Sex and age differences in social and cognitive function in offspring exposed to late gestational hypoxia.

BACKGROUND: Gestational sleep apnea is a hypoxic sleep disorder that affects 8-26% of pregnancies and increases the risk for central nervous system dysfunction in offspring. Specifically, there are sex differences in the sensitivity of the fetal hippocampus to hypoxic insults, and hippocampal impairments are associated with social dysfunction, repetitive behaviors, anxiety, and cognitive impairment. Yet, it is unclear whether gestational sleep apnea impacts these hippocampal-associated functions and if sex and age modify these effects. To examine the relationship between gestational sleep apnea and hippocampal-associated behaviors, we used chronic intermittent hypoxia (CIH) to model late gestational sleep apnea in pregnant rats. We hypothesized that late gestational CIH would produce sex- and age-specific social, anxiety-like, repetitive, and cognitive impairments in offspring. METHODS: Timed pregnant Long-Evans rats were exposed to CIH or room air normoxia from GD 15-19. Behavioral testing of offspring occurred during either puberty or young adulthood. To examine gestational hypoxia-induced behavioral phenotypes, we quantified hippocampal-associated behaviors (social function, repetitive behaviors, anxiety-like behaviors, and spatial memory and learning), hippocampal neuronal activity (glutamatergic NMDA receptors, dopamine transporter, monoamine oxidase-A, early growth response protein 1, and doublecortin), and circulating hormones in offspring. RESULTS: Late gestational CIH induced sex- and age-specific differences in social, repetitive, and memory functions in offspring. In female pubertal offspring, CIH impaired social function, increased repetitive behaviors, and elevated circulating corticosterone levels but did not impact memory. In contrast, CIH transiently induced spatial memory dysfunction in pubertal male offspring but did not impact social or repetitive functions. Long-term effects of gestational CIH on social behaviors were only observed in female offspring, wherein CIH induced social disengagement and suppression of circulating corticosterone levels in young adulthood. No effects of gestational CIH were observed in anxiety-like behaviors, hippocampal neuronal activity, or circulating testosterone and estradiol levels, regardless of sex or age of offspring. CONCLUSIONS: Our results indicate that hypoxia-associated pregnancy complications during late gestation can increase the risk for behavioral and physiological outcomes in offspring, such as social dysfunction, repetitive behaviors, and cognitive impairment, that are dependent on sex and age.

Sleep apnea during late pregnancy is a common pregnancy complication that can impact the brain development of children born to mothers with sleep apnea. Children with impaired brain development may present with decreased social skills, memory issues, anxiety, and compulsivity. It is unclear if there is a cause and effect relationship between sleep apnea during late pregnancy and behavioral changes in offspring. Additionally, it is unknown whether male or female sex or age of the offspring affects these relationships. In this study, we exposed pregnant rats to a model of sleep apnea called chronic intermittent hypoxia (CIH) within late gestation and examined the behavior of the offspring and brain activity during puberty and young adulthood. We found that CIH during late pregnancy had long-term effects in the offspring that were different in males and females. Notably, female offspring displayed social impairments in response to late gestation CIH, whereas male offspring displayed cognitive dysfunction.

Sex and age differences in social and cognitive function in offspring exposed to late gestational hypoxia.

Background: Gestational sleep apnea affects 8-26% of pregnancies and can increase the risk for autism spectrum disorder (ASD) in offspring. ASD is a neurodevelopmental disorder associated with social dysfunction, repetitive behaviors, anxiety, and cognitive impairment. To examine the relationship between gestational sleep apnea and ASD-associated behaviors, we used a chronic intermittent hypoxia (CIH) protocol between gestational days (GD) 15-19 in pregnant rats to model late gestational sleep apnea. We hypothesized that late gestational CIH would produce sex- and age-specific social, mood, and cognitive impairments in offspring. Methods: Timed pregnant Long-Evans rats were exposed to CIH or room air normoxia from GD 15-19. Behavioral testing of offspring occurred during either puberty or young adulthood. To examine ASD-associated phenotypes, we quantified ASD-associated behaviors (social function, repetitive behaviors, anxiety-like behaviors, and spatial memory and learning), hippocampal activity (glutamatergic NMDA receptors, dopamine transporter, monoamine oxidase-A, EGR-1, and doublecortin), and circulating hormones in offspring. Results: Late gestational CIH induced sex- and age-specific differences in social, repetitive and memory functions in offspring. These effects were mostly transient and present during puberty. In female pubertal offspring, CIH impaired social function, increased repetitive behaviors, and increased circulating corticosterone levels, but did not impact memory. In contrast, CIH transiently induced spatial memory dysfunction in pubertal male offspring but did not impact social or repetitive functions. Long-term effects of gestational CIH were only observed in female offspring, wherein CIH induced social disengagement and suppression of circulating corticosterone levels in young adulthood. No effects of gestational CIH were observed on anxiety-like behaviors, hippocampal activity, circulating testosterone levels, or circulating estradiol levels, regardless of sex or age of offspring. Conclusions: Our results indicate that hypoxia-associated pregnancy complications during late gestation can increase the risk for ASD-associated behavioral and physiological outcomes, such as pubertal social dysfunction, corticosterone dysregulation, and memory impairments.

