Molecular and behavioral timekeeping is regulated by the circadian system which includes the brain's suprachiasmatic nucleus (SCN) that translates environmental light information into neuronal and endocrine signals aligning peripheral tissue rhythms to the time of day. Despite the critical role of circadian rhythms in fertility, it remains unexplored how circadian rhythms change within reproductive tissues during pregnancy. To determine how estrous cycle and pregnancy impact phase relationships of reproductive tissues, we used PER2::Luciferase (PER2::LUC) circadian reporter mice and determined the time of day of PER2::LUC peak (phase) in the SCN, pituitary, uterus, and ovary. The relationships between reproductive tissue PER2::LUC phases changed throughout the estrous cycle and late pregnancy and were accompanied by changes to PER2::LUC period in the SCN, uterus, and ovary. To determine if the phase relationship adaptations were driven by sex steroids, we asked if progesterone, a hormone involved in estrous cyclicity and pregnancy, could regulate Per2‐luciferase expression. Using an in vitro transfection assay, we found that progesterone increased Per2‐luciferase expression in immortalized SCN (SCN2.2) and arcuate nucleus (KTAR) cells. In addition, progesterone shortened PER2::LUC period in ex vivo uterine tissue recordings collected during pregnancy. As progesterone dramatically increases during pregnancy, we evaluated wheel‐running patterns in PER2::LUC mice. We confirmed that activity levels decrease during pregnancy and found that activity onset was delayed. Although SCN, but not arcuate nucleus, PER2::LUC period changed during late pregnancy, onset of locomotor activity did not correlate with SCN or arcuate nucleus PER2::LUC period.

Previous studies have observed an association between maternal circadian rhythm disruption and preterm birth (PTB). However, the underlying molecular mechanisms and the potential of circadian clock genes to serve as predictors of PTB remain unexplored. We examined the association of 10 core circadian transcripts in maternal blood with spontaneous PTB (sPTB) vs term births using a nested case–control study design. We used a public gene expression dataset (GSE59491), which was nested within the All Our Babies (AOB) study cohort in Canada. Maternal blood was sampled in trimesters 2–3 from women with sPTB (n = 51) and term births (n = 106), matched for 5 demographic variables. In 2nd trimester maternal blood, only CLOCK and CRY2 transcripts were significantly lower in sPTB vs term (p = 0.02 ~ 0.03, FDR < 0.20). A change of PER3 mRNA from trimesters 2 to 3 was significantly associated with sPTB (decline in sPTB, p = 0.02, FDR < 0.20). When CLOCK and CRY2 were modeled together in 2nd trimester blood, the odds ratio of being in the low level of both circadian gene transcripts was greater in sPTB vs term (OR = 4.86, 95%CI = (1.75,13.51), p < 0.01). Using GSVA and Pearson correlation, we identified 98 common pathways that were negatively or positively correlated with CLOCK and CRY2 expression (all p < 0.05, FDR < 0.10). The top three identified pathways were amyotrophic lateral sclerosis, degradation of extracellular matrix, and inwardly rectifying potassium channels. These three processes have previously been shown to be involved in neuron death, parturition, and uterine excitability during pregnancy, respectively.

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Molecular and behavioral timekeeping is regulated by the circadian system represented on the cellular level by clock transcription factors, including Period2 (PER2), Bmal1, Clock and Cry. These transcription factors drive a daily ~24h rhythm in gene expression leading to tissue specific receptor expression optimizing sensitivity to hormones and drugs dependent on the time of day. To synchronize circadian rhythms in the body to the time of day, the brain’s suprachiasmatic nucleus (SCN) translates environmental light information into neuronal and endocrine signals allowing circadian rhythm synchrony. Despite the critical role of circadian rhythms in fertility, it remains unknown how circadian rhythms change within reproductive tissues during pregnancy, and how these adaptations might impact drug efficacy. Monitoring wheel-running patterns in circadian PER2::luciferase (PER2::LUC) reporter mice, we confirmed that pregnancy is associated with reductions of activity and identified a correlation between delayed onset of behavioral activity during late pregnancy and ex vivo SCN PER2::LUC period, whereas no correlation between arcuate nucleus PER2::LUC rhythms was identified. The time of day of peak PER2::LUC expression provides a time-stamp as to the circadian phase of a tissue. Pregnancy impacted circadian synchrony in the reproductive axis, with the most dramatic change within the uterus. To understand how circadian rhythms responded to input during pregnancy, we performed a pharmacology study and found that circadian rhythm generation in the uterus responded differentially to hormones regulating pregnancy and labor depending on gestational age and the time of day of drug administration. To test for functional changes in uterine contraction capacity, we recorded ex vivo uterine contractions. Our preliminary data reveal a circadian change in uterine function in the mouse, which impacted labor regulating drug efficacy depending on the time of day. Together our data show that pregnancy is associated with behavioral changes in locomotor activity patterns, as well as adaptations in phase-relationships in reproductive tissues. Our chrono-pharmacology study indicates that a better understanding of labor-regulating drug efficacy can potentially allow increased efficacy of currently used drugs in the clinic to both induce labor as well as halt preterm labor.

