Sex differences in HPA function may precipitate differences in the development and maintenance of mental disorders; however, the impact of changing sex hormones over the course of the menstrual cycle on HPA cortisol activity is poorly understood, and thus, motivated the current meta-analysis. The objective of this meta-analysis is to provide a comprehensive summary of the literature examining cortisol concentrations as a marker for HPA axis function across the menstrual cycle. Accordingly, we compared cortisol concentrations between the follicular and luteal phases, and between five more precise cycle phases. We additionally propose strategies for improving methodology and gaps to address in future studies.

The hypothalamic-pituitary-adrenal (HPA) axis is an essential system in the physiological response to stress exposure. Its primary end product -cortisol- increases cardiovascular activity and cognitive processes, while suppressing digestive, immune, and reproductive functions (Kudielka et al., 2012, Sapolsky et al., 2000). Given its influence on behavior and cognition, the HPA axis plays an important role in the development and maintenance of mental disorders (Bangasser and Valentino, 2014) and has therefore been of large interest in psychoneuroendocrinology research.

A distinction must be made between HPA axis activity and HPA axis reactivity when studying HPA axis functioning. HPA axis activity is measured through basal, unstimulated cortisol concentrations, whereas HPA axis reactivity is assessed by measuring cortisol concentrations in response to an acute stressor (e.g. Trier Social Stress Test (Kirschbaum et al., 1993)). Cortisol can be analyzed from various specimen types, such as blood, saliva, or urine. The testing method determines whether the sample measures cortisol bound to cortisol-binding globulin (blood), or reflects unbound free cortisol (saliva and urine, small amount in blood) (El-Farhan et al., 2017). Basal cortisol concentrations follow a circadian rhythm and are therefore most accurately assessed by repeated measurements throughout the day to characterize the diurnal profile. From these diurnal profiles, three established parameters can be retrieved to indicate HPA axis functioning according to Adam and Kumari (2009): the area under the curve (AUC), the slope and the cortisol awakening response (CAR). The CAR shows the rise and fall of cortisol after awakening (Stalder et al., 2016). Furthermore, many studies in the past have used overall means of diurnal profiles and 24-hour measurements or single measures.

Sex differences in HPA axis functioning have been demonstrated previously, especially regarding its reactivity. Liu et al. (2017) meta-analytically summarized sex differences of HPA axis reactivity in response to the gold standard laboratory stress task - Trier Social Stress Test - and reported higher cortisol peaks and faster recovery in male participants. Notably, within female participants, those on oral contraceptives showed lower concentrations than those who did not, indicating an impact of endogenous sex hormones on HPA axis reactivity. Other systematic reviews on sex differences in HPA axis functioning have reported mixed results, possibly due to differences in specimen, measurement parameter, as well as disparities in age, health status, and time of sampling (Goel et al., 2014, Kudielka and Kirschbaum, 2005). In summary, sex differences of HPA axis functioning have been studied, particularly regarding HPA axis reactivity in response to stress. The reviews discussed previously report high heterogeneity of findings, thus suggesting that further examination of the mechanisms underlying sex-differences in HPA axis functioning (e.g., sex hormone fluctuations) is warranted.

During the reproductive years starting at menarche, sex hormones (i.e., estradiol and progesterone) fluctuate in a characteristic pattern over the course of the menstrual cycle. This process is regulated by the hypothalamic–pituitary–gonadal (HPG) axis. The menstrual cycle is a finely coordinated process in which follicles grow and then release an oocyte through the fallopian tube into the uterus (ovulation) to enable fertilization and pregnancy. If no pregnancy occurs, menstrual bleeding starts in order to shed the endometrium along with the oocyte and the cycle starts anew.

A cascade of hormones regulates the menstrual cycle. The hormones mostly associated with these processes include estrogens (i.e., estradiol), progesterone (and metabolites such as allopregnanolone (ALLO)), luteinizing hormone (LH), and follicle stimulating hormone (FSH). These hormones show distinct secretion patterns across the menstrual cycle and thereby characterize the phases of the cycle (Bale and Epperson, 2017). Most studies that consider the menstrual cycle differentiate broadly between the follicular and luteal phases. Due to the hormonal fluctuations within these broader phases, an additional separation into more precise cycle phases is necessary to determine underlying hormonal patterns (Schmalenberger et al., 2019). As such, the follicular phase can be separated into the menstrual, mid-to-late follicular, and periovulatory phase. The follicular phase begins with the first day of menstrual bleeding (menstrual phase) and is characterized by low concentrations of progesterone and increasing concentrations of estradiol, especially in the mid-to-late follicular phase. The periovulatory phase is characterized by a peak of LH, FSH, and estradiol concentrations, which cause ovulation. The luteal phase can be separated into the early- to mid-luteal phase and the premenstrual phase. The luteal phase is characterized by rising concentrations of progesterone after ovulation (early- to mid-luteal phase) and a steep decline of progesterone and estradiol before the onset of bleeding (premenstrual phase) (Schmalenberger et al., 2021). Other hormones (e.g., testosterone and oxytocin) have been shown to fluctuate across the menstrual cycle, which show higher concentrations around the time of ovulation/mid-cycle (Bui et al., 2013, Engel et al., 2019).

