LAUSR

Are molecular clock genes (MCGs) expressed rhythmically in mouse placenta, and whether maternal circadian rhythm disruption (MCRD) is associated with intrauterine growth retardation (IUGR) through disturbing rhythmic expression of MCGs? Maternal circadian disruption causes impaired rhythmic expression of MCGs (Bmal1, Clock, Npas2, Per1, Per2, Per3, Cry1, and Cry2) and IUGR during placenta development in mice. The world economy is based on a 24/7 society and shift work or jet travel across time zones disrupts circadian rhythm in pregnant women. Evidence indicates that gestational chrono-disruption results in IUGR. Mature mouse and human placenta express MCGs. There is no information in the literature on whether the MCG expression in the placenta is rhythmic or not and whether the rhythmic expression of MCGs is impaired due to MCRD during pregnancy. Also, it is not known whether the association with MCRD and IUGR is related to MCGs. Young adult female BALB/c mice were paired with males until vaginal plug formation was verified. Females were randomly assigned to two groups: control and phase-advance. Controls remained on a constant 12-hr light:12-hr dark cycle, whereas phase-advanced mice were subjected to 6-hr advances in the LD cycle every 5 days. Placentae (n = 1329) and fetuses were obtained from 144 mice at Zeitgeber time (ZT)0, ZT6, ZT12, and ZT18 days 12, 14, and 16 of pregnancy. The following analysis was performed: (i) open field test was used for locomotor activity evaluations to confirm MCRD, (ii) placenta/fetus weight ratio for evaluation of IUGR development, (iii) morphometric evaluation of placental compartments utilizing H&E staining (iv) gene expression analysis of MCGs utilizing qRT-PCR. One-way and Two-way ANOVA test followed by Holm-Sidak posthoc test was used for multiple comparisons. Values are expressed as mean ± standard error, and values below p < 0.05 were considered statistically significant. Expression of MCGs (Bmal1, Clock, Npas2, Per1, Per2, Per3, Cry1, and Cry2) was rhythmic in the early and mature placenta development stages (days 12, 14, 16). Locomotor activity tests reveal that the total distance covered on the 16th day of pregnancy significantly decreased compared to the control group (p = 0.000158). The ratio of the time spent in the outer/inner quadrant, an anxiety indicator, significantly increased in the MCRD group on the 14th (p = 0.0351) and 16th days of pregnancy (p = 0.000329). While the number of fetuses was similar in both groups for all gestational days (p = 0.896), in the MCRD group, the fetus/placenta weight ratio decreased significantly on the 12th and 16th days of pregnancy (p < 0.001). Thus, IUGR developed due to MCRD. Histomorphometry analysis of the placental compartments revealed a significant reduction in the spongiotrophoblast layer’s size on all days of pregnancy and the labyrinth layer on day 16 (p < 0.05). Finally, the rhythmic expression MCGs were impaired in placentas obtained from MCRD groups on days 12th, 14th, 6th of pregnancy (p < 0.001). In conclusion, we found a robust relationship with the disturbed MCGs expression and occurrence of IUGR during a chrono-disrupted gestation. Since this study was conducted in mice, care should be taken when translating the results to humans. Wider implications of the findings: Our results in mice are important for initiating basic science knowledge regarding the outcomes of maternal chrono-disruption. Moreover, research in the placenta of gestational chrono-disrupted mothers, such as shift-workers, are urgently needed to translate our findings into the clinic. TUBITAK–119S121 and Akdeniz University Research Projects Unit TYL–2018–3960