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Understanding the Variations of Reproductive Hormones: Implications for Diagnostic Accuracy
Reproductive hormones play a crucial role in the functioning of the human body, influencing various physiological processes and reproductive health. Recent studies have shed light on the complex nature of these hormones, particularly how their levels fluctuate throughout the day and in response to various factors. This article aims to elucidate these dynamics in a manner understandable to a high school graduate, focusing on the implications these variations have on the diagnostic accuracy of reproductive hormone levels.Diurnal Rhythm and Nutrient Intake Effects on Hormone Levels
1. The Impact of Diurnal Rhythm
Reproductive hormones are not constant throughout the day. They follow a diurnal rhythm, meaning their levels change depending on the time of day. This rhythm significantly affects the accuracy of hormone diagnostics. For instance, testosterone levels in men peak early in the morning, between 05:30 and 08:00 hours, and reach their lowest in the evening, from approximately 5:30 PM to 8:00 PM. Consequently, for accurate assessment of male hypogonadism, it's recommended to measure testosterone levels in the morning.2. Nutrient Intake and Hormonal Fluctuations
The intake of food also influences hormone levels. Testosterone levels, for example, can decrease for more than two hours after eating. This effect is more pronounced after a mixed meal, with a reduction of about 34.3% in testosterone levels, compared to a 9.5% reduction with ad libitum feeding.Pulsatile Secretion of Hormones
Many hormones, including reproductive ones, are released in both a basal tonic manner and a pulsatile fashion. This pulsatile release has significant implications:1. Luteinizing Hormone (LH) Pulses
Luteinizing hormone exhibits pulses lasting 60–90 minutes. Therefore, a single measurement of LH can vary greatly depending on the specific time it's taken during these pulse cycles.2. Variations in LH and FSH Secretion
The frequency of Gonadotropin-releasing hormone (GnRH) pulses can alter the balance between LH and Follicle-stimulating hormone (FSH) secretion. Increased GnRH pulse frequency leads to LH-dominant secretion, whereas reduced frequency results in FSH-dominant secretion. This variation means that single measurements of LH and FSH can be misleading.Comparative Variability of Reproductive Hormones
1. Daily Hormonal Variations
The initial morning value of reproductive hormones is typically higher than the daily average. For instance, the morning level of LH is about 18.4% higher than its daily mean.2. Variability Among Different Hormones
Among reproductive hormones, LH shows the highest variability (CV 28%), followed by sex-steroid hormones (testosterone 12% and estradiol 13%). In contrast, FSH is the least variable (CV 8%).Testosterone Levels in Healthy Men
In healthy men, testosterone levels decrease by approximately 14.9% between 9:00 AM and 5:00 PM. Interestingly, morning testosterone levels correlate with late afternoon levels, indicating that morning levels can predict afternoon levels to some extent.Conclusion
The dynamics of reproductive hormones are complex and influenced by factors like time of day, nutrient intake, and pulsatile secretion patterns. This variability has critical implications for the diagnostic accuracy of these hormones. Understanding these dynamics is essential for accurate diagnosis and effective treatment of reproductive health issues. Medical professionals and patients alike should be aware of these factors to ensure that hormone levels are measured and interpreted correctly.STUDY DETAILS
Objective
To quantify how representative a single measure of reproductive hormone level is of the daily hormonal profile using data from detailed hormonal sampling in the saline placebo-treated arm conducted over several hours.
Design
Retrospective analysis of data from previous interventional research studies evaluating reproductive hormones.
Setting
Clinical Research Facility at a tertiary reproductive endocrinology center at Imperial College Hospital NHS Foundation Trust.
Patients
Overall, 266 individuals, including healthy men and women (n = 142) and those with reproductive disorders and states (n = 124 [11 with functional hypothalamic amenorrhoea, 6 with polycystic ovary syndrome, 62 women and 32 men with hypoactive sexual desire disorder, and 13 postmenopausal women]), were included in the analysis.
