4.1 High-temperature environment and menstrual disorders
High temperatures can result in heat stress (Horowitz 2002; Horowitz 2007), which is the sum of nonspecific responses occurring in humans or animals when an organism is exposed to excessive temperature stimuli exceeding its thermoregulatory capacity in a high-temperature environment. It is a stress factor that special operating populations have to guard against in high-temperature environments (Kovats and Hajat 2008). During heat stress, the normal thermal homeostasis system of the body may be disrupted, leading to the disruption of digestive somatic functions, impaired blood circulation, and disruption of the neuroendocrine functions, which together pose a severe threat to the development of reproductive health concerns in women working under high-temperature environments (Fan et al. 2019; Wang et al. 2016). HE can increase the risk of menstrual disorders and impact the reproductive function in women (An et al. 2020b). The characteristics of changes and regulatory mechanisms of menstrual disorders that appear at high temperatures remain unclear. We selected two regions with significant differences in their mean temperature and used a random, whole-group sampling method to conduct a cross-sectional survey of young women belonging to the same work category. In this study, the confounding factors were controlled accordingly. Only young migrant females rather than native youth were from both regions in this study. In addition, other variables including age, BMI, work intensity, and dietary habits were not significantly different between the two groups. Altitude and environmental temperature were also analyzed as climate variables. It was found that both regions were in the plains, while there were significant differences in environmental temperature. The control group was located at 43.88 ºN latitude, which belongs to the northern temperate zone (average annual temperature 0–10°C, summer temperature 15–25°C) and the high temperature group was located at 23.05 ºN latitude, belonging to the tropics (average annual temperature 20–28°C, summer temperature 28–38°C). Based on the analysis of population characteristics and environmental variables, we consider that it is some representative of these people in this study: young women who were migrant to both areas with the same work intensity.
This study found a significantly higher rate of menstrual cycle disorders in young women working in hot environments. These findings indicate that high-temperature environments are more likely to result in menstrual disorders and increase menstrual volume but has no significant effect on premenstrual syndromes. Anxiety, pressure, history of medication, low sleep quality, and high temperature were noted as influencing factors that affect menstruation and increase the risk of menstrual disorders in women. Hot environment as a stressor also had specific effects on the psychology of women working in such environments, mainly in the form of increased negative emotions such as tension, depression, anxiety, fear, and anger. Common menstrual problems are closely related to increased psycho-emotional changes, anxiety, and excessive psychological stress, which are critical factors affecting menstrual disorders and resulting in amenorrhea (Allsworth et al. 2007; Rafique and Al-Sheikh 2018). At the same time, emotions result in the release of hormones from the pituitary and hypothalamus (Matsuda et al. 1997). Menstruation occurs when there is a change in the regularity of hormones secreted by the ovaries and acting on the endometrium, which is controlled by hormones released by the hypothalamus (Shufelt et al. 2017). In addition, sleep deprivation, short sleep duration, low sleep quality, and altered circadian rhythms inhibit melatonin secretion, affecting ovarian function and reducing menstrual rate, thereby leading to menstrual disorders or dysmenorrhea in women (Czajkowska et al. 2019; Meers and Nowakowski 2020; Najafi et al. 2018). In summary, HE may affect women's emotions and alter hormone levels in them, which can interfere with the hypothalamic-pituitary-ovarian (HPO) axis and affect the normal appearance of menstruation. These changes induce ovulation and disruption of the menstrual cycle, thereby resulting in menstrual disorders.
4.2 High-temperature environment and salivary metabolites
Saliva is an information-rich biofluid that can noninvasively respond to human diseases. In this study, the expression levels of a total of 64 saliva metabolites were significantly altered in women with menstrual disorders in the H and C groups; of these, the expression of 34 metabolites was upregulated, whereas that of 30 was downregulated. These included amino acids, peptides, and carbohydrates, which can involve inflammatory response, immune regulation, amino acid metabolism, and membrane production in organisms.
Among the metabolites with upregulated expression levels, the levels of PC and N-acetylneuraminic acid were 3.39- and 3.06-times higher in the H group than in the C group, suggesting that HE induces inflammatory and immune response modulation. PC is a structural component of various prokaryotic and eukaryotic pathogens, with a surprising range of immunomodulatory properties that can benefit the infected host by targeting innate and adaptive immune responses. However, its broad immunomodulatory properties can harm the host through immunomodulation (Harnett and Harnett 1999). N-acetylneuraminic acid, a derivative of neuraminic acid collectively known as sialic acid (SA), is the major SA found in mammalian cells (Varki 1992). Changes in SA levels can trigger the development of various diseases, including inflammation, cardiovascular disease, neurological disorders, and endocrine disorders (Reuter and Gabius 1996). It has been suggested that elevated SA levels reflect acute phase response in the inflammatory process; that a positive correlation exists between tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which are essential mediators of the acute phase response; and that elevated inflammatory factors affect loops such as the hypothalamic–pituitary–adrenal (HPA) axis, which in turn affect reproductive function(Demir et al. 2018). Therefore, HE may induce inflammatory and immune response modulation, leading to menstrual disorders by affecting the HPA axis loop.
