Semen Levels of Spermatid-Specific Thioredoxin-3 Correlate with Pregnancy Rates in ART Couples

Semen Le els o Spe ma id-Speci ic Thio edoxin-3 Co ela e wi h P egnancy Ra es in ART Couples Clay on Buckman 1,2. , Ch is ophe Ozanon 5. , Jing Qiu 3 , Mi iam Su o sky 1,2 , Joyce Ann Ca a a 4{ , Vanesa Y. Rawe 6 , Gau ishanka Manandha 1 , An onio Mi anda-Vizue e 7" , Pe e Su o sky 1,2 1Di ision o Animal Sciences, Uni e si y o Missou i, Columbia, Missou i, Uni ed S a es o Ame ica, 2Depa men s o Obs e ics, Gynecology & Women’s Heal h, School o Medicine, Uni e si y o Missou i, Columbia, Missou i, Uni ed S a es o Ame ica, 3Depa men o S a is ics, Uni e si y o Missou i, Columbia, Missou i, Uni ed S a es o Ame ica, 4Cell and Immunology Co e, Uni e si y o Missou i, Columbia, Missou i, Uni ed S a es o Ame ica, 5Assis ed Fe iliza ion Medical Cen e NATECIA, NOVESCIA Labo a o y, Clinique NATECIA, Lyon, F ance, 6REPROTEC, Buenos Ai es, A gen ina, 7Ins i u o de Biomedicina de Se illa, Uni e sidad de Se illa, Se illa, Spain Abs ac Spe ma id speci ic hio edoxin-3 (SPTRX3 o TXNDC8) is a es is/male ge m line speci ic membe o hio edoxin amily ha accumula es in he supe luous cy oplasm o de ec i e human spe ma ozoa. We hypo hesized ha semen le els o SPTRX3 a e e lec i e o ea men ou come in assis ed ep oduc i e he apy (ART) couples ea ed by in i o e iliza ion (IVF) o in acy oplasmic spe m injec ion (ICSI). Rela ionship be ween SPTRX3 and ea men ou come was in es iga ed in 239 couples unde going ART a an in e ili y clinic. Spe m con en o SPTRX3 was e alua ed by low cy ome y and epi luo escence mic oscopy, and co ela ed wi h clinical semen analysis pa ame e s, and da a on emb yo de elopmen and p egnancy es ablishmen . High SPTRX3 le els (.15% SPTRX3-posi i e spe ma ozoa) we e ound in 51% o male in e ili y pa ien s (n = 72), in 20% o men om couples wi h unexplained, idiopa hic in e ili y (n = 61) and in 14% o men om couples p e iously diagnosed wi h emale-only in e ili y (n = 85). Couples wi h high SPTRX3 p oduced ewe wo- p onuclea zygo es and had a educed p egnancy a e (19.2% p egnan wi h .15% SPTRX3-posi i e spe ma ozoa s. 41.2% p egnan wi h ,5% SPTRX3-posi i e spe m; one-sided p,0.05). The a e age p egnancy a e o all 239 couples was 25.1%. Li e bi h a e was 19.2% and lowes a e age SPTRX3 le els we e ound in couples ha deli e ed wins. Men wi h .15% o SPTRX3-posi i e spe ma ozoa, a cu o alue es ablished by ROC analysis, had hei chance o a he ing child en by IVF o ICSI educed by nea ly wo- hi ds. The pe cen age o SPTRX3-posi i e spe ma ozoa had p edic i e alue o p egnancy a e ART. G adien pu i ica ion and spe m swim-up ailed o emo e all SPTRX3-posi i e spe ma ozoa om semen p epa ed o ART. In summa y, he ele a ed semen con en o SPTRX3 in men om ART couples coincided wi h educed incidence o p egnancy by IVF o ICSI, iden i ying SPTRX3 as a candida e bioma ke e lec i e o ART ou come. Ci a ion: Buckman C, Ozanon C, Qiu J, Su o sky M, Ca a a JA, e al. (2013) Semen Le els o Spe ma id-Speci ic Thio edoxin-3 Co ela e wi h P egnancy Ra es in ART Couples. PLoS ONE 8(5): e61000. doi:10.1371/jou nal.pone.0061000 Edi o : Joel R. D e e , Cle mon -Fe and Uni ., F ance Recei ed Decembe 10, 2012; Accep ed Ma ch 5, 2013; Published May 29, 2013 Copy igh : ß2013 Buckman e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Funding: This s udy was unded by g an numbe 1R21HD066333-01A1 om he Na ional Ins i u es o Heal h, NICHD, g an numbe CB000414 om he Resea ch Boa d o he Uni e si y o Missou i, seed unding om he Food o The 21s Cen u y P og am o he Uni e si y o Missou i and Unde g adua e Summe Resea ch In e nship, College o Ag icul u e, Food and Na u al Resou ces (CAFNR), Uni e si y o Missou i. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: D . Vanesa Rawe is employed by a comme cial company, REPROTEC, Buenos Ai es, ARGENTINA. The au ho s u he decla e ha his does no al e he au ho s’ adhe ence o all he PLOS ONE policies on sha ing da a and ma e ials. * E-mail: Su o skyP@missou i.edu .These au ho s con ibu ed equally o his wo k. "These au ho s also con ibu ed equally o his wo k. {Deceased. In oduc ion In e ili y and he pu suan assis ed ep oduc i e he apies (ART) a e on ise wo ldwide. A he same ime, oocy e/emb yo cul u e and manipula ion echnologies, and he diagnos ic echniques o emale in e ili y ha e been ad ancing s eadily. On he con a y, he and ological aspec o in e ile couple e alua ion s ill elies mos ly on adi ional, mic oscopy based semen analysis, in addi ion o clinical examina ions, ho monal p o iles, and cy ogene ic and Yq mic odele ion p o iles. To some ex en , he assessmen o he DNA agmen a ion o ch oma in damage, associa ed wi h oxida i e s ess, apop osis, o male geni al ac in ec ion a e used as diagnos ic ools. Exposu e o xenobio ics and ad anced pa e nal age a e possible isk ac o s associa ed wi h in e ili y. Spe m pa ame e s can be measu ed in specialized labo a o ies by he DNA-in eg i y based assays such as SCSA [1], Come [2] and TUNEL [3], bu he alue o such explo a ion is unce ain o he momen [4]. As he epe oi e o low cy ome ic semen e alua ion me hods inc eases, he challenge is on o ind new ools and e ili y ma ke s o eliably diagnose human male in e ili y [5]. In addi ion o DNA agmen a ion-based me hods, mi ochond ial memb ane po en ial p obes [6], lec ins [7], iabili y s ains [8], apop osis-de