Interventions
Data from 266 individuals who had undergone detailed hormonal sampling in the saline placebo-treated arms of previous research studies was used to quantify the variability in reproductive hormones because of pulsatile secretion, diurnal variation, and feeding using coefficient of variation (CV) and entropy.
Main Outcome Measures
The ability of a single measure of reproductive hormone level to quantify the variability in reproductive hormone levels because of pulsatile secretion, diurnal variation, and nutrient intake.
Results
The initial morning value of reproductive hormone levels was typically higher than the mean value throughout the day (percentage decrease from initial morning measure to daily mean: luteinizing hormone level 18.4%, follicle-stimulating hormone level 9.7%, testosterone level 9.2%, and estradiol level 2.1%). Luteinizing hormone level was the most variable (CV 28%), followed by sex-steroid hormone levels (testosterone level 12% and estradiol level 13%), whereas follicle-stimulating hormone level was the least variable reproductive hormone (CV 8%). In healthy men, testosterone levels fell between 9:00 AM and 5:00 PM by 14.9% (95% confidence interval 4.2, 25.5%), although morning levels correlated with (and could be predicted from) late afternoon levels in the same individual (r2 = 0.53, P<.0001). Testosterone levels were reduced more after a mixed meal (by 34.3%) than during ad libitum feeding (9.5%), after an oral glucose load (6.0%), or an intravenous glucose load (7.4%).
Conclusion
Quantification of the variability of a single measure of reproductive hormone levels informs the reliability of reproductive hormone assessment.
Diagnosis of most reproductive disorders includes assessment of reproductive hormone levels; however, because of practical limitations, a single measure of reproductive hormone levels is often used, often with confirmation on a second occasion in borderline cases (1). Notably, reproductive hormones vary during the day because of diurnal rhythm (2), pulsatile secretion (3), and nutrient intake (4), which has significant implications for the diagnostic accuracy of reproductive hormones. However, there is a scarcity of reports quantifying the variability of reproductive hormones to inform the accuracy of these measures for the diagnosis of reproductive disorders.
Many hormones exhibit both basal tonic secretion and pulsatile release (3, 5). For instance, the periodic release of gonadotropin-releasing hormone (GnRH) causes temporally-coupled luteinizing hormone (LH) release on the background of tonic LH secretion (6). Luteinizing hormone pulses typically have a duration of 60–90 minutes (6), and thus a single LH level will vary depending on the time point during the pulse cycle at which the measure is taken. Additionally, LH pulsatility may be altered in certain conditions, e.g., increased in polycystic ovary syndrome (PCOS) and decreased in functional hypothalamic amenorrhea (FHA) (7). Increased GnRH pulse frequency favors LH-predominant secretion, whereas reduced GnRH pulse frequency favors follicle-stimulating hormone (FSH)-predominant secretion. Therefore, a single LH and FSH level could vary with the number of pulses during the day; however, to date, there is only limited data describing the relationship between baseline reproductive hormone levels and LH pulsatility.
For the diagnosis of male hypogonadism, morning testosterone levels are recommended because testosterone is recognized to have diurnal variation, with levels peaking between 05:30 and 08.00 hours and reaching a nadir from approximately 5:30 PM to 8:00 PM (2). Indeed, up to 30% of men with a low testosterone level on the first measurement taken during the day demonstrate value within the reference range on a repeat measurement in the morning (8). Further, it is recommended to measure testosterone levels in the fasted state (1) because feeding can decrease serum testosterone concentrations for >2 hours (9, 10).
Quantification of the variability in reproductive hormone levels would therefore enable a more precise assessment of how closely they reflect the hormonal profile during the day, with relevance for the diagnosis of reproductive disorders and states. Here, we used data from detailed hormonal sampling conducted over several hours from 13 research studies (11–20) comprising 266 individuals to quantify how representative a single measure of reproductive hormone level is of the daily hormonal profile and to assess the impact of pulsatile secretion, diurnal rhythm, and nutrient intake.
