Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, the Sackler Faculty of Medicine,
Abstract
Substantial involvement of the Y chromosome in sexual development and spermatogenesis has been demonstrated. Over the last decade, varying extent of Y chromosome microdeletions have been identified among infertile patients with azoospermia or oligozoospermia. These microdeletions were clustered in three main regions named AZFa, AZFb, and AZFc. Analysis of the Y chromosome microdeletion was found to be of prognostic value in cases of infertility, both in terms of clinical management as well as for understanding the aetiology of the spermatogenesis impairment. However, the accumulated data are difficult to analyse, due to the variable extent of these deletions, the different sequence-tagged sites (STS) used to detect the microdeletions, and the non-uniformity of the histological terminology used by different investigators. This debate discusses the chances of finding testicular spermatozoa in men with a varying extent of Y chromosome microdeletions. The genotype and germ cell findings in men with AZFa microdeletions as well as those that include more than a single AZF region are reviewed, as is the effect of Y chromosome AZF microdeletions on the maturity of the Sertoli cells.
Key words: chromosomal instability/CK-18/infertility/spermatogenesis/Y chromosome microdeletion
Introduction
Research over the last 30 years has demonstrated a substantial involvement of the Y chromosome in sexual development and spermatogenesis. The long arm of the Y chromosome (Yq) harbours at least 15 gene families which have been proposed as being involved in the control of spermatogenesis (Ma et al., 1993; Reijo et al., 1995
; Jones et al., 1996
; Lahn and Page, 1997
). These findings are of great interest not only in the quest to understand the mechanism of the genetic control of spermatogenesis, but also in the clinical context in which there is a need for molecular tools that can assist in defining the aetiology and prognosis of infertile men.
Over the last decade, Y chromosome microdeletions of varying extent have been identified among infertile patients with azoospermia or oligozoospermia. These deletions were clustered in three main regions (AZFa, AZFb and AZFc) on the Yq arm (Vogt et al., 1996). In an early attempt to find the prognostic value of these microdeletions, a correlation between the deleted AZF region and the severity of the spermatogenesis impairment was proposed (Vogt et al., 1996
). However, additional reports of men with a range of microdeletions (see Tables I and II
) and the improved understanding of the histopathological nature of the testis have modified and shown this proposed correlation to be more complicated than originally thought (Silber et al., 1997
).
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While there is no doubt that Y microdeletions are involved in spermatogenesis impairment, it is now understood that the region and extent of the deletion play a major role in the magnitude of the impairment. No mature sperm cells were detected in most of the men with deletions that included more than one AZF region. Severe oligozoospermia was detected in only five out of 21 men (24%) in whom microdeletions involved more than one AZF region (Table I). Two of these men had partial AZFab deletions that included the DFFRY, DBY, BPY2 and CDY2 but apparently not the RBM1 gene. The third man had only a few sperm cells in the ejaculate (<1x103) and his deletion was a large one that included all the AZF regions. The fourth and the fifth men had an AZFbc deletion that included all the genes identified in both regions. The remaining 16 men were azoospermic with no germ cells identified in their testis.
Large microdeletions that include the heterochromatic Yq-tip (`terminal' deletions) were suggested to cause chromosomal instability and were shown to be prone to rearrangements or even to Y chromosome loss (Krausz et al., 1999a; Kirsch et al., 2000
). In our clinic, two patients with AZF terminal deletions were also found to be mosaic for the Y chromosome (46,X i(Yp)+ 45,X0).
The association between deletions that extend to a specific AZF region or to part of it and the semen quality or testicular histological findings is less clear. Deletions of the complete AZFc region (sY153sY158) are the most common type and are frequently associated with spermatogenesis impairment (from severe oligozoospermia to azoospermia with few mature spermatids or Sertoli cells only in the testicular tissue) (Reijo et al., 1996; Simoni et al., 1997
; Silber et al., 1998
; Kleiman et al., 1999a
). It has been estimated that mature spermatids can be found in ~50% of azoospermic men with AZFc microdeletions (Silber et al., 1998
). Consequently, the possibility of transmission of the AZFc deletion from father to male offspring has been demonstrated (Kent-First et al., 1996
; Vogt et al., 1996
; Mulhall et al., 1997
; Pryor et al., 1997
; Kleiman et al., 1999b
; Chang et al., 1999
; Page et al., 1999
). Recently, Y chromosome microdeletions, tested by polymerase chain reaction (PCR) and fluorescence in-situ hyridization (FISH), and germinal mosaicism (tested by FISH with DAZ probe) was analysed in infertile males (Le Bourhis et al., 2000
). A very low germ cell mosaicism (DAZ lacking) was found in spermatozoa from only two oligozoospermic men out of 27 infertile men analysed.
