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Fertilization potential of spermatozoa with abnormal morphology
Among the anomalies affecting sperm organelles, head defects are especially geneous and occasionally the defect does not affect 100 of the spermatozoa
What constitutes a normal seminal analysis? Semen parameters of
Normal sperm morphology is but one of many parameters for assessment of fertility Social alcohol consumption, cigarette smoking, and 'recent fever' did not
[PDF] Sperm pathology: a step beyond descriptive morphology Origin
shapes, which, with the exception of acrosome anomalies, do not atozoa adversely affects sperm morphology, motility, mitochon-
[PDF] Sperm Morphologic Characteristics and Their Impact on Embryo
Determination of normal sperm morphology is shows that sperm morphology does not in fl uence tion in the oocyte by ICSI does not affect the fer-
78220_714_suppl_1_47.pdf Human Reproduction, Vol. 14, (Suppl. 1), pp. 47-70, 1999 Fertilizatio
n potentia l of spermatozoa with abnorma l morpholog yN.Nikolettos
, W.Kiipker , C.Demirel , B.Schopper , C.Blasig ,R.Sturm
, R.Felberbaum , O.Bauer , K.Diedric h an d S.Al-Hasani 1Departmen
t of Obstetrics/Gynecology, Medical University Liibeck,Ratzeburge
r Alle e 160, 23538, Liibeck , German y 'T o who m correspondenc e shoul d be addressed On e of the best discriminators for the fertilization potential of human spermatozo a i s sperm morphology. The problem in the assessment of the sper m morphologica l characteristic s is their pleiomorphism. Examination of spermatozo a wit h the light microscope can provide only limited information o n thei r interna l structure . Mor e detaile d examinatio n o f sperm structure usin g electro n microscop y ca n revea l major , ofte n unsuspecte d ultrastructura l abnormalities . Result s an d cut-off values for sperm analysis depend on the criteri a for normal morphology. World Health Organizatio n recommenda - tion s provid e a classification suitable for clinical practice. Clinically reliable cut-of f limit s fo r normal sperm morphology according to strict Tygerberg criteri a wer e suggeste d t o b e 4% in in-vitro fertilization procedures. Patients wit h sever e sper m hea d abnormalitie s hav e a lower chance of establishin g successfu l pregnancies , eve n thoug h fertilizatio n may be achieved. The outcom e of intracytoplasmic sperm injection is not related to any of the standar d seme n parameter s or to sperm morphology. Sperm decondensation defect s an d DNA anomalies may be underlying factors for the unrecognize d derangement s of the fertilizing capacity of spermatozoa, regardless of sperm morphology . Centrosom e dysfunctio n ma y also represent a class of sperm defect s tha t canno t b e overcome simply by the insertion of a spermatozoon int o th e ooplasm. In this article a n overview on the composition and ultrastructur e of spermatozoa is presented, while emphasizing sperm ultra- structura l an d sperm DNA anomalies and their effects on fertilization. Key words: abnormal/fertilization/morphology/spermatozoa
Introductio
n A comprehensive high quality semen analysis is an essential first line investigatio n fo r the infertile couple. Semen quality is conventionally determined according t o th e number, motility and morphology of spermatozoa in an ejaculate. Of all © Europea n Societ y fo r Huma n Reproductio n an d Embryolog y 47Downloaded from https://academic.oup.com/humrep/article/14/suppl_1/47/635758 by guest on 15 August 2023
N.Nikolettos et al.
seme n parameters , sper m morpholog y turn s ou t t o b e th e bes t predicto r o f a man' s fertilizin g potentia l (Bostoft e et al, 1982; Rogers et al, 1983
; Kruge r et al, 1986
, 1988
; Cha n et al, 1989
; Engins u et al, 1991
; Ombele t et al,
1994,1997a
; Wichman n et al, 1994; Eggert-Krus e et al, 1996
) A lin k ha s bee n establishe d betwee n sper m morphologica l characteristic s an d infertilit y b y man y investigators . Th e presen t articl e provide s a n up-to-dat e revie w o f th e ultrastructura l abnormalitie s o f spermatozo a an d th e relationship s betwee n spermatozo a wit h abnorma l morpholog y an d th e outcom e o f fertilizatio n in vivo an d i n assiste d reproductiv e technologies .
