Environmental perturbation of the circadian clock disrupts pregnancy in the mouse

Abstract

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Figure 1. Phase delays or advances after copulation reduce the proportion of pregnancies carried to term.

(A) After copulation was verified via identification of vaginal plugs, mice were randomized into control (n = 12), phase-delay (n = 18) or phase-advance groups (n = 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, P<0.1; * Phase advances: χ² = 9.47, P<0.01).

Figure 2. Mice entrain more rapidly after phase delays than phase advances.

(A) Representative double-plotted actigrams from individually housed C57BL/6J females subjected to 6-hour phase advances (left) and phase delays (right) every 5–6 days. Time (48-hours) is shown on the x-axis and days are ordered sequentially down the y-axis. Each black tick mark indicates the break of an infrared beam within the animal’s cage. Arrows denote days when phase shifts occurred. (B) The number of days (mean ± s.e.m.) required for re-entrainment after a phase shift (for each group, n = 20 (5 animals×4 shifts)). Mice exposed to phase delays achieved entrainment to the new light cycle more rapidly than those exposed to phase advances (** P<0.001, * P<0.005; Student’s t-test).