Reproductive Technology

Prediction of Porcine Blastocyst Formation Using Morphological, Kinetic, and Amino Acid Depletion and Appearance Criteria Determined During the Early Cleavage of In Vitro-Produced Embryos

Abstract
The determination of non-invasive morphological and metabolic criteria in early cleavage-stage embryos that are predictive of blastocyst development and/or full-term viability remains an important research target. Here we report the derivation of a logistic regression model that predicts the probability of porcine blastocyst formation in vitro. Pig zygotes, derived by in vitro maturation and fertilization of slaughterhouse oocytes, were cultured in NCSU-23 medium supplemented with a mixture of 20 amino acids (NCSU-23_aa). On Day 1, at 21, 23, 25, 27, 29 and 31 h post-insemination, cleaving embryos were evaluated morphologically in terms of i) number of blastomeres, ii) evenness of division and iii) degree of fragmentation. These embryos were then placed in 1.5 µl NCSU-23_aa drops for 24 h, after which time the three morphological criteria were re-evaluated and 1.2 µl spent medium removed for analysis by HPLC for determination of the net rates of amino acid depletion and appearance. Embryos were then cultured singly in NCSU-23_aa by placing them between the filaments of a woven polyester mesh until Day 6 in order to permit identification of individual embryos. Of 256 cleaved embryos, 28.7±6.2% (n=5 replicates) developed into blastocysts. Discriminant analysis was used to select a subset of amino acids (threonine, valine, lysine and phenylalanine) that discriminated optimally between embryos that became blastocysts or degenerated. These discriminant scores were entered into the logistic regression. Significant univariate relationships were established between the probability of blastocyst development and amino acid score (0.53[0.40-0.69]<0.001) (odds ratio[±95% confidence intervals]P-value), cleavage time (0.79[0.71-0.87] <0.001), degree of fragmentation on Day 1 (0.55[0.35-0.84]0.009) and Day 2 (0.53[0.35-0.78]0.002), evenness of division on Day 2 (0.66[0.46-0.96]0.028) and categorical values of blastomere number on Day 2 (all P<0.02) although no variate, in isolation, could predict blastocyst formation accurately. However, multivariate analysis of cell numbers on Days 1 and 2 correctly classified 51.9% of predicted blastocysts. The inclusion of cleavage time in the regression raised this rate to 63.5% which was increased to 66.2% by the addition of evenness of division and degree of fragmentation. Finally, the full logistic regression model that incorporated amino acid score together with all the other morphological and kinetic variables correctly classified 80.8% of predicted blastocysts. This represented 51.2% of observed blastocysts. Our data are novel as they not only identify in a quantitative manner the influence of previously undescribed predictors of porcine blastocyst formation but provide a simple model of preimplantation development of reasonable predictive accuracy. This study also provides a basic model for the examination and incorporation of additional early morphological and metabolic correlates of developmental competence and could potentially be applied to the selection of human embryos for transfer in clinical IVF.

Key words: Embryo • Developmental biology • In vitro fertilization

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