Compositio
n an d ultrastructur e o f th e spermatozoo n Th e sper m cel l i s compose d o f head , neck , mid-piec e an d tail . Th e sper m plasm a membran e cover s th e hea d an d run s t o th e ti p o f th e tail . Th e majo r par t o f th e hea d i s occupie d b y a dens e compac t nucleu s containin g highl y condense d paterna l DN A which i s cappe d b y a n acrosom e an d th e plasma membrane . Th e acrosom e i s divide d int o a ca p regio n an d a n equatoria l regio n an d consist s o f inne r an d oute r acrosom e membrane s withi n whic h i s th e acrosoma l matrix . Th e acrosom e i s a Golgi-derive d organell e coverin g mor e tha n two-third s o f th e sper m nucleus . It s membrane s contai n hydrolyti c enzyme s whic h pla y a n essentia l rol e durin g th e acrosom e reactio n an d oocyt e penetration . Belo w th e acrosom e i s th e post-acrosoma l segmen t o f th e hea d wher e sper m eg g membran e fusio n usuall y occurs . Th e acrosom e reactio n usuall y take s plac e b y vesiculatio n o f surfac e membrane s whe n multipl e fusion s occu r betwee n th e plasm a membran e an d th e oute r acrosom e membrane . Th e majo r component s o f th e nec k ar e th e connectin g piec e jus t behin d th e nucleu s an d th e proxima l centriol e whic h consist s o f nin e triple t microtubule s (i.e . the sperm centriole has 9+0 organization of microtubules), forming a circle wit h dens e materia l withi n th e circl e an d outsid e th e centriole , calle d th e sper m centrosom e o r peri-centriola r material . Segmente d column s surroun d th e proxima l centriol e o n eithe r side . Th e centriol e is , afte r th e nucleus , th e mos t importan t sper m organell e fo r initiatio n o f th e intra-ooplasmi c fertilizatio n process , bein g responsibl e fo r th e formatio n o f th e sper m aste r (Va n Blerko m an d Davis , 1995). Th e sper m mid-piec e i s th e drivin g forc e o f th e spermatozoo n an d consist s o f a centra l axonem e consistin g o f microtubule s extendin g fro m th e proxima l centriol e t o th e dista l ti p o f th e tail . Th e microtubule s o f th e axonema l comple x ar e arrange d i n a characteristi c patter n 9 + 2 , i.e. nine sets of double microtubules a t th e peripher y surroundin g tw o singl e microtubule s i n th e centre . Outsid e th e periphera l doublet s ar e nin e dens e fibres . Cranially , thes e fibre s fus e wit h th e connecting piece . Th e fibre s facilitat e sper m movement , mediate d b y protei n phosphorylation (Tas h an d Means , 1983)
, an d serv e a s a protecto r agains t damag e durin g sper m transi t throug h th e mal e an d femal e reproductiv e tracts . Th e movemen t o f th e tai l i s mediate d throug h actio n o f th e dynei n arms , resultin g i n 4
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Fertilization potential of spermatozoa with abnormal morphology slidin g o f th e axonema l microtubule s alongsid e on e another . I n th e mid-piece , axonem e an d oute r dens e fibres ar e ensheathe d b y mitochondri a whic h ar e helicall y organize d i n 11-1 3 gyre s (Zambon i et al., 1971). Flagella r motilit y require s ATP , which originates from the mid-piece mitochondria and is hydrolysed b y ATPas e o f th e dynei n arm s i n th e presenc e o f magnesium . A cytoplasmi c drople t apparentl y indicate s immatur e spermatozoa . Th e mid-piec e end s wit h a thickenin g o f th e plasmalemma , th e annulus . Th e tai l consist s o f a principl e piec e an d th e termina l piec e o r end-piece . Th e proxima l regio n o f th e principl e piec e ha s a dens e fibrous sheat h an d seve n dens e fibres surroundin g th e axoneme . Th e fibres becom e reduce d i n thicknes s an d i n numbe r i n a dista l directio n throughou t th e principl e piece . Th e termina l piec e begin s a t th e en d o f th e fibrous sheat h o f th e principa l piece . I n th e proxima l par t o f th e termina l piec e th e doubl e tubule s o f axonem e remai n i n typica l arrangement . I n th e dista l par t o f th e termina l piec e th e 9 + 2 orde r o f th e axonem e disappear s an d th e tubule s for m a singl e bundl e withou t distinc t organizatio n (Pederse n 1970)
.
