Abstract
Background
Chronotype reflects individual preference for sleep–wake timing and is closely linked to circadian rhythm regulation. Greater eveningness tendency has been associated with adverse metabolic and endocrine outcomes; however, evidence regarding its relationship with reproductive hormones in women remains limited, particularly in Saudi Arabia. This study aimed to investigate the association between chronotype score and gonadal hormone profiles in young adult Saudi women.
Methods
In this cross-sectional study, 58 healthy Saudi female university students aged 18–23 years were recruited at Jazan University between September and October 2025. Blood samples were collected during the early follicular phase of the menstrual cycle (days 2–5). Chronotype was assessed using the Reduced Morningness–Eveningness Questionnaire (rMEQ). Serum estradiol and testosterone concentrations were measured using chemiluminescent immunoassays. Associations between rMEQ score and hormonal parameters were evaluated using Spearman’s rank correlation and partial Spearman correlation analyses.
Results
rMEQ score was negatively correlated with testosterone concentrations (ρ = −0.344, P = 0.008) and positively correlated with the estradiol-to-testosterone (E2/T) ratio (ρ = 0.338, P = 0.009), indicating that greater eveningness tendency was associated with a relatively more androgenic hormonal profile. These associations remained significant after adjustment for age, body mass index, and menstrual cycle day. No significant association was observed between rMEQ score and estradiol concentrations (ρ = 0.017, P = 0.897).
Conclusions
Lower rMEQ scores, indicating greater eveningness tendency, were associated with higher testosterone concentrations and a reduced E2/T ratio in healthy young Saudi women, suggesting a relatively more androgenic hormonal profile. These findings indicate that variation in chronotype may be linked to differences in reproductive hormone balance and may have implications for women’s reproductive and metabolic health.
Keywords: Chronotype, Testosterone, Estradiol, Circadian rhythms, women’s health, Saudi Arabia
Introduction
Chronotype, defined as an individual’s preferred timing of sleep, wakefulness, and daily activity, is a key behavioral characteristic related to circadian rhythm regulation. Individuals differ in their preferred timing of activity and sleep–wake patterns along a continuum ranging from greater morningness to greater eveningness [1]. Greater eveningness tendency is typically characterized by delayed sleep onset and wake times, often resulting in misalignment between the internal circadian clock and social or occupational schedules, a phenomenon commonly referred to as “social jetlag” [2]. Circadian misalignment has been associated with adverse metabolic and endocrine outcomes.
Physiologically, circadian rhythms are regulated by the central pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). The SCN synchronizes the peripheral circadian rhythms through neural and hormonal signaling pathways across multiple tissues, including those of the reproductive system. Light serves as the primary zeitgeber (time cue), entraining the SCN to the external day-night cycle [3]. In females, reproductive hormones, such as estradiol (E2) and testosterone, are regulated by both circadian and menstrual rhythms, largely through interactions with the hypothalamic–pituitary–gonadal axis [4].
Previous studies have suggested that sex hormones may, in turn, influence circadian function. Estradiol has been shown to affect sleep architecture, thermoregulation, and circadian gene expression, whereas androgens have been implicated in the modulation of melatonin suppression and SCN signaling [5]. Together, these findings support the existence of a bidirectional relationship between circadian rhythms and reproductive endocrinology. However, evidence regarding the relationship between chronotype and circulating gonadal hormone concentrations in women remains limited and inconsistent.
Recent evidence has explored the relationship between chronotype and estradiol levels, suggesting potential associations between circadian preference and estrogenic regulation in women [6]. In addition, large observational studies have demonstrated that sex hormone levels can be influenced by shift work, sleep deprivation, and irregular light exposure [7, 8]. However, relatively few studies have directly examined the relationship between chronotype and gonadal hormone profiles in young healthy women. Lower rMEQ scores, indicating greater eveningness tendency, have been associated with higher testosterone concentrations, whereas findings regarding estradiol levels have been inconsistent [9, 10]. Furthermore, a notable knowledge gap exists regarding chronotype–hormone interactions in culturally and behaviorally distinctive settings such as Saudi Arabia, where lifestyle patterns including late-night activity, prolonged screen exposure, and delayed sleep timing are prevalent and may influence circadian alignment and downstream hormonal regulation.
