What question did this study set out to answer?

Assess sequencing strategies to profile the uterine microbiome in endometrial cancer patients.

April 16, 2026Open Access

Evaluating Sequencing Strategies for Endometrial Microbiome Profiling in Endometrial Cancer: A Comparative Study of Short‐ and Long‐Read 16S rRNA Approaches

SBSophia BebelmanUniversity of Auckland AAAnastasiia ArtuyantsUniversity of Auckland BNBianca Nijmeijer

Key Points

Assess sequencing strategies to profile the uterine microbiome in endometrial cancer patients.
Piloted protocols for short-read and long-read sequencing methods.
Analyzed uterine and vaginal swabs for DNA recovery and sequencing success.
Evaluated the impact of sample handling on DNA extraction and sequencing efficiency.
ONT sequencing achieved better species-level resolution and identified low-abundance taxa.
Freezing samples before extraction increased bacterial 16S copy numbers.
Contamination issues were noted across platforms, particularly with low-bioburden samples.

Abstract

ABSTRACT Background Endometrial cancer (EC) is the most common gynaecological malignancy globally, with rising incidence and notable disparities in outcomes. In New Zealand, EC rates have increased significantly, particularly among Māori and Pacific women, who face higher risks of advanced disease and poorer outcomes. Microbial dysbiosis has been implicated in EC pathogenesis, but characterising the uterine microbiome is challenging due to low microbial biomass and high contamination risk. Aims This study aimed to pilot a protocol that could inform the preparation of a larger cohort trial. Short‐read Illumina MiSeq and long‐read Oxford Nanopore Technologies (ONT) 16S rRNA gene sequencing were investigated to profile the uterine microbiome in people with EC. Methods and Results Uterine and vaginal swabs were analysed to assess platform performance in terms of DNA recovery, sequencing success, diversity metrics, and taxonomic resolution. The impact of sample freezing or immediate lysis prior to DNA extraction was also evaluated. ONT sequencing provided enhanced species‐level resolution and improved detection of low‐abundance taxa but showed variable performance in low‐yield samples. Freezing prior to cell DNA extraction modestly increased bacterial 16S copy numbers and improved community consistency. Contamination was a problem across both platforms, particularly in low‐biomass samples, but can be minimised during data analysis. Conclusion This study provides practical guidance for sequencing platform selection and sample handling in uterine microbiome research. Our findings support future efforts to elucidate microbial contributions to EC pathogenesis and highlight the importance of rigorous contamination control. Importantly, this is the first presentation of a New Zealand cohort and contributes valuable data from an underrepresented population and informs future research in diverse clinical settings.

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