ec ing p obes [9], and an ibodies agains p o eins p esen p edominan ly in no mal o de ec i e spe ma o- zoa a e being adap ed o low cy ome ic semen analysis ( e iewed in [10]). Thio edoxins (TRX) a e a class o edox p o eins ha unc ion as gene al p o ein oxido educ ases by he e e sible oxida ion o PLOS ONE | www.plosone.o g 1 May 2013 | Volume 8 | Issue 5 | e61000 " * he cys eine esidues o hei ac i e si e whose conse ed sequence is Cys-Gly-P o-Cys. Thio edoxins a e main ained in hei educed ac i e o m by he la op o ein hio edoxin educ ase (TRXR) a he expense o he educing powe o NADPH [11]. The unc ions o hio edoxins a e ypically wide, mos ly elying on hei edox capabili ies, including an ioxidan de ense, modula ion o cell g ow h and di e en ia ion, egula ion o apop osis and ac i a ion o memb ane ion channels, among o he s [11,12]. Besides ubiqui ous hio edoxins ound bo h in cy oplasm and mi ochond ial ma ix in all cell ypes, mammals a e equipped wi h an addi ional se o hio edoxins wi h an exp ession pa e n es ic ed o male ge m cells [13]. One o such hio edoxins is Spe ma id-Speci ic Thio edoxin 3 [SPTRX3; HUGO nomencal- u e-TXNDC8 ( hio edoxin domain con aining 8)]. This hio - edoxin is ini ially obse ed in he Golgi appa a us o pachy ene spe ma ocy es; i is la e e ained pos -meio ically in ound spe ma ids in close associa ion wi h he de eloping ac osomal g anule, sugges ing a unc ion in ac osomal biogenesis [14]. Once he ac osome is o med, he esidual SPTRX3 accumula es in he cy oplasmic lobe o he elonga ing spe ma ids eady o be discha ged in he o m o esidual bodies. In e es ingly, SPTRX3 emains unde ec able in ully di e en ia ed no mal spe ma ozoa o a ious animal species, bu is e ained in he supe luous cy oplasm ound illing he nuclea acuoles and w apping a ound he spe m ail midpieces in mo phologically abe an human spe ma ozoa [14,15]. A dual low cy ome ic ial e ealed a obus posi i e co ela ion be ween spe m le els o SPTRX3 and ubiqui in (a alida ed de ec i e spe m bioma ke [16]) in a coho o 19 in e ile and 5 e ile subjec s [14]. A subsequen s udy used ImageS eam echnology combining low cy ome y and ligh mic oscopy, o di ec ly demons a e ha he low cy ome ic e en s wi h high SPTRX3 induced luo escence a e de ec i e spe ma ozoa wi h excess cy oplasm and/o nuclea acuoles [15]. In addi ion o spe ma id speci ic hio edoxins 1, 2 and 3 [14,17,18,19], he esidual cy oplasm ca ies cy oplasmic enzymes such as glucose-6-phospha e dehyd ogenase [20], supe oxide dismu ase [21], lac ic acid dehyd ogenase [22] and c ea ine phosphokinase [20,23]. Re en ion o esidual cy oplasm coincides wi h impai ed spe m unc ion in humans [24,25]. In mos mammals, he las emnan o spe ma id cy oplasm is ejec ed in he o m o a cy oplasmic d ople (CD). Human spe ma ozoa a e unique in ha hey do no de elop a ypical CD. The supe luous, edundan cy oplasm (o ‘‘excess esidual cy oplasm’’ [26]) in human spe ma ozoa is an al oge he di e en s uc u e, as i may no be ejec ed by he spe ma ozoon in a CD-like manne . I coincides wi h inc eased con en o eac i e oxygen species (ROS) in semen, which a high concen a ions a e cy o oxic [27]. The ROS gene a ed by excess spe m cy oplasm, o a leas by some ypes o i , can in lic damage by oxida ion o unsa u a ed a y acids in he spe m plasma memb ane, and by DNA b eakage, causing educed mo ili y, p egnancy loss, and emb yo mo bidi y [28]. We conduc ed he p esen s udy o de e mine he alue o he SPTRX3 as a p edic o o ART ea men ou come, se ing he s age o he alida ion o SPTRX3 as a bioma ke o human male in e ili y. Ou hypo hesis was ha in e ile men will ha e a signi ican ly highe con en o SPTRX3 p esen in hei spe m samples, compa ed o e ile males. We examined men om 239 couples unde going in e ili y ea men because o p e iously diagnosed emale, male, combined (male and emale) o idiopa hic in e ili y. We we e pa icula ly in e es ed in assessing possible e ec o high semen SPTRX3 le els on p egnancy a es. Resul s High Flow Cy ome ic SPTRX3 Values Coincide wi h Low P egnancy Ra es a e ART Spe m con en o SPTRX3 was e alua ed by low cy ome y and co ela ed wi h p egnancy a es in 239 couples ea ed by IVF and/o ICSI. The di ision o low cy ome ic his og ams in o ma ke s M1-M3 (Figu e 1) allowed us o dis inguish be ween cellula deb is (M1), cells no ca ying de ec able SPTRX3, wi h only a backg ound-le el o luo escence (M2) and SPTRX3-posi i e, de ec i e spe ma ozoa (M3). The a e age % o SPTRX3-posi i e cells (%M3) o he whole coho o 239 couples was 14.3%. The a e age p egnancy a e o all 239 couples en olled in his s udy was 25.1%. I was 29.8% o ICSI ea ed couples (n = 131) and 19.4% o IVF couples (n = 108). A e age p egnancy a e o couples abo e he a e age %M3 le el (n = 83) was 20.5%. A e age p egnancy a e o couples below he a e age %M3 (n = 156) was 27.6%. The median %M3 alue, i.e. he alue o %M3 a which hal he subjec s had highe and hal had lowe SPTRX3 le els, was 12.01%. A e age p egnancy a e o couples abo e he median was 20.2%. A e age p egnancy a e o couples below he median was 30.3%, which was signi ican ly highe han he a e age p egnancy a es o he abo e-median g oup (p = 0.04). The dosage e ec o SPTRX3 on p egnancy a es was cap u ed when all 239 couples we e di ided in o ou g oups based on %M3-SPTRX3 alues (less han 5% SPTRX3-posi i e spe ma o- zoa; 5–9.99% posi i e spe ma ozoa, 10–14.99% posi