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This work describes the use of mass spectrometry-based metabolomics as a non-invasive approach to accurately predict birth prior to embryo transfer (ET) starting from embryo culture media and plasma recipient. Metabolomics was used here as a predictive platform. Day-6 in vitro produced embryos developed singly in modified synthetic oviduct fluid culture medium (CM) drops for 24 h were vitrified as Day-7 blastocysts and transferred to recipients. Day-0 and Day-7 recipient plasma (N = 36 × 2) and CM (N = 36) were analyzed by gas chromatography coupled to the quadrupole time of flight mass spectrometry (GC-qTOF). Metabolites quantified in CM and plasma were analyzed as a function to predict pregnancy at Day-40, Day-62, and birth (univariate and multivariate statistics). Subsequently, a Boolean matrix (F1 score) was constructed with metabolite pairs (one from the embryo, and one from the recipient) to combine the predictive power of embryos and recipients. Validation was performed in independent cohorts of ETs analyzed. Embryos that did not reach birth released more stearic acid, capric acid, palmitic acid, and glyceryl monostearate in CM (i.e., (p < 0.05, FDR < 0.05, Receiver Operator Characteristic—area under curve (ROC-AUC)> 0.669). Within Holstein recipients, hydrocinnamic acid, alanine, and lysine predicted birth (ROC-AUC > 0.778). Asturiana de los Valles recipients that reached birth showed lower concentrations of 6-methyl-5-hepten-2-one, stearic acid, palmitic acid, and hippuric acid (ROC-AUC > 0.832). Embryonal capric acid and glyceryl-monostearate formed F1 scores generally >0.900, with metabolites found both to differ (e.g., hippuric acid, hydrocinnamic acid) or not (e.g., heptadecanoic acid, citric acid) with pregnancy in plasmas, as hypothesized. Efficient lipid metabolism in the embryo and the recipient can allow pregnancy to proceed. Changes in phenolics from plasma suggest that microbiota and liver metabolism influence the pregnancy establishment in cattle.

According to the World Health Organization, infertility affects up to 14% of couples under reproductive age, leading to an exponential rise in the use of assisted reproduction as a route for conceiving a baby. In the same way, thousands of embryos are produced in cattle and other farm animals annually, leading to increased numbers of individuals born. All reproductive manipulations entail deviations of natural phenotypes and genotypes, with in vitro embryo technologies perhaps showing the biggest effects, although these alterations are still emerging. Most of these indications have been provided by animal models, in particular the bovine species, due to its similarities to human early embryo development. Oocytes and embryos are highly sensitive to environmental stress in vivo and in vitro. Thus, during in vitro culture, a number of stressful conditions affect embryonic quality and viability, inducing subfertility and/or long-term consequences that may reach the offspring. A high proportion of the embryos produced in vitro are arrested at a species-specific stage of development during the first cell divisions. These arrested embryos do not show signs of programmed cell death during early cleavage stages. Instead, defective in vitro produced embryos would enter a permanent cell cycle arrest compatible with cellular senescence, in which they show active metabolism and high reactive oxygen species levels. Later in development, mainly during the morula and blastocyst stages, apoptosis would mediate the elimination of certain cells, accomplishing both a physiological role in to balancing cell proliferation and death, and a pathological role preventing the transmission of damaged cells with an altered genome. The latter would acquire relevant importance in in vitro produced embryos that are submitted to stressful environmental stimuli. In this article, we review the mechanisms mediating apoptosis and senescence during early embryo development, with a focus on in vitro produced bovine embryos. Additionally, we shed light on the protective role of senescence and apoptosis to ensure that unhealthy cells and early embryos do not progress in development, avoiding long-term detrimental effects.

In vitro produced (IVP) embryos show large metabolic variability induced by breed, culture conditions, embryonic stage and sex and gamete donors. We hypothesized that the birth potential could be accurately predicted by UHPLC-MS/MS in culture medium (CM) with the discrimination of factors inducing metabolic variation. Day-6 embryos were developed in single CM (modified synthetic oviduct fluid) for 24 h and transferred to recipients as fresh (28 ETs) or frozen/thawed (58 ETs) Day-7 blastocysts. Variability was induced with seven bulls, slaughterhouse oocyte donors, culture conditions (serum + Bovine Serum Albumin [BSA] or BSA alone) prior to single culture embryonic stage records (Day-6: morula, early blastocyst, blastocyst; Day-7: expanding blastocyst; fully expanded blastocysts) and cryopreservation. Retained metabolite signals (6111) were analyzed as a function of pregnancy at Day-40, Day-62 and birth in a combinatorial block study with all fixed factors. We identified 34 accumulated metabolites through 511 blocks, 198 for birth, 166 for Day-62 and 147 for Day-40. The relative abundance of metabolites was higher within blocks from non-pregnant (460) than from pregnant (51) embryos. Taxonomy classified lipids (12 fatty acids and derivatives; 224 blocks), amino acids (12) and derivatives (3) (186 blocks), benzenoids (4; 58 blocks), tri-carboxylic acids (2; 41 blocks) and 5-Hydroxy-l-tryptophan (2 blocks). Some metabolites were effective as single biomarkers in 95 blocks (Receiver Operating Characteristic – Area Under the Curve [ROC-AUC]: 0.700–1.000). In contrast, more accurate predictions within the largest data sets were obtained with combinations of 2, 3 and 4 single metabolites in 206 blocks (ROC-AUC = 0.800–1.000). Pregnancy-prone embryos consumed more amino acids and citric acid, and depleted less lipids and cis-aconitic acid. Big metabolic differences between embryos support efficient pregnancy and birth prediction when analyzed in discriminant conditions.

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