Enhanced progesterone support during stimulated cycles of transvaginal follicular aspiration improves bovine in vitro embryo production.

The in vitro production (IVP) of cattle embryos requires that germinal-vesicle stage oocytes undergo a period of maturation in vitro prior to fertilization and culture to the blastocyst stage. Success of IVP in taurine cattle is enhanced following ovarian stimulation prior to oocyte retrieval (OPU), particularly if preceded by a short period of FSH withdrawal ('coasting'). However, evidence regarding the importance of progesterone (P4) support during OPU-IVP is equivocal. The current study, therefore, determined the effects of increased peripheral P4 concentrations during FSH-stimulated ('coasted') cycles of OPU. Progesterone support was provided by either an active corpus luteum (CL) and/or one of two intravaginal P4 releasing devices (i.e., CIDR® [1.38 g P4] or PRID® Delta [1.55 g P4]). Expt. 1 established an initial estrus prior to OPU, allowing CL formation (single luteal phase) spanning the first two of five cycles of OPU; the remaining three cycles were supported by either a CIDR® or PRID® Delta. Expt. 2 commenced with two cycles of dominant follicle removal (including prostaglandin F2α) undertaken seven days apart prior to six cycles of OPU. The absence of a CL meant that these cycles were supported only by a CIDR® or PRID® Delta. As each experiment involved several sequential cycles of OPU, the cumulative effects of device use on vaginal discharges were also assessed. Each experiment involved 10 sexually mature Holstein heifers. In the absence of a CL, peak plasma P4 concentrations were greater (P = 0.002) for the PRID® Delta (4.3 ± 0.22) than for the CIDR® (2.9 ± 0.22). In Expt. 1 there was an interaction (P < 0.05) between CL presence at OPU and P4 device on Day 8 blastocyst yields, indicating an effect of P4 device only when the CL was absent. The percentage hatching/hatched blastocysts of matured oocytes for the CIDR® and PRID® Delta was 44.3 ± 5.04 and 41.0 ± 5.40 in the presence, and 17.1 ± 3.48 and 42.2 ± 3.76 in the absence, of a CL (P = 0.018). Combined analyses of data from Expt. 1 and 2, when no CL was present, confirmed that Day 8 blastocyst yields were greater (P = 0.022) for the PRID® Delta than the CIDR®. Vaginal discharge scores were higher (P < 0.001) for the PRID® Delta than the CIDR® in Expt. 1 but not in Expt 2; however scores were low, did not increase with repeated use, and thus were deemed of no clinical or welfare concern. In conclusion, enhanced P4 support during FSH-stimulated cycles of OPU-IVP can improve in vitro embryo development.

Periconceptional biomarkers for maternal obesity: a systematic review.

Periconceptional maternal obesity is linked to adverse maternal and neonatal outcomes. Identifying periconceptional biomarkers of pathways affected by maternal obesity can unravel pathophysiologic mechanisms and identify individuals at risk of adverse clinical outcomes. The literature was systematically reviewed to identify periconceptional biomarkers of the endocrine, inflammatory and one-carbon metabolic pathways influenced by maternal obesity. A search was conducted in Embase, Ovid Medline All, Web of Science Core Collection and Cochrane Central Register of Controlled Trials databases, complemented by manual search in PubMed until December 31st, 2020. Eligible studies were those that measured biomarker(s) in relation to maternal obesity, overweight/obesity or body mass index (BMI) during the periconceptional period (14 weeks preconception until 14 weeks post conception). The ErasmusAGE score was used to assess the quality of included studies. Fifty-one articles were included that evaluated over 40 biomarkers. Endocrine biomarkers associated with maternal obesity included leptin, insulin, thyroid stimulating hormone, adiponectin, progesterone, free T4 and human chorionic gonadotropin. C-reactive protein was associated with obesity as part of the inflammatory pathway, while the associated one-carbon metabolism biomarkers were folate and vitamin B12. BMI was positively associated with leptin, C-reactive protein and insulin resistance, and negatively associated with Free T4, progesterone and human chorionic gonadotropin. Concerning the remaining studied biomarkers, strong conclusions could not be established due to limited or contradictory data. Future research should focus on determining the predictive value of the optimal set of biomarkers for their use in clinical settings. The most promising biomarkers include leptin, adiponectin, human chorionic gonadotropin, insulin, progesterone and CRP.

Interspecific Variation in One-Carbon Metabolism within the Ovarian Follicle, Oocyte, and Preimplantation Embryo: Consequences for Epigenetic Programming of DNA Methylation.