Patients experiencing chronic and/or recurrent pain show a correlative decline in health and lifespan. In turn, declining health and sensory effects may contribute to persistent pain and poor response to analgesia. We therefore hypothesized that dietary alterations to improve health and stress reduction by mating may reduce chronic pain in male sickle mice. Methods. We used male HbSS-BERK (sickle) mice, which show hyperalgesia as compared to and HbAA-BERK (control) mice (Kohli et al., Blood 2010). Breeders and pups up to 12 wks of age were fed Sickle Mouse Diet (59M3, TestDiet) and 18% Protein Rodent Diet (2018, Harlan), thereafter, called ‘sickle’ and ‘rodent’ diet. Sickle diet contained 26.4% Protein, 11.1% fat and 27.5% and 26% kcal/g of each, respectively. Rodent diet contained 18.6% protein and 6.2% fat, and 24% and 18% kcal/g of each, respectively. Vitamins were about 2-fold higher in sickle as compared to rodent diet. Sickle mice showing hyperalgesia were recruited and treated as follows: [A] Rodent diet without mating (RD/M-); [B] Sickle diet with mating (SD/M+); [C] Rodent diet with mating (RD/M+); [D] Sickle diet without mating (SD/M-) and [E] mice on SD and mating for 4 weeks, were deprived of SD and mating and fed RD. Control mice which do not show hyperalgesia were fed RD without mating. Sensory testing was performed at baseline (BL) at recruitment and weekly, to evaluate mechanical hyperalgesia with von Frey filaments, thermal hyperalgesia in response to heat/cold and grip force for musculoskeletal/deep tissue hyperalgesia. Following the treatments, release of cytokines from skin biopsies was analyzed by cytokine arrays as described by us earlier (Vincent et al., Blood 2013), and spinal cords were analyzed for nociceptive signaling. Results. We did not observe difference in the body weight of mice between different groups at any time. White blood cell counts and spleen weight were significantly increased in group E following the withdrawal of SD and mating as compared to group C on SD/M+ (p<0.05), but the liver and kidney did not show any difference amongst groups. Groups B and C involving mating with SD/M+ or RD/M+, respectively, showed maximum decrease in mechanical, and thermal hyperalgesia following 3 weeks of treatment as compared to baseline (p<0.05 and 0.0001 for mechanical, p<0.001 and 0.0001 for thermal, respectively for B and C), suggesting that mating decreases sensitivity to noxious stimuli including touch and temperature. In group E (mice were in mating and with SD for 4 wks), withdrawal of sickle diet and mating for 4 weeks, led to a significant increase in thermal hyperalgesia as compared to the start of the withdrawal (p<0.05 for mechanical and heat). Group D (SD/M-) showed a significant decline thermal hyperalgesia one week after starting the sickle diet as compared to BL (p<0.0001 for cold and <0.005 for heat). Thus sickle diet in the absence of mating reduced hyperalgesia, but the reduction with sickle diet only (SD/M-) was significantly less than the reduction caused by mating (RD/M+) or by sickle diet with mating (SD/M+), suggesting that mating is more effective than diet in ameliorating pain. None of the groups demonstrated a decrease in grip force, suggesting that deep tissue pain was not influenced or that the strength of mice was not influenced and the influence is perhaps restricted to the periphery (skin). Cytokine profile of the skin showed a significant decrease in IL6, MIP-1a, RANTES, MCP-1 and GM-CSF in group B, (SD/M+) as compared to group A, (RD/M-), indicating that the diet and mating decrease inflammation in the periphery, thus reducing the activation of peripheral nerve fibers, resulting in reduced hyperalgesia. Consistent with reduced inflammation, spinal cords of group B (SD/M+) showed a significant decrease in nociceptive signaling of phosphorylation of Stat3 pathway as compared to group A (RD/M-). Since, Stat3 is associated with transcription of inflammatory cytokines, it likely reduces the inflammatory response in the spinal cord and inflammatory pain. These data suggest that high protein/high fat diet and mating reduce inflammation and hyperalgesia in sickle mice. Stress appears to be a critical factor in the perception of pain, because mating attenuated hyperalgesia. Thus, improving general health and happiness and stress reduction may reduce pain in SCD. Disclosures No relevant conflicts of interest to declare.