i e spe ma- ozoa; 15% o mo e SPTRX3-posi i e spe ma ozoa). In his pa i ion, he pe cen age o p egnan couples dec eased p og es- si ely wi h he inc eased SPTRX3 alues (Figu e 2A). The likelihood o p egnancy was u he educed i only he high SPTRX3 men om idiopa hic couples, we e analyzed (Figu e 2A; las column). A e age p egnancy a e o his g oup (n = 12) was only 16.7%. Compa ed o men wi h less han 5% SPTRX3- posi i e spe ma ozoa, hose ca ying mo e han 15% o such cells had hei chance o concei ing educed by a hal (41.2% p egnan , s. 19.2% p egnan ; one-sided p,0.05). The ROC analysis e ealed ha he cu o alues o 12.5% and 15% had a good balance o es sensi i i y and speci ici y (see Table S1 in File S1). The a e age p egnancy a e in couples ea ed by ICSI and ha ing .15%% SPTRX3-posi i e spe ma ozoa (n = 60) was 36.7%. A e age p egnancy a e was only 11.1% in IVF couples wi h .15% posi i e spe ma ozoa (n = 18). The IVF couples wi h ,15% SPTRX3-posi i e spe ma ozoa (n = 90) achie ed a p eg- nancy a e o 21.1%. Disc iminan analysis e ealed ha %M2 SPTRX3 was a signi ican posi i e p edic o o p egnancy in IVF couples and he median M3 was a signi ican nega i e p edic o o he ICSI g oup (see Supplemen al S a is ics – Sec ion ANALYSIS I & Table S11 in File S1). Based on clinical indica ion, SPTRX3 le els (%M3) we e signi ican ly highe (p,0.05) in couples wi h male and combined (male and emale) in e ili y, compa ed o couples wi h emale-only o idiopa hic in e ili y (Figu e 2B). Also, SPTRX3 le els we e signi ican ly highe (p,0.001) in ICSI couples compa ed o IVF couples (Figu e 2C). Wi hin he ICSI- ea ed g oup, couples ha achie ed p egnancy had nume ically highe %SPTRX3 posi i e spe ma ozoa, bu his inc ease was no s a is ically signi ican . The le els o spe m SPTRX3 we e no signi ican ly di e en be ween smoke s and non-smoke s when all couples we e e alua ed (Figu e S2). P egnancy ou comes we e con i med in 57/60 p egnan couples; 46 o hose couples deli e ed babies (con i med li e bi h a e o 19.2%)(Table S2A in File S1). High Semen SPTRX3 in In e ile Couples PLOS ONE | www.plosone.o g 2 May 2013 | Volume 8 | Issue 5 | e61000 Lowes a e age SPTRX3 le els we e ound in couples ha deli e ed wins (n = 12; Figu e 2E; Table S2B in File S1), compa ed o hose deli e ing a single on (n = 34) and hose ha misca ied (n = 10). In addi ion o p egnancy and li e bi h a es, pa ame e s o emb yo de elopmen and numbe s o oocy es and emb yos p oduced pe couple we e analyzed. Nume ically, couples in which men had less han 5% o SPTRX3 posi i e spe ma ozoa p oduced mos no mal wo-p onuclea zygo es and ans e able emb yos (Table S3 in File S1). Howe e , no signi ican co ela ions we e ound be ween pa ame e s o ea ly emb yo de elopmen and low cy ome ic SPTRX3 le els. Flow Cy ome ic SPTRX3 Le els Re lec Clinicians’ T ea men Decision, and Re eal Undiagnosed Male In e ili y in Idiopa hic In e ili y Cases The SPTRX3 le els in all 239 couples we e compa ed based on p egnancy ou come and clinical indica ion (Figu e 2D). Com- pa ed o emale in e ili y couples, he a e age %M3 alue o SPTRX3 in diagnosed male in e ili y cases was inc eased by 63%. Ele a ed SPTRX3 le els (.15% M3) coincided wi h he clinician’s decision o ea by ICSI. Consequen ly, logis ic eg ession and disc iminan unc ion analysis we e used o de e mine he ela ionship be ween ea men assignmen (ICSI o IVF) and semen SPTRX3 le els (Table S4 in File S1; Supplemen al S a is ics, sec ion ANALYSIS II & Table S12, S13 in File S1). Among 16 low-cy ome ic pa ame e s, only median M2, Mean M2 and %M1 we e no signi ican o p edic ing he ea men assignmen . Mean M1 and Median M1 had he la ges in luence on he odds o being assigned o IVF ea men . The odds o being assigned o IVF ea men inc eased by o e 34 old and 4 old o e e y uni o inc ease in Mean M1 and Median M1, espec i ely. Howe e , we no iced ha he anges o Mean M1 and Median M1 we e small ( hei alues a ied be ween 1 and 4). The nex h ee mos in luen ial p edic o s we e CV M1, %M2 and %M3. Fo e e y uni inc ease in CV M1 and %M3, he odds o being assigned o FIV ea men dec eased by 16.3% and 9.6%, espec i ely. Fo e e y uni inc eases in %M2, he odds inc eased by 14.1%. Disc iminan analysis iden i ied %M2 SPTRX3 as a posi i e disc iminan o IVF assignmen and Mean M3, Median M3 and % M3 SPTRX3 as posi i e disc iminan s o ICSI assignmen . Thus, inc eased SPTRX3 con en in spe ma ozoa can be used as an indica ion o ICSI ea men . Among he six con en ional semen pa ame e s, only spe m olume was no signi ican p edic o o ea men assignmen (Table S4 in File S1). Mo ili y pa ame e s, PR and M ga e he mos explana o y powe s wi h odds a io o 1.176 and 1.142 (i.e. he odds o being assigned o IFV ea men inc eases 17.6% and 14.2% o e e y uni inc ease in PR and Mob measu emen s, espec i ely). Flow Cy ome ic SPTRX3 Values Re lec Spe m SPTRX3 Re en ion Pheno ypes Obse ed by Epi luo escence Mic oscopy Immuno luo escence p ocessing and subsequen analysis o a subse o 150 samples by epi luo escence mic oscopy iden i ied a ied pheno ypes o SPTRX3- e en ion by de ec i e spe ma o- zoa, anging om spe ma ozoa bea ing a small do o SPTRX3 (Figu e 3A, 3E) o he highly SPTRX3-posi i e spe ma ozoa (Figu e 3D, 3G) and SPTRX3-con aining cellula deb is (Figu e 3F), eadily ob ious e en a low magni ica ion (compa e Figu e 1. Examples o low cy ome ic his og ams (A, B) and