One-carbon (1C) metabolism provides methyl groups for the synthesis and/or methylation of purines and pyrimidines, biogenic amines, proteins, and phospholipids. Our understanding of how 1C pathways operate, however, pertains mostly to the (rat) liver. Here we report that transcripts for all bar two genes (i.e., BHMT, MAT1A) encoding enzymes in the linked methionine-folate cycles are expressed in all cell types within the ovarian follicle, oocyte, and blastocyst in the cow, sheep, and pig; as well as in rat granulosa cells (GCs) and human KGN cells (a granulosa-like tumor cell line). Betaine-homocysteine methyltransferase (BHMT) protein was absent in bovine theca and GCs, as was activity of this enzyme in GCs. Mathematical modeling predicted that absence of this enzyme would lead to more volatile S-adenosylmethionine-mediated transmethylation in response to 1C substrate (e.g., methionine) or cofactor provision. We tested the sensitivity of bovine GCs to reduced methionine (from 50 to 10 µM) and observed a diminished flux of 1C units through the methionine cycle. We then used reduced-representation bisulfite sequencing to demonstrate that this reduction in methionine during bovine embryo culture leads to genome-wide alterations to DNA methylation in >1600 genes, including a cohort of imprinted genes linked to an abnormal fetal-overgrowth phenotype. Bovine ovarian and embryonic cells are acutely sensitive to methionine, but further experimentation is required to determine the significance of interspecific variation in BHMT expression.

Polycystic Ovary Syndrome: A Brain Disorder Characterized by Eating Problems Originating during Puberty and Adolescence.

Polycystic ovary syndrome (PCOS) is an endocrine condition associated with reproductive and psychiatric disorders, and with obesity. Eating disorders, such as bulimia and recurrent dieting, are also linked to PCOS. They can lead to the epigenetic dysregulation of the hypothalamic-pituitary-gonadal (HPG) axis, thereby impacting on ovarian folliculogenesis. We postulate that PCOS is induced by psychological distress and episodes of overeating and/or dieting during puberty and adolescence, when body dissatisfaction and emotional distress are often present. We propose that upregulated activation of the central HPG axis during this period can be epigenetically altered by psychological stressors and by bulimia/recurrent dieting, which are common during adolescence and which can lead to PCOS. This hypothesis is based on events that occur during a largely neglected stage of female reproductive development. To date, most research into the origins of PCOS has focused on the prenatal induction of this disorder, particularly in utero androgenization and the role of anti-Müllerian hormone. Establishing causality in our peripubertal model requires prospective cohort studies from infancy. Mechanistic studies should consider the role of the gut microbiota in addition to the epigenetic regulation of (neuro) hormones. Finally, clinicians should consider the importance of underlying chronic psychological distress and eating disorders in PCOS.

Comprehensive and quantitative profiling of B vitamins and related compounds in the mammalian liver.

A method for the simultaneous quantification of B vitamins and related amines in one-carbon (1C) metabolism would benefit the study of diet and genetic/epigenetic regulation of mammalian development and health. We present a validated method for the simultaneous quantitative analysis of 13 B vitamers and four related 1C-pathway amine intermediates in liver using hydrophilic interaction chromatography (HILIC) coupled to electrospray ionization tandem mass spectrometry. Frozen sheep liver samples (50 mg) were homogenized in cold 50% acetonitrile containing 1% acetic acid with the addition of two isotope labelled internal standards. Hot acid hydrolysis was applied to release the protein-bound forms. The separation of 17 analytes was achieved using a pHILIC column with a total run time of 13 min. Detection was achieved in electrospray positive ionisation mode. Limits of detection for the majority of analytes were within the range of 0.4-3.2 pmol/g. The method was applied to 266 sheep liver samples and revealed that adenosylcobalamin, methylcobalamin, pyridoxic acid, flavin adenine dinucleotide and thiamine were the major forms of the B vitamers present with pyridoxal 5'-phosphate and thiamine pyrophosphate being detected at lower concentrations. Trimethylglycine and methylglycine were the predominant 1C-related amines measured. As anticipated, the B vitamin status of individuals varied considerably, reflecting dietary and genetic variation in our chosen outbred model species. This method offers a simple sample extraction procedure and provides comprehensive coverage of B vitamins coupled with good sensitivity and reliability.

One-Carbon Metabolism: Linking Nutritional Biochemistry to Epigenetic Programming of Long-Term Development.

