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However, the subjectivity inherent in the morphological evaluation by embryologists can introduce inconsistencies that impact the optimal embryo choice for transfer. To provide a more comprehensive evaluation of embryo quality, we undertook the integration of embryo metabolomics alongside standardized morphokinetic classification. The culture medium of 55 embryos (derived from 21 couples undergoing ICSI) was collected at two timepoints (days 3 and 5). Samples were split into Good (n = 29), Lagging (n = 19), and Bad (n = 10) according to embryo morphokinetic evaluation. Embryo metabolic performance was assessed by monitoring the variation in specific metabolites (pyruvate, lactate, alanine, glutamine, acetate, formate) using 1H-NMR. Adjusted metabolite differentials were observed during the first 3 days of culture and found to be discriminative of embryo quality at the end of day 5. Pyruvate, alanine, glutamine, and acetate were major contributors to this discrimination. Good and Lagging embryos were found to export and accumulate pyruvate and glutamine in the first 3 days of culture, while Bad embryos consumed them. This suggests that Bad embryos have less active metabolic activity than Good and Lagging embryos, and these two metabolites are putative biomarkers for embryo quality. This study provides a more comprehensive evaluation of embryo quality and can lead to improvements in ARTs by enabling the selection of the best embryos. By combining morphological assessment and metabolomics, the selection of high-quality embryos with the potential to result in successful pregnancies may become more accurate and consistent.<\/jats:p>","DOI":"10.3390\/ijms25010491","type":"journal-article","created":{"date-parts":[[2023,12,29]],"date-time":"2023-12-29T11:19:21Z","timestamp":1703848761000},"page":"491","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Metabolomics Integration in Assisted Reproductive Technologies for Enhanced Embryo Selection beyond Morphokinetic Analysis"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8810-1978","authenticated-orcid":false,"given":"Soraia","family":"Pinto","sequence":"first","affiliation":[{"name":"Centre for Reproductive Genetics Alberto Barros, 4100-012 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3276-5094","authenticated-orcid":false,"given":"B\u00e1rbara","family":"Guerra-Carvalho","sequence":"additional","affiliation":[{"name":"LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9563-6364","authenticated-orcid":false,"given":"Lu\u00eds","family":"Cris\u00f3stomo","sequence":"additional","affiliation":[{"name":"Institute of Biomedicine, University of Turku, 20014 Turku, Finland"}]},{"given":"Ant\u00f3nio","family":"Rocha","sequence":"additional","affiliation":[{"name":"CECA\/ICETA\u2013Centro de Estudos de Ci\u00eancia Animal, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4200-135 Porto, Portugal"}]},{"given":"Alberto","family":"Barros","sequence":"additional","affiliation":[{"name":"Centre for Reproductive Genetics Alberto Barros, 4100-012 Porto, Portugal"},{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, University of Porto, 4200-135 Porto, Portugal"},{"name":"Department of Pathology, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7635-783X","authenticated-orcid":false,"given":"Marco G.","family":"Alves","sequence":"additional","affiliation":[{"name":"Institute of Biomedicine (iBiMED), Department of Medical Sciences, University of Aveiro, 3810-193 Aveiro, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4989-5699","authenticated-orcid":false,"given":"Pedro F.","family":"Oliveira","sequence":"additional","affiliation":[{"name":"LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2023,12,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"846","DOI":"10.1016\/j.fertnstert.2004.11.033","article-title":"Research on infertility: Which definition should we use?","volume":"83","author":"Larsen","year":"2005","journal-title":"Fertil. 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