Maternal-Fetal Circadian Communication during Pregnancy

Circadian rhythms in gene expression and hormones are ubiquitous across species and across cell types. Circadian rhythms depend on a transcription-translation negative feedback loop that drives many daily rhythms in behavior and physiological processes. Circadian rhythms regulate many aspects of reproduction, such as fetal development and birth timing. Mammalian pregnancy presents a unique situation with circadian rhythms in the mom and, eventually, in the fetus. Prior studies showed that circadian rhythms develop in utero and that maternal signals, such as glucocorticoids, may act to entrain or drive daily rhythms in the fetus. However, there remains considerable debate about when and where these daily molecular oscillations first arise in development and which signals may be involved. We hypothesized that fetal circadian rhythms arise during pregnancy and synchronize to the mother prior to birth via maternal-to-fetal circadian communication. We monitored fetal clock gene expression in utero and found that fetal daily rhythms arise by E9. Our results indicate rhythms develop much earlier in the fetus than previously thought. We also found fetal rhythms synchronize to the mother by E16. Furthermore, mistimed daily glucocorticoids disrupt this synchrony. Additionally, we showed that the placenta exhibits intrinsic circadian rhythms beginning as early as E9 and blocking glucocorticoid signaling disrupted synchrony within the placenta. We conclude that fetal and placental circadian rhythms arise early in pregnancy and glucocorticoids may act as important entraining signals for these tissues. We also wanted to better understand the role of circadian rhythms in birth timing. Previous research indicated that circadian rhythms ensured birth occurred at a specific time of day, depending on the species. We hypothesized that mutations in clock genes would alter birth timing and gestation length. To test this, we examined gestation length, birth timing, and fetal development from multiple circadian mutant mouse lines. We found that Period1 performs a necessary role in the circadian gating of birth as loss of Period1 caused mice to give birth at random times of day. On the contrary, Period2 modifies the time of day of birth as mutations in this gene led to a ~6-hour shift in the timing of birth. Secondarily, our results demonstrate the importance of the Period2 gene in regulating gestation length. We conclude that the Period genes act as important regulators of labor. Taken together, these results highlight the complex role circadian rhythms have in regulating reproduction and development

Glucocorticoids are lower at delivery in maternal, but not cord blood of obese pregnancies

Abstract Glucocorticoids are vital for lung maturation. We previously showed that cortisol is lower in obese pregnancy. Whether this is maintained at delivery is unknown but is clinically relevant as maternal and cord blood cortisol levels are correlated and offspring of obese are more likely to need neonatal respiratory support. We hypothesized that glucocorticoids are lower in maternal and cord blood at delivery in obese pregnancies. Glucocorticoids (cortisol and corticosterone) and their inactive versions (cortisone and 11-dehydrocorticosterone) were measured by LC-MS/MS in maternal and cord plasma from 259 Caucasian women at delivery (BMI 18–55 kg/m2). Analyses adjusted for labour status, delivery mode, offspring gender, birthweight and gestational age. Cortisol and corticosterone were significantly higher in maternal than cord blood. Inactive versions were significantly higher in cord than maternal blood. Increased maternal BMI associated with lower maternal cortisol, corticosterone and 11-dehydrocorticosterone. Despite significant positive correlations between maternal and cord blood glucocorticoid levels, increased maternal BMI was not associated with lower cord blood glucocorticoid levels. Conditions at delivery may overcome any potential negative effects of low maternal glucocorticoids on the fetus in the short-term. This may not preclude the longer-term effects of fetal exposure to lower glucocorticoid levels during obese pregnancy

Serotoninergic and Circadian Systems: Driving Mammary Gland Development and Function