Studies investigating the menstrual cycle use a variety of methods to determine the different phases. These methods differ substantially in their accuracy to correctly distinguish distinct hormonal patterns (Schmalenberger et al., 2021). The simplest methods are counting techniques, in which the day of the cycle is either determined by counting forward from the first day of menstruation (forward count) or counting back from the next menstruation (backward count). The counted cycle days are then assigned to a cycle phase. More precise cycle phase determination can be achieved by measuring ovulation. Ovulation can be indicated by its preceding LH peak, as determined directly via urine tests, or indirectly via basal body temperature (nadir before ovulation). Moreover, ovulation and cycle phase can be determined by measuring typical changes in sex hormones from blood serum, including the progesterone rise after ovulation or the estradiol peak right before ovulation (Schmalenberger et al., 2021).

The interaction of HPA and HPG axes has been studied for almost six decades. Carefully conducted meta-analyses and reviews have investigated overall sex differences of HPA axis functioning (Goel et al., 2014, Kudielka and Kirschbaum, 2005), the biological context underlying the associations between the two axes (Handa and Weiser, 2014, Heck and Handa, 2019, Oyola and Handa, 2017), and the interaction of specific sex hormones and their metabolites with the HPA axis (Crowley and Girdler, 2014, Wirth, 2011).

Sex hormones (i.e., estrogens, androgens, and progestogens) and their association with HPA axis regulation have been the focus of human and animal studies. The mechanisms underlying the interaction between the HPA- and HPG axes have been primarily examined in animal studies. These animal studies show interactions at each level of both axes via both organizational and activational effects of gonadal hormones and transcriptional regulation (for detailed reviews see Oyola and Handa, 2017, Handa and Weiser, 2014). However, studies on the HPA-HPG-interaction in humans are sparse and results are more heterogeneous (Heck and Handa, 2019, Kudielka et al., 2012, Kudielka and Kirschbaum, 2005).

Regarding estrogens, ovariectomy has been shown to reduce HPA axis activity, while estradiol treatment is reported to increase HPA axis reactivity in rodents (Lund et al., 2004, Serova et al., 2010, Viau and Meaney, 1991). This effect can be influenced by estrogen receptor signaling (ERα and ERb), which can have opposing influences on HPA axis functioning (reviewed by Handa & Weiser, 2014).

Regarding androgens, gonadectomy in males induces increased HPA axis reactivity, suggesting that androgens have an inhibitory influence on HPA axis functioning (Seale et al., 2004, Viau and Meaney, 2004). Further, androgen (e.g. testosterone) treatment consistently reduces HPA axis reactivity in animal and human studies (Handa et al., 1994; Rubinow et al., 2005, Toufexis and Wilson, 2012).

Regarding progestogens, an inverse relationship has been reported between progesterone and cortisol levels (Stephens et al., 2016). Animal studies suggest that this effect is mediated by the progesterone metabolite, allopregnanolone (3α-hydroxy-5αpregnan-20-one; ALLO), and its actions at GABAA receptors (Wirth, 2011). ALLO has therefore been studied extensively in this context and has repeatedly been shown to have inhibitory effects on HPA axis functioning (Crowley & Girdler, 2014; Handa et al., 1994; Viau, 2002).

Aside from specific relationships between sex steroids and HPA activity, the fluctuation of these sex hormones across the menstrual cycle have been investigated regarding their influence on HPA axis activity. Accordingly, the animal-derived glucocorticoid equivalent, corticosterone, showed increased concentrations during estrous cycle phases characterized by increased estradiol and progesterone (Handa and Weiser, 2014, Oyola and Handa, 2017), indicating that sex hormones and their fluctuation across the menstrual cycle can act as potent agents influencing HPA axis functioning. Even though cortisol has been measured across the menstrual cycle in humans, inconsistent study designs and hormone measurement has produced heterogeneous results, and a systematic analysis of these precise mechanisms is missing. To address this gap in the literature and further elucidate the relationship between sex hormones and HPA axis activity, we sought to meta-analytically investigate changes in cortisol concentrations across all menstrual cycle phases.

This study is part of a larger project that aims to investigate HPA axis functioning in all phases of the menstrual cycle using a comprehensive meta-analysis of longitudinal studies (i.e., repeated measurements in at least two cycle phases) in adult females across the reproductive lifespan. A previous meta-analysis compared basal cortisol between the follicular and luteal phase in a selected group of studies (k = 35), indicating slightly higher cortisol concentrations in the follicular relative to the luteal phase (Hamidovic et al., 2020). We expand upon this meta-analysis by 1) largely extending the number of included studies; 2) by not only comparing cortisol between the broader defined follicular vs. luteal phase, but between the more precise cycle phases which show distinct patterns of sex hormone concentrations; 3) by exclusively including longitudinal studies to permit robust statements about potential menstrual cycle-related changes in HPA axis activity.

The objectives and the following main review questions were defined:

(How) do basal cortisol concentrations, as a marker for HPA axis activity, change systematically between the follicular and luteal phase of the menstrual cycle?

(How) do basal cortisol concentrations, as a marker for HPA axis activity, change systematically between five finer-grained cycles of the menstrual cycle (menstrual phase, mid-to-late follicular phase, ovulatory phase, early- to mid-luteal phase, premenstrual phase)?