Liquid biopsies are becoming increasingly used for the detection and monitoring of disease states. While cell free DNA (cfDNA) in blood and urine have been well studied, much less is known about the composition of cfDNA in seminal fluid. We sought to characterize cfDNA in seminal fluid through tissue of origin studies using methylation analysis in men aged 21-60 yrs. We confirmed the observations of others that seminal fluid contains an abundance of cfDNA that is both nucleosomal and >1 kb. However, here we demonstrate for the first time that the high molecular weight (HMW) DNA harbors a lower sperm signal and higher somatic cell signal compared to the nucleosomal fraction. Prostate, granulocytes and kidney showed a mean predicted increased contribution of 6.2%, 4.9% and 2.9%, respectively in the HMW fraction. While sperm was the predominant signal in most men without vasectomies, the proportion of predicted prostate contribution reached as high as 26.5% in the HMW fraction. In other subjects without vasectomies, granulocyte cfDNA made up most of the signal. We also observed subject-specific cfDNA size distribution patterns that were reproducible over time, irrespective of abstinence times. These results suggest that seminal fluid is a rich source of cfDNA from various somatic cell types, and enriching for the HMW fraction would yield even higher sensitivity for somatic cfDNA detection. Considering these novel findings, it appears that seminal fluid may be able to serve as liquid biopsy for the detection and monitoring of prostate cancer, benign prostate hyperplasia, prostatitis and infertility.

S.H., J.C.H., J.F.S., S.B., L.R.Y., B.G. and K.M.C.L. are employees of Fellow Health Inc. and have stock ownership. S.H., J.C.H. and K.M.C.L. are inventors on patent applications related to cfDNA isolation from seminal plasma.

The study was funded by Fellow Health Inc.

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IRB of WIRB Copernicus Group (WCG IRB) gave ethical approval for this work.

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All data produced in the present study are available upon reasonable request to the authors.