1 Center for Male Reproductive Medicine and Microsurgery, Cornell Institute for Reproductive Medicine and Department of Urology, New York WeillCornell Medical Center, 525 East 68th Street, Starr 900, New York, NY 10021, 2 Population Council, Center for Biomedical Research, 1188 York Avenue, Room 514, New York, NY 10021 and 3 Center for Reproductive Medicine and Infertility, New York WeillCornell Medical Center, 505 East 70th Street, HT-340, New York, NY 10021, USA
4 To whom correspondence should be addressed at: New York WeillCornell Medical Center, 525 East 68th Street, Starr 900, New York, NY 10021, USA. e-mail: pnschleg{at}med.cornell.edu
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Abstract |
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Key words: AZF/genetics/male infertility/spermatogenesis/Y chromosome
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Introduction |
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Further analysis of Yq in 370 men with idiopathic azoospermia or severe oligozoospermia utilizing STS analysis of 76 DNA loci revealed de-novo microdeletions in 12 men and an inherited microdeletion in one individual (Vogt et al., 1996). In this sentinel study, the deletions localized to three non-overlapping regions in Yq, AZFa, AZFb and AZFc. The AZFc region corresponded with the DAZ region. However, the recent discovery that a large portion of euchromatic Yq consists of massive, nearly identical ampliconic repeats arranged in palindromes has elucidated the difficulty in precisely identifying borders of the AZF regions (Kuroda-Kawaguchi et al., 2001
; Repping et al., 2002
). Localization of breakpoints for the AZFb and c regions has demonstrated that these two regions overlap by 1.5 Mb, and the combined AZFb+c deletion has yet another distinct set of breakpoints in the same region (Repping et al., 2002
).
Vogt et al. (1996) first demonstrated that deletion of the three different AZF regions appears to have different effects on spermatogenesis. The correlation between detection of sperm within testicular tissue and specific AZF deletion has been made in only a few studies with small numbers of subjects. Whereas sperm have been detected within the testes of men with AZFc deletion by microdissection testicular sperm extraction (TESE) (Schlegel, 1999
), no spermatids were detected in men with deletion of the AZFb region for use in ICSI, suggesting that complete AZFb deletions are a significantly adverse prognostic finding for TESE (Girardi et al., 1997
; Brandell et al., 1998
; Silber et al., 1998
; Krausz et al., 2000
). A recent, thorough characterization of 42 men with AZFc deletion determined that 81% of men with this deletion produce sperm overall, 38% with sperm in the ejaculate and 67% of those that are azoospermic on diagnostic biopsy or with TESE (Oates et al., 2002
).
We have identified and characterized a large number of men with deletion of AZFa, AZFb and/or AZFc to determine the prevalence of complete spermatogenesis for each type of microdeletion by assessing the presence of sperm on semen analysis and the presence of intratesticular sperm identified either on diagnostic testis biopsy or with TESE. These findings provide prognostic information on the chance of successful sperm retrieval for these patients based upon the specific region of the Y chromosome deleted.
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Materials and methods |
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Y chromosome analysis
Genomic DNA was extracted from peripheral blood using two methods, the Stratagene DNA Extraction Kit (Stratagene, USA) and the Genomic DNA Purification Kit (Promega, USA). Thirty STS within the long arm of the Y chromosome were selected emphasizing the AZFa, b and c regions. Previously published primer sequences were used for each STS (Henegariu et al., 1994; Reijo et al., 1995
; 1996). Testing for the presence of the short arm of the Y chromosome (Yp) was performed with the STS sY14, located within the SRY (sex-determining region on the Y chromosome) gene. The most distal STS on Yq, sY160DYZ1, is located within the heterochromatic region, whereas all others are located within the euchromatic region. Multiplex PCR was performed for analysis of microdeletions. PCR products were run by electrophoresis on a 4% agarose gel impregnated with ethidium bromide for visualization under UV light. Failure of amplification for a given STS was confirmed twice with single primer PCR. DNA from a fertile male served as positive control, whereas water and DNA from a female served as negative control for multiplex and single primer PCR respectively.