co esponding ligh sca e diag ams (A’,B’) o wo male pa ien s wi h a ied SPTRX 3 le els. His og ams, ep esen a i e o SPTRX3 luo escene le els in 10,000 cells/sample a e di ided in o ma ke a eas M1 (low luo escen spe ma ozoa, spe m agmen s and cellula deb is), M2 (mos ly in ac spe ma ozoa wi h backg ound-le el luo escence) and M3 (de ec i e, SPTRX3-posi i e spe ma ozoa and imma u e spe m o ms wi h high luo escence). Pe cen age o SPTRX3-posi i e spe ma ozoa ou o o al spe m con en , including ma ke s M1, M2, and M3 is shown. Ga e R1 in sca e diag ams o isible ligh sepa a es spe ma ozoa (each do is one cell) om o he low cy ome ic e en s; esul an pe cen age is pe cen o high/low luo escen spe ma ozoa om o al numbe o low cy ome ic e en s measu ed pe sample in o de o acqui e 10,000 measu emen s. Sample B was dona ed by a pa ien p e iously diagnosed wi h male in e ili y (oligo-as heno- e a ozoospe mia; 5 million spe m/ml, 30% mo ili y, 22% no mal mo phology). Fla his og am-cu e and a seconda y peak wi hin ma ke a ea M3 coincide wi h 25.9% o SPTRX3–posi i e spe ma ozoa in his sample. Sample A was dona ed by a p esumably e ile man wi h accep able clinical semen pa ame e s (80 million/ml, 50% mo ili y, 74% no mal mo phology) whose emale pa ne su e ed om endome iosis. doi:10.1371/jou nal.pone.0061000.g001 High Semen SPTRX3 in In e ile Couples PLOS ONE | www.plosone.o g 3 May 2013 | Volume 8 | Issue 5 | e61000 Figu e 2. Diag ams o a e age SPTRX3 con en in spe ma ozoa o men om 239 in e ile couples. (A) A e age p egnancy a es a e in e ili y ea men (IVF o ICSI) dec eased g adually wi h he inc eased spe m SPTRX3-con en in 239 couples di ided by % o SPTRX3 spe ma ozoa ( low cy ome ic %M3 alue; x-axis) in o subg oups wi h less han 5%, 5–9.99%, 10–14.99%, and mo e han 15% o SPTRX3-posi i e spe ma ozoa. Compa ed o men wi h less han 5% SPTRX3-posi i e spe ma ozoa, hose ca ying mo e han 15% o such cells had hei chance o concei ing educed by a hal (41.2% p egnan , s. 19.2% p egnan ; one-sided p,0.05). (B) Low le els o spe m SPTRX3 we e ound in emale and idiopa hic High Semen SPTRX3 in In e ile Couples PLOS ONE | www.plosone.o g 4 May 2013 | Volume 8 | Issue 5 | e61000 Figu e 3C o Figu e 3B). Simila labeling was ob ained in spe ma ozoa immunolabeled wi hou ixa ion (Figu e 3H). Some SPTRX3 posi i e spe ma ozoa pe sis ed in semen samples pu i ied by g adien cen i uga ion and swim-up (Figu e 3 I–K). Non-spe m elemen s/bodies p esen in he analyzed samples did no display de ec able SPTRX3 luo escence. The epi luo escence mic oscopy-analysis o SPTRX3 co ela ed wi h low cy ome ic e alua ion o his subse o 150 pa ien s and was e lec i e o p egnancy a es (Figu e S3 in File S1; Table S5A, S5B in File S1). E ec o High SPTRX3 Con en on P egnancy Ra es a e Adjus men o Pa ne s’ Age Female pa ien s abo e 35 yea s o age end o p oduce ewe oocy es a e supe o ula ion, and hese oocy es a e pe cei ed o be o lesse quali y and de elopmen al po en ial. I was hus use ul o assess whe he high semen con en o SPTRX3 in male pa ne s o emale pa ien s 35 yea s o olde could u he agg a a e couples’ in e ili y. When he couples we e di ided based on emale age and male con en o SPTRX3 (Table S6 in File S1), and by age and indica ion (Table S7 in File S1), he e was a non-signi ican end owa ds highe p egnancy a es in low SPTRX3 couples in e ili y-couples, compa ed o male I an combined male & emale in e ili y-couples. Di e en supe sc ip s ma k s a is ically signi ican di e ences be ween columns a p,0.001. The mos signi ican di e ence was obse ed be ween emale and male in e ili y couples (p,0.001). (C) Couples ea ed by IVF had signi ican ly lowe a e age SPTRX3-le els han hose ea ed by ICSI, ega dless o whe he o no hey achie ed a p egnancy. Supe sc ip s a, b ma k s a is ical di e ence a p,0.05. The mos signi ican di e ence was ound be ween p egnan IVF couples and p egnan ICSI couples (p,0.001). (D) Couples wi h emale and idiopa hic in e ili y had signi ican ly lowe SPTRX3 le els (a e age %M3- alues) han couples p e iously diagnosed wi h male o combined (male and emale) in e ili y. (E) Dis ibu ion o SPTRX3 le els wi hin cu o s in pa ien s di ided based on p egnancy ou come. No e ha he couples deli e ing wins had he highes pe cen age o men wi h %M3 SPTRX below 15% and lowes e pe cen age o men wi h .15% M3 SPTRX3. doi:10.1371/jou nal.pone.0061000.g002 Figu e 3. Epi luo escence mic oscopy o spe m samples p ocessed o low cy ome ic measu emen s o SPTRX3 ( ed). The DNA-s ain DAPI (blue) was added a e low cy ome ic analysis. Epi luo escence images a e supe imposed on o pa ocal di e en ial in e e ence con as (DIC) images (g ay). (A) Examples o SPTRX3 labeling in samples om male in e ili y pa ien s. A owheads poin o mino ca yo e s o esidual cy oplasm in spe ma ozoa and imma u e spe m o ms; some o hese cy oplasmic s uc u es may no be de ec able by con en ional ansmi ed ligh mic oscopy, bu can be iden i ied accu a ely by SPTRX3 labeling. (B, C) The di e ence in SPTRX3 con en is ob ious e en a low magni ica ion be ween a sample om a p esumed e ile dono (B) and he one o a pa ien p e iously diagnosed wi h oligo-as heno- e a ozoospe mia (C). (D–G) High magni ica ion ende ings o a ious pa e ns o SPTRX3 e en ion in de ec i e spe ma ozoa, including a p edominan spe m head/nuclea labeling (D), mino SPTRX3 e en ion in he spe m head (E; a ows) and spe m