Eotaxin and Interleukin-4 Levels and Their Relation to Sperm Parameters in Infertile Men

ORİJİNAL ARAŞTIRMA Eotaxin and Interleukin-4 Levels and Their Relation to Sperm Parameters in Infertile Men Türkan YİĞİTBAŞI, MD, a Yasemin BASKIN, MD, b Gökhan AFACAN, MD, c Füsun KARAARSLAN, MD, d Cevat TAHERİ, e Dilek ASLAN, MD d a Clinical Biochemistry Clinic, Atatürk Training and Research Hospital, b Dokuz Eylül University Institute of Oncology, c Institute of Forensic Medicine, Adliye Sarayı, d Irenbe IVF Center, İzmir e Analiz Diagnostics Center, Kayseri Part of this study was presented on the 1th National Congress of The Society of Andrology at 2007/Ankara, Turkey. Ge liş Ta ri hi/re ce i ved: 24.06.2009 Ka bul Ta ri hi/ac cep ted: 28.10.2009 Ya zış ma Ad re si/cor res pon den ce: Türkan YİĞİTBAŞI, MD Atatürk Training and Research Hospital, Clinical Biochemistry Clinic, İzmir, TÜRKİYE/TURKEY turkanyigitbasi@gmail.com ABS TRACT Objective: Male factor infertility accounts for 30% to 50% of the total infertile couples seeking for infertility treatment. In about 40-60% of these men, a specific etiology can not be found. The aim of this study was to confirm the presence of eotaxin and interleukin-4 (IL-4) in human seminal plasma, to show the differences between eotaxin and IL-4 concentrations in fertile and infertile men, and to show the potential relationship between eotaxin and IL-4 levels in semen and spermiogram parameters. In literature, this is the first study that evaluates eotaxin in the human seminal plasma. Material and Methods: The participant of the study was 55 infertile males with abnormal semen parameters as study group and 16 healthy volunteers with normal sperm parameters as the control group. Semen samples were classified according to criteria of the World Health Organization Laboratory Manual. The Morphology of the smears was scored using Kruger s strict criteria. Seminal eotaxin and IL-4 levels were measured by bead based immunoassay multiplex methods. Results: Seminal eotaxin levels were significantly higher in infertilite group compared to fertile donors. There were negative correlations between eotaxin concentrations and parameters such as motility (r= -0.293, p< 0.01) and, +4 motility (r= -0.307, p< 0.01) in the study group. IL-4 levels were similar in fertile and infertile seminal plasma. There were positive correlations between eotaxin and IL-4 levels (r= 0.436 p< 0.01). The receiver operating characteristic curve (ROC) analysis revealed a diagnostic value for eotaxin activity with respect to male factor infetility in case group, with an area under curve of 0.69 (95% confidence interval= 0.55-0.84). Conclusion: Increased levels of eotaxin may play a role in the pathogenesis of male infertility. Key Words: Infertility; chemokines; interleukin-4; semen ÖZET Amaç: Erkek faktörüne bağlı infertilite, infertilite nedeniyle tedavi almakta olan tüm çiftlerin %30-50 sinden sorumludur. Bu erkeklerin yaklaşık% 40-60 ında belirli bir etyoloji bulunamamaktadır. Bu çalışmanın amacı insan seminal plazmasında eotaksin ve interlökin-4 (IL- 4) varlığını ortaya koymak, fertil ve infertil erkeklerde eotaksin ve IL-4 konsantrasyonları arasındaki farkı göstermek ve semendeki eotaksin ve interlökin-4 düzeyleri ile spermiogram parametreleri arasındaki olası ilişkiyi göstermektir. Yaptığımız literatür taramasına göre, bu çalışma insan semen plazmasında eotaksin düzeylerini değerlendiren ilk çalışmadır. Gereç ve Yöntemler: Çalışmaya anormal sperm parametreleri olan 55 infertil erkek çalışma grubu, normal sperm parametreleri olan 16 sağlıklı