Microdissection TESE technique
A semen specimen was obtained immediately prior to planned TESE to confirm azoospermia. If no sperm were identified within the pellet produced by centrifugation of the sample at 3000 g for 15 min, microdissection TESE was performed on either the same day of planned oocyte retrieval or 1 day prior to oocyte retrieval with incubation of the sperm overnight. Microdissection TESE was performed as previously described (Schlegel, 1999) utilizing the operating microscope and one transverse incision in the tunica albuginea through which spermatogenic tubules were selected for extraction.
Statistical analysis
Outcome variables (age, serum testosterone, serum FSH and LH, testicular volume) for azoospermic men with AZFc deletion with successful surgical sperm retrieval were compared to those with unsuccessful sperm retrieval using the MannWhitney U-test. Men with AZFc deletion and azoospermia were also compared to the group of AZFc-deleted men with oligozoospermia using the same variables and the MannWhitney U-test.
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Results |
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Age, serum testosterone, serum FSH and LH levels and testicular volume were correlated with surgical detection of sperm by TESE or diagnostic biopsy in azoospermic men with AZFc deletion for whom information on all parameters was available. No statistically significant differences were noted between median values of the variables between the two groups (Table IV). Comparison of men with AZFc deletion who were azoospermic to the group of AZFc-deleted men that were oligozoospermic also demonstrated no statistically significant differences in median values for age, testosterone, FSH, LH and testicular volume (Table V).
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Discussion |
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The most common microdeletion found in our patient population was AZFc. The association between AZFc deletion and impaired, but variable, spermatogenesis has been demonstrated previously (Mulhall et al., 1997a; Silber et al., 1998
; Oates et al., 2002
). Here, we report that 38% of men with isolated AZFc deletion had sperm present within the ejaculate and a surgical sperm retrieval rate of 56% overall (75% with TESE and 45% with diagnostic biopsy). These results are consistent with the characterization of a large cohort of men with AZFc deletion by Oates et al. (2002
), which showed that 38% of men with AZFc deletion had sperm in their ejaculate and 67% had some degree of complete spermatogenesis demonstrated either with TESE or diagnostic biopsy. Clearly men with AZFc deletion have variable capacity to produce sperm, some producing none within the seminiferous tubules, while some produce a quantity of sperm sufficient to survive epididymal transit and appear in the ejaculate (Silber et al., 1997
). Although rare cases of transmission of the AZFc deletion have been described from fertile father to infertile sons by natural conception (Chang et al., 1999
; Saut et al., 2000
), the majority of men with AZFc deletion have significantly compromised spermatogenesis, such that ICSI is necessary for sperm from these patients to produce biological offspring. The highest sperm concentration detected in an AZFc patient in this study was 8x106/ml. It is therefore unlikely that Y chromosome microdeletion testing in men with sperm concentration greater than this will yield a positive result.
Table III illustrates the success rates with which sperm were surgically retrieved from the testes by two techniques, standard diagnostic testis biopsy and microdissection TESE. Routine diagnostic biopsy is usually done by removal of a small piece of tissue from one superficial area of the testis, whereas microdissection TESE entails thorough microscopic assessment of a large area of seminiferous tubules accessed with one generous transverse incision within the tunica albuginea to identify spermatogenic tubules. Our results of sperm retrieval in men with AZFc deletion show that the microdissection TESE technique is nearly twice as effective when compared with routine biopsy technique for sperm retrieval, consistent with previous observations in men with nonobstructive azoospermia (Schlegel, 1999).
Age, serum testosterone, FSH, LH and testicular volume did not correlate with sperm retrieval in AZFc-deleted patients (Table IV). Success of surgical sperm retrieval in azoospermic men with AZFc deletion cannot be predicted using these variables. As previously noted by multiple investigators, FSH levels and testicular volume do not predict sperm retrieval as these factors reflect overall testicular function (Mulhall et al., 1997b; Schlegel et al., 1997
; Oates et al., 2002
). The single best region of spermatogenesis determines the chance of sperm retrieval and does not affect FSH or testicular size. Likewise, age, serum testosterone, FSH, LH and testicular volume did not correlate with the presence of sperm in the ejaculate in men with AZFc deletion (Table V). Mean age of the oligozoospermic group was not less than that of the azoospermic group. The capacity to maintain spermatogenesis at a level sufficient for spill into the ejaculate appears to vary between individuals in a manner independent of age, testosterone, FSH, LH and testicular volume.