Exposure to unmethylated CpG oligonucleotides disrupts blood pressure circadian rhythms and placental clock gene network in pregnant rats.

Bacterial infections and impaired circulating mitochondrial DNA dynamics are associated with adverse pregnancy outcomes. Unmethylated cytosine-guanine dinucleotide (CpG) motifs are common in bacterial and mitochondrial DNA and act as potent immunostimulators. We tested the hypothesis that exposure to CpG oligonucleotides (ODN) during pregnancy would disrupt blood pressure circadian rhythms and placental molecular clock network, mediating aberrant fetoplacental growth dynamics. Rats were repeatedly treated with CpG ODN in the third trimester [gestational days (GD) 14, 16, and 18] and euthanized on GD20 (near term) or treated with a single dose of CpG ODN on GD14 and euthanized 4 h after treatment. Hemodynamic circadian rhythms were analyzed via Lomb-Scargle periodogram analysis on 24-h raw data collected continuously via radiotelemetry. A P value ≥ 0.05 indicates the absence of a circadian rhythm. Following the first treatment with CpG ODN, maternal systolic and diastolic blood pressure circadian rhythms were lost (P ≥ 0.05). Blood pressure circadian rhythm was restored by GD16 and remained unaffected after the second treatment with CpG ODN (P < 0.0001). Diastolic blood pressure circadian rhythm was again lost after the last treatment on GD18 (P ≥ 0.05). CpG ODN increased placental expression of Per2, Per3, and Tnfα (P ≤ 0.05) and affected fetoplacental growth dynamics. Reduced fetal and placental weights were disproportionately associated with increases in the number of resorptions in ODN-treated dams compared with controls. In conclusion, gestational exposure to unmethylated CpG ODN dysregulates the placental molecular clock network and fetoplacental growth dynamics and disrupts blood pressure circadian rhythms.NEW & NOTEWORTHY Gestational exposure to unmethylated CpG ODN dysregulates placental molecular clock network and fetoplacental growth dynamics and disrupts blood pressure circadian rhythms. These findings provide novel insights into the relationship between circadian rhythms and immune responses in pregnancy and propose new mechanisms by which maternal responses to immune triggers could dictate circadian rhythms of cardiovascular processes and placental clock machinery function to determine fetal growth trajectories.

Gestational exposure to unmethylated CpG oligonucleotides dysregulates placental molecular clock network and fetoplacental growth dynamics, and disrupts maternal blood pressure circadian rhythms in rats.

Bacterial infections and impaired mitochondrial DNA dynamics are associated with adverse pregnancy outcomes. Unmethylated cytosine-guanine dinucleotide (CpG) motifs are common in bacterial and mitochondrial DNA and act as potent immunostimulators. Here, we tested the hypothesis that exposure to CpG oligonucleotides (ODN) during pregnancy would disrupt blood pressure circadian rhythms and the placental molecular clock machinery, mediating aberrant fetoplacental growth dynamics. Rats were repeatedly treated with CpG ODN in the 3 rd trimester (gestational day, GD, 14, 16, 18) and euthanized on GD20 (near term) or with a single dose of CpG ODN and euthanized 4 hours after treatment on GD14. Hemodynamic circadian rhythms were analyzed via Lomb-Scargle periodogram analysis on 24-h raw data collected continuously via radiotelemetry. A p -value ≥ 0.05 indicates the absence of a circadian rhythm. Following the first treatment with CpG ODN, maternal systolic and diastolic blood pressure circadian rhythms were lost ( p ≥ 0.05). Blood pressure circadian rhythm was restored by GD16 and remained unaffected after the second treatment with CpG ODN ( p < 0.0001). Diastolic blood pressure circadian rhythm was again lost after the last treatment on GD18 ( p ≥ 0.05). CpG ODN increased placental expression of Per2 and Per3 and Tnfα ( p ≤ 0.05) and affected fetoplacental growth dynamics, such as reduced fetal and placental weights were disproportionately associated with increases in the number of resorptions in ODN-treated dams compared to controls. In conclusion, gestational exposure to unmethylated CpG DNA dysregulates placental molecular clock network and fetoplacental growth dynamics and disrupts blood pressure circadian rhythms.