gönüllü de kontrol grubu olarak katıldı. Semen örnekleri, Dünya Sağlık Örgütü Laboratuvar Manual kriterlerine göre sınıflandırıldı. Morfoloji ve smear ler ve Kruger in kesin kriterleri kullanılarak skorlandı. Seminal eotaksin ve IL-4 düzeyleri multiplex immunoassay yöntemlerle ölçüldü. Bulgular: Seminal eotaksin seviyeleri infertilite grubunda fertil vericilere göre anlamlı oranda yüksek bulundu. Çalışma grubunda eotaksin konsantrasyonu ile motilite (r= -0.293, p< 0.01) ve +4 motilite (r= -0.307, p< 0.01) parametreleri arasında negatif korelasyonlar vardı. Çalışma grubundaki. IL-4 düzeyleri fertil ve infertil seminal plazmalarda benzerdi. Eotaksin ve IL- 4 düzeyleri arasında pozitif korelasyonlar (r= 0436 p< 0.01) vardı. Alıcı işletimli karakteristik ROC eğrisi, vaka grubuna kıyasla eotaksin aktivitesi için 0.69 eğrisi altında (%95 güvenlik indeksi= 0.55-0.84) tanısal bir değer ortaya çıkardı. Sonuç: Yükselmiş eotaksin düzeyleri erkek infertilitesinin patogenezinde rol oynayabilir. Anah tar Ke li me ler: Kısırlık; kemokinler; interlökin-4; semen Cop yright 2010 by Tür ki ye Kli nik le ri Turkiye Klinikleri J Med Sci 2010;30(5):1441-6 Turkiye Klinikleri J Med Sci 2010;30(5) 1441

Yiğitbaşı ve ark. a le fac tor in fer ti lity ac co unts for 30% to 50% of the to tal in fer ti le co up les se e king for in fer ti lity tre at ment. 1 In abo ut 40 60% of the se men, a spe ci fic eti o logy can not be fo und. 2 Cyto ki nes are re gu la tory pro te ins in vol ved in he ma to po i e sis, im mu ne cell de ve lop ment, inf lam - ma ti on and im mu ne res pon ses. Se ve ral cyto ki nes ha ve di rect ef fect on tes ti cu lar cell func ti ons, and a num ber of the se are pro du ced wit hin the tes tis even in the ab sen ce of inf lam ma ti on or im mu ne acti va ti on events. The re is com pel ling evi den ce that cyto ki nes, in fact, play an im por tant re gu la tory ro - le in the de ve lop ment and nor mal func ti on of the tes tis. 3 Eo ta xin is a mem ber of the CC che mo ki ne fa - mily of inf lam ma tory and im mu no re gu la tory cyto - ki nes. 4 Eo ta xin is synthe si zed by a num ber of dif fe rent cell types, and is sti mu la ted by in ter le u - kin-4 (IL-4) which is pro du ced by T-hel per 2 (Th- 2) lymphocy tes. 4-6 Its ro le in nu me ro us eo si nop hilas so ci a ted di sor ders such as fo od al lergy, gas tro en - te ri tis, pa ra si tic in fec ti ons, al ler gic co li tis and inf - lam ma tory bo wel di se a se has be en des cri bed. 4-7 Se mi nal plas ma is known to be res pon sib le for orc hes tra ting ma ting-in du ced im mu no mo du la ti on. Cen tral to this pro cess are nu me ro us cyto ki nes that mo du la te ute ri ne le u kocy te rec ru it ment and traf fic - king. Com pre hen si ve analy sis of the cyto ki ne pro fi - le of mu ri ne se mi nal flu id re ve a led that only few cyto ki nes inc lu ded IL-4 and eo ta xin le vels which we re sig ni fi cantly hig her in se mi nal plas ma when com pa red to tho se fo und in se rum. 8 To best of our know led ge, this is the first study that eva lu a tes eota xin in the hu man se mi nal plas ma. IL-4 is a T-lymphocy te-de ri ved 20-kDa glycop ro te in pos ses sing a bro ad spec trum of bi o lo gi cal ac ti vity. In ad di ti on to in du cing pro li fe ra ti on and dif fe ren ti a ti on of hu man B cells, it can sti mu la te the pro li fe ra ti on of a wi de ran ge of cells such as T- lymphocy tes, mast cells, and he mo po i e tic pro ge ni - tor cells. 9 The pre sen ce of in ter le u kins and se ve ral ot her cyto ki nes in se mi nal plas ma was re por ted in multip le stu di es, ho we ver the re was no ade qu a te in forma ti on in the li te ra tu re con cer ning IL-4. 