IntroductionInfertility is increasing globally, affecting one in six couples of reproductive age1. To address this issue, researchers have developed assisted reproductive technologies, such as In Vitro Fertilisation (IVF), resulting in about 12 million babies being born2. Despite being well-established, IVF is only successful in ~30% of cases3. One major limiting factor in modern IVF treatments is embryo development. IVF clinics often aim to have 30% of fertilised oocytes develop into high-quality embryos, although this success rate is not reached consistently4. Often, couples need several rounds of treatments to receive a baby5. In this context, the potential for male contributing factors to increase IVF success has remained unexplored.Accumulating evidence suggests that the sperm delivers not only genetic material but also small RNA (sRNA) to the oocyte. By definition, sRNA are short RNA molecules ranging from tens to a few hundreds of nucleotides. sRNA include microRNA (miRNA), originally discovered for their regulatory role in embryogenesis, as well as tRNA-derived fragments (tsRNA), ribosomal RNA-derived fragments (rsRNA), mitochondrial-derived RNA (mitosRNA), Piwi-interacting RNA (piRNA), and fragments from ribonucleoprotein associated RNA (ribonucleoprotein associated sRNA) such as Y-RNA and small nucleolar RNA6.Here, we show that by analysing sRNA in sperm samples retrieved during ongoing IVF treatment, we find subpopulations of sRNA to be biomarkers for sperm concentration, fertilisation rate, and embryo quality. Our work highlights the paternal role in IVF success and establishes a foundation for a new era in reproductive medicine.ResultsCharacteristics of the population studiedCouples undergoing IVF were recruited between the 18 November 2022 and 8 June 2023 at the Centre of Reproductive Medicine at University Hospital in Linköping, Sweden. Of the 84 couples assessed for eligibility, 72 were recruited and pseudonymized. One couple had an inadequate sperm count for sRNA sequencing and was excluded after sample collection. Two couples did not retrieve any oocytes and were excluded from further analysis. One couple provided two samples because they underwent two IVF treatments; both samples were included. Thus, 70 treatments from 69 couples were studied, with sperm samples sequenced from each of these treatments (Fig. 1). The median age of the men was 34 years, and their median BMI was 27. The median age of the women was 33, and their median BMI was 25. Median sperm concentration and progressive motility were 28 million sperm/mL and 50%, respectively (Table 1).Fig. 1: Study flow chart.IVF In vitro fertilisation. sRNA small RNA.Full size imageTable 1 Demographic and clinical characteristics of the couplesFull size tableSperm-borne sRNA is differentially expressed in IVF sperm samplesTo investigate whether there are molecular markers capable of predicting IVF outcome, leftover sperm was collected and sequenced. In total, 70 samples from 69 couples were sequenced for sRNA. Analysis of this data confirms previous findings that sperm contains a large and diverse repertoire of sRNA7,8 (Supplementary Data 1, and Supplementary Fig. 1).We performed differential expression analysis on sequenced sRNA and identified sRNA differing by sperm concentration, fertility rate, and rate of high-quality embryos. Specifically, differential expression analysis comparing samples with high ( >16 million sperm/mL) and low ( ≤16 million sperm/mL) sperm concentration (Supplementary Data 2) identified 563 (1.89%) significantly upregulated sRNA in samples of high concentration (Fig. 2a, Supplementary Data 3). Conversely, 640 (2.15%) sRNA were significantly downregulated. Of the identified upregulated sRNA, 72% were mitosRNA; of the downregulated sRNA, 48% were ribonucleoprotein associated sRNA (Fig. 2a). As sperm concentration and motility are used to determine sperm quality, we performed differential expression analysis of these two parameters. We identified a great overlap between significant sRNA in sperm concentration and sperm motility ( ≥5 million progressively motile sperm as high and /≤16 million sperm/mL). Pie charts represent up and downregulated sequences respectively, where the percentage of normalised sequences per biotype is shown. b Comparison of high and low fertilisation rate (≥/