Gestational hypoxia in late pregnancy differentially programs subcortical brain maturation in male and female rat offspring.

BACKGROUND: Hypoxia is associated with pregnancy complications, such as preeclampsia, placental abruption, and gestational sleep apnea. Hypoxic insults during gestation can impact the brain maturation of cortical and subcortical pathways, such as the nigrostriatal pathway. However, the long-term effects of in utero hypoxic stress exposure on brain maturation in offspring are unclear, especially exposure during late gestation. The purpose of this study was to determine the impact of gestational hypoxia in late pregnancy on developmental programming of subcortical brain maturation by focusing on the nigrostriatal pathway. METHODS: Timed pregnant Long-Evans rats were exposed to chronic intermittent hypoxia or room air normoxia from gestational day (GD) 15-19 (term 22-23 days). Male and female offspring were assessed during two critical periods: puberty from postnatal day (PND) 40-45 or young adulthood (PND 60-65). Brain maturation was quantified by examining (1) the structural development of the nigrostriatal pathway via analysis of locomotor behaviors and the substantia nigra dopaminergic neuronal cell bodies and (2) the refinement of the nigrostriatal pathway by quantifying ultrasonic vocalizations (USVs). RESULTS: The major findings of this study are gestational hypoxia has age- and sex-dependent effects on subcortical brain maturation in offspring by adversely impacting the refinement of the nigrostriatal pathway in the absence of any effects on the structural development of the pathway. During puberty, female offspring were impacted more than male offspring, as evidenced by decreased USV call frequency, chirp USV call duration, and simple call frequency. In contrast, male offspring were impacted more than female offspring during young adulthood, as evidenced by increased latency to first USV, decreased simple USV call intensity, and increased harmonic USV call bandwidth. No effects of gestational hypoxia on the structural development of the nigrostriatal pathway were observed. CONCLUSIONS: These novel findings demonstrate hypoxic insults during pregnancy mediate developmental programming of the cortical and subcortical pathways, in which male offspring exhibit long-term adverse effects compared to female offspring. Impairment of cortical and subcortical pathways maturation, such as the nigrostriatal pathway, may increase risk for neuropsychiatric disorders (e.g., mood disorders, cognitive dysfunction, brain connectivity dysfunction).

Exosomes facilitate intercellular communication between uterine perivascular adipose tissue and vascular smooth muscle cells in pregnant rats.

Perivascular adipose tissue (PVAT) is distinct from other adipose depots, as it has differential gene and protein profiles and vasoactive functions. We have shown that pregnancy affects the morphology of PVAT surrounding the uterine arteries (utPVAT) differentially than the morphology of nonperivascular reproductive adipose depots (i.e., periovarian adipose tissue, OVAT). Here, we hypothesized that pregnancy modifies the profile (size and molecular mass) of exosome-like extracellular vesicles released by utPVAT (Exo-utPVAT) compared with exosome-like extracellular vesicles released by OVAT (Exo-OVAT) and that primary uterine vascular smooth muscle cells (utVSMCs) can internalize Exo-utPVAT. Our findings indicate that utPVAT from pregnant and nonpregnant rats secrete exosome-like vesicles. Exo-utPVAT from pregnant rats were smaller (i.e., molecular size) and heavier (i.e., molecular mass) than those from nonpregnant rats, whereas pregnancy did not affect the size of Exo-OVAT. Immunocytochemistry and confocal microscopy showed that primary utVSMCs internalized Exo-utPVAT (both tissues from the same pregnant rat) labeled by the lipophilic tracer DiO. Treatment of isolated uterine arteries with Exo-utPVAT did not affect relaxation responses to acetylcholine in pregnant or nonpregnant rats. Collectively, these findings demonstrate a novel type of intercellular communication between Exo-utPVAT and utVSMCs and indicate pregnancy modulates the morphology and cargo of Exo-utPVAT.NEW & NOTEWORTHY Uterine perivascular adipose tissue secretes exosome-like vesicles, which are internalized by their adjacent uterine vascular smooth muscle cells. Consideration of the exosomal communication between adipose tissue and vascular smooth muscle cells in the uterine circulation in mathematical models and experimental designs may help us to improve understanding of mechanisms underlying uterine artery adaptive responses to a healthy pregnancy and during pregnancy complications.

