use of a highly sensitive quantitative telomerase assay in intracytoplasmic sperm injection...
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Use of a highly sensitive quantitative telomerase assayin intracytoplasmic sperm injection programmesfor the treatment of 47,XXY non-mosaic Klinefelter men*
Y. Yamamoto1, N. Sofikitis1,2, A. Kaponis1, J. Georgiou3, D. Giannakis3, CH. Mamoulakis3,D. Loutradis1, X. Yiannakopoulos2, Y. Mio3, I. Miyagawa1 and A. Chatzikyziakidou2
1Tottori University School of Medicine, Tottori, Japan; 2Ioannina University School of Medicine, Ioannina, Greece;3MFC Clinic, Yonago, Japan
Key words. ICSIKlinefelters syndromespermatozoontelomerasetestis
Summary. We evaluated the role of the sensi-tive quantitative telomerase assay (SQTA) in themanagement of men with non-mosaic Klinefelterssyndrome (KS). Diagnostic testicular biopsy (DTB)was performed in 24 men with KS. A part of theDTB was stained and the remaining fragment wasprocessed for the SQTA. After 318 months, atherapeutic testicular biopsy (TTB) was performedin the same testicle and the recovered specimenswere processed to identify spermatozoa. Men with aSQTA outcome equal to 0.00 Units lg)1 protein(n 7) demonstrated therapeutic testicular biopsymaterial that was negative for spermatogenic cells.In five men with a SQTA outcome of 8.1138.03 Units lg)1, the most advanced germ cell wasthe spermatogonium primary spermatocyte. In theremaining 12 men, the most advanced spermato-genic cell in the TTB was the spermatozoon. Inthese men, the SQTA outcome was equal to 25.7692.68 Units lg)1 protein. Using 39.00 Units lg)1
protein as a cut-off value, the accuracy of theSQTA in identifying men positive for spermatozoawas 91.6%. It appears that the SQTA has a role foridentifying non-mosaic KS men who have testicularspermatozoa.
The introduction of ooplasmic injections of testicu-lar spermatozoa or spermatids has revolutionizedthe treatment of nonobstructed azoospermic (NOA)men (Silbert et al., 1995; Silbert, 1996; Sofikitis et al.,1998a). One of the most perplexing problems inassisted reproduction programs dealing with NOA-men is the lack of parameters that can be used topredict the presence or absence of foci of testicularspermatozoa. It is widely accepted that the histo-logical results of the diagnostic testicular biopsy(DTB; tissue stain), the peripheral serum levels offollicle stimulating hormone (FSH) and the testicularsize are not highly accurate markers in predictingthe presence or absence of spermatozoa in thetesticles of NOA men (for review see Sofikitis et al.,1998a). Occasionally, men with small testicles, highperipheral serum FSH profiles, and histologicalimages of Sertoli cellonly syndrome are positive fortesticular foci of spermatozoa (Sofikitis et al., 1998a).It is of great clinical importance to find newparameters that can predict the presence absenceof foci of advanced spermatogenesis in a therapeutictesticular biopsy [tissue cutting mincing and pro-cessing for assisted reproduction trials; therapeutictesticular biopsy (TTB)] sample. Evaluation of suchparameters may have a role in the therapeuticmanagement of men with Klinefelters syndrome(KS) known to have small testicles (Foss & Lewis,1971; Laron et al., 1982; Terzoli et al., 1992; Bourneet al., 1997; Tournaye et al., 1997a; Nodar et al.,1998; Palermo et al., 1998; Reubinoff et al., 1998;Ron-El et al., 1999). In these men, prediction of theabsence of spermatozoa in a TTB is of paramountimportance in avoiding unnecessary TTB that
Correspondence: Prof. Nikolas Sofikitis, Department of Urol-ogy, Tottori University School of Medicine, Nishimachi 361,Yonago 683, Japan. Tel.: +81 859 348119; Fax: +81 859348074; e-mail: email@example.com*A part of this study was presented at the 55th Annual Meetingof The American Society of Reproductive Medicine in SanFrancisco, CA, October 49, 1998.
andrologia 34, 218226 (2002) Accepted: January 10, 2002
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would further reduce testicular volume. Consideringthat (i) KS is a common genetic disorder found in1 : 1000 live born males and in 3.1% of infertilemen (Sharara, 1998), and (ii) few pregnancies havebeen achieved with assisted reproduction in coupleswith KS (for review see Sharara, 1998); it is notsurprising that men with KS often appear in assistedreproduction centres. These men currently repre-sent a significant subpopulation of those infertilemen for whom an assisted reproduction trial is theonly hope of fathering their own children. Aprognostic parameter that would accurately indicatethose men with KS who were negative for testicularspermatozoa and could not be candidates forassisted reproduction programs would avoid theunnecessary expense, and associated risks, of ovar-ian stimulation for the female partners.