In the Saudi context, social norms, academic pressures, and late-night screen exposure may reinforce eveningness among university students [11, 12]. Such behaviors can alter melatonin secretion patterns and light exposure timing, thereby potentially influencing the regulation of reproductive hormones. Given increasing concerns regarding fertility trends and metabolic disorders in the region, understanding hormonal variation in relation to chronotype may have important clinical and public health implications [13, 14].
In addition to intrinsic circadian mechanisms, a range of environmental and lifestyle factors may influence both circadian timing and hormonal regulation. Behavioral determinants such as sleep timing, light exposure, dietary patterns, and daily activity rhythms are known to modulate circadian alignment, primarily through effects on melatonin secretion and downstream hypothalamic–pituitary–gonadal axis activity [15]. Seasonal variation and environmental light conditions may further influence endocrine rhythms, although their effects may vary according to geographic location. These factors are particularly relevant in young adult populations, in whom late-night activities and irregular sleep schedules may contribute to circadian misalignment and subsequent hormonal alterations [3].
In this study, we investigated the relationship between chronotype score and gonadal hormone profiles, specifically estradiol, testosterone, and the estradiol-to-testosterone (E2/T) ratio, in young Saudi women from the Jazan region. By providing region-specific data on chronotype–hormone associations, this study aimed to improve current understanding of endocrine variability related to circadian preference and its potential implications for women’s health in Middle Eastern populations.
Methods
Study design and participants
This cross-sectional laboratory-based study was conducted in September 2025 in the College of Nursing and Health Sciences at Jazan University (Jazan, Saudi Arabia). Fifty-eight healthy Saudi female university students aged 18–23 years were enrolled in this study. Participants were recruited through voluntary participation using announcements distributed within the university. All eligible students who met the inclusion criteria were invited to participate. All participants were in the early follicular phase of their menstrual cycle (days 2–5), a phase known for relatively stable hormonal baseline levels [16]. Exclusion criteria included pregnancy, lactation, current use of hormonal medications, endocrine disorders, or any chronic illness affecting hormone metabolism. Eligibility was assessed using a structured screening questionnaire that captured medical history, medication use, and menstrual cycle status prior to enrollment. Ethical approval was obtained from the Jazan University Research Ethics Committee (approval No.: REC-47/04/1624). All participants provided written informed consent prior to participation, and the study was conducted in accordance with ethical standards for research involving human subjects.
The study was conducted in Jazan, Saudi Arabia, during the period from September to October 2025. This region is characterized by a relatively stable photoperiod with limited seasonal variation in daylight duration. Participants were full-time university students with predominantly regular daytime schedules. Although factors such as shift work, night work, recent transmeridian travel (jet lag), and fasting practices were not formally assessed as exclusion criteria, these conditions are uncommon in the studied population and were therefore unlikely to have systematically influenced the results.
Participants first completed the rMEQ questionnaire, followed by anthropometric measurements. Blood samples were subsequently collected on the same day during the early follicular phase.
Chronotype assessment
Chronotype was evaluated using the validated Reduced Morningness–Eveningness Questionnaire (rMEQ), which consists of five questions assessing individual preferences regarding sleep–wake timing, morning alertness, and peak performance periods [17]. The rMEQ is widely used in clinical and epidemiological chronobiology research owing to its established psychometric properties.
The continuous rMEQ score was used as the primary chronotype measure in all analyses, with lower scores indicating greater eveningness tendency and higher scores indicating greater morningness tendency.
Anthropometric measurements
Body weight and height were measured using calibrated equipment, with the participants wearing light clothing and no shoes. The body mass index (BMI) (kg/m2) was calculated as weight in kilograms (kg) divided by the square of the height in meters (m).
Sample collection and processing
Venous blood samples (5 mL) were drawn between 8:00 AM and 10:00 AM under aseptic conditions to minimize diurnal hormonal fluctuations. Samples were collected using standard red-top vacutainers and serum separator tubes. After clotting at room temperature, samples were centrifuged at 3,000 rpm for 15 min. Serum was separated and stored at − 20 °C in polypropylene tubes until biochemical analysis. Strict cold-chain protocols were maintained to preserve hormonal integrity.