Cyclooxygenase-dependent mechanisms mediate in part the anti-dilatory effects of perivascular adipose tissue in uterine arteries from pregnant rats.

Uterine perivascular adipose tissue (PVAT) contributes to uterine blood flow regulation in pregnancy, at least in part, due to its effects on uterine artery reactivity. We tested the hypothesis that uterine PVAT modulates the balance between the contribution of nitric oxide synthase (NOS)- and cyclooxygenase (COX)-dependent pathways to acetylcholine (ACh)-induced relaxation in isolated uterine arteries. Concentration-response curves to ACh (1 nM - 30 µM) were performed on uterine arteries from pregnant and non-pregnant rats. Arteries were exposed to Krebs-Henseleit solution (control) or PVAT-conditioned media (PVATmedia) in the presence of the following inhibitors: L-NAME (NOS inhibitor), indomethacin (COX inhibitor), SC560 (COX-1 inhibitor), NS398 (COX-2 inhibitor), SQ 29,548 (thromboxane receptor (TP) inhibitor). In arteries incubated with PVATmedia, the presence of indomethacin increased ACh-induced relaxation, reversing the anti-dilatory effect of PVATmedia. NOS inhibition reduced ACh-induced relaxation in uterine arteries from pregnant rats, and exposure to PVATmedia did not change this effect. Selective inhibition of COX-1 but not COX-2 suppressed relaxation responses to ACh in control arteries. The presence of PVATmedia abolished the effect of COX-1 inhibition. Incubation of uterine arteries from pregnant rats with PVATmedia increased production of thromboxane B2 (TxB2, p = 0.01) but thromboxane receptor (TP) inhibition did not affect the anti-dilatory properties of PVATmedia. In conclusion, inhibition of COX signaling suppressed the anti-dilatory effects of PVATmedia, while PVATmedia had no effect on the contribution of the NOS/NO pathway to ACh-induced relaxation in uterine arteries from pregnant rats, indicating that the anti-dilatory effects of uterine PVAT are mediated in part by COX-dependent mechanisms.

Uterine perivascular adipose tissue is a novel mediator of uterine artery blood flow and reactivity in rat pregnancy.

KEY POINTS: In vivo, uterine perivascular adipose tissue (PVAT) potentiates uterine artery blood flow in pregnant rats, although not in non-pregnant rats. In isolated preparations, uterine PVAT has pro-contractile and anti-dilatory effects on uterine arteries. Pregnancy induces changes in uterine arteries that makes them responsive to uterine PVAT signalling. ABSTRACT: An increase in uterine artery blood flow (UtBF) is a common and necessary feature of a healthy pregnancy. In the present study, we tested the hypothesis that adipose tissue surrounding uterine arteries (uterine perivascular adipose tissue; PVAT) is a novel local mediator of UtBF and uterine artery tone during pregnancy. In vivo experiments in anaesthetized Sprague-Dawley rats showed that pregnant animals (gestational day 16, term = 22--23 days) had a three-fold higher UtBF compared to non-pregnant animals. Surgical removal of uterine PVAT reduced UtBF only in pregnant rats. In a series of ex vivo bioassays, we demonstrated that uterine PVAT had pro-contractile and anti-dilatory effects on rat uterine arteries. In the presence of PVAT-conditioned media, isolated uterine arteries from both pregnant and non-pregnant rats had reduced vasodilatory responses. In non-pregnant rats, these responses were mediated at the level of uterine vascular smooth muscle, whereas, in pregnant rats, PVAT-media reduced endothelium-dependent relaxation. Pregnancy increased adipocyte size in ovarian adipose tissue but had no effect on uterine PVAT adipocyte morphology. In addition, pregnancy down-regulated the gene expression of metabolic adipokines in uterine but not in aortic PVAT. In conclusion, this is the first study to demonstrate that uterine PVAT plays a regulatory role in UtBF, at least in part, as a result of its actions on uterine artery tone. We propose that the interaction between the uterine vascular wall and its adjacent adipose tissue may provide new insights for interventions in pregnancies with adipose tissue dysfunction and abnormal UtBF.

A free-choice high-fat, high-sucrose diet induces hyperphagia, obesity, and cardiovascular dysfunction in female cycling and pregnant rats.

