Use of a highly sensitive quantitative telomerase assay in intracytoplasmic sperm injection programmes for the treatment of 47,XXY non-mosaic Klinefelter men
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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: firstname.lastname@example.org*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)
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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 participant. The averagefor each participant was then calculated.
The SQTA was performed by The JapaneseSpecial Reference Laboratory (Matsue, Japan) aspreviously described (Yamamoto et al., 1999a). Analiquot of extract containing 1 lg protein was usedfor each TRAP assay. Aliquots of the extracts wereincubated with 0.1 ng Cy-5 labeled TS primer(TRAP-eze). Following a 30-min incubation at30C, a polymerase chain reaction (PCR) wasperformed at 94C (30 s), 60C (30 s) and 72C(45 s) for 30 cycles (Yamamoto et al., 2000a). Theexternal control, a TSR8 (TRAP-eze), was used as apositive control (Hisatomi et al., 1997). The productswere diluted with an equal volume of formamide dyesolution, heated at 94C for 5 min and applied(5 ll lane)1) to a 10% denaturing gel containing 6 murea fitted to an automatedDNAsequencer (ALF redTM DNA Sequencer: Pharmacia Biotech, Uppsala,Sweden). The temperature of the gel was kept at45C during electrophoresis at 45 W. The data fromtheALF red TMDNA Sequencer were collected andanalyzed automatically by Fragment Manager V1.1(Pharmacia Biotech, Osaka, Japan). Each peak wasquantified in terms of size, peak height and peak area.The quantification of telomerase activity was deter-mined by the mathematic formula described byHisatomi et al. (1997) as we previously reported(Yamamoto et al., 1999a, 1999b). The SQTA out-come values were expressed as TPG (Total ProductGenerated) Units lg)1 protein. Quantification oftelomerase activity was determined by the followingformula ( 100):
fmeasured total area of telomerase activity(50 bp, 56 bp, 62 bp, 68 bp ...) g=fmeasuredarea of internal control (36 bp)g of the sample
fmeasured total area of telomerase activity (50 bp,56 bp, 62 bp, 68 bp...)g=fmeasured area of internalcontrol (36 bp)g of the external control TSR8
Processing of therapeutic testicular biopsy material
The testicular tissue was washed three times withnormal saline. The seminiferous tubules were thenwashed in Dulbeccos phosphate buffered saline(DPBS; Sigma Co., St. Louis, MI) containing5.6 mm glucose and 5.8 mm sodium lactate (modi-fied DPBS; Sofikitis et al. 1998a) and subsequentlyminced into small pieces. Samples were observedvia a dissecting microscope (Olympus SZ-STS;Olympus, Tokyo, Japan) during the tissue mincingprocess. The overall mincing process lasted 1 h.The samples were then centrifuged at 500 g for30 min, the sedimented pieces of tissue and cellswere resuspended in modified DPBS, and thesamples were passed through a filter paper of3040lm pore size (Whatman Co., New York, NY)(Sofikitis et al., 1998b). The total volume of testicu-lar cell suspension, which passed through the filter,was 818 ml. The filtration procedure was per-formed at room temperature and lasted 1525 min.A minor part of the filtrate (Fraction-1) wascollected, centrifuged at 750 g for 30 min, and thesedimented cells were resuspended in modifiedDPBS. Some of the sedimented cells were thenobserved via CSLM-CAS. The remaining sedi-mented cells were processed for FISH. The majorpart of the filtrate was filtered again (secondfiltration; via an 1112 lm pore size filter). Filtra-tion via the 3040 lm-pore size filter and the12 lm-pore size filter removed the small pieces ofseminiferous tubuli and most of the Sertoli cellfragments, debris, tissue fragments and spermato-gonia primary spermatocytes (if present) from thefinal (second) filtrate. Thus in men with KS positivefor testicular foci of round spermatids and sperma-tozoa, the final filtrate contained mainly spermatidsand spermatozoa.
A small part (Fraction-2) of the final filtrate wasprocessed for observation via CSLM-CAS andFISH techniques. The major part of the finalfiltrate was processed for sperm identification.
Confocal-scanning laser microscope-computer assistedsystem
CSLM-CAS is powerful in the field of microscopy(Sofikitis et al., 1994, 1996a, 1996b; Yamanaka et al.,1997; Sofikitis et al., 1998a, b, c; Yamamoto et al.,2001). The capacity of the CSLM-CAS to accu-rately identify subtypes of germ cells has beenpreviously confirmed using FISH or transmissionelectron microscopy as a control (Yamanaka et al.,1997; Sofikitis et al., 1998a, c; Yamamoto et al.,1999a, 2001). The stage of the most advancedspermatogenic cell in Fraction-1 or Fraction-2 noted
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during observation (and quantitative morphometricanalysis) via CSLM-CAS was recorded.
Fluorescent in-situ hybridization techniques
FISH techniques were applied as an additionalmethodology to define the most advanced germ cellin Fractions-1 and -2 of the minced testicularsamples. Three colour FISH was performed usingpreviously described methodology (Harper et al.,1994; Sofikitis et al., 1998c; Yamamoto et al.,1999a). The probe kit was a combination of Yspectrum Red Pink, X spectrum Yellow, and 18spectrum Green Blue fluorescently labelled DNAprobes specific for chromosomes Y, X and 18,respectively.
Spermatogonia primary spermatocytes were46-XY1818-germ cells or 47-XXY1818 germcells. Secondary spermatocytes were consideredX18-(2C)-DNA cells or Y18-(2C)-DNA cells (2Cindicates a diploid amount of DNA; Alberts et al.,1994; Sofikitis et al., 1998c) and...