Metabolites Secreted by Bovine Embryos In Vitro Predict Pregnancies That the Recipient Plasma Metabolome Cannot, and Vice Versa.

Enrique Gomez,Antonio Murillo,Adrià Cereto,Marta Muñoz,Susana Carrocera,Adrià Cereto,Pol Herrero,Pol Herrero,David Martin-Gonzalez,Antonio Murillo,Nuria Canela,Isabel Gimeno

doi:10.3390/metabo11030162

Abstract

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.

Highlights

Developing “a robust, non-invasive test with which to select single embryos for transfer into the uterus” remains one of the major challenges for in vitro embryo technologies [1]to predict embryo quality and pregnancy outcome [2]
The gas chromatography coupled to quadrupole time of flight mass spectrometry (GC-qTOF) analysis led to identification and quantification of N = 37 metabolites in culture medium (CM) (Supplemental Table S1) and N = 71 metabolites in plasma (Supplemental Table S2)
A complete description of each embryo transfer (ET) performed in this study and its gestational trajectory is shown in Supplemental Table S3

Summary

Introduction

Developing “a robust, non-invasive test with which to select single embryos for transfer into the uterus” remains one of the major challenges for in vitro embryo technologies [1]to predict embryo quality and pregnancy outcome [2]. Developing “a robust, non-invasive test with which to select single embryos for transfer into the uterus” remains one of the major challenges for in vitro embryo technologies [1]. The interest of identifying viable embryos increases as embryonic competence decreases, since embryo transfer (ET) with the less competent embryos means using more recipients per born calf. Predictive studies typically selected either embryos or recipients for pregnancy and birth, while the other factor of the pregnancy equation was assumed to be viable (the embryo) or competent (the recipient). The need for extracting information from both embryo and recipient to accurately profile pregnancy was stated almost two decades ago [8], but no solution has yet leveraged the predictive power of both sources in a single tool

Methods

Results

Conclusion