Determining embryo developmental competence by measuring expressivity of the paternal genome

Abstract

To profile the transcriptome of men with unexplained infertility and to identify key sperm-specific RNAs that contribute to post-fertilization embryonic developmental competence. Sequencing and differential expression of sperm-specific RNAs in men with unexplained infertility. Next generation sequencing (RNA-Seq) was performed on ejaculated semen samples provided by consenting men undergoing infertility screening. All men had normal semen parameters and their female partners <35 years had normal infertility screening. RNA was isolated from 25x106 spermatozoa using a hybrid isolation protocol with TRIzol® Reagent (ThermoFisher Scientific, USA) and a spin column kit (RNeasy Mini Kit, QIAGEN, Germany). Pilot sequencing was carried out at 2x36bp to determine library quality and expanded to 50-60M reads at 2x76bp utilizing an Illumina platform (NextSeq 500). Expression values were calculated in FPKM (Fragments Per Kilobase of Exon Per Million Fragments Mapped). The expression of RNA transcripts was compared between men who were and were not able to achieve conception with intracytoplasmic sperm injection (ICSI). The expression profiles of these RNA transcripts were then compared to embryo development parameters. The 25 consenting men had the following semen parameters: 37.3±17 x 106/mL (concentration), 46.6±10% (motility), and normal morphology. The average concentration of RNA extracted was 14.3±6 ng/μL. The age demographics were comparable among couples who conceived i.e., fertile (men=37.6±3 years; women=34.8±3 years) and did not conceive i.e., infertile (men=38.3±5 years; women=33.6±7 years) with ICSI. The fertilization rates with ICSI and number of embryos transferred were also similar. RNA expression analysis revealed 25 differentially expressed genes between the groups (P<0.001): 10 overexpressed and 15 underexpressed. The large majority of differentially expressed sperm-specific RNAs (23/25) were particularly important for the development of embryos from the 1-cell to 4-cell stage. A higher embryonic fragmentation rate (14.3%) was noted in the infertile group, which corresponded to a higher expression of BOK RNA transcripts in the sperm of infertile patients (P<0.001). PLK4 and BUB1, which dictate chromosome stability and mitotic segregation by regulating centrosome development, had decreased expression in the infertile group (P<0.001). Transcriptome profiling of human spermatozoa using RNA-Seq can reveal genes products that are critical to embryonic development. Our results suggest that sperm-specific RNAs are vital for embryonic development between the 1-cell to 4-cell stage.