16S rRNA long-read nanopore sequencing is feasible and reliable for endometrial microbiome analysis.

RESEARCH QUESTION: Full-length 16S rRNA gene sequencing using nanopore technology is a fast alternative to conventional short-read 16S rRNA gene sequencing with low initial investment costs that has been used for various microbiome studies but has not yet been investigated as an alternative approach for endometrial microbiome analysis. Is in-situ 16S rRNA gene long-read sequencing using portable nanopore sequencing technology feasible and reliable for endometrial microbiome analysis? DESIGN: A prospective experimental study based on 33 patients seeking infertility treatment between January and October 2019. A 16S rRNA gene long-read nanopore sequencing protocol for analysing endometrial microbiome samples was established, including negative controls for contamination evaluation and positive controls for bias evaluation. Contamination caused by kit and exterior sources was identified and excluded from the analysis. Endometrial samples from 33 infertile patients were sequenced using the optimized long-read nanopore sequencing protocol and compared with conventional short-read sequencing carried out by external laboratories. RESULTS: Of the 33 endometrial patient samples, 23 successfully amplified (69.7%) and their microbiome was assessed using nanopore sequencing. Of those 23 samples, 14 (60.9%) were Lactobacillus-dominated (>80% of reads mapping to Lactobacillus), with 10 samples resulting in more than 90% Lactobacillus reads. Our long-read nanopore sequencing revealed results similar to two conventional short-read sequencing approaches and to long-read sequencing validation carried out in external laboratories. CONCLUSION: In this pilot study, 16S rRNA gene long-read nanopore sequencing was established to analyse the endometrial microbiome in situ that could be widely applied owing to its cost efficiency and portable character.

Abnormal Extracardiac Development in Fetuses With Congenital Heart Disease.

BACKGROUND: Knowledge about extracardiac anomalies (ECA) in fetal congenital heart disease (CHD) can improve our understanding of the developmental origins of various outcomes in these infants. The prevalence and spectrum of ECA, including structural brain anomalies (SBA), on magnetic resonance imaging (MRI) in fetuses with different types of CHD and at different gestational ages, is unknown. OBJECTIVES: The purpose of this study was to evaluate ECA rates and types on MRI in fetuses with different types of CHD and across gestation. METHODS: A total of 429 consecutive fetuses with CHD and MRI between 17 and 38 gestational weeks were evaluated. ECA and SBA rates were assessed for each type of CHD and classified by gestational age (<25 or ≥25 weeks) at MRI. RESULTS: Of all 429 fetuses with CHD, 243 (56.6%) had ECA on MRI, and 109 (25.4%) had SBA. Among the 191 fetuses with normal genetic testing results, the ECA rate was 54.5% and the SBA rate 19.4%. Besides SBA, extrafetal (21.2%) and urogenital anomalies (10.7%) were the most prevalent ECA on MRI in all types of CHD. Predominant SBA were anomalies of hindbrain-midbrain (11.0% of all CHD), dorsal prosencephalon (10.0%) development, and abnormal cerebrospinal fluid spaces (10.5%). There was no difference in the prevalence or pattern of ECA between early (<25 weeks; 45.7%) and late (≥25 weeks; 54.3%) fetal MRI. CONCLUSIONS: ECA and SBA rates on fetal MRI are high across all types of CHD studied, and ECA as well as SBA are already present from midgestation onward.