Biochemical analysis
Serum estradiol (E2) and testosterone (T) concentrations were measured using a chemiluminescent immunoassay on the UniCel DxI 600 Access Immunoassay System (Beckman Coulter, Fullerton, CA, USA). This platform uses paramagnetic particles and chemiluminescent signals to quantify hormone levels in a competitive binding format. Both assays demonstrated intra- and inter-assay coefficients of variation below 10%, indicating acceptable analytical precision.
Hormone concentrations were expressed in nmol/L, and the E2/T ratio was calculated by dividing serum estradiol concentration by testosterone concentration. Values below the lower limit of detection were assigned the minimum detectable value (0.1 nmol/L), a commonly used approach to avoid bias from exclusion of low-level measurements.
Statistical analysis
Statistical analyses were performed using JASP software (version 0.95.4.0). Data distribution was assessed using the Shapiro–Wilk test. Given the relatively small sample size and non-normal distribution of multiple variables, non-parametric methods were applied throughout to ensure analytical consistency and avoid assumptions of normality.
Continuous variables are presented as median (interquartile range [IQR]) and minimum–maximum values. Associations between continuous rMEQ score and clinical and hormonal variables were assessed using Spearman’s rank correlation coefficient (ρ).
To evaluate whether the observed associations were independent of potential confounding factors, partial Spearman correlations were performed between rMEQ score and hormonal parameters after adjustment for age, BMI, and menstrual cycle day. A two-sided P-value < 0.05 was considered statistically significant.
Results
Participant characteristics
The baseline demographic, anthropometric, and hormonal characteristics of the study participants are summarized in Table 1. The median age was 21 (IQR 21–22) years, and the median body mass index (BMI) was 22.3 (IQR 19.0–25.6) kg/m², consistent with the healthy weight range for young adult females. Median serum estradiol (E2) and testosterone (T) concentrations were 0.13 (IQR 0.10–0.16) nmol/L and 1.35 (IQR 0.86–1.99) nmol/L, respectively, resulting in a median estradiol-to-testosterone (E2/T) ratio of 0.10 (IQR 0.06–0.20). The median rMEQ score was 15.5, indicating an overall predominance of intermediate chronotype characteristics within the study population. Overall, anthropometric variables demonstrated relatively limited variability, whereas hormonal parameters exhibited greater inter-individual variation.
Table 1.
Baseline demographic, anthropometric, and hormonal characteristics of the study participants
| Variable | Total (n = 58) |
|---|---|
| Age (years) | 21.00 (21.00–22.00) [19–23] |
| Weight (kg) | 53.00 (47.00–61.00) [33–92] |
| Height (cm) | 156.0 (152.0–160.0) [148–167] |
| Body mass index (kg/m²) | 21.55 (19.00–25.55) [15.10–40.90] |
| Menstrual cycle day | 3.00 (2.00–4.00) [2–5] |
| Testosterone (nmol/L) | 1.40 (0.86–2.03) [0.10–3.30] |
| Estradiol (nmol/L) | 0.13 (0.10–0.16) [0.06–0.31] |
| Estradiol/testosterone ratio | 0.105 (0.06–0.21) [0.02–2.50] |
| rMEQ score | 15.50 (12.00–19.00) [9–22] |
Data are presented as median (interquartile range) [minimum–maximum]
BMI body mass index, rMEQ Reduced Morningness–Eveningness Questionnaire
Correlation analyses
The spearman correlation matrix (Table 2) demonstrated no significant association between rMEQ score and estradiol concentrations (ρ = 0.017, P = 0.897). In contrast, rMEQ score was negatively correlated with serum testosterone concentrations (ρ = −0.344, P = 0.008), indicating higher testosterone levels among individuals with greater eveningness tendency (lower rMEQ scores). Similarly, the E2/T ratio showed a significant positive correlation with rMEQ score (ρ = 0.338, P = 0.009), suggesting a relatively more estrogenic hormonal balance with increasing morning preference. Additionally, a strong inverse correlation was observed between testosterone concentration and E2/T ratio (ρ = −0.884, P < 0.001). No other significant correlations were identified among the studied variables (Table 2).
Table 2.