RESULTS
The summary statistics for each cohort are presented for LH in Table 1, FSH in Table 2, and testosterone and estradiol in Supplemental Table 2 (21). The initial morning values of reproductive hormones were typically higher than the mean value throughout the day (percentage decrease: LH 18.4%, FSH 9.7%, testosterone 9.2%, and estradiol 2.1%) (Supplemental Tables 3 to 5).
Determining overall variability in the daily reproductive hormone profile
The variability of reproductive hormone levels was quantified using CV and entropy. Across the entire cohort (both men and women), FSH level was the least variable reproductive hormone (CV 8%) (Table 2), followed by sex-steroid hormone (testosterone 12% and estradiol 13%) levels (Supplemental Table 2) (21), whereas LH level was the most variable (CV28%) (Table 1). Testosterone levels were highly variable and pulsatile in some individuals, with levels varying by as much as 15 nmol/L within 30 minutes (Supplemental Fig. 1, available online) (21). Women with FHA had the highest entropy for LH (1.31–1.32 nats) (Table 1) and FSH (0.80 nats) (Table 2). Healthy men had consistently high entropy for sex-steroid hormones between 0.79 and 0.87 nats (Supplemental Table 2) (21). Estradiol had the highest entropy (0.79–0.92 nats) in healthy women and women with FHA, and the lowest entropy (0.07 nats) in postmenopausal women, where estradiol levels were low and undetectable( Supplemental Table 2) (21). Similarly, postmenopausal women also had the lowest entropy for LH (0.72 nats) (Table 1) and FSH (0.38 nats) (Table 2).
Diurnal variation in reproductive hormones
To assess diurnal variation, the percentage change of LH, FSH, and testosterone levels (from 9:00 AM to 5:00 PM) was analyzed in healthy men. Testosterone levels fall during the day, with other studies conducted over a 24-hour interval indicating that the nadir for testosterone levels is detected between 07:00 PM and 9:00 PM in young men. Thus, testosterone levels had yet to reach their nadir by the end of the sampling period, and thus we used the latest value available at 5:00 PM within the limits of the duration of the sampling conducted. Baseline 9:00 AM values of LH (P= .011), FSH (P<.0001), and testosterone (P =.0043) were significantly higher than their respective 5:00 PM measures (Fig. 1A to C). The largest percentage decrease in testosterone levels compared with the baseline value was 14.9% (7.7 hours after the 9:00 AM baseline value) (Fig. 1C). Notably, baseline testosterone levels were significantly correlated with the testosterone levels at 5:00 PM (r2=.53, P<.0001) (Fig. 1D). The pattern of decline in both LH and testosterone levels likely indicates evidence of diurnal variation (Fig. 1A and C), although not in FSH levels, which fell within the first hour after sampling and then remained relatively steady (Fig. 1B).
A small but significant change in LH levels between the baseline value and the 5:00 PM value was also observed in healthy women (P=.037) (Supplemental Fig. 2A) (21). However, there was no significant change in either FSH or estradiol levels (Supplemental Fig. 2B and C) (21).
Effect of nutrient interventions on LH, FSH, and testosterone levels
The effects of four nutrient-intake interventions—ad libitum feeding, MM, OGL, and IVGL—on LH, FSH, and testosterone levels over a 1.25-hour monitoring period were examined in healthy men.
The median nadir of LH level was 26.4% at 0.75 hours after ad libitum feeding, 24.0% at 1.25 hours after an MM, 21.2% at 1 hour after an OGL, and 16.7% at 0.67 hours after an IVGL (Fig. 2A). There was no significant difference in the change in LH levels between these interventions (Fig. 2B).
The median nadir of FSH level was 13.4% at 1.25 hours after ad libitum feeding, 6.1% at 1 hour after a MM, 4.9% at 0.5 hours after an OGL, and 5.4% at 0.23 hours after an IVGL (Fig. 2C). Percentage FSH level changes after ad libitum feeding was greater after a MM (P=.0052), OGL (P=.0051), or IVGL (P=.0018) (Fig. 2D)
Healthy men had the greatest fall in testosterone levels after a MM (median nadir 34.3% at 0.5 hours after ingestion) compared with 9.5% during ad libitum feeding at 1.25 hours, 6.0% after OGL at 1.25 hours, and 7.4% at 0.23 hours after IVGL (Fig. 2E and F).
