The development of immunoassays enables more sophisticated studies of the associations between protein concentrations and pregnancy outcomes, allowing early biomarker identification that can improve neonatal outcomes. The aim of this study was to explore associations between selected mid-trimester amniotic fluid proteins and (1) overall gestational duration and (2) spontaneous preterm delivery. A prospective cohort study, including women undergoing mid-trimester transabdominal genetic amniocentesis, was performed in Gothenburg, Sweden, 2008-2016 (n = 1072). A panel of 27 proteins related to inflammation was analyzed using Meso-Scale multiplex technology. Concentrations were adjusted for gestational age at sampling, experimental factors, year of sampling, and covariates (maternal age at sampling, parity (nulliparous/multiparous), smoking at first prenatal visit, and in vitro fertilization). Cox regression analysis of the entire cohort was performed to explore possible associations between protein concentrations and gestational duration. This was followed by Cox regression analysis censored at 259 days or longer, to investigate whether associations were detectable in women with spontaneous preterm delivery (n = 47). Finally, linear regression models were performed to analyze associations between protein concentrations and gestational duration in women with spontaneous onset of labor at term (n = 784). HMG-1, IGFBP-1, IL-18, MIP-1α, MIP-1β, S100A8, and thrombospondin-1 were significantly associated with gestational duration at term, but not preterm. Increased concentrations of thrombospondin-1, MIP-1β, and S100A8, respectively, were significantly associated with decreased gestational duration after the Holm-Bonferroni correction in women with spontaneous onset of labor at term. This adds to the concept of a pregnancy clock, where our findings suggest that such a clock is also reflected in the amniotic fluid at early mid-trimester, but further research is needed to confirm this.

BCNatal Barcelona Center for Maternal Fetal and Neonatal Medicine Barcelona Spain

Biomedical Research Center University Hospital Hradec Kralove Hradec Kralove Czech Republic Department of Obstetrics and Gynecology University Hospital Hradec Kralove Charles University Faculty of Medicine in Hradec Kralove Hradec Kralove Czech Republic

Center for Neonatal Screening Department of Congenital Disorders Statens Serum Institut Copenhagen Denmark

Department of Mathematical Sciences Chalmers University of Technology Gothenburg Sweden Department of Pathology and Genetics Institute of Biomedicine Sahlgrenska Academy University of Gothenburg Gothenburg Sweden

Department of Obstetrics and Gynecology Sahlgrenska University Hospital Östra Gothenburg Sweden Department of Obstetrics and Gynecology Institute of Clinical Sciences Sahlgrenska Academy University of Gothenburg Gothenburg Sweden

Department of Obstetrics and Gynecology Sahlgrenska University Hospital Östra Gothenburg Sweden Department of Obstetrics and Gynecology Institute of Clinical Sciences Sahlgrenska Academy University of Gothenburg Gothenburg Sweden Department of Genetics and Bioinformatics Division of Health Data and Digitalisation Institute of Public Health Oslo Norway

Department of Obstetrics and Gynecology Sahlgrenska University Hospital Östra Gothenburg Sweden School of Maths and Stats Victoria University of Wellington Wellington New Zealand

Department of Pediatrics Institute of Clinical Sciences Sahlgrenska Academy University of Gothenburg Gothenburg Sweden Department of Neonatology The Queen Silvia Children's Hospital Sahlgrenska University Hospital Gothenburg Sweden

Stockholm South General Hospital Stockholm Sweden Department of Obstetrics and Gynecology Sahlgrenska University Hospital Östra Gothenburg Sweden

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