Obesity and oocyte quality: significant implications for ART and emerging mechanistic insights.

The prevalence of obesity in adults worldwide, and specifically in women of reproductive age, is concerning given the risks to fertility posed by the increased risk of type 2 diabetes, metabolic syndrome, and other noncommunicable diseases. Obesity has a multi-systemic impact in female physiology that is characterized by the presence of oxidative stress, lipotoxicity, and the activation of pro-inflammatory pathways, inducing tissue-specific insulin resistance and ultimately conducive to abnormal ovarian function. A higher body mass is linked to Polycystic Ovary Syndrome, dysregulated menstrual cycles, anovulation, and longer time to pregnancy, even in ovulatory women. In the context of assisted reproductive technology (ART), compared to women of normal body mass index, obese women have worse outcomes in every step of their journey, resulting in reduced success measured as live birth rate. Even after pregnancy is achieved, obese women have a higher chance of miscarriage, gestational diabetes, pregnancy complications, birth defects, and most worryingly, a higher risk of stillbirth and neonatal death. The potential for compounding effects of ART on pregnancy complications and infant morbidities in obese women has not been studied. There is still much debate in the field on whether these poorer outcomes are mainly driven by defects in oocyte quality, abnormal embryo development, or an unaccommodating uterine environment, however the clinical evidence to date suggests a combination of all three are responsible. Animal models of maternal obesity shed light on the mechanisms underlying the effects of obesity on the peri-conception environment, with recent findings pointing to lipotoxicity in the ovarian environment as a key driver of defects in oocytes that have not only reduced developmental competence but long-lasting effects in offspring health.

OXIDATIVE STRESS AND REPRODUCTIVE FUNCTION: Reactive oxygen species in the mammalian pre-implantation embryo.

In brief: Reactive oxygen species are generated throughout the pre-implantation period and are necessary for normal embryo formation. However, at pathological levels, they result in reduced embryo viability which can be mediated through factors delivered by sperm and eggs at conception or from the external environment. Abstract: Reactive oxygen species (ROS) occur naturally in pre-implantation embryos as a by-product of ATP generation through oxidative phosphorylation and enzymes such as NADPH oxidase and xanthine oxidase. Biological concentrations of ROS are required for crucial embryonic events such as pronuclear formation, first cleavage and cell proliferation. However, high concentrations of ROS are detrimental to embryo development, resulting in embryo arrest, increased DNA damage and modification of gene expression leading to aberrant fetal growth and health. In vivo embryos are protected against oxidative stress by oxygen scavengers present in follicular and oviductal fluids, while in vitro, embryos rely on their own antioxidant defence mechanisms to protect against oxidative damage, including superoxide dismutase, catalase, glutathione and glutamylcysteine synthestase. Pre-implantation embryonic ROS originate from eggs, sperm and embryos themselves or from the external environment (i.e. in vitro culture system, obesity and ageing). This review examines the biological and pathological roles of ROS in the pre-implantation embryo, maternal and paternal origins of embryonic ROS, and from a clinical perspective, we comment on the growing interest in combating increased oxidative damage in the pre-implantation embryo through the addition of antioxidants.

Dynamics of Mitochondrial DNA Copy Number and Membrane Potential in Mouse Pre-Implantation Embryos: Responses to Diverse Types of Oxidative Stress.

