The Effects of In Utero Fetal Hypoxia and Creatine Treatment on Mitochondrial Function in the Late Gestation Fetal Sheep Brain.

Near-term acute hypoxia in utero can result in significant fetal brain injury, with some brain regions more vulnerable than others. As mitochondrial dysfunction is an underlying feature of the injury cascade following hypoxia, this study is aimed at characterizing mitochondrial function at a region-specific level in the near-term fetal brain after a period of acute hypoxia. We hypothesized that regional differences in mitochondrial function would be evident, and that prophylactic creatine treatment would mitigate mitochondrial dysfunction following hypoxia; thereby reducing fetal brain injury. Pregnant Border-Leicester/Merino ewes with singleton fetuses were surgically instrumented at 118 days of gestation (dGa; term is ~145 dGA). A continuous infusion of either creatine (n = 15; 6 mg/kg/h) or isovolumetric saline (n = 16; 1.5 ml/kg/h) was administered to the fetuses from 121 dGa. After 10 days of infusion, a subset of fetuses (8 saline-, 7 creatine-treated) were subjected to 10 minutes of umbilical cord occlusion (UCO) to induce a mild global fetal hypoxia. At 72 hours after UCO, the fetal brain was collected for high-resolution mitochondrial respirometry and molecular and histological analyses. The results show that the transient UCO-induced acute hypoxia impaired mitochondrial function in the hippocampus and the periventricular white matter and increased the incidence of cell death in the hippocampus. Creatine treatment did not rectify the changes in mitochondrial respiration associated with hypoxia, but there was a negative relationship between cell death and creatine content following treatment. Irrespective of UCO, creatine increased the proportion of cytochrome c bound to the inner mitochondrial membrane, upregulated the mRNA expression of the antiapoptotic gene Bcl2, and of PCG1-α, a driver of mitogenesis, in the hippocampus. We conclude that creatine treatment prior to brief, acute hypoxia does not fundamentally modify mitochondrial respiratory function, but may improve mitochondrial structural integrity and potentially increase mitogenesis and activity of antiapoptotic pathways.

Novel Lipidomic Signature Associated With Metabolic Risk in Women With and Without Polycystic Ovary Syndrome.

CONTEXT: Dyslipidemia is a feature of polycystic ovary syndrome (PCOS) and may augment metabolic dysfunction in this population. OBJECTIVE: Using comprehensive lipidomic profiling and gold-standard metabolic measures, we examined whether distinct lipid biomarkers were associated with metabolic risk in women with and without PCOS. METHODS: Using preexisting data and biobanked samples from 76 women (n = 42 with PCOS), we profiled > 700 lipid species by mass spectrometry. Lipids were compared between women with and without PCOS and correlated with direct measures of adiposity (dual x-ray absorptiometry and computed tomography) and insulin sensitivity (hyperinsulinemic-euglycemic clamp), as well as fasting insulin, HbA1c, and hormonal parameters (luteinizing and follicle-stimulating hormones; total and free testosterone; sex hormone-binding globulin [SHBG]; and free androgen index [FAI]). Multivariable linear regression was used with correction for multiple testing. RESULTS: Despite finding no differences by PCOS status, lysophosphatidylinositol (LPI) species esterified with an 18:0 fatty acid were the strongest lipid species associated with all the metabolic risk factors measured in women with and without PCOS. Across the cohort, higher concentrations of LPI(18:0) and lower concentrations of lipids containing docosahexaenoic acid (DHA, 22:6) n-3 polyunsaturated fatty acids were associated with higher adiposity, insulin resistance, fasting insulin, HbA1c and FAI, and lower SHBG. CONCLUSION: Our data indicate that a distinct lipidomic signature comprising high LPI(18:0) and low DHA-containing lipids are associated with key metabolic risk factors that cluster in PCOS, independent of PCOS status. Prospective studies are needed to corroborate these findings in larger cohorts of women with varying PCOS phenotypes.

Creatine Metabolism in Female Reproduction, Pregnancy and Newborn Health.

