Environmental Perturbation of the Circadian Clock Disrupts Pregnancy in the Mouse

The circadian clock has been linked to reproduction at many levels in mammals. Epidemiological studies of female shift workers have reported increased rates of reproductive abnormalities and adverse pregnancy outcomes, although whether the cause is circadian disruption or another factor associated with shift work is unknown. Here we test whether environmental disruption of circadian rhythms, using repeated shifts of the light:dark (LD) cycle, adversely affects reproductive success in mice.Young adult female C57BL/6J (B6) mice were paired with B6 males until copulation was verified by visual identification of vaginal plug formation. Females were then randomly assigned to one of three groups: control, phase-delay or phase-advance. Controls remained on a constant 12-hr light:12-hr dark cycle, whereas phase-delayed and phase-advanced mice were subjected to 6-hr delays or advances in the LD cycle every 5–6 days, respectively. The number of copulations resulting in term pregnancies was determined. Control females had a full-term pregnancy success rate of 90% (11/12), which fell to 50% (9/18; p<0.1) in the phase-delay group and 22% (4/18; p<0.01) in the phase-advance group.Repeated shifting of the LD cycle, which disrupts endogenous circadian timekeeping, dramatically reduces pregnancy success in mice. Advances of the LD cycle have a greater negative impact on pregnancy outcomes and, in non-pregnant female mice, require longer for circadian re-entrainment, suggesting that the magnitude or duration of circadian misalignment may be related to the severity of the adverse impact on pregnancy. These results explicitly link disruptions of circadian entrainment to adverse pregnancy outcomes in mammals, which may have important implications for the reproductive health of female shift workers, women with circadian rhythm sleep disorders and/or women with disturbed circadian rhythms for other reasons

Caloric intake data from experiment 1.

<p>Standard deviation (StDev) and standard error (<i>StErr</i>) of Desynchronized Feeding (DF) and Synchronized Feeding (SF).</p

Caloric intake data from experiment 2.

<p>Standard deviation (StDev) and standard error (<i>StErr</i>) of Desynchronized Feeding (DF) and Synchronized Feeding (SF).</p

Summary cartoon depicting the results of experiment 3.

<p><i>Ob/ob</i> mice fed in the light phase and receiving leptin in the dark phase (DF-DL) gain the most amount of weight over the 2-week period. Conversely, <i>ob/ob</i> mice fed in the dark and receiving leptin in the dark (SF-SL) gain the least amount of weight. Of the intermediate groups, (SF-DL and DF-SL), <i>ob/ob</i> mice feeding during the light phase gain more weight. Cheeseburgers represent the availability of the high fat diet. Blue lines represent plasma leptin levels within the groups.</p

Phase delays or advances after copulation reduce the proportion of pregnancies carried to term.

<p>(A) After copulation was verified via identification of vaginal plugs, mice were randomized into control (<i>n</i> = 12), phase-delay (<i>n</i> = 18) or phase-advance groups (<i>n</i> = 18). Zero to four days after copulation, mice were transferred to new light-tight cabinets, each with 12-hr light and 12-hr dark light cycles, but differing in the time of light onset and offset. Control females remained in this chamber on a constant 12∶12 light (yellow bar):dark (black bar) cycle that matched the preceding one during mating, whereas females in the experimental groups were exposed to either 6-hour delays or advances in the light cycle, which was repeated by switching cabinets every 5–6 days for the duration of gestation. (B) The number of copulations successfully carried to term in each of the groups was recorded via daily visual inspection. Data comparisons were made using Pearson’s chi-square test (Phase delays: χ² = 3.41, <i>P</i><0.1; * Phase advances: χ² = 9.47, <i>P</i><0.01).</p

Body weight gains of <i>ob/ob</i> mice administered two different doses of leptin or saline over 7 days.

<p>The low-dose of leptin injection (100 ug/kg/day) was sufficient to reduce body weight in <i>ob/ob</i> mice (dotted line) compared to the saline control (solid line). The high-dose of leptin injection (1 mg/kg/day) greatly reduced weight gain (dashed) compared to saline control.</p

Circulating plasma leptin levels hours after low-dose leptin injection.

<p>After the 11^th 100 ug/kg/day injection, circulating leptin was found to be absent from the plasma of <i>ad libitum</i> fed animals within 4–6 hours regardless of whether the injection occurred during the circadian light phase at ZT 5 (dotted line) or during the circadian dark phase at ZT 17 (solid line).</p

Body weights of <i>ob/ob</i> mice fed <i>ad libitum</i> and treated with a once daily low-dose leptin injection.

<p>When <i>ob/ob</i> mice are allowed free access to food, a single 100 ug/kg/day leptin injection during the circadian light at ZT 5 (dotted line) or during the circadian dark phase at ZT 17 (solid line) does not result in differential weight gain.</p

Experiment 2: Body weight gain and total activity in <i>ob/ob</i> mice treated with constant leptin during desynchronized feeding.

<p>(<b>A</b>) Body weight gain over the two-week period. No significant differences were observed in weight gain in <i>ob/ob</i> mice treated with constant non-rhythmic leptin. (<b>B</b>) Total activity over the two-week period as measured by IR-beam breaks. No significant differences were observed in total cumulative activity between DF and SF groups. While not statistically significant, the SF group appears to be more active during the circadian dark phase as compared to DF group.</p

Caloric intake data from experiment 3.

<p>Standard deviation (StDev) and standard error (StErr) of Desynchronized Feeding with Desynchronized Leptin (DF-DL), Desynchronized Feeding with Synchronized Leptin (DF-SL), Synchronized Feeding with Desynchronized Leptin (SF-DL), Synchronized Feeding with Synchronized Leptin (SF-SL).</p