Spearman correlation matrix showing associations between rMEQ score, hormonal parameters, and clinical variables
| Variable | rMEQ score | Testosterone (nmol/L) | Estradiol (nmol/L) | E2/T ratio | BMI (kg/m²) | Age (years) | Cycle day |
|---|---|---|---|---|---|---|---|
| rMEQ score | 1.00 | - | - | - | - | - | - |
| Testosterone (nmol/L) |
−0.344 (0.008) |
1.00 | - | - | - | - | - |
| Estradiol (nmol/L) |
0.017 (0.897) |
0.093 (0.487) |
1.00 | - | - | - | - |
| E2/T ratio |
0.338 (0.009) |
−0.884 (< 0.001) |
0.303 (0.021) |
1.00 | - | - | |
| BMI (kg/m²) |
0.009 (0.944) |
−0.114 (0.392) |
0.027 (0.842) |
0.068 (0.612) |
1.00 | - | - |
| Age (years) |
−0.220 (0.097) |
0.127 (0.343) |
0.073 (0.587) |
−0.044 (0.741) |
−0.057 (0.673) |
1.00 | - |
| Cycle day |
0.033 (0.804) |
0.123 (0.356) |
−0.050 (0.712) |
−0.131 (0.327) |
−0.135 (0.314) |
0.046 (0.731) |
1.00 |
Values are Spearman’s correlation coefficients (p-values in parentheses). Bold values indicate statistical significance (P< 0.05)
Adjusted correlations
To account for potential confounding factors, partial Spearman correlations were performed between rMEQ score and both testosterone and the E2/T ratio, adjusting for age, BMI, and cycle day (Table 3). The negative association between rMEQ score and testosterone remained significant after adjustment (ρ = −0.336, P = 0.012). Likewise, the positive association between rMEQ score and E2/T ratio also remained significant (ρ = 0.346, P = 0.010). These findings indicate that the observed relationships between chronotype and hormonal parameters are independent of age, BMI, and menstrual cycle timing.
Table 3.
Adjusted correlations between rMEQ score and hormonal parameters
| Variable | Adjusted ρ | P-value |
|---|---|---|
| Estradiol (nmol/L) | 0.037 | 0.791 |
| Testosterone (nmol/L) | −0.336 | 0.012 |
| E2/T ratio | 0.346 | 0.010 |
Partial Spearman correlations adjusted for age, BMI, and cycle day. Bold values indicate statistically significant correlations (P< 0.05)
Discussion
This study provides new evidence linking chronotype score to gonadal hormone profiles in healthy young women from Saudi Arabia. Lower rMEQ scores, indicating greater eveningness tendency, were associated with higher serum testosterone concentrations and lower estradiol-to-testosterone (E2/T) ratio. These associations remained significant after adjustment for age, BMI, and menstrual cycle day, suggesting that chronotype-related variation in hormonal profile is independent of these potential confounding factors. In contrast, no significant association was observed between rMEQ score and estradiol concentrations.
The observed positive association between rMEQ score and E2/T ratio suggests that increasing morningness tendency may be associated with a relatively more estrogenic hormonal balance, whereas greater eveningness tendency may reflect a relatively more androgenic endocrine profile. In this context, the E2/T ratio may provide an integrated indicator of the balance between estrogenic and androgenic activity, potentially capturing subtle endocrine variation that may not be apparent when evaluating individual hormones independently [18].
Our findings align with emerging evidence suggesting associations between chronotype and reproductive hormonal regulation, although results remain heterogeneous across populations. Recent studies have also reported associations between chronotype and estradiol levels, indicating that circadian preference may influence estrogenic regulation in women [6]. Observational studies have reported that individuals with a greater eveningness tendency tend to exhibit higher testosterone levels, particularly in males, supporting a role for circadian preference in androgen regulation [19, 20]. In women, however, findings are less consistent, with some studies reporting higher testosterone levels in individuals with lower rMEQ scores [19, 21], while others found no significant association [9]. Additionally, earlier increases in estradiol concentrations have been observed in women with morning preference, suggesting a link between circadian timing and ovarian hormone rhythms [8]. Notably, studies examining the relationship between chronotype and the E2/T ratio are scarce. In this context, the present study adds novel evidence by demonstrating a lower E2/T ratio in individuals with lower rMEQ scores, indicating a relative shift toward androgenic dominance.