Diurnal variation
*In the present study, we found evidence of a diurnal fall in both testosterone and LH levels in healthy men, with baseline (9:00 AM) testosterone levels being correlated with that at 5:00 PM in each man. The initial 12.5% drop in FSH levels occurred within the first hour, followed by relatively steady levels, suggesting that this fall could be more consistent with a stress response e.g., because of cannulation rather than a diurnal effect.
Nutrient intake
In the present study, an MM resulted in the largest negative impact on testosterone levels, with a median fall of 34.3%at 0.5 hours after ingestion. This was greater than those observed after ad libitum feeding (9.5%), OGL (6.0%), or IVGL (7.4%), indicating that other components of nutrient intake aside from glucose, such as fat (38, 39) or protein(40), contribute to the fall in testosterone levels. These results corroborate those of a randomized control trial whereby ingestion of protein caused a greater decline in serum testosterone levels than glucose levels (4). A further report also found that MM decreased serum testosterone levels by 26%, whereas OGL decreased by only 18% (9). One proposed theory is that ingestion of amino acids such as leucine causes upregulation of androgen receptors, increasing muscle tissue uptake of testosterone, and consequently decreasing serum testosterone levels (4).
It is feasible that oral ingestion can stimulate gut hormone secretion, with protein and fat components recognized to increase postprandial rises of gastrointestinal incretin hormones such as gastric inhibitory peptides more than glucose load (41). However, recent studies were unable to demonstrate that glucagon-like peptide 1 (14), peptide YY (42), or glucagon(43) had acute effects on reproductive hormone secretion in healthy men. Another proposed mechanism is due to a rise in insulin inhibiting the hypothalamic-pituitary-gonadalaxis and decreasing testosterone production (44), although this is inconsistent with the lesser impact of an IVGL than an MM. The constancy of LH and FSH levels in the fed and fasted states could indicate that the effect of nutrient intake could be mediated via testicular sensitivity to gonadotropins rather than central suppression of pituitary LH secretion, although the absence of a compensatory increase in LH levels in response to decreased testosterone levels could suggest a central component.
Pulsatility
LH pulsatility is a sensitive marker of gonadal status, being altered in several endocrine disorders (45). A significant relationship between baseline LH levels and LH secretion rate was present in our female cohorts. Postmenopausal women displayed a positive relationship between FSH level predominance and LH secretion rate, consistent with increased GnRH pulsatility secondary to a lack of estradiol-induced negative feedback (46); however, the inverse was true in premenopausal women.
Strengths of this study include a comprehensive assessment of variability in hormonal profiles (not usually conducted in clinical practice) of a relatively large sample of both healthy individuals and those with reproductive disorders and states. The hormonal assays were conducted in the same laboratory with frequent quality controls, minimizing variability because of the use of different assays between studies. The use of entropy and bootstrapping of 95% confidence intervals increased the robustness of the assessment . Limitations include the retrospective study design, different. sampling intervals, and durations between some cohorts. Because of the small number of individuals with specific reproductive conditions, such as PCOS, additional data are needed to fully capture whether there are differences in the viability of reproductive hormone levels in women with different PCOS phenotypes. Therefore, future studies with larger subgroup sizes could be more conclusive. The sampling after nutrient-intake interventions was of short duration; therefore, the full effect of nutrient-intake may not have been captured. All feeding studies were conducted only in healthy men; however, the ad libitum group did not have any constraints around feeding time or amount and were not the same individuals who had dedicated feeding interventions, which could result in some residual unidentified confounding.
CONCLUSIONS
Collectively, we have quantified the variability in reproductive hormone levels over the day in both healthy men and women, as well as those with reproductive disorders. These data could inform the timing, frequency, and reliability of hormonal assessments when evaluating patients in different reproductive states. Further, these data can inform guideline writers regarding the reliability of a single measure of reproductive hormones in the assessment of hypogonadism.
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