Ultrastructura
l abnormalitie s o f spermatozo a Ligh t microscop y ca n revea l majo r abnormalitie s o f th e spermatozoo n aberration s i n hea d shap e an d gros s morpholog y o f th e mid-piec e an d tai l ma y b e visualised .Nowadays
, electro n microscopi c investigation s ca n augmen t ligh t microscopi c findings. Transmissio n electro n microscop y permit s usefu l evaluatio n o f cyto - logica l detail s an d shoul d ideall y b e th e metho d o f choic e fo r investigatio n o f fertilit y disturbance s (Bisso n an d Escalier , 1978). A n in-dept h evaluatio n o f th e qualit y o f seme n usin g transmissio n electro n microscop y ca n improv e th e diagnosi s o f mal e subfertilit y an d provid e substantia l informatio n abou t th e fertilizin g competenc e o f spermatozoa .
Ultrastructural
abnormalities of the sperm head Amon g th e anomalie s affectin g sper m organelles , hea d defect s ar e especiall y common . Thes e includ e double-hea d forms , taperin g forms , amorphou s forms , elongate d head s an d acrosom e defects . Th e mai n structura l acrosom e defect s ar e partia l lac k o f acrosome , complet e lac k o f acrosome , intra-organella r inclusions , degeneratio n an d hypoplasi a o f th e acrosom e (Kiipke r et al., 1998). Disorganiza - tio n o f th e acrosoma l membran e ofte n lead s t o a chang e o f th e nuclea r shape .
Inclusion
s ar e characterize d b y th e presenc e o f pleiomorphi c structure s withi n th e organelle . Thes e structure s probabl y originat e a s remnant s o f Golg i elements . Thes e structura l abnormalitie s ar e assume d t o b e associate d wit h inabilit y o f th e spermatozoo n t o underg o th e acrosom e reactio n o r t o penetrat e th e zon a pellucida .Complet
e lac k o f th e acrosom e change s th e nuclea r shape , resultin g i n th e characteristi c roun d sper m head . Globozoospermi a o r round-heade d sper m syndrom e i s rar e an d th e spermatozo a ar e motile , lac k acrosom e an d postacrosoma l 49Downloaded from https://academic.oup.com/humrep/article/14/suppl_1/47/635758 by guest on 15 August 2023
N.NikoIettos et aL
sheat h an d hav e anomalou s mid-piece s an d mitochondria . Globozoospermi a i s suspecte d t o b e inherite d a s a geneti c defec t (Florke-Gerlof f et aL, 1984).
Globozoospermi
a i s th e onl y pathologica l conditio n o f sper m morpholog y tha t i s associate d wit h markedl y reduce d succes s afte r intracytoplasmi c sper m injectio n (ICSI) , even though the karyotypes of these spermatozoa appear to be normal (Rybouchki n et aL, 1996). Sper m hea d defect s ma y b e marker s fo r othe r sper m defect s tha t significantl y impai r fertility . Sper m nucleu s defect s hav e bee n associate d wit h infertilit y (Zamboni , 1987)
. A nuclea r defec t ma y explai n th e apparen t failur e o f th e inseminate d oocyte s t o continu e t o develo p owin g t o th e absenc e o f sperm - derive d factor s whic h appea r t o b e involve d i n th e developmenta l progressio n o f th e penetrate d oocyte , an d whic h ar e release d int o th e ooplas m durin g nuclea r expansio n an d DN A decondensatio n (Dozortse v et aL, 1995)
. A failur e o f th e conventiona l seme n parameter s t o predic t fertilizatio n indicate s tha t patient s wit h mal e facto r infertilit y posses s hidde n anomalie s i n th e compositio n o f thei r sper m nuclei, displayin g highe r level s o f loosel y package d chromati n an d damage d DN A (Evenso n et aL, 1980
; Bianch i et aL, 1996)
.