Mitochondria undergo a myriad of changes during pre-implantation embryo development, including shifts in activity levels and mitochondrial DNA (mtDNA) replication. However, how these distinct aspects of mitochondrial function are linked and their responsiveness to diverse stressors is not well understood. Here, we show that mtDNA content increased between 8-cell embryos and the blastocyst stage, with similar copy numbers per cell in the inner cell mass (ICM) and trophectoderm (TE). In contrast, mitochondrial membrane potential (MMP) was higher in TE than ICM. Culture in ambient oxygen (20% O2) altered both aspects of mitochondrial function: the mtDNA copy number was upregulated in ICM, while MMP was diminished in TE. Embryos cultured in 20% O2 also exhibited delayed development kinetics, impaired implantation, and reduced mtDNA levels in E18 fetal liver. A model of oocyte mitochondrial stress using rotenone showed only a modest effect on on-time development and did not alter the mtDNA copy number in ICM; however, following embryo transfer, mtDNA was higher in the fetal heart. Lastly, endogenous mitochondrial dysfunction, induced by maternal age and obesity, altered the blastocyst mtDNA copy number, but not within the ICM. These results demonstrate that mitochondrial activity and mtDNA content exhibit cell-specific changes and are differentially responsive to diverse types of oxidative stress during pre-implantation embryogenesis.

Arrdc4-dependent extracellular vesicle biogenesis is required for sperm maturation.

Extracellular vesicles (EVs) are important players in cell to cell communication in reproductive systems. Notably, EVs have been found and characterized in the male reproductive tract, however, direct functional evidence for their importance in mediating sperm function is lacking. We have previously demonstrated that Arrdc4, a member of the α-arrestin protein family, is involved in extracellular vesicle biogenesis and release. Here we show that Arrdc4-mediated extracellular vesicle biogenesis is required for proper sperm function. Sperm from Arrdc4-/- mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as observed by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and two-cell embryo production. We found a significant reduction in extracellular vesicle production by Arrdc4-/- epididymal epithelial cells, and further, supplementation of Arrdc4-/- sperm with additional vesicles dampened the acrosome reaction defect and restored zona pellucida binding. These results indicate that Arrdc4 is important for proper sperm maturation through the control of extracellular vesicle biogenesis.

A Hyperandrogenic Environment Causes Intrinsic Defects That Are Detrimental to Follicular Dynamics in a PCOS Mouse Model.

Polycystic ovary syndrome (PCOS) is a common cause of female infertility. Hyperandrogenism is both a major symptom and key diagnostic trait of PCOS; however, the direct impact of this androgen excess on ovarian dynamics is unclear. By combining a DHT-induced PCOS mouse model with an ex vivo follicle culture system, we investigated the impact of hyperandrogenism on ovarian function. Ovaries from PCOS mice exhibited the characteristic polycystic ovary morphology with numerous large cystic follicles and no corpora lutea present. Isolation and individual culture of preantral and antral follicles from PCOS mice resulted in slower growth rates during 5 days compared with the follicles isolated from control mice (P < 0.01). In contrast, preovulatory follicles from PCOS mice exhibited a significant increase in growth rate compared with controls (P < 0.01). Preantral follicles from PCOS ovaries maintained comparable follicular health as control follicles, but antral and preovulatory PCOS follicles exhibited reduced follicle health (P < 0.01) and survival rates (P < 0.01). Compared with controls, PCOS females also exhibited a poorer response to hyperstimulation (P < 0.01), impaired oocyte function evident by increased levels of reactive oxygen species (P < 0.01), and a reduction in on-time embryo development (P < 0.01). These results demonstrate that prolonged exposure to androgen excess leads to aberrant follicle development, which persists even after removal from the hyperandrogenic environment, causing perturbed follicular developmental trajectories. These findings indicate that an in vivo hyperandrogenic environment in patients with PCOS may intrinsically induce detrimental effects on follicles and oocytes.

Inflammatory markers in human follicular fluid correlate with lipid levels and Body Mass Index.