10-15 Zhang Biyokimya et al has fo und that the con tent of IL-4 in the se m- i nal plas ma of the in fer ti le gro up was sig ni fi cantly lo wer than that of the nor mal gro up (p<0.01). 16 Pa - ra di si et al. re por ted the lack of IL-4 in se mi nal plas ma. 17 The aim of this study was to con firm the pre s- en ce of eo ta xin and IL-4 in hu man se mi nal plas ma, to show dif fe ren ces bet we en eo ta xin and IL-4 concen tra ti ons in fer ti le and in fer ti le men and to show the po ten ti al re la ti ons hip bet we en eo ta xin and IL- 4 le vels and the sper mi og ram pa ra me ters. MA TE RI AL AND MET HODS 1. SUB JECTS AND SE LEC TI ON The study po pu la ti on inc lu ded 55 ma les of in fer ti - le co up les who we re tre a ted in Iren be IVF cen ter. Six te en he althy vo lun te ers who had nor mal sperm pa ra me ters we re en rol led as the con trol gro up. All the par ti ci pants ga ve their in for med con sents to par ti ci pa te in the study, which had be en ap pro ved by the lo cal Et hics Com mit te e (Iz mir Te pe cik Tra - i ning and Re se arch Hos pi tal lo cal Et hic Com mit - te e: 30.03.2007-67/11). No ne of the par ti ci pants re ce i ved any me di ca ti on or vi ta min supp le men ta - ti on. Ins tan ces of le u kocy tos per mi a and vis co us se - men samp les we re exc lu ded from the study. 2. SE MEN COL LEC TI ON AND PRE PA RA TI ON Se men spe ci mens we re col lec ted by mas tur ba ti on af ter 48 to 72 ho urs of se xu al abs ti nen ce. The spe ci - mens un der went comp le te li qu e fac ti on at 37 o C for 20 mi nu tes. All se men samp les we re co un ted in a Mack ler co un ting cham ber (Se fi Me di cal Ins tru - ments, Re ho vot, Is ra el). Samp les we re clas si fi ed accor ding to cri te ri a of the World He alth Or gani za ti on La bo ra tory Ma nu al, 4th edi ti on. 18 The mo - ti lity of sperm was clas si fi ed as +1,+2,+3,+4 mo ti le. Morp ho logy of the sme ars we re sco red using Kru - ger s strict cri te ri a. 19 All samp les we re cen tri fu ged at 1000 g for 10 mi nu tes. Cle ar se mi nal plas ma was sto red at 80 C un til analy sis. 3. ME A SU RE MENT OF EO TA XIN AND IL-4 IN SE MI NAL PLAS MA Ins tru ment: Lu mi nex 100 or 200 re a ders (Lu mi - nex Corp., Aus tin, TX) with da ta ac qu i si ti on soft- 1442 Turkiye Klinikleri J Med Sci 2010;30(5)

Biochemistry wa re ver si on 1.7, IS2.3, or xpo NENT, Mil li po re va cu um pump (Ca ta log# WP6111560) and Mil li - po re Mul tis cre en RE SIST va cu um ma ni fold (Ca talog# MAVM0960R) HU MAN CYTO KI NE LIN COp lex KIT 96 Well Pla te As say (Cat. #HCYTO-60K) As say pla te: Mil li po re Mul tis cre en HTS, BV, 96-well fil ter pla te (Ca ta log# MSBVN1250) was used for all as says. An ti bod y Con ju ga ted Be ads: Car boxy la ted polys trye ne mic - rosp he res we re purc ha sed from Lu mi nex Corp. (Aus tin, TX). An ti bo di es we re eit her de ve lo ped inter nally at Mil li po re or purc ha sed com mer ci ally. As say Met ho do lo gi es: All as says we re bu ilt on sand wich for mat with two an ti bo di es for each analy te. Phyco eryt hrin was used as the re por ter on the sur fa ce of mic rosp he res. Samp le Re qu i re ment: 25 μl of samp les was used per well. Da ta Analy sis: Stat LI A soft wa re (Bren dan Sci en ti fic., Inc.) was used for samp le cal cu la ti ons with a we igh ted 2-pa - ra me ter lo gis tic cur ve-fit ting met hod (Eo ta xin sensi ti vity 1,23 pg/ml); (IL-4 sen si ti vity 0,57pg/ml). 