Since lactation is one of the most metabolically demanding states in adult female mammals, beautifully complex regulatory mechanisms are in place to time lactation to begin after birth and cease when the neonate is weaned. Lactation is regulated by numerous different homeorhetic factors, all of them tightly coordinated with the demands of milk production. Emerging evidence support that among these factors are the serotonergic and circadian clock systems. Here we review the serotoninergic and circadian clock systems and their roles in the regulation of mammary gland development and lactation physiology. We conclude by presenting our hypothesis that these two systems interact to accommodate the metabolic demands of lactation and thus adaptive changes in these systems occur to maintain mammary and systemic homeostasis through the reproductive cycles of female mammals

​​The influence of maternal obesity on placental fatty acid composition, synthesis of inflammatory mediators and pregnancy outcomes

​Introduction: Obesity is a global issue with ever increasing prevalence, having detrimental effects on the health of the population. Maternal obesity has been identified to significantly increase the risk of prolonged and dysfunctional labour, labour induction and emergency caesarean delivery. An established rodent model of diet induced obesity has been utilised to unravel the potential mechanisms behind uncoordinated myometrial contractile function labour. In this model a high-fat, high-cholesterol (HFHC) diet successfully induces maternal obesity, significantly altering maternal fatty acid status leading to un-coordinated contractions that significantly prolongs labour. Potential mechanisms identified to be behind dysfunctional labour is a decrease in the uterine expression of key contractile associated proteins (CAPs), an increase in progesterone concentrations, the hormone that suppresses expression of the contractile proteins, and decreased circulating PGF2α that stimulates myometrial contractions during labour.   ​ ​ Aims: The aim of this thesis was to first evaluate the impact the HFHC diet may have on the placenta in the maternal obesity model, focusing on its role in the initiation of labour. The second aim was to assess if the human placenta from obese women had the same characteristics. The third aim was to evaluate how these fatty acid profile changes may affect trophoblast cell's ability to produce pro-labour mediators, in vitro. Finally, the last chapter aimed to evaluate non-communicable disease and diet alongside obesity on pre-term birth, mode of delivery and birthweight, in a setting where obesity, NCDs and adverse pregnancy outcomes are extremely prevalent. Two of the most common NCDs associated with obesity and placental pathologies, hypertension and type 2 diabetes were evaluated alongside obesity, looking at how these effect pregnancy outcomes. The final prospective study aimed to evaluate any dietary patterns associated with pregnancy outcomes. ​ ​ ​Methods: Fatty acid profiles of the placental tissue from both the rat study and from human participants of different BMI were analysed through GCMS and RTqPCR to compare the fatty acid composition and gene expression profiles respectively. Gene expression profiles were centred around the placentas role in initiation of labour including hormone synthesis and signalling (e.g corticotrophin releasing hormone -CRH, Oestrogen receptor 1 -ER1 and cytochrome P450 family 17 subfamily A - CYP17), prostaglandin synthesis (e.g cyclooxygenase 2 - COX2), inflammatory regulators (e.g nuclear factor kappa B - NFκB and Sirtuin 1 SIRT1) and peroxisome proliferator-activated receptors - PPARs. HTR8-SVneo trophoblast cell line was treated with increasing doses of Oleic acid and pro-inflammatory cytokine interleukin 6 (IL-6) and prostaglandins PGE2 and PGF2a were measured using ELISA. Prostaglandin synthesis enzymes and PPARg mRNA levels were also investigated to attempt to elucidate a signalling pathway. Multinomial logistic regression was used to assess the joint impact of obesity and NCDs on mode of delivery, preterm birth and birthweight. A food frequency questionnaire was also used to assess dietary patterns and multinomial logistic regression used to understand the effects they may have on pregnancy outcomes. ​ ​ ​ Results: This experiment found significant alterations in the fatty acid composition of the placenta from obese pregnant rats and women. In the obese rat placentae, there was a significant decrease in saturated fatty acids, and an increase in monounsaturated fatty acids (MUFAs) as well as decreased desaturase enzyme activity. Similar results were found in placental tissue from obese pregnant women, although to a much lesser extent. Interestingly, only one of the genes quantified was differentially expressed. Placental expression of PPARg within the placenta was found to be significantly higher in HFHC fed rats but this was expressed any differently within the placenta of pregnant women of different BMI. In the cell culture study oleic acid at the highest physiological doses of (400µM) significantly decreased the synthesis of all the markers measured. Only one enzyme, responsible for producing PGE2 was found to decrease in a similar pattern with increasing oleic acid treatment. The retrospective cohort study found obesity to increase the risk of operative deliveries and remained significant after adjusting for confounding variables, for emergency CS. Once hypertension and type 2 diabetes were added into the model this no longer remained significant and neither of these had any significant effect on pregnancy outcomes looked at. No dietary patterns were significantly associated with any of these. ​ ​ Discussion: A diet high in saturated fats may create an anti-inflammatory response, seeing an increase in monounsaturated fatty acids, namely oleic acid within the placenta. The decrease in expression of genes associated with the prostaglandin biosynthetic pathway by high physiological levels of oleic acid may explain the reduced circulating PGF2a, and reduction in the expression of CAPs and dysfunctional contractions seen in the translational model. In conclusion the research findings from this thesis suggest further study into the effects of diet and fatty acid profiles as a risk factors or potential biomarkers for negative pregnancy outcomes are warranted

