BackgroundMetabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts.ResultsBovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological (“BASAL”) concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological (“HIGH COMBI”) concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders.ConclusionsOverall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.

Although fragmented and sometimes inconsistent, the proof of a vital link between the importance of the physiological status of the mother and her subsequent reproductive success is building up. High-yielding dairy cows are suffering from a substantial decline in fertility outcome over past decades. For many years, this decrease in reproductive output has correctly been considered multifactorial, with factors including farm management, feed ratios, breed and genetics and, last, but not least, ever-rising milk production. Because the problem is complex and requires a multidisciplinary approach, it is hard to formulate straightforward conclusions leading to improvements on the 'work floor'. However, based on remarkable similarities on the preimplantation reproductive side between cattle and humans, there is a growing tendency to consider the dairy cow's negative energy balance and accompanying fat mobilisation as an interesting model to study the impact of maternal metabolic disorders on human fertility and, more specifically, on oocyte and preimplantation embryo quality. Considering the mutual interest of human and animal scientists studying common reproductive problems, this review has several aims. First, we briefly introduce the 'dairy cow case' by describing the state of the art of research into metabolic imbalances and their possible effects on dairy cow reproduction. Second, we try to define relevant in vitro models that can clarify certain mechanisms by which aberrant metabolite levels may influence embryonic health. We report on recent advances in the assessment of embryo metabolism and meantime critically elaborate on advantages and major limitations of in vitro models used so far. Finally, we discuss hurdles to be overcome to successfully translate the scientific data to the field.

STUDY QUESTION Does oocyte maturation under lipolytic conditions have detrimental carry-over effects on post-hatching embryo development of good-quality blastocysts after transfer? SUMMARY ANSWER Surviving, morphologically normal blastocysts derived from bovine oocytes that matured under lipotoxic conditions exhibit long-lasting cellular dysfunction at the transcriptomic and metabolic levels, which coincides with retarded post-hatching embryo development. WHAT IS KNOWN ALREADY There is increasing evidence showing that following maturation in pathophysiologically relevant lipotoxic conditions (as in obesity or metabolic syndrome), surviving blastocysts of good (transferable) morphological quality have persistent transcriptomic and epigenetic alteration even when in vitro embryo culture takes place under standard conditions. However, very little is known about subsequent development in the uterus after transfer. STUDY DESIGN, SIZE, DURATION Bovine oocytes were matured in vitro in the presence of pathophysiologically relevant, high non-esterified fatty acid (NEFA) concentrations (HIGH PA), or in basal NEFA concentrations (BASAL) as a physiological control. Eight healthy multiparous non-lactating Holstein cows were used for embryo transfers. Good-quality blastocysts (pools of eight) were transferred per cow, and cows were crossed over for treatments in the next replicate. Embryos were recovered 7 days later and assessed for post-hatching development, phenotypic features and gene expression profile. Blastocysts from solvent-free and NEFA-free maturation (CONTROL) were also tested for comparison. PARTICIPANTS/MATERIALS, SETTING, METHODS Recovered Day 14 embryos were morphologically assessed and dissected into embryonic disk (ED) and extraembryonic tissue (EXT). Samples of EXT were cultured for 24 h to assess cellular metabolic activity (glucose and pyruvate consumption and lactate production) and embryos’ ability to signal for maternal recognition of pregnancy (interferon-τ secretion; IFN-τ). ED and EXT samples were subjected to RNA sequencing to evaluate the genome-wide transcriptome patterns. MAIN RESULTS AND THE ROLE OF CHANCE The embryo recovery rate at Day 14 p.i. was not significantly different among treatment groups (P &gt; 0.1). However, higher proportions of HIGH PA embryos were retarded in growth (in spherical stage) compared to the more elongated tubular stage embryos in the BASAL group (P &lt; 0.05). Focusing on the normally developed tubular embryos in both groups, HIGH PA exposure resulted in altered cellular metabolism and altered transcriptome profile particularly in pathways related to redox-regulating mechanisms, apoptosis, cellular growth, interaction and differentiation, energy metabolism and epigenetic mechanisms, compared to BASAL embryos. Maturation under BASAL conditions did not have any significant effects on post-hatching development and cellular functions compared to CONTROL. LARGE-SCALE DATA The datasets of RNA sequencing analysis are available in the NCBI’s Gene Expression Omnibus (GEO) repository, series accession number GSE127889 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127889). Datasets of differentially expressed genes and their gene ontology functions are available in the Mendeley datasets at http://dx.doi.org/10.17632/my2z7dvk9j.2. LIMITATIONS, REASONS FOR CAUTION The bovine model was used here to allow non-invasive embryo transfer and post-hatching recovery on Day 14. There are physiological differences in some characteristics of post-hatching embryo development between human and cows, such as embryo elongation and trophoblastic invasion. However, the main carry-over effects of oocyte maturation under lipolytic conditions described here are evident at the cellular level and therefore may also occur during post-hatching development in other species including humans. In addition, post-hatching development was studied here under a healthy uterine environment to focus on carry-over effects originating from the oocyte, whereas additional detrimental effects may be induced by maternal metabolic disorders due to adverse changes in the uterine microenvironment. RNA sequencing results were not verified by qPCR, and no solvent control was included. WIDER IMPLICATIONS OF THE FINDINGS Our observations may increase the awareness of the importance of maternal metabolic stress at the level of the preovulatory oocyte in relation to carry-over effects that may persist in the transferrable embryos. It should further stimulate new research about preventive and protective strategies to optimize maternal metabolic health around conception to maximize embryo viability and thus fertility outcome. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Flemish Research Fund (FWO grant 11L8716N and FWO project 42/FAO10300/6541). The authors declare there are no conflicts of interest.