20 4. STA TIS TI CAL ANALY SIS Da ta dif fe ren ces bet we en the study and the con trol gro up we re analy zed by using Stu dent s t-test.the Kol mo go rov-smir nov test was used to de ter mi ne nor ma lity. Co ef fi ci ents of cor re la ti on we re cal cu - la ted using Spe ar man s cor re la ti on analy sis. All hypot he sis tests we re two-ta i led with sta tis ti cal sig ni fi can ce as ses sed at the p va lu e < 0.01 le vel with 95% con fi den ce in ter vals. The da ta we re ex pres sed as the Me an ± SD. Sta tis ti cal com pu ta ti ons we re cal cu la ted using SPSS 11.0 for win dows soft wa re (SPSS Inc, Chi ca go, IL, USA). The are a un der the re ce i ver ope ra ting cha rac te ris tic cur ve (ROC) was used to as sess the dis cri mi na ti ve abi lity of eo ta xin le vels in pa ti ent with ma le in fer ti lity. RE SULT De mog rap hic cha rac te ris tics, se mi nal eo ta xin and IL-4 le vels in the study and con trol gro ups we re sum ma ri zed in Tab le 1. The re was not any sig ni fi - cant dif fe ren ce in terms of age bet we en the gro ups. Se mi nal eo ta xin le vels we re sig ni fi cantly hig her in in fer ti le gro up as com pa red to fer ti le do nors (Tab- le 1). Yiğitbaşı et al TABLE 1: Demographics, semen characteristics, seminal eotaxin and IL-4 levels of infertile men and fertile donors. Fertil donors Infertile men (n= 16 ) (n= 55) P Age (years) 33,2±6,0 33,5±6,8 0.857 Semen Parameters Concentrations ( 10 6 /ml) 49,9±28 33,2±28 0.043 Motility (% motile sperm) 55,8±7,2 34,8±12 <0.001 Morphology (Kruger s criteria 15,9±5,8 6,0±2,2 <0.001 % normal sperm) +4 Motility (%motile sperm) 25,6±5,0 8,6±4 <0.001 Eotaxin(pg/ml) 9,7 ± 8,7 20,2± 15,7 0.023 IL-4(pg/ml) 13,2±12,3 11,6±12,4 0.669 Values are Means + SD. The mean difference is significant at the.01 level. The re we re ne ga ti ve cor re la ti ons bet we en eota xin le vels and sperm pa ra me ters such as mo ti lity and +4 mo ti lity (p<0.01). The re we re si mi lar IL-4 le vels in fer ti le and in fer ti le se mi nal plas ma. Ho w- e ver the re we re po si ti ve cor re la ti ons bet we en eota xin and IL-4 le vels (r= 0,436; p<0.01). ROC analy sis re ve a led a high di ag nos tic va lu - e for eo ta xin le vels with res pect to ma le fac tor infer ti lity, with an are a un der cur ve (AUC) of 0.69 [95% con fi den ce in ter val (CI) =0.55-0.84], sen si ti - vity = 68% and spe ci fi city= 43% with a cut off va - lu e of 5.13 (greater than the va lu e that was re la ted to ma le fac tor in fer ti lity) (Fi gu re 1). DIS CUS SI ON In fer ti lity con ti nu es to be a highly pre va lent condi ti on. The pri mary prob lem re si des exc lu si vely in the ma le part ner in 30-50% of in fer ti le co up les. A spe ci fic ca u se of in fer ti lity is not de ter mi nab le in 40 60% of the in fer ti le men. 1,2 Cyto ki nes rep re sent a wi dely de fi ned gro up of bi o ac ti ve poly pep ti des in vol ved in the com mu ni - ca ti on net work of im mu ne-com pe tent cells. 21 In ad di ti on, cyto ki nes ha ve de ci si ve ac ti vi ti es out si de of the im mu ne system whe re they func ti on as re g- u la tors of tes ti cu lar ste ro id hor mo ne pro duc ti on. Cyto ki nes ha ve al so be en imp li ca ted as no vel growth and dif fe ren ti a ti on fac tors in vol ved in the re gu la ti on of cells in both the en doc ri ne and the tu bu lar com part ment of the tes tis. 22 Turkiye Klinikleri J Med Sci 2010;30(5) 1443

