNA in a mesh pattern[9], which is a cell death different from apoptosis. Studies have shown that there are two forms of NETosis: NADPH-dependent NETosis (induced by NAPDH oxidase, a ROS produced in neutrophils), which is accompanied by cell death, and NADPH-independent NETosis, which does not involve plasma membrane disruption[39–41]. Previous studies have reported that NADPH-independent NETosis is associated with estrogen (ERα, ERβ) and membrane estrogen receptors (GPR30)[42], which are also expressed in neutrophils, as well as estradiol agonists and GPR30 NETosis and the suppression of dependent NETosis by suppressing ROS production in neutrophils with increased estradiol[43]. In contrast, progesterone, which increases in the luteal phase, antagonizes the NETosis-promoting effect of estradiol (by promoting phagocytic activity)[44]. This suggests that estradiol alone has the effect of increasing cfDNA (especially NADPH-independent cfDNA) levels. Although estradiol levels are also higher in the luteal phase when progesterone levels increase, the increase in cfDNA levels after exercise is lower than that in the ovulatory phase due to the antagonistic effect of progesterone on estradiol. In contrast, the anti-inflammatory and immunomodulatory effects of progesterone are associated with the inhibition of proinflammatory cytokines[45]. Progesterone primarily reduces the amount of proinflammatory cytokines and induces a certain amount of anti-inflammatory cytokines. In both in vivo and in vitro models, progesterone induces more anti-inflammatory cytokines such as IL-10 and IL-4 and decreases the production of inflammatory cytokines such as IL-6, TNF-α, IL-1β and IL-6, TNF-α, and IL-1β[46–49]. Assuming that cfDNA in the present study is a marker of acute-phase inflammation during exercise, progesterone may exhibit anti-inflammatory effects. In the present study, the change between Post 0-Pre in P4/E2 in the luteal phase showed a significant negative correlation with cfDNA Post 0-Pre. The luteal phase is when progesterone and estradiol levels are significantly increased, and these ovarian hormones are also shown to be significantly increased immediately after exercise. Therefore, unlike in the menstrual and ovulatory phases, progesterone levels also increase immediately after exercise, and the increase in P4/E2 may indicate a relationship in which the cfDNA levels decrease. On the other hand, although a negative correlation could be observed in the ovulatory phase based on the same theory, the correlation between the change in Post 0-Pre was r =-0.64 (p = 0.096), which did not lead to the conclusion that there was a significant difference. In the present study, estradiol levels in the ovulatory phase were not high enough to differ significantly from those in the menstrual or luteal phases. However, if such a hormonal state can be described, increased estradiol levels after exercise may lead to decreased P4/E2 and increased cfDNA levels.
To the best of our knowledge, this is the first report on the dynamics of cfDNA secretion during exercise. The relationship between cfDNA and ovarian hormone levels before and after exercise revealed the possible involvement of progesterone in inflammation. This suggests that cfDNA may be useful as an indicator of the immediate response to exercise in women and men, but post-exercise changes may be influenced by differences in female hormone concentrations, which should be considered when using cfDNA as an indicator. Unfortunately, we could not obtain significantly higher estradiol levels during the ovulatory phase relative to those during the menstrual phase. Therefore, the results of this study are insufficient to validate the results for the period when estradiol levels are independently higher than those during the menstrual and luteal phases. In addition, ROS, myeloperoxidase (MPO), and inflammatory cytokine levels were not measured. Estradiol suppresses NADPH-dependent NETosis by suppressing ROS production, whereas MPO and other substances are released during NAPDH-independent NETosis, increasing cfDNA concentration. Although the present study discusses the effects on cfDNA derived from NETosis based on previous studies, we have not yet measured these parameters. Therefore, it is necessary to measure them in the future.