Transposon Insertion Profiling By Sequencing (Tipseq) Identifies Novel Line-1 Insertions In Human Sperm

Abstract Study question Do human sperm contain novel LINE–1 insertions and are they affected by paternal age? Summary answer Human sperm contain novel LINE–1 insertions. Their location or number are not affected by paternal age. What is known already LINE–1 comprises 17% of the human genome and some LINE–1s are the only autonomous retrotransposons in humans. Retrotransposons influence genomic instability and/or regulation if new retrotransposition events disrupt coding or regulatory regions in the host genome. Demethylation during germ cell development de-represses retrotransposons. Advanced paternal age is associated with genomic instability. Previously we showed that sperm LINE–1 copy number decreases with paternal age. We hypothesize that human sperm exhibit de novo retrotransposition and that sperm from older men contain increased novel LINE–1 insertions. Study design, size, duration Cross-sectional case–control study with semen samples collected between February to July 2020. Participants/materials, setting, methods Normospermic sperm samples (n = 10; 5 <35 years old and 5 ≥45 years old) obtained from consenting men undergoing IVF at NYU Fertility Center were submitted to a novel method, single cell Transposon Insertion Profiling by Sequencing (scTIPseq) to identify and map LINE–1 insertions in human sperm. TIPseqHunter, a custom bioinformatics pipeline, compared the architecture of sperm LINE–1 to known LINE–1 insertions from the European database of human specific LINE–1 (L1Hs) retrotransposon insertions in humans (euL1db). Main results and the role of chance TIPseq identified 17 novel insertions in sperm, 8 from older (≥ 45 years) and 9 in younger men (<35 years). New insertions were mainly intergenic or intronic, including AC007402 (2/10), TMEM163 (2/7), CTTNBP2NL (3/5), AC107023 (3/3), TMC2 (2/19), MacroD2 (2/6), RAB3C (3/4), LINC02664 (1/1), AC079052 (2/3) and AC017091 (4/4). One novel insertion (<35 years old) hits a known regulatory element. Only one sample (≥ 45 years old) did not exhibit any new insertion. The location or number of novel insertions did not differ by paternal age. Limitations, reasons for caution The small sample-size and use of normospermic specimens limit interpretation of paternal age effect on LINE–1. Besides, the novel insertions could be polymorphic sites that have low allele frequency and thus have not yet been described. Wider implications of the findings: This study for the first time reports novel LINE–1 insertions in human sperm, demonstrating that scTIPseq method is a feasible technique, and identifying new contributions to genetic diversity in the human germ line. Further studies are needed to evaluate the impact of these insertions on sperm function. Trial registration number Not applicable