Yiğitbaşı ve ark. FIGURE 1: ROC of eotaxin levels in patient with male infertility. The inf lam ma ti on du e to ge ni tal or syste mic in fec ti ons can ca u se al te ra ti ons in the tes ti cu lar func ti on. The re cog ni ti on of in tra tes ti cu lar an ti - gens pro vo kes the pro duc ti on of an ti bo di es by B lymphocy tes. Then, the im mu ne system in du ces a cel lu lar res pon se, by cyto ki ne sec re ti on, ac ti va ti on of comp le ment and T lymphocy tes ac ti va ti on. 23 Mo re o ver, the se are pro du ced wit hin the tes tis even in the ab sen ce of inf lam ma ti on or im mu ne acti va ti on events. The re is com pel ling evi den ce that cyto ki nes, in fact, play an im por tant re gu la tory ro - le in the de ve lop ment and nor mal func ti on of the tes tis. 3 Post-ma ting inf lam ma tory res pon se has be en des cri bed in mi ce and pigs. 24,25 The cel lu lar chan ges are ini ti a ted when se mi nal mo i e ti es in te ract with cer vi cal and ute ri ne epit he li al cells to in du ce a sur - ge in synthe sis of cyto ki nes. 24 The ac ti va ti on of the ex pres si on of ute ri ne cyto ki nes and le u kocy te traffic king that has be en imp li ca ted in pre-imp lan ta ti - on emb ryo de ve lop ment. 24-26 Go pic han dran et al. fo und that le vels of IL-4, G-CSF, eo ta xin, KC and RAN TES in flu id drawn from the se mi nal ve sic les of sing le mi ce we re sig ni - fi cantly hig her when com pa red to tho se fo und in se rum. 8 Ba sed on the se fin dings, aut hors pro po sed Biyokimya a mo del of ma ting-in du ced im mu no mo du la ti on that imp li ca ted se mi nal eo ta xin, RAN TES and MIP-1 in the re lo ca ti on and con cen tra ti on of extra va sa ted mig ra ting en do met ri al eo si nop hils to the lu mi nal epit he li um. 8 Eo ta xin (con den sed from eo si nop hil che mo - ta xin) mic ro se qu en cing re ve a led a no vel 73 ami - no acid C-C che mo ki ne. The pu ri fi ed pro te in was a highly po tent eo si nop hil che mo at trac tant in vit - ro and in vi vo, but had no sig ni fi cant ef fect on ne - ut rop hils. The eo ta xins are unu su al (but not uni qu e) in sig na ling vi a a sing le re cep tor: CCR3. Many cell types in the lung ap pe ar to be ca pab le of synthe si zing eo ta xin (eg air way epit he li al cells, air way smo oth musc le cells, vas cu lar en dot he li al cells and mac rop ha ges, as well as eo si nop hils them sel ves) 4. Thus, cyto ki nes that are synthe si zed by Th2 lymphocy tes, such as IL-4, ha ve be en inves ti ga ted as po ten ti al in ter me di a ri es in eo ta xin pro duc ti on. 27 In our study, se mi nal eo ta xin le vels we re signi fi cantly hig her in in fer ti le gro up when com pa - red to fer ti le do nors (Tab le 1). It has be en shown that mo ti lity and +4 mo ti lity dec re a sed as eo ta xin le vels in cre a sed. In our study ROC analy sis re ve a led a high diag nos tic va lu e for eo ta xin le vels with res pect to ma le fac tor in fer ti lity, with an are a un der cur ve (AUC) of 0.69 ]95% con fi den ce in ter val (CI) =0.55-0.84], sen si ti vity = 68% and spe ci fi city= 43% with a cut off va lu e of 5.13 (greater than the va lu e that was re la ted to ma le fac tor in fer ti lity) (Fi gu re 1). A li te ra tu re re vi ew re ve a led that no da ta existed regar ding this mat ter. IL-4 can in du ce eo ta xin mrna ex pres si on in hu man vas cu lar en dot he li al cells. 5 In our study, the re was a po si ti ve cor re la ti on bet we en eo ta xin and IL-4 le vels. In vit ro stu di es of Te ran et al. 6 de - mons tra ted that IL-4 syner gi zes with the pro-inf - lam ma tory cyto ki ne tu mor nec ro sis fac tor-α to in cre a se eo ta xin pro duc ti on from lung fib rob - lasts. The le vels of LIF, IL-4, IL-10 and M-CSF produ ced by de ci du al T cells of wo men suf fe ring from 1444 Turkiye Klinikleri J Med Sci 2010;30(5)

