Aberrant methylation of the TDMR of the GTF2A1L promoter does not affect fertilisation rates via TESE in patients with hypospermatogenesis

Abstract

Increasing evidence shows a relationship between epigenetic regulation and male infertility. The GTF2A1L gene promoter contains the DNA methylation site of a tissue-specific differentially methylated region (TDMR). Eighty-six patients with non-obstructive azoospermia were assessed for the DNA methylation state of CpG islands in the GTF2A1L promoter using testicular genomic DNA. Based on histological criteria, 26 of the 86 patients had normal spermatogenesis (controls), 17 had hypospermatogenesis and 26 had a Sertoli cell-only phenotype or tubular sclerosis. GTF2A1L TDMR methylation was significantly lower in testes DNA from control samples than from hypospermatogenic samples (P=0.029). Patients with hypospermatogenesis were divided into two subgroups: high DNA methylation (HM, n=5) and low DNA methylation (LM, n=12). The GTF2A1L TDMR methylation rate differed significantly between the HM and LM groups (P=0.0019), and GTF2A1L expression was significantly higher among the LM than in the HM patients (P=0.023). High TDMR methylation was correlated with low GTF2A1L gene expression levels. Both groups demonstrated relatively good outcomes with respect to sperm retrieval, fertilisation, pregnancy and childbirth rates. We observed that aberrant GTF2A1L gene expression was not correlated with fertilisation rates. The testicular sperm extraction (TESE) technique may be used to overcome male infertility due to aberrant TDMR methylation.

Figure 1

Tissue-specific expression of GTF2A1L in normal human tissues. cDNAs were prepared from total RNA isolated from testis, kidney, muscle, heart, brain and colon tissues. GAPDH was used as the internal standard for comparison.

Figure 2

Chromosomal location of GTF2A1L. This map is based on the March 2006 human reference sequence (NCBI Build 36.1). The GTF2A1L gene is shown as an arrow, with an arrowhead indicating the direction of transcription. CpG islands are shown with the number of CpGs. The amplicon was generated from the −258 bp to +41 bp (299 bp) CpG region near the transcription start site.

Figure 3

Rate of methylation status in patients characterized histological diagnosis. Normal spermatogenesis controls (NS, closed circles, n=26), hypospermatogenesis (HS, closed squares, n=17), maturation arrest phenotype (MA, closed triangles, n=17), Sertoli cell only phenotype (SCO, inverted closed triangles, n=21) and tubular sclerosis (TS, closed diamonds, n=5). The asterisk indicates a statistically significant difference between values (P<0.05).

Figure 4

Classification of high- and low-methylation TDMR groups in patients with hypospermatogenesis. (a) Box–Whiskers plot (min. to max.) of patients with hypospermatogenesis subdivided according to the GTF2A1L methylation rate: low methylation (LM, n=12) and high methylation (HM, n=5). (b) The relative expression level of GTF2A1L in patients with hypospermatogenesis and low methylation (LM, n=12) or high methylation (HM, n=5). The asterisk indicates a statistically significant difference between values (P<0.05). TDMR, tissue-specific differentially methylated region.