Daily-, estral- and age-dependent regulation of RFRP-3 neurons and their role in luteinizing hormone secretion in female mice

Female reproductive success relies on proper integration of circadian- and ovarian- signals to the hypothalamic-pituitary-gonadal axis in order to synchronize the pre-ovulatory LH surge with the onset of the main active period. In this study, we assessed in female mice whether hypothalamic neurons expressing the gonadotropin inhibitory peptide, RFRP-3, exhibit daily-, estrous stage-, and age-dependent variations. Furthermore, we investigated whether arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP), two circadian peptides produced by the suprachiasmatic nucleus, regulate RFRP-3 neuronal activity. In young mice, the number of activated (c-Fos–positive) RFRP-3 neurons was reduced at the day-to-night transition with no difference between diestrus and proestrus. Contrastingly, RFRP neuron firing rate was higher in proestrus as compared to diestrus, independently of the time of the day. AVP fibers contacted RFRP neurons with the highest density observed during the late afternoon of diestrus and proestrus, while AVP application increased RFRP neuron firing in the afternoon of diestrus, but not of proestrus. By contrast, we found no daily variation and no effect of VIP on RFRP neurons. Moreover, we demonstrated an age-dependent decrease in the total and activated number of RFRP-3 neurons. Finally, we reported that the daily variations in the activated RFRP-3 neurons and number of RFRP-3 neurons with close AVP and VIP fiber appositions were abolished in old mice. In conclusion, RFRP neurons integrate both daily and estrogenic signals in young, but not in old mice, a phenomenon which could be implicated in the aging of the reproductive axis