We investigated whether and to which extent plasma non-esterified fatty acids (NEFAs) are reflected in oviduct fluid (OF) using an improved ex vivo flushing method. OF and plasma NEFA concentrations were respectively 0.29±0.19 and 0.31±0.14mmol/L, they didn't differ significantly (P=0.13) and tended to be positively correlated (Pearson correlation coefficient=0.56; P=0.07). Results suggest that OF NEFAs mirror the concentrations seen in plasma of healthy cattle.

A negative energy balance in metabolically compromised high producing dairy cows has been shown to influence oocyte and embryo quality. However, the possible involved pathways needed more attention to better understand specific deleterious effects. Oocyte maturation is the first process to be scrutinized. Because many possible metabolic factors might directly impact oocyte quality, systematic in vitro approaches were used to investigate the effects of oocyte maturation under elevated NEFA concentrations. Blastocysts originating from NEFA-exposed oocytes showed a lower cell number, an increased apoptotic cell index, signs of glucose intolerance, sensitive to oxidative stress and mitochondrial dysfunction. Defining these embryos' transcriptome and epigenome signatures revealed changes in DNA methylation patterns. Long-term exposure of developing murine follicles to elevated NEFA concentrations showed to impair oocyte developmental competence even more. While little is known on how the oviductal microenvironment can change as a consequence of a negative energy balance, a validated in vitro bovine oviduct model offered some valuable insights on how NEFAs disturb oviductal cell physiology. NEFA exposure reduces cell proliferation, cell migration, sperm binding capacity and monolayer integrity. In addition, oviductal cells seem to play an active role in regulating luminal NEFA-concentrations through increased permeability, intracellular lipid accumulation and fatty acid metabolism. This might favour early embryo development. The establishment of a successful pregnancy largely depends on the ability of the embryo to interact with a properly prepared endometrium. Because suboptimal physiological conditions influence oocyte maturation and embryo development to the extent that epigenetic consequences are unavoidable, the question arises if these changes hamper embryo implantation and subsequent development. Gene expression studies on epithelial endometrial cells brought in contact with in vitro embryos cultured for 4 days under suboptimal conditions reveal that the embryo-endometrial signaling is affected. Transfer of bovine embryos derived from compromised oocytes showed disturbed embryo development following recovery at day 14 with a negative impact on IFNt secretion and therefore suggesting carry-over effects from suboptimal culture conditions. The current paper will document the most important recent findings and comment on extrapolation possibilities from in vitro studies to field conditions in daily dairy practice. In addition, the possibility of remediating approaches will be discussed to see how this knowledge might generate insights on possible alleviating strategies.