Comparison of testosterone fractions between Framingham Heart Study participants and Japanese participants

13301甲第4105号博士（医学）金沢大学博士論文要旨Abstract 以下に掲載：International Journal of Urology 21(7) pp.689-695 2014. Wiley. 共著者：Masaki Taya, Eitetsu Koh, Kouji Izumi, Masashi Iijima, Yuji Maeda, Tomohiko Matsushita, Teruaki Iwamoto, Mikio Namik

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

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. © 2013 AJA, SIMM & SJTU. All rights reserved

Comparison of testosterone fractions between Framingham Heart Study participants and Japanese participants

Objectives: To determine testosterone fractions in Japanese men and to compare these values with those of Framingham Heart Study participants. Methods: We enrolled 498 healthy Japanese men. Total testosterone was assayed by liquid chromatography tandem mass spectrometry, sex hormone-binding globulin was assayed by immunoassay and free testosterone was calculated by a laboratory at the Boston Medical Center. Analog-based free testosterone and immunoassay-based total testosterone were determined by immunoassay. We compared mass spectrometry assay-based total testosterone and calculated free testosterone values in the Japanese participants with values in the American Framingham Heart Study third generation cohort. Results: The mean serum mass spectrometry assay-based total testosterone, sex hormone-binding globulin, and calculated free testosterone values were 439.4±167ng/dL, 65.34±30.61nmol/L, and 58.75±20.0pg/mL, respectively. The correlation coefficients with age for mass spectrometry assay-based total testosterone, sex hormone-binding globulin, and calculated free testosterone were 0.0010, 0.5041, and -0.496, respectively. There were no age-related changes in mass spectrometry assay-based total testosterone values in healthy men (P=0.981), whereas sex hormone-binding globulin and calculated free testosterone levels showed similar age-related changes (P<0.0001). Serum analog-based free testosterone levels (8.24±2.9pg/mL) showed age-related changes (P<0.0001) regardless of immunoassay-based total testosterone levels (P=0.828). Serum immunoassay-based total testosterone values (486.1±162.5ng/dL) correlated with serum mass spectrometry assay-based total testosterone values (r=0.740, 95% confidence interval 0.6965-0.7781, P<0.0001). Similarly, analog-based free testosterone and calculated free testosterone values showed a highly significant correlation (r=0.706, 95% confidence interval 0.6587-0.7473, P<0.0001). The analog-based free testosterone values were approximately 10% of the calculated free testosterone values. Conclusions: In contrast to the Framingham Heart Study cohort, total testosterone values in Japanese men are not associated with advancing age; thus, they cannot be used to diagnose late-onset hypogonadism in Japan. The analog-based free testosterone value can be considered instead as a suitable biochemical determinant for diagnosing late-onset hypogonadism syndrome. © 2014 The Japanese Urological Association

New molecular diagnostic kit to assess Y-chromosome deletions in the Japanese population

Objectives: Deletions in the azoospermia factor regions are the most common known molecular genetic cause of human male infertility involving spermatogenetic failure. Testing for these deletions in Japanese DNA samples using conventional sequence-tagged site probes occasionally lead to considerable non-specific or faint products in the Japanese population. The aim of the present study was to evaluate the sensitivity and specificity of a newly developed kit for the detection of azoospermia factor microdeletions in the Japanese population. Methods: Sequence-tagged site probes were reselected and the Luminex suspension array assay was carried out. Validation was retrospectively carried out with 2014 DNA sequences with known microdeletions, which were divided into four categories. Results: Category1 deletions that corresponded to the conventional classification of azoospermia factor deletion were present in 83 men (4.2%), which can result in intrachromosomal homologous recombination. Kit data confirmed the presence of deletions of this type in DNA sequences known to harbor the azoospermia factor deletions. Category2 deletions involved cytogenetic abnormalities in 28 men (1.4%), whereas category3 deletions in 759 men (37.7%) were atypical classifications including the gr/gr deletion. As these deletions are thought to be a result of palindromic units and non-homologous recombination, these microdeletions might impact in the interpretation of some clinical findings. The rest of the 1145 cases (56.8%) were assigned to category4 as normal variants (polymorphism/no deletion). Conclusions: The present findings show that this new kit offers good sensitivity and specificity with the advantage of saving in terms of cost and time. © 2014 The Japanese Urological Association

A novel y chromosome microdeletion with the loss of an endogenous retrovirus related, testis specific transcript in AZFb region

Purpose: We identified the endogenous retroviruses associated with TTYs (testis specific transcripts linked to the Y) in the AZFb region. We evaluated the relationship between endogenous retroviruses, and TTY expression patterns and function in spermatogenesis. Materials and Methods: We identified family members of TTYs in the AZFb region using computational screening. After investigating the relationship between the endogenous retrovirus genome and TTY expression patterns we screened genomic polymerase chain reaction products from TTY13 amplified from 790 Japanese men, including 275 with azoospermia, 285 with oligozoospermia and 230 who were fertile. Results: Computational screening revealed that 3 members of the TTY family, TTY9, 10 and 13, were regulated by endogenous retroviruses in the AZFb region. Homologous recombination between long terminal repeat of the TTY13 associated human endogenous retrovirus-K14C resulted in TTY13 deletion events. These deletions were more common in patients with azoospermia and oligozoospermia than in fertile males. Specifically 15.63% of the azoospermia group, 10.88% of the oligozoospermia group and 0% of fertile controls had only the deletion variant, indicating an association between the homologous recombination rate and the severity of spermatogenesis failure that was statistically significant (p <0.05). Conclusions: Because of the finding of what are to our knowledge novel microdeletions due to endogenous retrovirus in the AZFb region, our study raises the possibility that specific variations in genomic structure may contribute to some forms of human idiopathic male infertility. © 2011 American Urological Association Education and Research, Inc