One-carbon (1C) metabolism comprises a series of interlinking metabolic pathways that include the methionine and folate cycles that are central to cellular function, providing 1C units (methyl groups) for the synthesis of DNA, polyamines, amino acids, creatine, and phospholipids. S-adenosylmethionine is a potent aminopropyl and methyl donor within these cycles and serves as the principal substrate for methylation of DNA, associated proteins, and RNA. We propose that 1C metabolism functions as a key biochemical conduit between parental environment and epigenetic regulation of early development and that interindividual and ethnic variability in epigenetic-gene regulation arises because of genetic variants within 1C genes, associated epigenetic regulators, and differentially methylated target DNA sequences. We present evidence to support these propositions, drawing upon studies undertaken in humans and animals. We conclude that future studies should assess the epigenetic effects of cumulative (multigenerational) dietary imbalances contemporaneously in both parents, as this better represents the human experience.

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver.

BACKGROUND: Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown. METHODS: In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses. RESULTS: In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1). CONCLUSIONS: Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism.

Impact of di-ethylhexylphthalate exposure on metabolic programming in P19 ECC-derived cardiomyocytes.

Di(2-ethylhexyl)phthalate (DEHP) is the most common plasticizer in plastic devices of everyday use. It is a ubiquitous environmental contaminant and primarily known to impair male gonadal development and fertility. Studies concerning the long-term effects of prenatal DEHP exposure on certain diseases [The Developmental Origins of Health and Disease paradigm (DOHaD) hypothesis] are scarce although it is proven that DEHP crosses the placenta. Rising environmental pollution during the last centuries coincides with an increasing prevalence of cardiovascular and metabolic diseases. We have investigated the effects of an early embryonic DEHP exposure at different developmental stages on cardiomyogenesis. We used an in-vitro model, the murine P19 embryonic carcinoma cell line (P19 ECC), mimicking early embryonic stages up to differentiated beating cardiomyocytes. P19 ECC were exposed to DEHP (5, 50, 100 µg ml(-1)) at the undifferentiated stage for 5 days and subsequently differentiated to beating cardiomyocytes. We analyzed the expression of metabolic (Pparg1, Fabp4 and Glut4), cardiac (Myh6, Gja1) and methylation (Dnmt1, Dnmt3a) marker genes by quantitative real-time PCR (qRT-PCR), beating rate and the differentiation velocity of the cells. The methylation status of Pparg1, Ppara and Glut4 was investigated by pyrosequencing. DEHP significantly altered the expression of all investigated genes. The beating rate and differentiation velocity were accelerated. Exposure to DEHP led to small but statistically significant increases in methylation of specific CpGs within Ppara and Pparg1, which otherwise were generally hypomethylated, but methylation of Glut4 was unaltered. Early DEHP exposure of P19 ECC alters the expression of genes associated with cellular metabolism and the functional features of cardiomyocytes.

Parental diet, pregnancy outcomes and offspring health: metabolic determinants in developing oocytes and embryos.

The periconceptional period, embracing the terminal stages of oocyte growth and post-fertilisation development up to implantation, is sensitive to parental nutrition. Deficiencies or excesses in a range of macro- and micronutrients during this period can lead to impairments in fertility, fetal development and long-term offspring health. Obesity and genotype-related differences in regional adiposity are associated with impaired liver function and insulin resistance, and contribute to fatty acid-mediated impairments in sperm viability and oocyte and embryo quality, all of which are associated with endoplasmic reticulum stress and compromised fertility. Disturbances to maternal protein metabolism can elevate ammonium concentrations in reproductive tissues and disturb embryo and fetal development. Associated with this are disturbances to one-carbon metabolism, which can lead to epigenetic modifications to DNA and associated proteins in offspring that are both insulin resistant and hypertensive. Many enzymes involved in epigenetic gene regulation use metabolic cosubstrates (e.g. acetyl CoA and S-adenosyl methionine) to modify DNA and associated proteins, and so act as 'metabolic sensors' providing a link between parental nutritional status and gene regulation. Separate to their genomic contribution, spermatozoa can also influence embryo development via direct interactions with the egg and by seminal plasma components that act on oviductal and uterine tissues.

The periconceptional period, reproduction and long-term health of offspring: the importance of one-carbon metabolism.

