Light exposure during night work suppresses melatonin production, and night work has been associated with an increased cancer risk. There is little information, however, about the interrelationships of night work, urinary melatonin levels, and levels of plasma steroid hormones in women. We examined the reproducibility of morning urinary measurements of 6-sulfatoxymelatonin over a 3-year period in 80 premenopausal women. We assessed correlations between average urinary melatonin and plasma steroid hormone levels and evaluated potential associations between night work and hormone levels, using current and long-term shift work information from two large, prospective cohorts, the Nurses' Health Study cohorts. The intraclass correlation for creatinine-adjusted 6-sulfatoxymelatonin was 0.72 (95% confidence interval, 0.65, 0.82). We found significantly increased levels of estradiol after longer durations of night work (geometric mean levels of estradiol, 8.8 pg/mL for women who never worked night shifts versus 10.1 pg/mL for women who worked 15 or more years of night shifts; P for trend = 0.03). We observed a significant inverse association between increasing number of nights worked within the 2 weeks preceding urine collection and urinary melatonin levels (r = −0.30, P = 0.008), but no association of recent night work with estradiol (r = 0.10, P = 0.41). A single morning urinary melatonin measurement is a reasonable marker for long-term melatonin levels among premenopausal women. Women who work on rotating night shifts seem to experience changes in hormone levels that may be associated with the increased cancer risk observed among night-shift workers.

A spot morning urine sample was collected an estimated 7 to 9 days before the anticipated start of the next menstrual cycle (luteal phase) in all three instances, and urine samples were timed (nurses provided information on the onset of their period following urine collection). The samples were then returned by overnight mail, with a frozen water bottle to keep them cool. On arrival in the laboratory, the samples were aliquoted into labelled cryotubes of 4.5 mL volume each. Without the addition of any preservative, all of the tubes were then stored in liquid nitrogen freezers.

Each woman was sent a questionnaire on which to record the dates of collection of the urine sample. In addition, information was gathered on the numbers of nights worked in the 2 weeks before urine collection, the participant's current weight, and other lifestyle variables. Information on more detailed lifestyle factors associated with health, including reproductive history, were available for each participant from the NHS II questionnaires completed in 1997.

We conducted a small pilot study to investigate whether levels of urinary aMT6-s would remain stable when processing was delayed for 24 and 48 hours. Fifteen premenopausal women from the greater Boston area were recruited, and a first morning spot urine sample from the luteal phase of their menstrual cycle was sampled for each. The unpreserved samples were split (to assess laboratory reproducibility) and processed immediately or exactly as described above (which included simulating transport in a cooler with a frozen water bottle). In this pilot, there was significant batch-to-batch variation; batch-wise within-batch coefficients of variation for aMT6-s splits were 10.6%, 15.3%, and 19.0%.

Previous reports on the effects of transport of whole blood on several analytes have shown that overnight shipping is feasible. In this study, we found that delayed processing of urine samples also had only a modest effect on aMT6-s levels, with slightly lower levels of aMT6-s associated with delayed processing. When measured 3 years apart, the ICC for creatinine-adjusted aMT6-s was 0.72 in a sample of premenopausal women. To our knowledge, this is the first report assessing aMT6-s reproducibility among premenopausal women. The ICCs observed in this study are comparable in magnitude to those of a number of other exposure measures commonly used in epidemiology and indicate that a single morning spot urine sample can reasonably represent aMT6-s levels over at least a 3-year period. Thus, first spot morning urine melatonin measurements are a feasible biomarker in observational studies.

The hypothalamus and the hypophysis constitute a complex axis that regulates a variety of circulating steroid hormone levels through feedback loops. It is unclear, however, to what extent, if any, melatonin, a hormone produced in the pineal gland, is involved in these feedback loops. The observation of an increased breast cancer risk among women exposed to light at night has fueled speculations about reduced melatonin levels among night workers and an inverse correlation between melatonin and estrogen production. The results of our study revealed several interesting associations and shed new light on these hypotheses.

We found recent night work (within the last 2 weeks) to be associated with a 56% significant reduction of urinary aMT6-s, and aMT6-s levels were inversely associated with levels of bioavailable estradiol. There was no direct association between recent night work and estrogen levels; long-term night work, however, did seem to increase estrogen levels among postmenopausal women. We further noted a positive association between aMT6-s and progesterone levels, which is novel and needs further evaluation.

A potential limitation of our study is that urine collections were done during a woman's luteal phase. We did not observe important variations of aMT6-s levels within the luteal phase. According to a previous report, the correlation of aMT6-s excretion with plasma melatonin is higher during the follicular phase (r = 0.89) than during the luteal phase (r = 0.73) of a woman's cycle. However, these data need to be confirmed before any conclusions can be drawn about the optimal timing of urine collection for aMT6-s studies in premenopausal women. We also cannot rule out the possibility of individual susceptibility to the effects of exposure to light at night that we were not able to account for. For example, those with lower baseline melatonin levels may be more susceptible to the additional effects of light exposure at night in lowering their melatonin levels. Finally, season of the year is known to affect the increase of melatonin. However, we were able to take into account the effects of season of the year in our analyses and did not detect a significant influence.

Our results provide some evidence for a potential link between night-shift work (a surrogate for exposure to light) and melatonin suppression, including possible long-term effects of night work on circulating estrogen levels. Further studies are needed to determine the optimal timing within the menstrual cycle to measure urinary aMT6-s among premenopausal women. Future research should address individual susceptibility and the question whether women with lower melatonin levels choose to do night work more often than do women with higher melatonin levels. Finally, associations between melatonin levels and breast cancer risk need to be evaluated.

In summary, our data support a potential association between night work and cancer risk through the melatonin pathway. Breast cancer, in particular, may be influenced by relations between melatonin and estrogen levels.