Structura
l abnormalitie s o f th e nucleu s includ e incomplet e o r impaire d chromati n condensatio n an d nuclea r vacuole s an d inclusions . Thes e defect s ofte n occu r i n associatio n wit h alteration s i n th e structur e o f th e acrosome . Karyolyti c change s o r th e presence o f larg e intranuclea r lacuna e o r vacuole s ar e th e morphologica l manifestation s o f underlyin g biochemica l alterations . I t i s possibl e tha t abnorma l sper m hea d morpholog y reflect s abnormalit y i n spermatogenesi s tha t i s manifeste d b y embryo s wit h a lo w potentia l fo r establishin g a norma l pregnanc y (Tasdemi r et aL, 1997). Sperm DNA anomalies Durin g th e lat e stage s o f spermiogenesis , elongatio n an d progressiv e condensatio n o f th e chromati n take s place , which , wit h simultaneou s acrosom e attachment , result s i n th e typica l shap e o f th e sper m head . Chromati n condensatio n i s associate d wit h biochemica l change s suc h a s replacemen t o f histone s whic h bin d t o DNA , firs t b y transitio n protein s an d the n b y arginine-ric h protamine s an d th e formatio n o f chromatin-stabilizin g disulphid e bond s (Zamboni , 1987
; Gree n et aL, 1994)
. War d an d Coffe y (1991 ) sugges t tha t th e protamine s bin d i n th e mino r groov e o f th e DN A heli x an d thi s protamine-DN A comple x fit s int o th e majo r groov e o f a n adjacen t strand , formin g a tightl y packe d arra y (War d an d
Coffey
, 1991). Thi s proces s result s i n a smalle r sper m cel l whic h require s les s energ y t o suppor t motility . Th e hig h degre e o f chromati n aggregatio n protect s th e matur e spermatozoo n agains t physica l an d chemica l damage . I t i s onl y withi n th e ooplas m o f a n activate d oocyt e tha t th e sper m chromati n become s decondense d a s a resul t o f th e cleavag e o f disulphid e bond s an d th e substitutio n o f protamine s b y oocyte-derive d histone s (Longo , 1985)
. Du e t o th e tigh t packagin g afforde d b y th e protamines , an y modificatio n o r absenc e o f thes e protein s coul d lea d t o a n anomal y i n th e packaging proces s o f sper m nucle i an d influenc e sper m qualit y 5
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Fertilization potential of spermatozoa with abnormal morphology an d fertilizin g capacity . On e o f th e majo r factor s leadin g t o chromati n packagin g problem s i n ejaculate d huma n spermatozo a coul d b e fault y protamin e depositio n durin g spermatogenesis . A n associatio n betwee n abnorma l sper m chromati n packagin g an d th e presenc e o f DN A stran d break s ha s bee n show n t o exis t an d i t ha s bee n postulate d tha t thes e anomalie s ma y aris e du e t o fault s i n th e mechanism s tha t packag e an d protec t th e sper m chromati n durin g spermatogenesi s (Manicard i et ah, 1995; Saile r et al., 1995)
. Spermatozo a wit h incomplet e chromati n condensatio n apparentl y mor e ofte n displa y single-strande d rathe r tha n double-strande d DN A (Pedersen , 1987
) o r posses s chromosoma l abnormalitie s (Abramsso n et al., 1982)
. A numbe r o f studie s hav e show n tha t spermatozo a wit h abnorma l nuclea r chromati n organizatio n ar e mor e frequen t i n infertil e me n tha n i n fertil e me n (Evenso n et al, 1980
; Forest a et al., 1992)
. Alteration s i n sper m chromati n migh t resul t i n defectiv e decondensatio n an d DN A activatio n durin g fertilization , leadin g t o a dela y i n th e formatio n o f th e mal e pronucleu s and/o r th e firs t division . On e consequenc e o f thi s migh t b e earl y embryoni c wastag e o r poo r embryoni c developmen t (Hamama h et al., 1997)
. Th e accessibilit y o f specifi c dye s an d fluorochrom e t o DN A ca n giv e clue s t o th e packagin g o f th e chromati n whic h occur s durin g spermiogenesis . Method s use d t o evaluat e sper m chromati n structur e includ e stainin g fo r histone s wit h
Anilin
e Blu e (Haid l an d Schill , 1994), stainin g o f huma n seme n sample s wit h
Acridin
e Orang e base d o n th e amoun t o f denature d DN A i n spermatozo a (Evenso n et al., 1980; Claassen s et al, 1992
) an d Chromomyci n A 3 (CMA 3 ) staining , whic h provide s evidenc e o f poo r chromati n packagin g i n spermatozo a an d allow s indirec t visualizatio n o f protamine-deficienc y (Bianch i et al., 1993)
.