Creatine metabolism is an important component of cellular energy homeostasis. Via the creatine kinase circuit, creatine derived from our diet or synthesized endogenously provides spatial and temporal maintenance of intracellular adenosine triphosphate (ATP) production; this is particularly important for cells with high or fluctuating energy demands. The use of this circuit by tissues within the female reproductive system, as well as the placenta and the developing fetus during pregnancy is apparent throughout the literature, with some studies linking perturbations in creatine metabolism to reduced fertility and poor pregnancy outcomes. Maternal dietary creatine supplementation during pregnancy as a safeguard against hypoxia-induced perinatal injury, particularly that of the brain, has also been widely studied in pre-clinical in vitro and small animal models. However, there is still no consensus on whether creatine is essential for successful reproduction. This review consolidates the available literature on creatine metabolism in female reproduction, pregnancy and the early neonatal period. Creatine metabolism is discussed in relation to cellular bioenergetics and de novo synthesis, as well as the potential to use dietary creatine in a reproductive setting. We highlight the apparent knowledge gaps and the research "road forward" to understand, and then utilize, creatine to improve reproductive health and perinatal outcomes.

Creatine metabolism in the uterus: potential implications for reproductive biology.

Creatine is an amino acid derivative synthesized from arginine, glycine and methionine. It serves as the substrate for the creatine kinase system, which is vital for maintaining ATP levels in tissues with high and fluctuating energy demand. There exists evidence that the creatine kinase system operates in both the endometrial and myometrial layers of the uterus. While use and regulation of this system in the uterus are not well understood, it is likely to be important given uterine tissues undergo phases of increased energy demand during certain stages of the female reproductive cycle, pregnancy, and parturition. This review discusses known adaptations of creatine metabolism in the uterus during the reproductive cycle (both estrous and menstrual), pregnancy and parturition, highlighting possible links to fertility and the existing knowledge gaps. Specifically, we discuss the adaptations and regulation of uterine creatine metabolite levels, cell creatine transport, de novo creatine synthesis, and creatine kinase expression in the various layers and cell types of the uterus. Finally, we discuss the effects of dietary creatine on uterine metabolism. In summary, there is growing evidence that creatine metabolism is up-regulated in uterine tissues during phases where energy demand is increased. While it remains unclear how important these adaptations are in the maintenance of healthy uterine function, furthering our understanding of uterine creatine metabolism may uncover strategies to combat poor embryo implantation and failure to conceive, as well as enhancing uterine contractile performance during labor.

Vitamin D-Binding Protein in Pregnancy and Reproductive Health.

Vitamin D-binding protein (VDBP), the main carrier of vitamin D, has recently been implicated in reproductive health and pregnancy outcomes including endometriosis, polycystic ovary syndrome (PCOS), pre-eclampsia, and gestational diabetes mellitus (GDM). Improved methods for measuring VDBP and an increased understanding of its role in biological processes have led to a number of newly published studies exploring VDBP in the context of pregnancy. Here, we synthesize the available evidence regarding the role of VDBP in reproductive health and pregnancy, and we highlight areas requiring further study. Overall, low levels of maternal serum VDBP concentrations have been associated with infertility, endometriosis, PCOS and spontaneous miscarriage, as well as adverse pregnancy outcomes including GDM, pre-eclampsia, preterm birth and fetal growth restriction. However, increased VDBP concentration in cervicovaginal fluid has been linked to unexplained recurrent pregnancy loss and premature rupture of membranes. Some genetic variants of VDBP have also been associated with these adverse outcomes. Further studies using more accurate VDBP assays and accounting for ethnic variation and potential confounders are needed to clarify whether VDBP is associated with reproductive health and pregnancy outcomes, and the mechanisms underlying these relationships.

The Effects of Early-Onset Pre-Eclampsia on Placental Creatine Metabolism in the Third Trimester.

Creatine is a metabolite important for cellular energy homeostasis as it provides spatio-temporal adenosine triphosphate (ATP) buffering for cells with fluctuating energy demands. Here, we examined whether placental creatine metabolism was altered in cases of early-onset pre-eclampsia (PE), a condition known to cause placental metabolic dysfunction. We studied third trimester human placentae collected between 27-40 weeks' gestation from women with early-onset PE (n = 20) and gestation-matched normotensive control pregnancies (n = 20). Placental total creatine and creatine precursor guanidinoacetate (GAA) content were measured. mRNA expression of the creatine synthesizing enzymes arginine:glycine aminotransferase (GATM) and guanidinoacetate methyltransferase (GAMT), the creatine transporter (SLC6A8), and the creatine kinases (mitochondrial CKMT1A & cytosolic BBCK) was assessed. Placental protein levels of arginine:glycine aminotransferase (AGAT), GAMT, CKMT1A and BBCK were also determined. Key findings; total creatine content of PE placentae was 38% higher than controls (p < 0.01). mRNA expression of GATM (p < 0.001), GAMT (p < 0.001), SLC6A8 (p = 0.021) and BBCK (p < 0.001) was also elevated in PE placentae. No differences in GAA content, nor protein levels of AGAT, GAMT, BBCK or CKMT1A were observed between cohorts. Advancing gestation and birth weight were associated with a down-regulation in placental GATM mRNA expression, and a reduction in GAA content, in control placentae. These relationships were absent in PE cases. Our results suggest PE placentae may have an ongoing reliance on the creatine kinase circuit for maintenance of cellular energetics with increased total creatine content and transcriptional changes to creatine synthesizing enzymes and the creatine transporter. Understanding the functional consequences of these changes warrants further investigation.

