Mosaicism for 45,X cell line may accentuate the severity of spermatogenic defects in men with AZFc deletion. (Letters to the editor).(Letter to the Editor)

EDITOR--Over the past 10 years, several authors have reported microdeletions in the long arm of the Y chromosome (Yq) in men with idiopathic, non-obstructive azoospermia or severe oligospermia. These microdeletions were clustered on the Yq fragment previously described as the azoospermia factor region (AZF). (1) More recently, a number of genes expressed specifically in the testes and mapping to AZFa, AZFb, or AZFc subregions have been cloned. (2-4) One of the approaches to understanding the role of these genes in human spermatogenesis is to look for a correlation between the lack of given AZF genes and the particular spermatogenic defect in the phenotypes of the patients. However, attempts to find such a correlation have failed so far. Instead, a broad spectrum of phenotypes ranging clinically from azoospermia to severe oligospermia and histologically from Sertoli cell only syndrome (SCOS) to hypospermatogenesis has been described in association with AZFc deletions. (5 6)

Maciej Kotecki

A recent study found chromosomal aberrations in 15% of azoospermic patients. (7) However, in papers focusing on the analysis of AZF microdeletions in patients with idiopathic infertility, (2 5 8-30) systematic, bilateral, histological, molecular, and cytogenetic analyses in the same large group of patients was rarely carried out, thus limiting information on the coexistence of AZF deletions and chromosomal aberrations.

In this study, we propose and test the hypothesis that chromosomal defects may often accompany AZF deletions and cause the lack of a genotype-phenotype correlation in human male idiopathic infertility. We also attempt to evaluate the nature of the spermatogenetic failure associated with isolated AZFc deletions. For this purpose, we performed a dual genetic analysis of karyotypes and molecular status of the AZF region along with bilateral testicular histological evaluation in 94 patients with non-obstructive, idiopathic infertility and azoospermia, severe oligospermia, or oligospermia.

Material and methods

Sixty five men with azoospermia (lack of sperm cells in semen), 23 men with severe oligospermia (fewer than 5 x 106 sperm cells/ml semen), and six with oligospermia (5-10 x 106 sperm cells/ml semen), all of them of Polish origin, were included in the study.

Histological analyses of biopsies from both testes of 77 patients were performed in formalin fixed paraffin embedded tissue blocks. Sections were cut at 4 pm thickness and stained with haematoxylin-eosin.

Chromosome studies were carried out on peripheral blood lymphocytes of 93 out of 94 patients using GTG, FPG, CBG, and QFQ banding. Karyotypes were analysed in at least 100 metaphases.

DNA was isolated from 10 ml of peripheral blood leucocytes of the patients and, when available, also from the fathers or other male relatives on the paternal side. For molecular analysis, genomic DNA was amplified by PCR using primers specific for 23 Y chromosome specific sequence tagged site (STS) markers (19 mapping to AZFa, AZFb, and AZFc and four mapping to short arm of the Y chromosome) according to conditions described in the Genebank entry.

Results

HISTOLOGICAL PHENOTYPES

The histological evaluation of testicular biopsies was performed in all 77 patients, 62 with azoospermia, 14 with severe oligospermia, and one with oligospermia. Histological abnormalities were detected in 63 patients and in 14 patients the histology was normal. The histological phenotypes are summarised in table 1. Among 14 patients with normal histology, 10 were azoospermic, three severely oligospermic, and one was oligospermic.

AZF DELETIONS

Based on the PCR amplification of 23 STS markers specific to the Y chromosome (mostly AZF region), deletions in six patients (IHG8, IHG18, IHG22, IHG67, IHG82, and IHG120) were detected (table 2). In two cases, IHG8 and IHG22, the deletions were large, terminal, and similar in size encompassing AZFb, AZFc, and the heterochromatin region. Thus, they were detectable cytogenetically. Four other deletions, IHG18, IHG67, IHG82, and IHG120, were small, interstitial, and similar in size and spanned the AZFc subregion (table 2).

In four cases (IHG22, IHG67, IHG82, and IHG120), deletions were found to be de novo, and all STS markers absent in the patients were present in their father. DNA samples of close male relatives of patients IHG8 and IHG18 were not available and therefore the de novo status could not be tested.

KARYOTYPING

Patients IHG8, IHG22, and IHG120 were mosaic: They were carrying one cell line 46,XY with a large terminal deletion (IHG8 and IHG22) or small interstitial deletion (IHG120) and a second cell line (45,X) lacking the entire Y chromosome (table 2). Among patients with no AZF deletions, chromosomal aberrations were detected in 30% of cases. In 18 subjects, aberrations of the sex chromosomes were found including 10 47,XXY cases, whereas autosomal aberrations were present in 10 males (Wojda et al, submitted).

GENOTYPE-PHENOTYPE CORRELATION

Both patients with large terminal deletions (IHG8 and IHG22) were found to have azoospermia and complete maturation arrest lacking secondary spermatocytes, whereas three patients with small interstitial AZFc deletions (IHG18, IHG67, and IHG82) had milder phenotypes: azoospermia or severe oligospermia with incomplete maturation arrest (IHG18 and IHG67) or hypospermatogenesis (IHG82) (table 3). In addition, the pattern of incomplete maturation arrest of patient IHG18 was focal and not generalised (table 3). Despite significant representation of the 45,X cell line, especially in patients IHG8 and IHG22 (table 2), no gonadal degeneration, short stature, webbing of the neck, lymphoedema,…