ail connec ing piece (E; a owhead), spe m-de i ed cellula deb is ee o DNA/ch oma in (F), imma u e, spe ma id-like o m (G; op), and SPTRX3- illed la ge nuclea acuole (G; bo om). (H) Immunolabeling o SPTRX3 in a ozen- hawed semen sample labeled acco ding o a simpli ied p o ocol omi ing spe m ixa ion and washing. (I–K) De ec ion o SPTRX3 be o e (I) and a e semen pu i ica ion on Pu eSpe m g adien (J), and in a sample pu i ied by swim-up a e Pu eSpe m ea men (K). De ec i e spe ma ozoa a e no emo ed comple ely by his wo-s ep p ocedu e. Scale ba s a e 10 mm in all panels excep B, C, whe e hey ep esen 20 mm. doi:10.1371/jou nal.pone.0061000.g003 High Semen SPTRX3 in In e ile Couples PLOS ONE | www.plosone.o g 5 May 2013 | Volume 8 | Issue 5 | e61000 ega dless o emale age. Male age i sel did no show co ela ion wi h p egnancy a es, SPTRX3 le els o con en ional semen pa ame e s. The e was a mode a e- o-high co ela ion be ween low cy ome ic da a and se e al pa ame e s o con en ional, clinical semen analysis (Table S8 in File S1). Nex , he logis ic eg ession analysis was applied o es he ela ionship o p egnancy a e wi h he spe m quali y pa ame e s, aking in o conside a ion o emale age and ea men assignmen s (Supplemen al S a is ics – ANALYSIS III in File S1). Conside ing only he pa ien s assigned o IVF ea men , bo h %M2 and %M3 SPTRX3 we e signi ican p edic o s o p egnancy a e, wi h o wi hou adjus men o he emale age ac o . Impo an ly, none o he con en ional semen pa ame e s, o o he emaining 14 low cy ome ic SPTRX3 pa ame e s, was signi ican e en wi h adjus men o he emale age. The odds o ge ing p egnan in he IVF g oup we e signi ican ly educed by inc eased %M3 alue, wi h (p,0.05) o wi hou (p,0.05) adjus men o ma e nal age. The %M2 was also a signi ican p edic o o p egnancy a e o IVF g oup (p,0.02/p,0.05 wi h/wi hou age adjus men ). Fo he ICSI g oup, he p egnancy a e was no a ec ed by any o he low cy ome ic o con en ional spe m quali y pa ame e s o he emale age ac o s. This is no su p ising since ICSI can o e come a wide ange o spe m anomalies ha would hinde con en ional IVF ea men . When conside ing all pa ien s oge he , %M2 and %M3 SPTRX3 we e no signi ican p edic o s o p egnancy a e (p.0.1). Howe e , when we ca ego ized he %M3 le els in o ou g oups (below 5%, be ween 5–10%, be ween 10–15%, abo e 15%), he es ima ed odds a io be ween he below 5% g oup and he abo e 15% g oup had a p- alue = 0.06. A e adjus ing o emale age abo e/below 35 yea s, he adjus ed odds a io o he ,5% SPTRX3 g oup o e he .15% SPTRX3 g oup had a p,0.05. Using 85% as a cu o o %M2 (no mal spe ma ozoa), he below 85% g oup and abo e 85% g oup had signi ican ly di e en p egnancy a es when conside ing all pa ien s oge he . The odds o ge ing p egnan o he .85% %M2 g oup we e signi ican ly highe han ha o he ,85% g oup (p,0.05) and he p egnancy a e was 41.6% e sus 22.72%. A e he adjus men o he emale age abo e 35, he odds a io emained signi ican (p,0.05). Rela ionship be ween Flow Cy ome ic SPTRX3 Pa ame e s, Clinical Semen Pa ame e s and Clinical Diagnosis o Male In e ili y This analysis was pe o med using logis ic eg ession analysis (SAS p oc log) o SPTRX3 pa ame e s and con en ional semen pa ame e s including spe m concen a ion (C), semen olume (V), % no mal spe ma ozoa (NX), p og essi e mo ili y (PR), % mo ile (M) and % nec o ic (NEC)(Table 1). All pa ame e s excep V we e signi ican p edic o s o he clinical diagnosis o male in e ili y (Table 2). NX, PR, M and C we e nega i e p edic o s o male in e ili y. Among 16 low cy ome ic pa ame e s (Table 2), Mean M1, Median M1, CV M1, %M2 and %M3 we e he mos sensi i e p edic o s o male in e ili y. Disc iminan unc ion analysis iden i ied PR, M, NX, C, %To al and %M2 SPTRX3 as he disc iminan s co ela ed posi i ely wi h spe m quali y (Supplemen al S a is ics – Sec ion ANALYSIS IV, Table S14, S15 in File S1). Nega i e disc iminan s included %M3, Mean M3 and Median M3 SPTRX3. Analysis based on e iology/ es iculopa hy (Table 3) e ealed highes le els o SPTRX3 in men om male in e ili y couples (n = 72) diagnosed wi h oligo-as henozoospe mia. Couples wi h as heno- e a ospe - mia had he lowes SPTRX3 and highes p egnancy a es. Al oge he , 97.2% o male-only in e ili y couples we e ea ed by ICSI. Spea man ank co ela ion was used o con i m he ela ionship be ween spe m SPTRX3 alues and con en ional semen pa ame e s. Fi e mos in o ma i e low cy ome ic SPTRX3 pa ame e s (Mean M1, Median M1, CV M1, %M2, %M3) we e signi ican ly co ela ed wi h con en ional semen pa ame e s, pa icula ly wi h mo ili y pa ame e s M and PR (Table S9 in File S1). The %M3 pa ame e , cap u ing cells wi h high SPTRX3 con en , also co ela ed nega i ely wi h % no mal spe ma ozoa (NX) and spe m coun (C), and posi i ely wi h % nec o ic spe ma ozoa (NEC). Discussion P esen s a is ical analyses indica e ha high semen con en o SPTRX3 in in e ile men ansla es in o a educed chance o imp egna ing hei emale pa ne s ia ART. In ac , couples’ p egnancy a es we e signi ican ly lowe o men wi h .15% SPTRX3 spe m compa ed o men wi h ,5% SPTRX3 cells. Since he ea men decision/assignmen in he examined 239 cases was in pa based on semen analysis, i is no su p ise ha men om IVF couples had signi ican ly lowe a e age SPTRX3 le els han men om couples ha we e ea ed by ICSI. Tha he inc eased le els o SPTRX3-posi i e spe ma ozoa p edispose couples