BACKGROUND Most reproductive failures originate during the periconceptional period and are influenced by the age and the lifestyle of parents-to-be. We advance the hypothesis that these failures can arise as a partial consequence of derangements to one-carbon (1-C) metabolism (i.e. metabolic pathways that utilize substrates/cofactors such as methionine, vitamin B12, folate). 1-C metabolic pathways drive the synthesis of proteins, biogenic amines and lipids required for early growth, together with the synthesis and methylation of DNA and histones essential for the regulation of gene expression. We review how deficiencies in periconceptional 1-C metabolism affect fertility and development together with underlying mechanisms derived from animal studies. METHODS A literature search was performed using PubMed and bibliographies of all relevant original research articles and reviews. RESULTS We define 'periconception' as a 5-6-month period in women embracing oocyte growth, fertilization, conceptus formation and development to Week 10 of gestation (coinciding with the closure of the secondary palate in the embryo). During this period significant epigenetic modifications to chromatin occur that correspond with normal development. Subtle variations in 1-C metabolism genes and deficiencies in 1-C substrates/cofactors together with poor lifestyle, such as smoking and alcohol consumption, disturb 1-C metabolism and contribute to subfertility and early miscarriage and compromise offspring health. Procedures used in assisted reproduction can also disturb these metabolic pathways and contribute to poor pregnancy outcomes. CONCLUSIONS Evidence presented indicates that parental nutrition and other lifestyle factors during the periconceptional period can affect reproductive performance via 1-C metabolic pathways. This knowledge provides opportunities for treatment and prevention of reproductive failures and future non-communicable diseases.

A mathematical model of the bovine oestrous cycle: simulating outcomes of dietary and pharmacological interventions.

A mathematical model was constructed to simulate the bovine oestrous cycle by using nonlinear differential equations to describe the biological mechanisms which regulate the cycle. The model predicts circulating concentrations of gonadotrophin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, oestradiol, inhibin and progesterone. These hormones collectively provide control and feedback mechanisms between the hypothalamus, pituitary gland and ovaries, which regulate ovarian follicular dynamics, corpus luteum function and ovulation. When follicular growth parameters are altered, the model predicts that cows will exhibit either two or three follicular waves per cycle, as seen in practice. Changes in other parameters allow the model to simulate: effects of nutrition on follicle recruitment and size of the ovulatory follicle; effects of negative energy balance on postpartum anoestrus; and effects of pharmacological intervention on hormone profiles and timing of ovulation. It is concluded that this model provides a sound basis for exploring factors that influence the bovine oestrous cycle in order to test hypotheses about nutritional and hormonal influences which, with further validation, should help to design dietary or pharmacological strategies for improving reproductive performance in cattle.

The expression, regulation and function of secreted protein, acidic, cysteine-rich in the follicle-luteal transition.

The role of the tissue remodelling protein, secreted protein, acidic, cysteine-rich (SPARC), in key processes (e.g. cell reorganisation and angiogenesis) that occur during the follicle-luteal transition is unknown. Hence, we investigated the regulation of SPARC in luteinsing follicular cells and potential roles of SPARC peptide 2.3 in a physiologically relevant luteal angiogenesis culture system. SPARC protein was detected mainly in the theca layer of bovine pre-ovulatory follicles, but its expression was considerably greater in the corpus haemorrhagicum. Similarly, SPARC protein (western blotting) was up-regulated in luteinising granulosa but not in theca cells during a 6-day culture period. Potential regulatory candidates were investigated in luteinising granulosa cells: LH did not affect SPARC (P>0.05); transforming growth factor (TGF) B1 (P<0.001) dose dependently induced the precocious expression of SPARC and increased final levels: this effect was blocked (P<0.001) by SB505124 (TGFB receptor 1 inhibitor). Additionally, fibronectin, which is deposited during luteal development, increased SPARC (P<0.01). In luteal cells, fibroblast growth factor 2 decreased SPARC (P<0.001) during the first 5 days of culture, while vascular endothelial growth factor A increased its expression (P<0.001). Functionally, KGHK peptide, a SPARC proteolytic fragment, stimulated the formation of endothelial cell networks in a luteal cell culture system (P<0.05) and increased progesterone production (P<0.05). Collectively, these findings indicate that SPARC is intricately regulated by pro-angiogenic and other growth factors together with components of the extracellular matrix during the follicle-luteal transition. Thus, it is possible that SPARC plays an important modulatory role in regulating angiogenesis and progesterone production during luteal development.

Effects of omega-3 and -6 polyunsaturated fatty acids on ovine follicular cell steroidogenesis, embryo development and molecular markers of fatty acid metabolism.

