Up to 30% of men evaluated for infertility have severe oligospermia or azoospermia without a clear etiological explanation. Cytogenetic analysis of azoospermic men have revealed deletions of the distal long arm of Y chromosome and the concept of an Azoospermia Factor, AZF, has been defined as a region required for spermatogenesis. 1 Since then, smaller interstitial deletions detected at a molecular level on the Y chromosome of infertile men have been reported by many investigators suggesting that this region of Yq carries important genetic information essential for normal spermatogenesis. The advancement of molecular techniques and increasing knowledge of the DNA sequences of the Y chromosome led to the identification of three different microdeleted regions along Yq designated initially by Vogt as AZFa, AZFb and AZFc. 2, 3 Wthin AZF regions many candidate genes for spermatogenesis have been identified . 4, 5 However, recent discoveries that large portion of euchromatic Yq consists of masive, nearly identical ampliconic repeats arranged in palindromes has elucidated the difficulty in precisely identifying borders of the AZF microdeletions. 6, 7 Localization of breakpoints for AZFb and AZFc regions has demonstrated that those two regions overlap by 1.5Mb and the combined AZFb+c deletion has yet another distinct set of breakpoints in the same region. 6, 7 Using detaieled mapping of proximal and distal breakpoints of Y chromosme microdeletion alternative nomenclature has been proposed which better represents the actual physical map of Y: P5/proximal-P1 (for previously AZFb); b2/b4 (AZFc); P5/distal-P1 (AZFb+c). Furthermore, three other deletions within AZFb+c region have been identified: gr/gr deletion with high prevalence, b2/b3 and b1/b3 has very low frequency deletion with still unknown phenotypic effect. The gr/gr deletion is observed in fertile and infertile men. 8
Although presence of Y chromosome microdeletion has detrimental effect on spermatogenesis, as presence of AZFa or AZFb deletion is associated with complete lack of germ cells, sperm can be found in over 50 % of men with AZFc deletion. Thus screening for Y chromosome microdeletion has significant clincial implications. 9, 10 Although men with AZFc deletion can father children through intracytoplasmic sperm injection (ICSI), presence of deletion will be transferred vertically to the offsprins raising concern that genetic causes of male infertility may be transmitted through ICSI to the offspring. 11 These children may be expected to experience fertility problems similar to those of their fathers. There is no evidence that vertical transmission of AZFc deletionts will lead to any other phenotypical problems.
Approximately 7% of men presenting for severe infertility will have clinically significant deletions within Y chromosome. Those deletions are too small to be detected by routine karyotype analysis. 12-14 These submicroscopic deletions are associated with severe spermatogenic defects and may be detected by microdeletion screening test.
No other clinical parameters such as testis volume or hormonal evaluation can accurately predict presence of Y chromosome microdeletions.
Thus all men with low sperm density (less than 5 mil/ml) should be screened for Y chromosome microdeletion in addition to karyotype. If Y chromsome microdeletion is identified, comprehensive genetic counseling should be offered especially especially if they are considering in vitro fertilization. In addition, men who do not elect IVF might also benefit from Y chromosome microdeletion testing to determine the underlying cause of their infertility.
Over the last 14 years we have used 31 STSs within Y chromosome region spanning the area of interest. However, recent analysis of our 2400 patients showed that all clinically relevant deletions can be detected using 11 proposed STSs. Proposed number of STSs exceeds established standards for Y chromosome microdeletion testing in Europe and USA. European Academy of Andrology recommends using only 8 STSs for Y chromosome microdeletion screening.
The Y microdeletion screening described in this protocol uses panel of 11 Yq chromosome specific markers referred as sequenced tagged sites, STSs, whose presence is determined in patient’s DNA by polymerase chain reaction (PCR). These STSs have been shown to be consistently present in large population of males of proven fertility. 15 Absence of the expected STS is interpreted as deletion of the corresponding Y locus.
In previous protocols, the presence of area of interest on Y chr. was verrified by electrophoresis of the amplicon product on gel stained with ethidium bromide.
The protocol required that PCR products were mixed with loading buffer and transferred to the gel for electrophoresis. Using ethidium bromide gel creates enviromental and health problems as EB is a known carcinogen. In addition transfer of PCR products from open PCR plates creates high risk of cross-contamination.
The proposed new method based on real time PCR technology is a result of our efforts on designing a close system method which would be sensitive and at the same time, decrease the risk of amplicon cross-contamination.
High-Resolution Melting Real Time PCR technique with ResoLight dye is a closed method and it is especially usefull for Y chromosome microdeletion screening as it minimizes the risk of amplicon contamination as this system uses sealed plates. Amplified product is never transferred or pipetted which greatly decrease time from preparation to analysis.
Our Laboratory has extensive experience in performing Y chromosome microdeletion testing using traditional method. So far we have screened more than 2400 patients and have been able to detect continuous regions of deletions in 250 patients.
1. Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet 1976;34:119-24.
2. Kamp C, Huellen K, Fernandes S, et al. High deletion frequency of the complete AZFa sequence in men with Sertoli-cell-only syndrome. Mol Hum Reprod 2001;7:987-94.
3. Vogt PH, Edelmann A, Kirsch S, et al. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 1996;5:933-43.
4. Ma K, Inglis JD, Sharkey A, et al. A Y chromosome gene family with RNA-binding protein homology: candidates for the azoospermia factor AZF controlling human spermatogenesis. Cell 1993;75:1287-95.
5. Reijo R, Lee TY, Salo P, et al. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet 1995;10:383-93.
6. Repping S, Skaletsky H, Lange J, et al. Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet 2002;71:906-22.
7. Kuroda-Kawaguchi T, Skaletsky H, Brown LG, et al. The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet 2001;29:279-86.
8. Repping S, Skaletsky H, Brown L, et al. Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection. Nat Genet 2003;35:247-51.
9. Palermo GD, Schlegel PN, Hariprashad JJ, et al. Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum Reprod 1999;14:741-8.
10. Schlegel PN, Palermo GD, Goldstein M, et al. Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia. Urology 1997;49:435-40.
11. Oates RD, Silber S, Brown LG, Page DC. Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome, and of 18 children conceived via ICSI. Hum Reprod 2002;17:2813-24.
12. Brandell RA, Mielnik A, Liotta D, et al. AZFb deletions predict the absence of spermatozoa with testicular sperm extraction: preliminary report of a prognostic genetic test. Hum Reprod 1998;13:2812-5.
13. Girardi SK, Mielnik A, Schlegel PN. Submicroscopic deletions in the Y chromosome of infertile men. Hum Reprod 1997;12:1635-41.
14. Hopps CV, Mielnik A, Goldstein M, Palermo GD, Rosenwaks Z, Schlegel PN. Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions. Hum Reprod 2003;18:1660-5.
15. Kostiner DR, Turek PJ, Reijo RA. Male infertility: analysis of the markers and genes on the human Y chromosome. Hum Reprod 1998;13:3032-8.
16. Palermo GD, Schlegel PN, Sills ES, et al. Births after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter's syndrome. N Engl J Med 1998;338:588-90.
17. Pryor JL, Kent-First M, Muallem A, et al. Microdeletions in the Y chromosome of infertile men. N Engl J Med 1997;336:534-9.