Pseudopregnancy and reproductive cycle synchronisation cannot be induced using conventional methods in the spiny mouse (Acomys cahirinus).

The menstruating spiny mouse is the first rodent identified to exhibit natural spontaneous decidualisation, cyclical endometrial shedding and regeneration. While the spiny mouse shares several primate-like characteristics in its reproductive biology, it has not been established whether pseudopregnancy can be induced or if its cycles can be synchronised as in non-human mammals. Here we describe attempts to induce pseudopregnancy and synchronisation of menstrual cycles (i.e. Whitten effect) in spiny mice. Virgin females (n=3-8 per group) underwent one of the following procedures to induce pseudopregnancy: daily vaginal lavage only (control), progesterone injection, mechanical stimulation of the cervix and sterile mating. A separate cohort was also exposed to male-soiled bedding to assess the Whitten effect. Pseudopregnancy was deemed successful if females presented with extended (>12 consecutive days) leukocytic vaginal cytology. No female from any method of induction met this criterion. In addition, the menstrual cycles of a group of six females could not be synchronised, nor immediate ovulation induced via exposure to male-soiled bedding. These responses indicate that the spiny mouse does not behave as a typical rodent. Like higher-order primates, the spiny mouse exhibits a relatively rare reproductive strategy, of failure to show pseudopregnancy or cyclical synchronisation. This is further endorsement of the use of this species as a versatile animal model for translational studies of menstruation and fertility.

Dysregulation of the placental renin-angiotensin system in human fetal growth restriction.

Fetal growth restriction (FGR) is a pregnancy complication wherein the foetus fails to reach its growth potential. The renin-angiotensin system (RAS) is a critical regulator of placental function, controlling trophoblast proliferation, angiogenesis and blood flow. The RAS significantly influences uteroplacental blood flow through the balance of its vasoconstrictive and vasodilatory pathways. Although the RAS is known to be dysregulated in placentae from women with preeclampsia, the expression of the RAS has not yet been studied in pregnancies compromised by FGR alone. This study investigated the mRNA expression and protein levels of RAS components in placentae from pregnancies compromised by FGR. Angiotensin II type 1 receptor (AGTR1) and angiotensin-converting enzyme 2 (ACE2) mRNA levels were reduced in FGR placentae compared with control (P = 0.012 and 0.018 respectively). Neprilysin (NEP) mRNA expression was lower in FGR placentae compared with control (P = 0.004). mRNA levels of angiotensinogen (AGT) tended to be higher in FGR placentae compared with control (P = 0.090). Expression of prorenin, AGT, angiotensin-converting enzyme (ACE) or ACE2 proteins were similar in control and FGR placentae. The renin-AGT reaction is a first order reaction so levels of expression of placental AGT determine levels of Ang II. Decreasing levels of ACE2 and/or NEP by limiting the production of Ang-(1-7), which is a vasodilator, and increasing placental Ang II levels (vasoconstrictor) may result in an imbalance between the vasoconstrictor and vasodilator arms of the placental RAS. Ultimately this dysregulation of the placental RAS could lead to reduced placental perfusion that is evident in FGR.

UNICORN Babies: Understanding Circulating and Cerebral Creatine Levels of the Preterm Infant. An Observational Study Protocol.

