STRUCTURAL AND GENOMIC CHARACTERIZATION OF GLOBOZOOSPERMIC MEN

Abstract

To characterize the structural abnormalities, genetics, and transcriptomic profile of globozoospermic (GZ) men. During the last 8 years, 11 GZ men undergoing treatment at our center were screened by ultrastructural analysis and categorized as having partial (PG) or complete (CG) globozoospermia. CG men, according to their ability to sustain a term pregnancy, underwent whole genome karyotyping, gene mutation analysis, and transcriptomic profiling. GZ men underwent standard semen analysis, and ultrastructural details were evaluated by transmission electron microscopy (TEM). Aniline blue staining measured histone content (normal threshold ≤20%). SCF was performed by TUNEL (normal threshold ≤15%). PLCζ was detected with immunofluorescence staining (normal threshold ≥30%). Genome and transcriptome analyses were obtained by DNA- and RNA-seq, respectively. A total of 11 men were diagnosed as PG (N=4) and CG (N=7) by TEM, revealing 98 ± 1% and 100 ± 0% occurrence of round heads, respectively (P=0.01). PG men (32.6±4 years) had a sperm concentration of 31 ± 42 x106/ml, 15 ± 12% motility, and normal morphology of 0.7 ± 0.5%; CG men (36.1 ± 4 years) had a sperm concentration of 36.5 ± 39 x106/ml, 24.4± 21% motility, and 0% normal morphology. Residual histones were 40±19% and 53.8±24% and SCF was 16.3 ± 2 and 19.5 ± 12 in PG and CG men, respectively. PLCζ was 34 ± 4 in PG men and 7.4 ± 4 in CG men (P=0.001). Aneuploidy was 3.6% in PC men and 8.2% in CG men. Genetic analysis of 3 men showed common mutations in DPY19L2, SPATA16, and PICK1 genes, which are all associated with spermiogenesis. A patient unable to achieve a pregnancy (CG1) was found to have a mutation on NLRP5, which is essential for zygote development. In a patient whose female partner suffered a miscarriage (CG2), exome sequencing revealed a heterozygous deletion on BSX, which is associated with neonatal development. Genetic assessment of a patient whose female partner had a term pregnancy (CG3) showed a single nucleotide insertion on PIWIL1, which is related to sperm phenotype. CG1 had 71 imbalanced genes involved in reproductive processes, with 19 associated with spermatogenesis (KLHDC3/chr 6, KLHL10/chr 17, MORN2/chr 2, PAPPA/chr 9, UBR2/chr6), sperm maturation (ADAM21/chr 14, CNBD2/chr 20, SPEM1/chr17, TXNRD3/chr 3), DNA condensation (H1FNT/chr 12, NEK2/chr 1, RNF8/chr 6), fertilization (CALR3/chr 19, HSPA1L/chr 6, IQCF1/chr 3), and embryo development/implantation (OVGP1/chr 1, STC2/chr 5, WNT9B/chr 17). CG2 had 37 imbalanced genes, with 13 down-regulated ones involved with spermatogenesis (CSF1/chr 1, NAMPT/chr 7, SEMG1/chr 20, SEMG2/chr 20, SMCP/chr 1), DNA double-strand break repair (RMI1/chr 9), and embryo development (ACVR1C/chr 2, CBX2/chr 17, CRHR1/chr 17, TGFB3/chr 14). CG3 had only 2 underexpressed genes involved with cell adhesion, migration, and signaling receptor binding: HAS2/Chr 8 and TGFB2/Chr 19. Globozoospermia is a rare but severe form of male infertility that requires extensive structural and genomic analysis to identity treatment options and the ability to support a term pregnancy.