The observed associations may be explained by both behavioral and physiological mechanisms. Evening chronotypes are more likely to experience social jetlag, reduced sleep duration, and irregular light exposure, all of which can disrupt circadian alignment and affect hypothalamic–pituitary–gonadal axis function [22, 23]. Experimental studies have shown that circadian misalignment alters the pulsatile secretion of gonadotropin-releasing hormone and downstream gonadotropins, including luteinizing hormone and follicle-stimulating hormone, thereby influencing ovarian steroidogenesis [4]. In addition, altered melatonin secretion, a characteristic feature of circadian phase delay, may influence androgen synthesis through interactions with pituitary and adrenal pathways [5].
From a clinical perspective, elevated testosterone in females is a hallmark of polycystic ovary syndrome (PCOS), a condition associated with infertility, metabolic syndrome, and cardiovascular risk [24]. Although participants in the present study were healthy, the androgenic shift observed among individuals with lower rMEQ scores may reflect a subclinical hormonal pattern. Supporting this interpretation, previous studies have reported a higher prevalence of evening chronotypes among women with PCOS and hyperandrogenism [25]. These findings suggest a potential link between circadian timing and reproductive endocrine health.
Behavioral patterns common in Saudi Arabia, such as delayed sleep–wake schedules, daytime sleep, and prolonged nighttime screen exposure, may further contribute to circadian misalignment, particularly in young adults [26, 27]. These factors may amplify the association between chronotype and hormonal regulation. In this context, the present findings provide region-specific insight into the relationship between circadian preference and endocrine variability in young Saudi women.
An additional consideration is the potential influence of circadian phase differences between chronotypes. Evening chronotypes typically exhibit a delayed circadian phase; therefore, fixed morning sampling may capture different phases of hormonal secretion across individuals [9]. Consequently, the observed differences may partly reflect phase-related variation rather than absolute differences in hormone production. This highlights the importance of time-resolved or repeated hormonal measurements in future studies.
This study also highlights the potential clinical relevance of chronotype assessment. Given its non-invasive nature, the rMEQ may serve as a useful tool for evaluating circadian-related influences on endocrine function and hormonal variability in women. In addition, chronobiological interventions, such as optimization of light exposure and modification of sleep timing, may represent potential areas for future investigation aimed at improving circadian alignment and endocrine health [28].
However, several limitations should be considered. First, the cross-sectional design precludes causal inference regarding the relationship between chronotype and hormonal parameters. Second, hormonal measurements were obtained at a single time point and may not fully capture diurnal or intra-cycle hormonal variability, despite standardized sampling during the early follicular phase and morning collection period. In addition, lifestyle-related factors such as dietary habits, physical activity, sleep duration, and light exposure were not formally assessed and may have influenced both chronotype and endocrine function. The use of voluntary participation may also have introduced selection bias, and the size of the initially approached population was not recorded. Finally, the relatively small sample size may limit the generalizability of the findings to broader populations.
Conclusions
Lower rMEQ scores, indicating greater eveningness tendency, were associated with higher testosterone concentrations and a reduced estradiol-to-testosterone (E2/T) ratio in healthy young Saudi women, suggesting a relatively more androgenic hormonal profile. These findings indicate that variation in chronotype may be linked to differences in reproductive hormone balance. However, given the cross-sectional design, causal relationships cannot be established, and further longitudinal studies are required to confirm these associations and clarify their potential clinical implications.
Acknowledgements
The authors thank the participants and staff of Jazan University for their cooperation and support. The authors also acknowledge Editage for English language editing.
Authors’ contributions
RIS, BHAL, MYD, and RMB contributed to participant recruitment and data collection. MHM and MAJ contributed to laboratory analyses and methodological oversight. YAM and YAK conceptualized the study, supervised the project, and contributed to data interpretation. RIS and YAK drafted the manuscript. All authors read and approved the final manuscript.
Funding
No external funding was received for this study.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
This study was approved by the Jazan University Research Ethics Committee (approval No.: REC-47/04/1624). All participants provided written informed consent prior to participation. The study was conducted in accordance with the Declaration of Helsinki.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.