Isolated microdeletion of the AZFa region is relatively rare, whereas deletion of the AZFb region occurs more commonly, either in isolation or in conjunction with deletion of the AZFc region (Figure 1). We have identified nine men with deletion of the seven STS spanning the AZFb region only, whereas an additional seven patients had the entire AZFb region deleted plus the proximal six (out of a total of eight) STS of the AZFc region. Although the STS used in our study are different from those used by Repping et al. (2002), the deletion pattern of these seven patients may correlate with the P5/distal-P1 type of deletion pattern in which the distal portion of AZFc is spared. Several deletion patterns involving the AZFb and c regions are evident in Figure 1, and although spermatogenesis is variable with deletion of the AZFc region alone, none of the patients in this study with deletion of the AZFb region, either isolated, in continuity with the AZFc region, or as part of Yq deletion demonstrated evidence of mature spermatid production.
Incomplete spermatogenesis has been demonstrated for men with deletions involving the AZFa and AZFb regions on testis histology (Vogt et al., 1996; Pryor et al., 1997
; Martinez et al., 2000
). Kamp et al. (2001
) described in detail a series of nine patients with complete AZFa deletion; all patients had a Sertoli cell-only pattern on histology with no sperm identified. Consistent with reported testis biopsy findings, attempted TESE in men with complete deletions of the AZFa or AZFb regions has been unsuccessful. Brandell et al. (1998
) described seven azoospermic patients with deletions including the entire AZFb region; all underwent TESE with no evidence of elongating spermatids or sperm. Silber et al. (1998
) did not detect sperm by TESE in five azoospermic patients with deletions encompassing the AZFa and/or AZFb regions. To our knowledge, completion of spermatogenesis in men with deletions of the full AZFa or AZFb regions has not been demonstrated. Partial deletions within an AZF region are not prognostically revealing.
A total of seven men with deletion patterns that include deletion of the AZFa region (three with deletion of AZFa alone + four with Yq deletion) and 23 men with deletion including the AZFb region (nine with AZFb deletion, 10 with AZFb+c deleted and four with Yq deletion) underwent either diagnostic testis biopsy or TESE and all had evidence of abnormal spermatogenesis with complete absence of elongating or mature spermatids. The majority of men with isolated AZFc deletion, however, did have adequate sperm production so that sperm from the ejaculate or surgically retrieved from the testis were available for ICSI. Vertical transmission of the deletion has been demonstrated, and all sons inherit the same deletion, likely to cause defective spermatogenesis, though to an uncertain degree (Chang et al., 1999; Saut et al., 2000
; Oates et al., 2002
). Boys with inherited AZFc deletion are otherwise healthy, and girls are unaffected. Couples in which the male partner has AZFc deletion should be informed of this inheritance pattern prior to the decision to proceed with ICSI.
Karyotype analysis demonstrated that large chromosome abnormalities can coexist with Y chromosome microdeletions, most commonly sex chromosome mosaicism. The five men with Yq deletion by microdeletion testing were found to lack the entire Yq on karyotype analysis. If the first genetic test done is that of the karyotype, and this demonstrates absence of Yq (i.e. 46,XX male), then microdeletion testing is likely unnecessary. We did not observe translocation of Yq regions to other chromosomes in this study.
In conclusion, deletions of the AZFa, b and c regions are associated with abnormal spermatogenesis, and certain deletions are of prognostic value. Microdeletion of either the entire AZFa or AZFb regions of the Y chromosome heralds an exceptionally poor prognosis for sperm retrieval, whereas the majority of men with AZFc deletion have sperm retrieved successfully for use in IVF with ICSI. The prognostic significance of complete deletions involving either the AZFa or AZFb regions should be discussed with patients prior to attempted TESE.
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Submitted on February 26, 2003; accepted on May 9, 2003.