| Background:The acquisition and development of infant microbiome are key to establishing a healthy host-microbiome symbiosis.Despite the fact that whether the first microbiota colonization occurs in utero remains debatable,emerging evidence continues to mount suggesting that infant's intrinsic host microenvironmental factors(e.g.,the gestational age)combining with exogenous factors(e.g.,the mode of delivery)drive early life formation and maturation of microbial,and intrinsic factors appear to exert more crucial roles.Thus,prenatal stage is pivotal,in which the development of biological system may determine the selection and formation of infant's microbiome and influence immune system development,metabolic function and potentially future health.Considerable efforts have focused on cataloguing infant gut microbiome and investigating its relation to childhood development and health.However,to the best of our knowledge,no published study has specifically investigated the gut microbiome in infants conceived by the assisted reproductive technology(ART).ART,a technique that uses medical techniques and methods manually manipulate gametes,zygotes,and embryos to achieve the purpose of conception,has become a standard and common practice in reproductive medicine clinics for infertile couples worldwide.The total number of newborns in the world through ART has exceeded five million.It is yet not clear whether early microbiota composition is significantly different in infants conceived by ART when compared with those who were conceived spontaneously.Additionally,the impact of neonatal microbial composition on their growth and development is still mostly unknown.The bacteria found in neonates'first meconium reflects the earliest colonization and formation of microbiota,and then neonates'gut microbiota is gradually shaped by dietary,nutrition and medical factors in the following days.Objective:We aimed to profile the microbiome in ART individual samples.We also comprehensively compared the gut microbiome composition between ART-and spontaneous-neonates.16S r RNA gene amplicon sequencing analysis was conducted to identify bacteria in individual samples.Moreover,we characterized the determinants of the gut microbiome in ART newborns and examined the potential impact of early microbiota composition on neonatal growth and development.Methods:We conducted longitudinal and prospective study that included a total of 118 ART families and 91 spontaneous pregnancy families.The participants were from the China National Birth Cohort(CNBC)Study,which is a prospective and longitudinal study of both assisted reproductive technology(ART)pregnancies and spontaneous pregnancies.The CNBC Study was mainly designed for comprehensively comparing the birth outcomes of ART and spontaneous-conceived pregnancies and systematically evaluating the environmental and genetics factors that may influence birth outcomes.In the present study,we enrolled 228 healthy singleton newborns who were born to a cohort of women after 35 gestational weeks,and had first-pass meconium samples.Newborns who were diagnosed of congenital abnormalities or their mothers had significant pregnancy complications were not recruited in this study.We further excluded newborns from this study if key maternal(e.g.,ART treatments,BMI)or infant(e.g.,birth weight,42-day weight)data were missing(n=19).In total,209newborns were included in the analytical population,among which 118 were born after ART and 91 were born after spontaneous conception.ART-treated and spontaneous-conceived women completed standardized and structured questionnaires by face-to-face interview to collect their demographic information and reproductive history.During parturition(Delivery Visit),pregnancy outcomes were collected from the medical records and neonates'anthropometric parameters were measured.All children visited the clinic for health examination and anthropometric measurement approximately 42 days after the birth and then 6 months of age.In addition to the first-pass meconium,fecal samples were also collected in the following three days after birth from each newborns during hospitalization if available.For 16s r RNA sequencing data,operational Taxonomic Units(OTUs)were generated at 97%identity threshold.The seed sequences of each OTU were chosen for taxonomic classification.The total diversity in the OTU profile was decomposed into?-diversity and?-diversity.We assessed the dissimilarity of group by permutational ANOVA(PERMANOVA).Signature microbiota taxa between ART and spontaneous-conceived groups were identified by one-way ANOVA and linear discriminant analysis(LDA).We used the Sorenson index to measure the similarity between microbiota at different days of intra-individual.Finally,we used association analysis to characterize the determinants of the gut microbiome in ART newborns and examined the potential impact of early microbiota composition on neonatal growth and development.Results:We observed a declined?-diversity(Simpson index)in the first meconium from ART neonates,compared with spontaneous conception group(Simpson index:0.59±0.29for ART versus 0.68±0.28 for spontaneous,P=0.03).Shannon index and Chao1index were not significantly different in the two groups.We specifically examined microbiota?-diversity in the neonates born after caesarean section and still observed lower Simpson index in the ART neonates(0.60±0.29 for ART conception versus0.78±0.28 for spontaneous conception,P<0.001).Principal coordinate analysis showed that the meconium microbiota in ART offspring appeared to cluster separately from that in neonates conceived spontaneously(PERMANOVA on Bray-Curtis dissimilarity,P=0.001).In the neonates born following spontaneous conception,the most abundant phylum in their meconium samples was the Proteobacteria(mean±SD relative abundance:41.4%±36.3%)and then the Firmicutes(37.3%±32.8%),while the majority of OTUs belong to the Firmicutes in ART neonates(48.7%±35.8%),and then followed by the Proteobacteria(36.3%±36.0%).The third most dominant phylum was the Bacteroidetes in both populations,whereas it showed only 50%amount of mean relative abundance in the ART conception versus the spontaneous conception group(5.5%±8.4%for ART conception versus 10.5%±15.7%for spontaneous conception,P=0.01 after Bonferroni Correction).At the genus level,we observed over2-fold increase in the relative abundance of Staphylococcus but more than 50%reduce of Bacteroides(phylum Bacteroidetes)in ART neonates meconium,compared with spontaneous conception(Staphylococcus:19.4%±33.0%for ART conception versus7.9%±18.7%for spontaneous conception,P=0.03 after Bonferroni Correction;Bacteroides:1.7%±3.5%for ART conception versus 4.5%±10.2%for spontaneous conception,P=0.04 after Bonferroni Correction).Among ART neonates,gravidity showing a marginal association with the relative abundance of Bacteroidetes(Ever pregnant versus never pregnant:?adj=-0.05,95%CI=-0.11,-0.002,P=4.11?10-2).We found that the usage of vaginal suppositories for luteal phase support exhibited a strong and positive association with the relative abundance of Bacteroidetes(?adj=0.11,95%CI=0.04,0.17,P=1.79?10-3),compared with non-vaginal corpus luteum support drugs.In contrast,antagonists usage during ART process and frozen embryo transfer significantly reduced Bacteroidetes abundance in ART-neonates(antagonists regimen versus agonist long regimen:?adj=-0.09,95%CI=-0.17,-0.02,P=1.90?10-2,frozen versus fresh embryo:?adj=-0.06,95%CI=-0.12,-0.01,P=2.89?10-2).We observed that gut microbiota?-diversity and the relative abundance of Bacteroidetes in ART newborns were inversely associated with their weight gain rate.Newborns who had reduced?-diversity(Simpson index)and declined relative abundance of Bacteroidetes showed rapid 42-day weight gaining(Simpson index:?adj=-11.0,95%CI=-18.3,-3.63,P=4.13?10-3;Bacteroidetes relative abundance:?adj=-56.4,95%CI=-80.7,-32.0,P=1.42?10-5).In spontaneously-conceived newborns,the association of 42-day weight gain rate was significantly inverse with Bacteroidetes relative abundance(?adj=-39.2,95%CI=-54.2,-24.2,P=1.83?10-6),but was null with overall Simpson index(?adj=-7.20,95%CI=-17.5,3.12,P=1.75?10-1).Conclusion:In this longitudinal and prospective study,we identified features and determinants of the gut microbiota composition in the ART newborns,and provided evidence for the importance of microbiota composition in infant growth. |
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