A possible decrease of sperm count over time in men with AZFc deletions has been proposed based on the ejaculated sperm concentration that deteriorated from severe oligozoospermia to azoospermia over time (13 years) in two men (Girardi et al., 1997; Simoni et al., 1997
). These findings would be of clinical relevance if they could be demonstrated to be related to AZFc deletions in a larger group of men.
The impact of AZFb deletions in infertility has been widely discussed (see Krausz et al., 2000): in essence, in case of deletions which remove the whole AZFb region (complete AZFb deletions) the probability of finding testicular spermatozoa for successive ICSI is virtually zero. The associated phenotype is mainly spermatocyte arrest.
Isolated AZFa deletions are rare in comparison with AZFb and AZFc ones. While they are usually associated with azoospermia and testicular histology of Sertoli cell-only, severe oligozoospermia (three out of 11 men) or azoospermia and severe hypospermatogenesis in the testicular tissue (one out of 11 men) were also identified in AZFa-deleted patients (Table II). Reanalysing the data by looking at the deletion of genes that have been recently mapped to the AZFa region revealed that mature spermatids or spermatozoa can be detected occasionally when only the DFFRY gene was deleted (Table II
). Moreover, a de-novo point mutation was identified in the DFFRY gene of an azoospermic man with testicular histology of severe hypospermatogenesis (Sun et al., 1999
). Only one man with severe oligozoospermia and an AZFa deletion that included, in addition to DFFRY, also DBY and UTY genes, has been reported thus far (Table II
; Ferlin et al., 1999).
Are Sertoli cells affected by the absence of the AZF regions?
Sertoli cells play a key role in triggering and regulating the process of spermatogenesis. We studied the involvement of Sertoli cells in different spermatogenesis impairment by an immunohistomorphometric technique using cytokeratin-18 (CK-18) as a marker for immature Sertoli cells and vimentin (Bar-Shira Maymon et al., 2000). CK-18 and vimentin are typically co-expressed in Sertoli cells cytoplasm during prenatal and pubertal differentiation (Stosiek et al., 1990
; Aumuller et al., 1992
). CK-18 is normally lost at puberty and the predominant intermediate filament in mature Sertoli cells of adults is of the vimentin type. High CK-18 expression was detected in testicular biopsies of 22 azoospermic men who showed mixed atrophy (NB: different spermatogenesis impairments can be found in adjacent seminiferous tubules, ranging from Sertoli cells only to complete spermatogenesis). A lower expression of CK-18 was found in 12 biopsies showing Sertoli cells only, and minimal residual staining was detected in the group of biopsies showing normal spermatogenesis (six men with obstructive azoospermia) (Bar-Shira Maymon et al., 2000
).
Examination of testicular biopsies obtained from nine men with microdeletions in the AZF regions revealed that, despite the differences in the specific AZF deleted region and its extent, they were either negative for CK-18 expression or showed only weak and insignificant residual staining. The specimens examined included those of four men with AZFa-c deletion accompanied with Sertoli cell-only in their testicular histology, of one man with AZFb deletion that included the RBM1 gene and exhibited spermatocyte maturation arrest, and of four men with AZFc deletion showing progression of spermatogenesis up to spermatids. These findings suggest that AZF deletions have no impact on the Sertoli cell maturation process even when some of the deleted genes, e.g. DFFRY and DBY, are expressed in the somatic cells of the testis (S.E.Kleiman, unpublished data).
Conclusions
Y chromosome AZF microdeletions have no apparent effect on the maturity of the Sertoli cells, as histochemically tested by the expression of cytokeratin-18 and vimentin. Analysis of Y chromosome microdeletion is of clinical importance in cases of azoospermia and oligozoospermia, not only in terms of defining the aetiology of the spermatogenesis impairment but also for its clinical prognostic value. The chances of finding spermatozoa in the testes of men with Y chromosome microdeletions of different regions and to different extents vary. In addition, the possibility of chromosomal instability occurring as a consequence of some microdeletions and the possibility of a decline in sperm production over time in patients with AZFc microdeletions are important considerations in clinical decision-making and appropriate genetic counselling. Today, we might just be observing the tip of the iceberg.
Notes
1 To whom correspondence should be addressed at: Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel. E-mail: ser{at}tasmc.health.gov.il
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