Hypothalamic pituitary adrenal axis dysregulation in obese pregnancy

There has been a global rise in obesity in the last three decades, and at present one in five women are obese at antenatal booking. Maternal obesity is associated with an increased risk of adverse pregnancy outcomes, including increased fetal size and prolonged pregnancy. In the longer-term, offspring of obese are at increased risk of premature death from a cardiovascular event in their adulthood. One mechanism that has been linked to these outcomes is fetal exposure to glucocorticoids in utero. During normal pregnancy, the maternal hypothalamic pituitary adrenal (HPA) axis undergoes major changes, resulting in exponentially increasing levels of the major circulating glucocorticoid cortisol, and other HPA axis hormones, such as corticotrophin releasing hormone (CRH). Cortisol and CRH are vital for normal fetal growth and length of gestation, but in excess they are associated with fetal growth restriction and preterm labour. In non-pregnant obesity, it is thought that the HPA axis is dysregulated, although evidence is inconclusive. Little is known about the effects of maternal obesity in pregnancy on the HPA axis. The work in this Thesis used clinical studies to test the hypothesis that the HPA axis is dysregulated in obese pregnant women with altered release, clearance and placental metabolism of cortisol. Associations with clinical outcomes related to fetal size and length of gestation were also studied. The HPA axis activity during pregnancy was investigated in a prospective case-control study cohort. Fasting serum cortisol levels were measured at 16, 28 and 36 weeks of gestation (obese n=276, lean n=135). In a subset (obese n=20, lean n=20), corticosteroid binding globulin (CBG), CRH, estrogens and progesterone were measured. Salivary cortisol was measured in samples collected at bedtime, waking and 30 minutes after waking at 16 weeks. Urinary glucocorticoid metabolites were measured at 19 weeks and 36 weeks (obese n=6, lean n=5) and non-pregnant (obese n=7, lean n=7) subjects. All circulating hormone levels rose similarly in obese and lean during pregnancy, but were significantly lower in obese women. The diurnal rhythm of cortisol was maintained. Urinary glucocorticoids increased with gestation in lean, but not in obese, indicating a lesser activation of the HPA axis in obese compared with lean pregnancy. These findings associated with increased birthweight and longer gestation in obese pregnancy, suggesting that decreased HPA axis activity may underlie these obese related adverse pregnancy outcomes. Whether or not lower glucocorticoids in obese pregnancies are maintained at delivery was investigated by measuring active glucocorticoids (cortisol and corticosterone) and their inactive versions (cortisone and 11- dehydrocorticosterone, respectively) from matched maternal and cord plasma samples (n=259, BMI 18 – 55 kg/m2). Active glucocorticoids were significantly higher in maternal than cord blood, and inactive versions were significantly higher in cord than maternal blood. Increased maternal BMI associated with lower maternal cortisol, corticosterone and 11-dehydrocorticosterone. Despite significant correlations between maternal and cord blood glucocorticoid levels, increased maternal BMI did not associate with lower cord blood glucocorticoids. This suggests that conditions at delivery may overcome any potential negative effects of low maternal glucocorticoids on the fetus in the short-term. However, it may not preclude the longer-term effects of fetal exposure to lower glucocorticoid levels during obese pregnancy, and offspring follow-up studies are required. Potential mechanisms leading to altered HPA axis activity in obese pregnancy were explored by studying the pulsatile release and placental metabolism of glucocorticoid hormones. Glucocorticoid pulsatility is thought to be important for transcriptional regulation of glucocorticoid responsive genes, and disruptions to pulsatility have been reported in some disease processes. Glucocorticoids were measured in 10-minute serum sampling between 08.00h-11.00h and 16.00h- 19.00h. Peripheral tissue cortisol was measured from 20-minute sampling of interstitial fluid, over 24-hours, at 16-24 weeks and 30-36 weeks (obese n=7, lean n=8), and non-pregnant controls (obese n=4, lean n=3). Total circulating serum cortisol levels were higher in pregnancy than non-pregnancy in lean and obese, and increased significantly with advancing gestation in lean but not in obese. Pulsatility of cortisol was demonstrated in interstitial fluid in both non-pregnancy and pregnancy. In obese pregnancy, interstitial fluid pulse frequency was lower with advancing gestation. This may be a novel mechanism underlying the observed decreased HPA axis activity in obese pregnancy. Placental cortisol metabolism and transport was studied using an ex vivo placental perfusion model, perfused with a deuterium-labelled cortisol tracer combined with computational modeling. The findings challenge the concept that maternal cortisol diffuses freely across the placenta, but confirmed that 11β- HSD2 acts as major ‘barrier’ to cortisol transfer to the fetus, protecting the fetus from the high maternal circulating cortisol levels. In addition we showed preliminary evidence of local cortisol production within the placenta. The model is able to predict maternal-fetal cortisol transfer and can now be used in future experimental design. In conclusion, in obese pregnancy, lower maternal cortisol and urinary clearance suggested reduced HPA axis activity. Altered glucocorticoid pulsatility may underlie this change. Future studies of placental cortisol metabolism in maternal obesity could be conducted using an ex vivo perfusion model. The lower HPA axis activity in obese pregnancy represents a novel pathway underlying increased fetal growth