Biochemistry unexp la i ned spon ta ne o us abor ti on are lo wer than tho se of he althy preg nant wo men, and this can indi ca te that the se cyto ki nes may con tri bu te to the ma in te nan ce of preg nancy. T cells from the cu mu - lus oop ho rus sur ro un ding the pre imp lan ta ti on emb ryo pro du ce LIF and IL-4. The se fin dings sug gest that cyto ki nes pro du ced by ma ter nal T cells cre a te a su i tab le mic ro en vi ron ment for pre imp lan ta ti on emb ryo de ve lop ment and ma in te nan ce of preg - nancy. 28,29 Se mi nal flu id was al so cha rac te ri zed by hig her le vels of IL-4 and G-CSF com pa red to se rum, sug ges ting thatthe se to o, may ha ve a ro le, as yet unde fi ned, in ma ting-in du ced im mu no mo du la ti on. In man, IL-4 is tho ught to be ne ces sary for the es tab - lish ment and ma in te nan ce of preg nancy, by avo i - ding the harm ful ef fects of cell-me di a ted im mu nity in the vi ci nity of pu ta ti ve emb ryo imp lan ta ti on si tes and at the fe to-ma ter nal in ter fa ce. 8 Zhang et al. fo und that the con tent of IL-1 be ta in the se mi nal plas ma of the in fer ti le gro up was ob vi o usly hig her, but the con tent of IL-4 and IL-10 le vels we re sig ni fi cantly lo wer than that of the nor mal gro up. 16 Pa ra di si et al. re por ted the lack of IL-4 in se mi nal plas ma. 17 The se fin dings might in di ca te that furt her stu di es are ne ces sary to cla rify the ro le of IL-4 in pa ti ents with ma le infer ti lity. Yiğitbaşı et al In our study, we fo und si mi lar IL-4 le vels in fer ti le and in fer ti le se mi nal plas ma. It has be en shown that the ro le of cyto ki nes at post-ma ting inf lam ma tory res pon se is im por tant. Ho we ver in li te ra tu re, the re are no studies con cer - ning the le vels of eo ta xin in hu man se mi nal plas - ma. Our study is the first study that eva lu a tes eo ta xin le vels in the se mi nal plas ma of in fer ti le men using mul tip lex im mu no as says. In conc lu si on, our re sults de mons tra ted that eo ta xin le vels we re sig ni fi cantly hig her in pa ti ents with ma le in fer ti lity, and cor re la ted with the sperm pa ra me ters such as mo ti lity and +4 mo ti lity. In cre a sed le vels of eo ta xin may play a ro le in the pat ho ge ne sis of ma le in fer ti lity. Ack now ledg ments Many thanks to Mr.Mark Kop nitsky, Ze us Sci en ti fic app li ca ti on spe ci a list, for IFA met hod app li ca ti on and eva l- u a ti on. Spe ci al thanks to Le vent Guc lu er for his tech ni cal as sis tan ce. Thanks for sta tis ti cal eva lu a ti ons of Dr. Ca gin Kan de mir Ca vas. 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A no vel hu man CC che mo ki ne, eo ta xin-3, which is ex pres sed in IL-4-sti mu la ted vas cu lar en dot he li al cells, exhi bits po tent ac ti vity to ward eo si nop hils. J Immu nol 1999;163(3):1602-10. REFERENCES 6. Te ran LM, Moc hi zu ki M, Bar tels J, Va len ci a EL, Na ka ji ma T, Hi ra i K, et al. Th1- and Th2- type cyto ki nes re gu la te the ex pres si on and pro duc ti on of eo ta xin and RAN TES by hu man lung fib rob lasts. Am J Res pir Cell Mol Bi ol 1999;20(4):851-4. 7. Oh HE, Chetty R. Eo si nop hi lic gas tro en te ri tis: a re vi ew. J Gas tro en te rol 2008;43(10):741-50. 8. Go pic han dran N, Ek bo te UV, Wal ker JJ, Bro - o ke D, Or si NM. Mul tip lex de ter mi na ti on of mu ri ne se mi nal flu id cyto ki ne pro fi les. Rep ro - duc ti on 2006;31(3):613-21. 9. Mi re-slu is AR, Thor pe R. 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