The detrimental consequences of obesity on female fertility are well known, but the functional changes that occur in the ovary in response to elevated BMI are not clear. Obesity induces multiple components of a systemic inflammatory state that is a key pathway by which it initiates tissue dysfunction in adipose, liver and muscle; however whether obesity induces similar inflammatory changes in the ovary has not been fully investigated. This is important to understand because it is increasingly clear that obesity at conception impacts not only pregnancy rates but also influences pre-implantation embryo development. To further understand the characteristics of inflammation in the ovaries of obese women we analysed a panel of cytokines (IL6, IL10 and TNFα), adipokines (adiponectin, leptin and monocyte chemotactic factor 1 (MCP-1)) and acute phase proteins (C-Reactive Protein (CRP) and sICAM-1) in the ovarian follicular fluid obtained at oocyte aspiration from women (n = 48) who were lean, overweight or obese. We hypothesised that adipokines and pro-inflammatory cytokines would be correlated with and/or dysregulated by increasing Body Mass Index (BMI). Surprisingly however, the majority were not related to BMI but instead were positively correlated with lipid levels in follicular fluid, namely triglycerides and free fatty acids. Further, as is typical of metabolic inflammation, the inflammatory markers that were associated with intra-follicular lipids included both pro-inflammatory (CRP, IL6, TNFα) and anti-inflammatory (adiponectin, IL10) mediators. The direct consequences of an ovarian microenvironment containing high levels of lipids and inflammatory mediators are not known but could impact luteinisation, ovulation and/or oocyte developmental competence.

Regulation of the ovarian inflammatory response at ovulation by nuclear progesterone receptor.

PROBLEM: The nuclear progesterone receptor (PGR) transcription factor is essential for ovulation; however, the exact mechanisms by which PGR controls ovulation are not known. The aim of this study was to determine whether PGR regulates inflammatory mediators in the ovary. METHOD OF STUDY: Ovaries from mice lacking PGR (PRKO) and heterozygous PR+/- littermates were subjected to microarray analysis of a large panel of inflammatory genes. Immune cell subsets were detected by gene expression; and neutrophils by immunohistochemistry and chemotaxis assay. RESULTS: PRKO ovaries exhibited dysregulated expression of vasodilator (Edn1), cytokine (Il-6, Tgfb1), adhesion receptor (Cd34), apoptotic factor (Bax) and transcription factors (Nfkb2, Socs1, Stat3). Ptgs2 was also reduced in PRKO ovaries, but mRNA and protein were not different in granulosa cells. There were reduced neutrophils in ovaries of PRKO mice at ovulation; however, chemotaxis assays showed PRKO neutrophils migrate normally and that PRKO ovarian extracts exhibit chemotactic properties in vitro. CONCLUSION: Specific inflammatory mediators are altered in the ovaries of PRKO mice indicating that progesterone regulates features of inflammation at ovulation.

Periconception onset diabetes is associated with embryopathy and fetal growth retardation, reproductive tract hyperglycosylation and impaired immune adaptation to pregnancy.

Diabetes has been linked with impaired fertility but the underlying mechanisms are not well defined. Here we use a streptozotocin-induced diabetes mouse model to investigate the cellular and biochemical changes in conceptus and maternal tissues that accompany hyperglycaemia. We report that streptozotocin treatment before conception induces profound intra-cellular protein ß-O-glycosylation (O-GlcNAc) in the oviduct and uterine epithelium, prominent in early pregnancy. Diabetic mice have impaired blastocyst development and reduced embryo implantation rates, and delayed mid-gestation growth and development. Peri-conception changes are accompanied by increased expression of pro-inflammatory cytokine Trail, and a trend towards increased Il1a, Tnf and Ifng in the uterus, and changes in local T-cell dynamics that skew the adaptive immune response to pregnancy, resulting in 60% fewer anti-inflammatory regulatory T-cells within the uterus-draining lymph nodes. Activation of the heat shock chaperones, a mechanism for stress deflection, was evident in the reproductive tract. Additionally, we show that the embryo exhibits elevated hyper-O-GlcNAcylation of both cytoplasmic and nuclear proteins, associated with activation of DNA damage (É£H2AX) pathways. These results advance understanding of the impact of peri-conception diabetes, and provide a foundation for designing interventions to support healthy conception without propagation of disease legacy to offspring.