Creatine is an essential metabolite for brain function, with a fundamental role in cellular (ATP) energy homeostasis. It is hypothesized that preterm infants will become creatine deplete in the early postnatal period, due to premature delivery from a maternal source of creatine and a limited supply of creatine in newborn nutrition. This potential alteration to brain metabolism may contribute to, or compound, poor neurological outcomes in this high-risk population. Understanding Creatine for Neurological Health in Babies (UNICORN) is an observational study of circulating and cerebral creatine levels in preterm infants. We will recruit preterm infants at gestational ages 23+0-26+6, 27+0-29+6, 30+0-32+6, 33+0-36+6, and a term reference group at 39+0-40+6 weeks of gestation, with 20 infants in each gestational age group. At birth, a maternal capillary blood sample, as well as a venous cord blood sample, will be collected. For preterm infants, serial infant plasma (heel prick), urine, and nutrition samples [total parenteral nutrition (TPN), breast milk, or formula] will be collected between birth and term "due date." Key fetomaternal information, including demographics, smoking status, and maternal diet, will also be collected. At term corrected postnatal age (CPA), each infant will undergo an MRI/1H-MRS scan to evaluate brain structure and measure cerebral creatine content. A general movements assessment (GMA) will also be conducted. At 3 months of CPA, infants will undergo a second GMA as well as further neurodevelopmental evaluation using the Developmental Assessment of Young Children - Second Edition (DAYC-2) assessment tool. The primary outcome measures for this study are cerebral creatine content at CPA and plasma and urine creatine and guanidinoacetate (creatine precursor) concentrations in the early postnatal period. We will also determine associations between (1) creatine levels at term CPA and neurodevelopmental outcomes (MRI, GMA, and DAY-C); (2) dietary creatine intake and circulating and cerebral creatine content; and (3) creatine levels and maternal characteristics. Novel approaches are needed to try and improve preterm-associated brain injury. Inclusion of creatine in preterm nutrition may better support ex utero brain development through improved cerebral cellular energy availability during a period of significant brain growth and development. Ethics Ref: HDEC 18/CEN/7 New Zealand. ACTRN: ACTRN12618000871246.

First evidence of a menstruating rodent: the spiny mouse (Acomys cahirinus).

BACKGROUND: Advances in research relating to menstruation and associated disorders (eg, endometriosis and premenstrual syndrome) have been hindered by the lack of an appropriate animal model. Menstruation, the cyclical shedding of the decidualized endometrium in the absence of pregnancy, is believed to be limited to 78 higher-order primates (human beings and Old World monkeys), 4 species of bat, and the elephant shrew. This represents only 1.5% of the known 5502 mammalian species and <0.09% of these are nonprimates. Thus, many aspects of menstruation remain poorly understood, limiting the development of effective treatments for women with menstrual disorders. Menstruation occurs as a consequence of progesterone priming of the endometrial stroma and a spontaneous decidual reaction. At the end of each infertile cycle as progesterone levels decline the uterus is unable to maintain this terminally differentiated stroma and the superficial endometrium is shed. True menstruation has never been reported in rodents. OBJECTIVE: Here we describe the first observation of menstruation in a rodent, the spiny mouse (Acomys cahirinus). STUDY DESIGN: Virgin female spiny mice (n = 14) aged 12-16 weeks were sampled through daily vaginal lavage for 2 complete reproductive cycles. Stage-specific collection of reproductive tissue and plasma was used for histology, prolactin immunohistochemistry, and enzyme-linked immunosorbent assay of progesterone (n = 4-5/stage of the menstrual cycle). Normally distributed data are reported as the mean ± SE and significant differences calculated using a 1-way analysis of variance. Nonnormal data are displayed as the median values of replicates (with interquartile range) and significant differences calculated using Kruskal-Wallis test. RESULTS: Mean menstrual cycle length was 8.7 ± 0.4 days with red blood cells observed in the lavages over 3.0 ± 0.2 days. Cyclic endometrial shedding and blood in the vaginal canal concluding with each infertile cycle was confirmed in all virgin females. The endometrium was thickest during the luteal phase at 322.6 µm (254.8, 512.2), when plasma progesterone peaked at 102.1 ng/mL (70.1, 198.6) and the optical density for prolactin immunoreactivity was strongest (0.071 ± 0.01 arbitrary units). CONCLUSION: The spiny mouse undergoes spontaneous decidualization, demonstrating for the first time menstruation in a rodent. The spiny mouse provides a readily accessible nonprimate model to study the mechanisms of menstrual shedding and repair, and may therefore be useful in furthering studies of human menstrual and pregnancy-associated disorders.