MYO and D-sorbitol were mainly enriched in the membrane transport pathway. MYO has high biological importance and is widely distributed in nature; it belongs to the glycan family. Its derivatives are important components of the cell membrane structural phospholipids and are precursors of second messengers of metabolic pathways (Milewska et al. 2016). The results suggested that menstrual cycle disorders are associated with endocrine disruption and maintenance of cell membrane stability. MYO deficiency and impairment of the MYO-dependent pathways might play crucial roles in the pathogenesis of insulin resistance and hypothyroidism. Insulin and thyroxine are important components of the HPO axis. In a previous study, the kinetic cycle was disturbed and insulin levels were reduced in female rats after prolonged heat stress (An et al. 2020a). Abnormalities in insulin metabolism also underlie several clinical diseases (MacFarlane and Di Fiore 2018). Heat stress can downregulate the expression of inositol-requiring enzyme 1α (IRE1α), leading to termination of the IRE1α signaling pathway, which causes an unfolded protein response in cells and affect the production of cell membrane proteins (Homma and Fujii 2016). The expression of D-sorbitol, a metabolite that was positively correlated with MYO, was also increased. Appropriate D-sorbitol supplementation can significantly reduce the formation of blastocysts and increase the apoptosis index (Lin et al. 2015). Therefore, long-term exposure to high temperatures and HE may result in an imbalance in the metabolism of insulin and thyroxine. Cell membrane stability is reduced, which may increase apoptosis and cause menstrual disorders.
Among the metabolites with reduced expression in this study, the expression levels of MVA and tyramine were 0.3 and 0.57 times higher in the H group than in the C group. Reduced expression of MVA affects the MVA pathway, which is an essential metabolic pathway for the organism and is critical for cell survival and function (Ermini et al. 2017; Mullen et al. 2016). MVA biosynthetic intermediates are crucial regulators of intrinsic immunity in bovine endometritis (Mullen et al. 2016). Tyramine is a biological trace amine that is generated through decarboxylation of the amino acid tyrosine, and substantial evidence suggest that tyramine is a neuroactive chemical exhibiting multiple physiological effects (Lange 2009); it can affect various physiological mechanisms, exhibits neuromodulatory properties and cardiovascular and immunological effects (Andersen et al. 2019), stimulates the insulin-IGF-1 signaling (IIS) pathway, and blocks the induction of stress response genes by activating adrenergic-like receptors in the intestine. Tyrosine can directly or indirectly act on the ovaries to inhibit luteal function, thereby affecting reproduction. Therefore, changes in N-acetylneuraminic acid levels, MYO, and tyramine in salivary metabolites at high-temperature environment may affect the menstrual cycle through inflammatory responses, influence membrane production, and participate in immune regulation, leading to the development of menstrual disorders.
In this study, KEGG pathway enrichment revealed the involvement of several metabolites in carbohydrate metabolism, mainly fructose and mannose, galactose, and amino and nucleotide sugar metabolism. Glucose metabolism is a core component of energy metabolism, essential in the maintenance of normal physiological functions of the body. Abnormal glucose metabolism is closely associated with metabolic syndrome and diseases such as cancer (Andersen et al. 2013). Additionally, abnormal glucose metabolism is associated with several gynecological disorders (Ferreira and Motta 2018). Insulin resistance, as a pathogenic base of glucose metabolism abnormalities, affects the action of sex hormones on the ovaries and endometrium through insulin-like growth factor-1 receptor (IGF-1R), leading to anovulation or endometrial lesions (Li et al. 2012). Therefore, abnormal glucose metabolism triggers gynecologically related diseases and also induce menstrual disorders.
4.3 High-temperature environment and serum neurotransmitters
Neurotransmitter is a chemical "messenger" molecule that transmits signals between synapses. The secretion of neurotransmitters promotes the balance of amino acid metabolism in the body, regulate body's immune functions and cardiovascular activities, and mediate smooth muscle contraction. In this study, serum targeted metabolomics revealed increased expression of HA in the H group, which was 2.077 times higher than that in the C group. HA is present in the mammalian myocardium, mast cells, basophils, skin, gastrointestinal tract, and lungs, as well as in the central nervous system. Central HA, as a central neurotransmitter, is related to obesity, diabetes, and endocrine (Watanabe and Yanai 2001). Plasma HA is mainly used as an inflammatory mediator and immune substance. Studies have confirmed that HA released by mast cells can stimulate histamine type 2 receptor (H2R) in the rat kidney as an inflammatory mediator. The release of renin and under stress conditions resulted in increased HA levels in the hypothalamus and periphery in mice(He et al. 2009). This is consistent with the results of our saliva metabolomics analysis, in which inflammatory reactions occurred under high-temperature environments. In contrast, the expression of 5-HIAA, which is a primary end product of 5-hydroxy tryptamine (5-HT) metabolism and plays an emotional regulation role, decreased under high-temperature environment (Elghozi and Laude 1989). Dysfunction of 5-HT can cause different mental diseases, including depression, impulsive aggression, and anxiety, which is also consistent with our previous questionnaire results. Thus, prolonged hot environments may increase anxiety in humans and affect the endocrine system. Therefore, changes in serum neurotransmitter levels provide better corroboration for our salivary metabolomics results.