Fungal DNA profiles across maternal and neonatal biological sites: findings from an exploratory pilot study

The neonatal mycobiome remains an underexplored component of early-life microbial ecology. Although fungi may contribute to immune maturation and host-microbe interactions, fungal DNA profiles across maternal and neonatal body sites are poorly characterized. This exploratory pilot study aimed to characterize fungal DNA profiles across multiple maternal and neonatal biological compartments during early life.

Ten mother-infant dyads were enrolled at ≥37 weeks' gestation. Three focal dyads (one complete and two near complete) were selected for detailed mycobiome sequencing, while additional breast milk samples expanded the dataset to 30 analyzed specimens. Maternal (vaginal, rectal, breast milk) and neonatal (skin, oral cavity, stool) samples were collected at 48-72 h after delivery and 6 weeks postpartum. Fungal DNA was analyzed using Oxford Nanopore long-read 18S rRNA sequencing. Taxonomic classification was performed with Kraken2 using the NCBI RefSeq Targeted Loci fungal 18S rRNA database. Alpha- and beta-diversity analyses were conducted in R using the phyloseq package.

A total of 428,555 fungal reads representing 107 genus-level operational taxonomic features were retained. Fungal DNA profiles differed across biological sites, with a greater relative abundance of Malassezia in neonatal skin and more heterogeneous profiles in gastrointestinal-associated samples. Alpha diversity did not differ significantly between sampling sites, whereas beta diversity suggested exploratory site-associated differences in community composition. Richness-based metrics were higher at 6 weeks than during early sampling, although this trend was not robust after accounting for sequencing depth, and beta-diversity analyses showed no clear temporal shift. Because negative controls were not sequenced, contamination from laboratory or reagent-derived fungal DNA cannot be excluded, and the findings should not be interpreted as evidence of viable fungi or stable colonization.

This exploratory pilot study suggests that fungal DNA profiles differ across maternal and neonatal body sites and that detectable fungal richness may increase by 6 weeks postpartum, although this finding was not robust to sequencing depth. Larger longitudinal studies incorporating ITS-based sequencing, strain-level analyses, sequenced technical controls, and functional profiling are needed to validate these observations and clarify the biological significance of early-life fungal communities.