57 Non-targeted metabolomic profiles within the uterine milieu of porcine pregnancies containing populations of uniform or diverse spherical, ovoid, or tubular conceptuses during initiation of embryo elongation

Abstract

Alterations in the signalling of critical molecular factors within the uterine milieu result in deficiencies in embryo elongation, leading directly to embryonic loss as well as delayed elongation. The objective of this study was to identify metabolites within the uterine environment from populations of uniform and diverse porcine conceptuses as they transition between spherical, ovoid, and tubular conceptuses during the initiation of embryo elongation. White crossbred gilts (n=38) were bred at standing oestrus (designated Day 0) and again 24h later and randomly assigned to collection group. At Day 9, 10, or 11 of gestation, reproductive tracts were collected immediately following harvest and flushed with 40mL of RPMI-1640 media. Conceptus morphologies were assessed from each pregnancy to assign to 1 of 5 treatment groups based on these morphologies: (1) uniform spherical (n=8); (2) diverse spherical and ovoid (n=8); (3) uniform ovoid (n=8); (4) diverse ovoid and tubular (n=8); and (5) uniform tubular (n=6). Subsequently uterine flushings from these pregnancies were submitted for non-targeted profiling by gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) techniques. Raw spectral data were processed using the XCMS package in R (R Foundation for Statistical Computing, Vienna, Austria) and features were clustered using RAMclustR. Unsupervised multivariate principal component analysis was performed in R using pcamethods package, and univariate ANOVA was performed in R with a Benjamini-Hochberg false discovery rate adjustment. Principal component analysis of the GC-MS and UPLC-MS data identified 153 and 104 metabolites, respectively. Of the identified metabolites, 51 and 71 metabolites from the GC-MS and UPLC-MS analysis, respectively, corresponded to known compounds. After false discovery rate adjustment of the GC-MS and UPLC-MS data, 38 and 59 metabolites from the GC-MS and UPLC-MS analysis, respectively, differed (P<0.05) in uterine flushings from pregnancies for the 5 conceptus stages. Some metabolites were greater (P<0.05) in abundance for uterine flushings containing earlier stage conceptuses (i.e. spherical) such as uric acid, tryptophan, 5-hydroxy-L-tryptophan, and L-tryosine. In contrast, some metabolites were greater (P<0.05) in abundance for uterine flushings containing later stage conceptuses (i.e. tubular) such as creatinine, serine, isovaleryl-I-carnitine, and lauric diethaolamide. These data illustrate several putative metabolites that change within the uterine milieu as porcine embryos transition between spherical, ovoid, and tubular conceptuses. Funding was provided by USDA-NIFA-AFRI Grant no. 2017-67015-26456.