o ICSI ea men is ob ious in spi e o he ac ha se e al couples we e ini ially ea ed by IVF, wi hou success, be o e he ICSI ea men was applied. Al oge he , p egnancy a es ell p og essi ely wi h inc easing semen SPTRX3 le els. When b oken down in o ICSI and IVF g oups, couples di ided by %M3 SPTRX3 showed high p egnancy a es in ICSI g oup wi h ,5 %M3. On a e age, ICSI and male in e ili y cases had signi ican ly highe %M3 alues bu also sligh ly highe p egnancy a es han IVF and emale-only in e ili y cases. This is unde - s andable because mos o hese cases we e oligo-as heno- e a ozoospe mic and he e o e ea ed by ICSI. The IVF pa ien s had o e whelmingly emale-only in e ili y, wi h poo oocy e quali y being he likely con ibu o o a high incidence o ailed IVF ea men (75%). Howe e , his could also ha e been compounded by undiagnosed male in e ili y, indica ed by high SPTRX3 le els in 20% o hose p esumed emale-only in e ili y couples. Consequen ly, he pe cen age o ma u e oocy es ha o med wo p onuclei a e assis ed e iliza ion dec eased in a s epwise manne , in pa allel wi h inc easing %M3 SPTRX3 in bo h he IVF- ea ed and ICSI- ea ed couples. Al oge he , high spe m SPTRX3 le els seem o coincide wi h clinical decision o ea by ICSI. The SPTRX3-le els co obo a e p e ious clinical diagnosis o male in e ili y, and e eal undiagnosed male in e ili y in a ac ion o cases wi h idiopa hic and emale-only in e ili y. While he majo i y o he 239 couples in his s udy we e assigned o IVF o ICSI based on hei clinical es esul s, ou SPTRX3 esul s indica e ha app oxima ely 20% o IVF couples would ha e p o i ed om ICSI because o undiagnosed male in e ili y. Table S3 in File S1 shows ha spe m SPTRX3-le els bu no couples’ clinical diagnosis, in pa based on subjec i e semen analysis, coincided wi h success ul e iliza ion and o ma- ion o wo no mal p onuclei in he esul an zygo es. While couples wi h ,5 %M3 alues o SPTRX3 p oduced 2-PN zygo es in 86.2% o e ilized oocy es, idiopa hic couples wi h .15 %M3 did so in only 69.7% o oocy es (see Table S3A in File S1; column 9). No such di e ences we e obse ed based on clinical diagnosis/indica ion (Table S3B in File S1). The e was no signi ican di e ence in %M3 alues o SPTRX3 be ween emale and idiopa hic cases. Howe e , 20% o he idiopa hic cases showed high SPTRX3 alues (.15% M3), indica ing ha hese High Semen SPTRX3 in In e ile Couples PLOS ONE | www.plosone.o g 6 May 2013 | Volume 8 | Issue 5 | e61000 Table 1. A e age clinical semen pa ame e s in 239 male pa ien s di ided by %M3 SPTRX3 and p egnancy. %M3 SPTRX3 N % p egnan Semen olume Spe m concen a ion % dead spe ma ozoa % mo ile spe ma ozoa % p og essi ely mo ile spe ma ozoa % no mal spe ma ozoa , 5% 16 41.2% Mean 2.4 95.2 22 49 28 48 SE 0.3 21.3 2 2 3 4 5–9.99% 57 29.8% Mean 3.2 83.6 27 46 27 46 SE 0.2 7.1 1 1 1 2 10–14.99% 88 23.9% Mean 3.3 71.1 28 42 24 42 SE 0.2 5.0 1 1 1 2 . 15% 78 19.2% Mean 3.3 40.6 33 33 16 35 SE 0.2 5.2 2 2 1 2 . 15% idiopa hic 12 16.7% Mean 3.2 64.0 30 44 24 45 SE 0.3 18.4 4 2 2 4 P egnan 60 100% Mean 3.2 65.8 29 38 20 38 SE 0.2 7.5 1 2 1 2 Non-p egnan 179 0% Mean 3.2 65.8 29 41 23 43 SE 0.1 4.0 1 1 1 1 The only g oup ha shows somewha educed a e age clinical semen pa ame e s a e he men wi h highes SPTRX3 le els (.15% M3). Idiopa hic in e ili y pa ien s wi h appa en ly high SPTRX3 le el (.15% idiopa hic; bo om ow) shows accep able, no mal clinical semen pa ame e s. Da id’s classi ica ion scheme o spe m mo phology was employed [32,33]. SE = s anda d e o . doi:10.1371/jou nal.pone.0061000. 001 High Semen SPTRX3 in In e ile Couples PLOS ONE | www.plosone.o g 7 May 2013 | Volume 8 | Issue 5 | e61000 we e in ac male in e ili y cases. Simila ly, 20% o p esumed e ile males in couples diagnosed wi h emale-only in e ili y also had .15% SPTRX3-posi i e spe ma ozoa. Toge he wi h he e ec o SPTRX3 on p egnancy a es, hese da a illus a e he p edic i e alue o SPTRX3-based semen analysis o ea men ou come. Table 2 and Table S8 in File S1 e eal ela ionship be ween clinically diagnosed male in e ili y and SPTRX3 pa ame e s. Fu he mo e, Table 1 shows ha men wi h high SPTRX3 le els show accep able clinical semen pa ame e s, bu low p egnancy a es a e achie ed by hei emale pa ne s. As shown in Table S8 in File S1, he le els o co ela ion ( - alues) be ween low cy ome ic pa ame e s (%M3; %TOTAL, GeoMean M3) and con en ional semen pa ame e s a e a ied, depending on how he pa ien pool was subdi ided based on indica ion, ea men decision and ea men ou come. All h ee low cy ome ic pa ame e s show median le el o co ela ion wi h some clinical semen pa ame e s in some subg oup bu no in o he s. This indica es ha spe m SPTRX3 alues a e ela i ely independen o con en ional semen pa ame e s. The low cy ome ic pa ame e mos closely co ela ed wi h clinical semen pa ame e s is he %TOTAL, which e lec s he pe cen age o low cy ome e - egis e ed e en s co esponding o in ac spe ma ozoa (bo h mo phologically no mal and abno mal) ou o all objec s passing h ough a low cy ome e , including spe m agmen s and deb is p esen in semen sample. This is based on isible ligh sca e o egis e ed e en s a he han on SPTRX3-induced luo escence. The p oposi ion o a spe m quali y es ha is ela i ely independen o con en ional semen pa ame e s bu p edic i e o p egnancy ou come a i es a a ime o enewed calls o mo e accu a e semen analysis. Some ecen s udies e en go as a as implying ha e en he s ic es , mos accu a e mo phological c i e ia could lead o diagnosis o e a ozoospe mia ha o ces unnecessa y ICSI ea men in couples ea able by IVF [29]. No single bioma ke is a uni e sal indica o o male in e ili y and ea men ou come; howe e , he SPTRX3-based assay could be combined wi h o he immunocy ochemical and cy ochemical es s such as he ubiqui in-based spe m quali y assay [16] o a es o spe m ac osomal s a us based on lec in labeling [7]. SPTRX3 is a unique ma ke because i is a well cha ac e ized, ge mline- speci ic p o ein wi h a dis inc localiza ion pa e n in spe ma ozoa, and he e is no need o measu e i s ela i e abundance in indi idual cells o decide which cells a e ‘‘posi i e’’ and which a e ‘‘nega i e.’’ Mos no mal spe ma ozoa do no con ain amoun s o SPTRX3 de ec able by immuno luo escence. Simply coun ing posi i e and nega i e spe ma ozoa is s aigh - o wa d and, i desi ed, such a es gi es numbe s ha could be used o se up clea cu o s o no mal and in e ile males. Howe e , h esholds will be di e en o manual, epi luo escence mic oscopy based assess- men s as opposed o au oma ed low cy ome ic measu emen . Disc iminan unc ion analysis o low cy ome ic da a and clinical diagnosis showed ha he mean % o SPTRX3-pos i i e spe ma ozoa (%M3) was o 11.6% in p esumed e ile men s. 18.6% in he clinically diagnosed in e ile men (Supplemen al S a is ics – ANALYSIS IV in File S1). The cu o alues o 12.5 and 15% we e ound o ha e a good balance o sensi i i y and speci ici y in ROC analysis (Table S1 in File S1). A 10% cu o alue had sensi i i y close o 90%, bu his was a he expense o speci ici y. The h eshold/cu o le els o an in e ile male could also di e be ween IVF pa ien s (e.g. 7.5 o 10% posi i e) and ICSI pa ien s (e.g.15 o 17.5%). Thus, SPTRX3-based es could be used as a decision making ool when physicians ha e o decide whe he o use in au e ine insemina ion, IVF, o ICSI. Fo example, a ea men wi h in au e ine insemina ion could be used in cases when a male pa ne ’s SPTRX3 le els a e below 10% posi i e spe ma ozoa, IVF o men wi h be ween 10–15% SPTRX3 posi i e spe ma ozoa, and ICSI o men wi h .15% posi i e spe ma ozoa. Simila o low cy ome ic SPTRX3 analysis, he simpli ied, ligh mic oscopic analysis e ealed highe p egnancy a es in couples wi h low SPTRX3 le els (Table S6 in File S1). While he mo e sensi i e low cy ome ic analysis would be p e e able, he simpli ied ligh mic oscopic es could become a aluable ool o clinical semen e alua ion whe e e low cy ome y is no con enien o a ailable. This could be done ei he by epi luo es- cence mic oscopy (di ec conjuga ion o a luo escen p obe o an i-SPTRX3 an ibody), o by simple ansmi ed ligh mic osco- py using an i-SPTRX3 an ibody conjuga ed o ho se adish pe oxidase and de eloped by applying alkaline phospha ase o DAB. To u he acili a e sample s aining, spe m sp eads would be ixed by me hanol and d ied di ec ly on he mic oscopy slides. E en so, de elopmen o simpli ied p o ocols o low cy ome ic semen analysis is necessa y. As shown in Fig. 3 H, spe ma ozoa can be labeled wi h an ibodies wi hou p e ious ixa ion, i.e. by a Table 2. Rela ionship be ween he clinically diagnosed male in e ili y and indi idual, con en ional and low cy ome ic SPTRX3 pa ame e s e lec i e o spe m quali y. Pa ame e p- alue Odds a io* V 0.9833 NA PR ,0.0001 0.677 NX ,0.0001 0.903 NEC ,0.0001 1.07 M,0.0001 0.772 C,0.0001 0.948 Median ALL 0.0007 1.055 Median M3 ,0.0001 1.028 Median M2 0.0176 1.074 Median M1 ,0.0001 0.08 Mean all ,0.0001 1.055 Mean M3 ,0.0001 1.022 Mean M2 0.0249 1.084 Mean M1 ,0.0001 0.014 To al ,0.0001 0.915 %M3 ,0.0001 1.12 %M2 ,0.0001 0.869 %M1 0.8212 NA CVALL 0.0005 1.007 CVM3 0.0131 1.012 CVM2 0.3455 NA CVM1 ,0.0001 1.241 An odds a io smalle han 1 indica es ha he pa ame e is nega i ely associa ed wi h he odds o being diagnosed wi h male in e ili y. A la ge han 1 odds a io indica es a posi i e ela ionship be ween he pa ame e and he clinical diagnosis o male in e ili y. An odds a io la ge han 1, e.g. 1.12 is in e p e ed as ‘‘ he odds o being diagnosed wi h male in e ili y inc ease by 12% o e e y uni o inc ease in he pa ame e measu emen s.’’ An odds a io smalle han 1, e.g., 0.014, is in e p e ed as ‘‘ he odds o being diagnosed wi h male in e ili y dec ease by 98.6% o e e y uni o inc ease in he measu ed pa ame e .’’ Abb e ia ions: C = spe m coun ; V = semen olume; M = o al mo ili y; PR = p og essi e mo ili y; NX = mo phology/% no mal spe ma ozoa; NEC = pe cen o nec o ic spe ma ozoa assessed by Williams es . doi:10.1371/jou nal.pone.0061000. 