The main objective of these studies was to characterize metabolic, body composition, and cardiovascular responses to a free-choice high-fat, high-sucrose diet in female cycling and pregnant rats. In the nonpregnant state, female Sprague-Dawley rats offered a 3-wk free-choice high-fat, high-sucrose diet had greater energy intake, adiposity, serum leptin, and triglyceride concentrations compared with rats fed with standard chow and developed glucose intolerance. In addition, choice-diet-fed rats had larger cardiac ventricular weights, smaller kidney and pancreas weights, and higher blood pressure than chow-fed rats, but they did not exhibit resistance artery endothelial dysfunction. When the free-choice diet continued throughout pregnancy, rats remained hyperphagic, hyperleptinemic, and obese. Choice pregnant rats exhibited uterine artery endothelial dysfunction and had smaller fetuses compared with chow pregnant rats. Pregnancy normalized mean arterial blood pressure and pancreas weights in choice rats. These studies are the first to provide a comprehensive evaluation of free-choice high-fat, high-sucrose diet on metabolic and cardiovascular functions in female rats, extending the previous studies in males to female cycling and pregnant rodents. Free-choice diet may provide a new model of preconceptual maternal obesity to study the role of increased energy intake, individual food components, and preexisting maternal obesity on maternal and offspring physiological responses during pregnancy and after birth.

Mechanisms of Sex Disparities in Cardiovascular Function and Remodeling.

Epidemiological studies demonstrate disparities between men and women in cardiovascular disease prevalence, clinical symptoms, treatments, and outcomes. Enrollment of women in clinical trials is lower than men, and experimental studies investigating molecular mechanisms and efficacy of certain therapeutics in cardiovascular disease have been primarily conducted in male animals. These practices bias data interpretation and limit the implication of research findings in female clinical populations. This review will focus on the biological origins of sex differences in cardiovascular physiology, health, and disease, with an emphasis on the sex hormones, estrogen and testosterone. First, we will briefly discuss epidemiological evidence of sex disparities in cardiovascular disease prevalence and clinical manifestation. Second, we will describe studies suggesting sexual dimorphism in normal cardiovascular function from fetal life to older age. Third, we will summarize and critically discuss the current literature regarding the molecular mechanisms underlying the effects of estrogens and androgens on cardiac and vascular physiology and the contribution of these hormones to sex differences in cardiovascular disease. Fourth, we will present cardiovascular disease risk factors that are positively associated with the female sex, and thus, contributing to increased cardiovascular risk in women. We conclude that inclusion of both men and women in the investigation of the role of estrogens and androgens in cardiovascular physiology will advance our understanding of the mechanisms underlying sex differences in cardiovascular disease. In addition, investigating the role of sex-specific factors in the development of cardiovascular disease will reduce sex and gender disparities in the treatment and diagnosis of cardiovascular disease. © 2019 American Physiological Society. Compr Physiol 9:375-411, 2019.

Effects of low-dose aspirin on maternal blood pressure and vascular function in an experimental model of gestational hypertension.

Daily intake of low-dose aspirin after 12weeks of gestation is currently recommended as a preventative intervention in pregnancies in high risk of developing preeclampsia. This recommendation is based on epidemiological evidence, whereas experimental studies investigating the exact mechanisms of aspirin action during pregnancy are lacking. We previously showed that treating pregnant rats with a synthetic mimetic of unmethylated CpG DNA (bacterial DNA) caused preeclampsia-like characteristics such as maternal hypertension and increased cyclooxygenase (COX) expression and activity. In this study, we tested the hypothesis that daily maternal treatment with low-dose aspirin would prevent the development of maternal hypertension, reduce COX activity and thromboxane A2 (TxA2) production, and improve maternal vascular function in pregnant rats exposed to CpG ODN during gestation. Pregnant rats were treated with ODN2395 (synthetic CpG DNA) or saline (vehicle) on gestational days (GD) 14, 16, 18. Daily low-dose aspirin treatment (1.5mg/kgBW) started on GD10 and continued throughout gestation. Pregnant rats treated with ODN2395 had greater systolic blood pressure compared to controls (120±4mmHg vs. 100±5mmHg, p=0.03) and aspirin did not prevent this increase (p=0.86). Aspirin prevented ODN2395-induced increases of TxB2 (TxA2 metabolite) in serum and mesenteric arteries. ODN2395 increased expression of COX-1 and COX-2 in mesenteric and uterine arteries and aspirin abolished these effects. Aspirin reduced contractile responses to phenylephrine and U46619 (TxA2 mimetic) in mesenteric arteries from control rats but not from ODN2395-treated rats. In conclusion, treatment with low-dose aspirin reduced systemic and vascular COX expression and activity but did not prevent the development of maternal hypertension induced by exposure to unmethylated CpG DNA (bacterial DNA).