We previously reported increased follicular fluid progesterone (P(4)) concentrations in ewes fed an n-3 compared to an n-6 polyunsaturated fatty acid (PUFA)-enriched diet, but detected no differential effect of n-3 and n-6 PUFA-enriched high-density lipoproteins (HDL) on granulosa cell (GC) steroidogenesis in vitro. Moreover, net n-6 PUFA-enriched HDL reduced early embryo development, but in the absence of a net uptake of FA. Consequently, we hypothesised that a) effects of n-3 PUFA on ovarian steroidogenesis are mediated by theca rather than GCs and b) during embryo culture lipids are acquired solely from the albumin fraction of serum, so that albumin-delivered n-3 and n-6 PUFA exert a greater differential effect on embryo development than either low-density lipoprotein (LDL)- or HDL-delivered PUFA. Data confirmed that n-3 PUFA increases P(4) production solely in theca cells and that this is associated with an increase in STAR transcript expression. Furthermore, LDL- and HDL-delivered n-3 PUFA are equally efficacious in this regard during the first 96 h of culture, but thereafter only HDL-delivered n-3 PUFA induces this effect in partially luteinised theca cells. We also demonstrate that albumin is the sole serum fraction that leads to a net uptake of FA during embryo culture. PUFA-enriched serum and albumin increased the yield of morphologically poorer quality blastocysts with increased transcript expression for the antioxidant enzyme SOD1. Important differential effects of n-3 and n-6 PUFA on ovarian steroidogenesis acting solely on theca cells are identified, but differential effects of PUFA on embryo development are less apparent.

Preconception folic acid use modulates estradiol and follicular responses to ovarian stimulation.

BACKGROUND: Folate is a methyl donor. Availability of folate affects DNA methylation profiles and thereby gene expression profiles. We investigated the effects of low-dose folic acid use (0.4 mg/d) on the ovarian response to mild and conventional ovarian stimulation in women. METHODS: In a randomized trial among subfertile women, 24 and 26 subjects received conventional and mild ovarian stimulation, respectively. Blood samples were taken during the early follicular phase of the cycle prior to treatment and on the day of human chorionic gonadotropin administration for determination of serum total homocysteine, anti-Müllerian hormone (AMH), estradiol, and folate. Folic acid use was validated by questionnaire and serum folate levels. Preovulatory follicles were visualized, counted, and diameters recorded using transvaginal ultrasound. The relation between folic acid use and ovarian response was assessed using linear regression analysis. RESULTS: Folic acid use modified the ovarian response to ovarian stimulation treatment. The estradiol response was higher in nonfolic acid users receiving conventional treatment [ß(interaction) = 0.52 (0.07-0.97); P = 0.03], and this effect was independent of serum AMH levels and the preovulatory follicle count. In the conventional treatment, the mean follicle number was also greater in nonusers compared with the users group (14.1 vs. 8.9, P = 0.03). CONCLUSION: Low-dose folic acid use attenuates follicular and endocrine responses to conventional stimulation, independent of AMH and follicle count. The nature of this observation suggests that the effect of folic acid is most prominent during early follicle development, affecting immature follicles. Deleterious effects of folate deficiency, like DNA hypomethylation and oxidative stress, can help to explain our observations.

B-vitamin and homocysteine status determines ovarian response to gonadotropin treatment in sheep.

Maternal B-vitamin status and homocysteinemia can affect fertility and pregnancy establishment, although the direct effects on ovarian follicle and oocyte development are not known. We report on the effects of restricting the supply of vitamin B(12) and methionine from the diet of mature female sheep on ovarian folliculogenesis following follicle-stimulating hormone (FSH) stimulation. The study was split into three batches and involved 76 animals. Surprisingly, the number of growing, estrogen-active antral follicles following FSH treatment was enhanced (P = 0.005) following this dietary intervention. This increase occurred even in the presence of modest live-weight loss (batch 1 only) and depressed plasma insulin concentrations, suggesting a breakdown in the regulation of follicular responsiveness to FSH. This dietary intervention also increased plasma homocysteine concentrations. Physiological concentrations of homocysteine increased granulosa cell proliferation (P < 0.001), estradiol production (P = 0.05), and FSHR transcript expression (P = 0.017) during culture. Transcript levels for growth differentiation factor 9 and bone morphogenetic protein 15 in oocytes from treated ewes were increased (P < 0.05) in the first two batches. Furthermore, regression of BMP receptor 2 (BMPR2) transcript expression and diet on follicle number revealed a significant interaction (P = 0.01); BMPR2 transcript expression was associated with follicle number only in vitamin B(12)/methionine-restricted animals. Because FSHR transcript expression also was positively (P = 0.007) related to follicle number, the effects of diet may have arisen through enhanced FSH and BMP signaling. Although this remains to be confirmed, the data support an intraovarian impact of vitamin B(12)/methionine-deficient diets.