002 High Semen SPTRX3 in In e ile Couples PLOS ONE | www.plosone.o g 8 May 2013 | Volume 8 | Issue 5 | e61000 mild pe mebiliza ion and immunolabeling. Pe mebiliza ion kills spe ma ozoa bu in his case, i canno be omi ed because he SPTRX3 an igen is concealed unde he plasma memb ane o he memb ane-in ac spe ma ozoa. This p o ocol will add ess he majo disad an age o SPTRX3 es compa ed o o he low cy ome y based spe m es such as SCSI o TUNEL, i.e. he leng hy, mul i-s ep p ocessing. This app oach would bene i he new gene a ion o dedica ed spe m analysis low cy ome e s such as Easy Cy e Plus, a capilla y low-based de ice wi h p e-se spe m es s, used o analyze small olumes o spe m suspensions [30]. The SPTRX3 based es may be a aluable addi ion o he a o emen ioned low cy ome ic es s. Ou o i e male in e ili y pa ien s es ed by SCSI in he p esen coho , none had he DNA agmen a ion index (DFI) abo e he h eshold le el o 20% (Mean DFI = 7.862.2%; ange 2–14%). Howe e , he a e age %M3 SPTRX3 o hese i e pa ien s was 25.167.0% ( ange 7.8- 50.0%), compa ed o 18.761.2 %M3 o he en i e coho o male in e ili y pa ien s. Fu u e ials will be designed o examine he ela ionship be ween SPTRX3 e en ion and abe an spe m ch oma in s uc u e. The SPTRX3-posi i e spe ma ozoa mos likely co espond o spe ma ozoa commonly e e ed o as ‘‘imma u e spe m’’ [31]. Supe luous cy oplasm can be e y conspicuous in some o hese spe ma ozoa, bu di icul o de ec by con en ional ligh mic oscopy in o he s, e en wi h high magni ica ion lenses and DIC con as op ics. The use o SPTRX3 as a bioma ke could allow clinicians o iden i y de ec i e spe ma ozoa du ing semen analysis ha would be unde ec able o con en ional semen e alua ion. The SPTRX3 based me hod is obus , and as demons a ed he e, capable o de ec ing spe m de ec s in men om couples diagnosed wi h idiopa hic in e ili y. Al oge he , he p esen s udy documen s he ela ionship be ween SPTRX3 le els and p egnancy a es a e ART, and he e ec o SPTRX3 on quali y o zygo es (numbe o p onuclei); i also iden i ied di e ences in semen con en o SPTRX3 and pheno ypes o i s e en ion be ween male pa ien s om couples ea ed by ICSI and hose ea ed by con en ional IVF. Consequen ly, i may be possible, and necessa y o se he h eshold le els o no mal/ ele a ed semen SPTRX3 con en di e en ly o hese wo coho s o in e ili y pa ien s. Al oge he , SPTRX3 p o ein is a candida e bioma ke o spe m dys unc ion ha is ela i ely independen o clinical semen pa ame e s, and ele an o decision making in ART. Ma e ials and Me hods Semen Samples and S udy Design Semen samples we e dona ed by men om 239 consen ing couples, ee o HIV and any o he de ec able con agious diseases, p esen ing o in e ili y ea men a he Assis ed Fe iliza ion Medical Cen e , Clinique Monplaisi , Lyon, F ance. Semen was collec ed on he day o IVF/ICSI and he le o e was sample c yop ese ed wi hin wo a e collec ion hou s. Iden i ies o dono s we e no disclosed o esea che s in ol ed in his s udy. All couples also consen ed o sha ing o diagnos ic da a and ea men ou come-da a. Couples we e classi ied and assigned as ollows: Female in e ili y included hyd osalpinx, endome iosis and polycys ic o a y synd ome (PCOS); hese couples we e assigned o IVF since hei spe m pa ame e s we e wi hin accep able limi s by WHO c i e ia. This g oup also included o a ian insu iciency e ealed by ul asonog aphic baseline an al ollicle coun , associa ed wi h o he pa hologies; such cases we e ea ed by ICSI. Male and combined male and emale in e ili y was diagnosed based on semen analysis, as desc ibed below. Clinical indica ions o male in e ili y, as de ined by WHO, included as henozoospe mia (A), as heno- e a ozoospe mia (AT), oligo- as henozoospe mia (OA) and oligo-as heno- e a ozoospe mia (OAT). Cases o obs uc i e azoospe mia (AZ), obs uc i e OAT, e a ozoospe mia, obs uc i e OT, azoospe mia due o asec omy, a icocele, and an i-spe m an ibodies we e p esen . In asec omy cases and cases o obs uc i e OAT due o in lamma- ion, spe ma ozoa o ICSI we e collec ed om es is. Fi e male in e ili y pa ien s we e es ed o ch oma in s uc u e/DNA agmen a ion by SCSI a a comme cial labo a o y, bu all i e had accep able DNA- agmen a ion index (,20% DFI). Gene - ally, IVF was used in cases wi h accep able p og essi e mo ili y, while cases o OAT we e ea ed by ICSI. Idiopa hic in e ili y (no clinical symp oms o pa hologies, and no p e ious his o y o and ological p oblems in male pa ne s) we e ea ed by ICSI i hey epo ed p e ious ailu es wi h in au e ine insemina ion (IUI). I he e was no his o y o IUI, couples we e assigned o IVF Table 3. SPTRX3 le els and ART ou comes in 72 male in e ili y pa ien s di ided by e iology. Ae hiology A AT OA OAT AZ* O he ** AVG/To al Mean %M3 SPTRX3 18.4 14.8 28.2 18.4 25.6 17.6 18.7 SE 2.6 2.5 7.2 1.6 8.4 3.9 1.2 N 1494333972 P egnan / o al (%) 2/14 (14%) 4/9 (44%) 0/4 (0%) 12/33 (36%) 3/3 (100%) 2/9 (22%) 23/72 (32%) ICSI- ea ed/ o al (%) 12/14 (86%) 9/9 *** (100%) 4/4 (100%) 33/33 (100%) 3/3 (100%) 9/9 (100%) 70/72 (97%) Mos equen e iologies included as henozoospe mia (A), as heno- e a ozoospe mia (AT), oligo-as henozoospe mia (OA), and oligo-as heno- e a ozoospe mia (OAT). One pa ien had obs uc i e azoospe mia and one had obs uc i e oligo- e a ozoospe mia. O he , less equen e iologies included e a ozoospe mia (2 pa ien s), obs uc i e (1), asec omy (1), a icocele (1), an i-spe m an ibodies (2) and as henozoospe mia wi h an i-spe m an ibodies (1). One couple epo ed ailed IVF be o e being ea ed by ICSI. Couples diagnosed wi h combined male & emale, emale-only o idiopa hic in e ili y excluded om his analysis/ able. A = as henozoospe mia. AT = as heno- e a ozoospe mia. OA = oligo-as henozoospe mia. OAT = oligo-as heno- e a ozoospe mia. *Obs uc i e. **O he = e a ozoospe mia (2 pa ien s), oligo- e a ozoospe mia, obs uc i e (1), asec omy (1), a icocele (1), an i-spe m an ibodies (2), as henozoospe mia wi h an i- spe m an ibodies (1). ***One couple epo ed ailed IVF be o e being ea ed by ICSI. doi:10.1371/jou nal.pone.0061000. 003 High Semen SPTRX3 in In e ile Couples PLOS ONE | www.plosone.o g 9 May 2013 | Volume 8 | Issue 5 | e61000