Comprehensive characterization of maternal, fetal, and neonatal microbiomes supports prenatal colonization of the gastrointestinal tract

www.nature.com/scientificreports OPEN Comprehensive characterization of maternal, fetal, and neonatal microbiomes supports prenatal colonization of the gastrointestinal tract Jee Yoon Park 1,2,7, Huiyoung Yun 5,6,7, Seung‑been Lee 5,6, Hyeon Ji Kim 2, Young Hwa Jung 3,4, Chang Won Choi 3,4, Jong‑Yeon Shin 5,6, Joong Shin Park 1* & Jeong‑Sun Seo 5,6* In this study, we aimed to comprehensively characterize the microbiomes of various samples from pregnant women and their neonates, and to explore the similarities and associations between mother-neonate pairs, sample collection sites, and obstetrical factors. We collected samples from vaginal discharge and amniotic fluid in pregnant women and umbilical cord blood, gastric liquid, and meconium from neonates. We identified 19,597,239 bacterial sequences from 641 samples of 141 pregnant women and 178 neonates. By applying rigorous filtering criteria to remove contaminants, we found evidence of microbial colonization in traditionally considered sterile intrauterine environments and the fetal gastrointestinal track. The microbiome distribution was strongly grouped by sample collection site, rather than the mother-neonate pairs. The distinct bacterial composition in meconium, the first stool passed by newborns, supports that microbial colonization occurs during normal pregnancy. The microbiome in neonatal gastric liquid was similar, but not identical, to that in maternal amnionic fluid, as expected since fetuses swallow amnionic fluid in utero and their urine returns to the fluid under normal physiological conditions. Establishing a microbiome library from various samples formed only during pregnancy is crucial for understanding human development and identifying microbiome modifications in obstetrical complications. Abbreviations AF Amniotic fluid ANCOM Analysis of the composition of microbiomes ART Assisted reproductive technology ASVs Amplicon sequence variants BMI Body mass index CB Umbilical cord blood CLR Centered log-ratio DNA Deoxyribonucleic acid GL Gastric liquid IUI Intrauterine insemination IVF-ET In vitro fertilization and embryo transfer M Meconium NCBI National Center for Biotechnology Information NGS Next-generation sequencing 1 Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Republic of Korea. 2Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Gyeonggi‑do, Republic of Korea. 3Department of Pediatrics, Seoul National University College of Medicine, Seoul, Republic of Korea. 4Department of Pediatrics, Seoul National University Bundang Hospital, Gyeonggi‑do, Republic of Korea. 5Precision Medicine Center, Seoul National University Bundang Hospital, Gyeonggi‑do, Republic of Korea. 6Macrogen Inc, Seoul, Republic of Korea. 7These authors contributed equally: Jee Yoon Park and Huiyoung Yun. *email: ; Scientific Reports | (2023) 13:4652 | https://doi.org/10.1038/s41598-023-31049-1 1 Vol.:(0123456789) www.nature.com/scientificreports/ NICU Neonatal intensive care unit PCoA Principal coordinates analysis PCR Polymerase chain reaction PERMANOVA Permutational multivariate analysis of variance RNA Ribosomal ribonucleic acid VD Cervicovaginal discharge The human microbiome potentially carries the answer to many secrets of the human body. It has been linked to maintaining homeostasis in health and is associated with numerous diseases1,2. Recent research has shifted to explore the microbiome in less-studied populations, such as infants or pregnant women, to better understand its role in human development. Microbiome development is likely to start from the in-utero environment and changes in a lifetime, continuously affecting the immune system and metabolism. Pregnancy has been shown to alter microbial populations within the maternal body and may impact future maternal, fetal, and neonatal health3. Pregnancy allows temporary immunotolerance to a foreign body, facilitating microbiome remodelling and potential adaptations to the immune system and m etabolism4. Some microbiome studies in pregnancy have proposed that fetal environments, including placenta and amniotic fluid, traditionally known as sterile, contain several characteristic microbiotas not identified in routinely performed culture t echniques5,6. However, the biomass of these microbiotas is small and the reliability of the sequencing methods and potential for contamination have been criticized. The association between those microbiota and specific obstetric conditions has not yet been proven and warrants further investigation. The vagina is the most commonly studied site of bacteria in the female reproductive organ, as it is connected to the uterus through the cervix and is exposed to the skin. Microbiome research in pregnancy, however, has advanced slowly due to ethical concerns and difficulties in accessing samples. Aagaard et al. found that the vaginal microbiome changes during pregnancy based on gestational age and that Lactobacillus species play a role in preventing pathogenic bacterial g rowth7. More specifically, pregnancy leads to decreased diversity, increased proportion of Lactobacillus, and higher stability in the vaginal m icrobiome8,9. Some vaginal bacteria have been linked to preterm birth via intrauterine inflammation or infection10–14, yet there are no clinical guidelines for testing or monitoring these microbiota. Other sites that had been evaluated for microbiome in pregnancy are maternal15, oral cavity16, placenta5, amniotic fluid17,18, and neonatal gut19; but previous studies were fragmentary and more systematic research is needed. In this study, we have comprehensively characterized the microbiome in vaginal discharge (VD) and amniotic fluid (AF) from pregnant women and in umbilical cord blood (CB), gastric liquid (GL), and meconium (M) from their neonates. The goal was to determine the relationships between these samples and various obstetric conditions. Results Description of the study populations and clinical characteristics. A total of 141 low-risk pregnant women were enrolled sequentially and 178 neonates were born from the study population with 37 cases being twin pregnancies. All women were of Asian ethnicity (Korean), and the median age was 34 (interquartile range 31–37) years (Table 1). The proportion of nulliparity was slightly over half of the population (67%), and the median values of height, weight, and body mass index (BMI) were 162 cm, 70 kg, and 27 kg/m2, respectively. About 30% were conceived by assisted reproductive technology (ART), including intrauterine insemination (IUI) and in vitro fertilization with embryo transfer (IVF-ET). As mentioned above, twin pregnancy was approximately one-fourth of the total population, and among them, 19% were monochorionic. The median gestational age at delivery was 37.7 weeks (interquartile range 36.9–38.6), and preterm birth before 37 weeks of gestation was 26.2% (37/141). The rate of (...truncated)