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© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. To the Editor Amniotic fluid (AF) has been considered sterile for nearly a century because no microorganisms were identified by traditional culture methods [1]. However, this opinion paradigm has been challenged by recent studies based on culture-independent sequencing techniques [2–4] and AF proteomics [5]. Recently, a conclusion that no microorganisms were present in the mid-trimester AF of healthy pregnancies was reached using culture-independent sequencing techniques [6] and seemed to settle the argument [7]. However, it could not explain why nonhuman proteins were identified in normal human AF supernatants [8] and why microbial exposure primes fetal immune cells in fetal tissues during fetal development [9]. Intra-amniotic infection caused by microbial invasion of the amniotic cavity (MIAC) was associated with adverse pregnancy outcomes, when the bacteria were at high concentrations [10]. This may be a reason why people think that AF is sterile and that bacteria in AF are abnormal because of their negative effects. Meanwhile, we could identify bacterial proteins in the AF proteomics database that were consistent with the results of 16S ribosomal RNA (rRNA) gene sequencing. Nine amniotic fluid samples were collected from 9 pregnant women and the pregnancy outcomes of the participants were followed. No bacteria were found by cultivation, but a sparse microbial presence was found by proteomics analysis and 16S rRNA gene sequencing approach. The 148 microbes found in the human AF proteomics database were consistent with the microbes found in the 16S rRNA gene sequencing database. The species composition and the structure of communities in the normal age (< 35 years old) and advanced maternal age (AMA) (> 35 years old) pregnancies differed significantly. However, all of the newborns were healthy and had no allergic reactions. Proteomics analysis has identified nearly 2000 proteins in AF during the past 20 years [11], including many nonhuman proteins [8]. In a recent study, 7 normal AF samples were used to generate human AF proteomes, which were divided into 4 groups: original proteins, bound proteins, flow-through proteins, and iTRAQ-labeled individual/mixed digested peptides [11]. We reanalyzed the human AF protein database and found that a part of the non-human proteins were derived from microorganisms. A total of 148 microbial-associated proteins in the normal human AF proteome, which could potentially play important roles in cellular/metabolic processes and binding/catalytic activity (Additional file 1). Notably, all of the microbial-associated proteins were present at a low level in human AF. These data suggest that there may be low concentrations of microorganisms in normal human AF samples. To test our hypothesis, we conducted a study to investigate the presence of microorganisms in the mid-trimester AF of 9 women [4 normal age [normal] and 5 advanced Open Access Journal of Translational Medicine