Telomeres are specialized structures at the endsof eukaryotic chromosomes that appear to functionin chromosome stabilization, positioning, and rep-lication (Blackburn, 1991). Telomeres stabilizenatural chromosome ends and inhibit aberrantfusions and rearrangements that occur on brokenchromosomes (Blackburn, 1991). The length of thetelomere, which contains TTAGGG repeats, pro-gressively decreases with cell division (Morin, 1989;Prowse et al., 1993). Telomere repeats are synthes-ized de novo onto chromosome ends by the enzymetelomerase. Telomerase, an RNA-dependent DNApolymerase, is the only enzyme to compensate forthe telomeric losses of DNA that occur at each celldivision (Hisatomi et al., 1997, 1999). It is wellknown that telomerase activity is expressed inimmortal cell lines and most human tumours, butis inactive in most normal somatic cells, tissuesadjacent to tumours, or benign growths (Kim et al.,1994; Kim, 1995). Telomerase activity has alsobeen demonstrated in human germ lines andtesticles (Wright et al., 1996; Yamamoto et al.,1999a, 2000a). Some investigators have reportedpositive telomerase activity in mouse, rat, andhuman spermatogonia primary spermatocytes, sec-ondary spermatocytes, and round spermatids,whereas testicular and epididymal spermatozoaare negative for telomerase activity (Prowse et al.,1993; Eisenhauer et al., 1997; Yamamoto et al.,1999a,b). Telomerase is inactivated during sper-miogenesis (Yamamoto et al., 1999b). The telom-erase hypothesis suggests that telomerase activity isgreater in embryonic cells and decreases in somatictissues during development and differentiation(Eisenhauer et al., 1997). In a previous study, thesensitive quantitative telomerase assay (SQTA) wasused in DTB material collected from men withSertoli cellonly syndrome (Yamamoto et al.,1999a) and showed a high sensitivity, specificity,positive predictive value, and negative predictive
value, for the identification of Sertoli cellonlysyndrome men with foci of haploid cells in theTTB. That study muted an important role for theSQTA in DTB for the prediction of testicular fociof haploid cells in the TTB of NOA men. Ourhypothesis that severe defects in spermatogenesisare accompanied by a reduction in testicular tissuetelomerase activity is consistent with the findingthat men with testicles with active spermatogenesis(i.e. men with obstructive azoospermia with normalspermatogenesis) have significantly larger SQTAprofiles than men with Sertoli cellonly syndrome(with or without foci of haploid cells) (Yamamotoet al., 1999a). SQTA was performed to quantifytelomerase activity (Hisatomi et al., 1997, 1999;Yamamoto et al., 1999a) and has been proven to bea highly sensitive and quantitative assay (Hisatomiet al., 1997; Yamamoto et al., 1999a,b, 2000a,b).
The objective of the present study was toevaluate the role of the SQTA in predicting thepresence or absence of spermatozoa in TTBmaterial recovered from men with non-mosaicKS. The SQTA is the gold standard for quantifyingtelomerase activity and this is the first publishedstudy to use SQTA for assessing the testicles of menwith KS. Additionally, we assessed the fertilizingpotential and the reproductive capacity of sperma-tozoa obtained from the testicular tissue of non-mosaic KS men. However, the results of the assistedreproduction are reported in another communica-tion (Yamamoto et al., 2001).
Participants and methods
Twenty-four men with non-mosaic KS werereferred to our facilities (age 2345; FSH:1456 IU L)1). Normal levels of FSH for males ofreproductive age in our facilities ranged from3-11 IU l)1. All these men were negative forspermatozoa in centrifuged semen samples. Theirwives were 2041 years old. A DTB was per-formed. A part of the DTB material (58 mg) wasprocessed for haematoxylin-eosin staining. Theremaining piece (5 mg) was frozen and processedfor the SQTA. After 318 months, all men under-went a TTB (in the same testicle that had undergoneDTB) during assisted reproduction trials.
The TTB material (148194 mg) was processedfor mincing, filtering and dispersion extraction ofspermatogenic cells. During mincing filtering of theTTB material, fractions of recovered cells wereobserved via a confocal scanning laser microscope-computer assisted system (CSLM-CAS). Othertesticular cellular fractions (from the TTB material)
Klinefelters syndrome and telomerase 219
ANDROLOGIA 34, 218226 (2002)
were processed for fluorescent in situ hybridization(FISH). Recovered spermatozoa were processed forassisted reproduction and cryopreservation.
Highly sensitive quantitative telomerase assay (SQTA)
Details on the application of the SQTA have beendescribed previously (Hisatomi et al., 1997, 1999;Yamamoto et al., 1999a,b, 2000a). Previous experi-ments have shown that the SQTA is a quantitativeassay (Yamamoto et al., 1999a). Twenty aliquots ofextracts (each containing 1 lg protein) from each5-mg piece of testicular tissue from each participantwere prepared and processed for 20 TRAP assays(Yamamoto et al., 2000a). Thus, 20 TRAP assayswere performed for each participa