Bianch
i et al. (1996 ) investigate d th e associatio n betwee n anomalie s i n sper m chromati n packaging , morpholog y an d fertilizatio n i n patient s undergoin g routin e in-vitr o fertilizatio n (IVF ) o r subzona l inseminatio n (SUZI ) usin g CMA 3 , a fluorochrom e specifi c fo r guanine-cytosin e ric h sequence s o f DN A (Bianch i et al., 1996). The y suggeste d tha t lowe r packagin g qualit y i n morphologicall y norma l spermatozo a ma y represen t a majo r limitin g facto r i n th e fertilizin g abilit y o f mal e facto r patients . The y foun d tha t a hig h percentag e o f CMA 3 positivit y wa s presen t i n certai n form s o f mal e facto r infertilit y an d tha t suc h a tes t ma y b e use d t o distinguis h separat e population s amon g morphologicall y norma l spermatozoa . Sakka s et al. (1996) , investigate d whethe r morpholog y an d chromati n anomalie s i n huma n spermatozo a ca n influenc e fertilizatio n afte r ICSI . The y examine d unfertilize d oocyte s an d assesse d sper m chromati n packaging usin g th e CMA 3 fluorochrom e (Sakka s et al., 1996)
. Althoug h the y di d no t postulat e tha t th e failur e o f fertilizatio n i s entirel y du e t o sper m defects , i t seem s likel y tha t poo r chromati n packagin g and/o r damage d DN A ma y contribut e t o failur e o f sper m decondensatio n afte r ICS I an d t o a resultan t failur e o f fertilization . Th e qualit y o f sper m chromati n (DNA ) i s a n importan t facto r i n th e fertilizatio n proces s an d i s especiall y critica l wher e on e spermatozoo n i s artificiall y selecte d fo r fertilizin g a n oocyte , a s occur s i n th e cas e o f ICSI . T o evaluat e th e chromati n qualit y i n huma n samples , Gola n et al. (1997 ) use d flo w cytometr y afte r stainin g o f huma n spermatozo a wit h Acridin e Orang e t o evaluat e chromati n qualit y i n 5
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N.Nikolettos et al.
seme n sample s (Gola n et al., 1997). The y foun d tha t thi s techniqu e provide d a quantitativ e assessmen t o f chromati n condensatio n i n spermatozoa , an d als o showe d tha t th e swim-u p an d Percol l gradien t centrifugatio n method s improv e th e percentag e o f spermatozo a wit h norma l chromati n structur e i n som e sample s wit h poo r initia l quality .
Accordin
g t o th e availabl e data , sper m decondensatio n defect s an d DN A anomalie s ma y resul t i n unrecognize d dearrengemen t o f th e spermatozoa , regardles s o f sper m morpholog y whic h i s know n t o influenc e th e fertilization .Consequently
, i n case s o f sever e mal e facto r infertility , studie s o f sper m DN A statu s ar e warranted .Ultrastructural
abnormalities of the mid-piece A s i n mos t mammals , huma n centriole s an d centrosome s ar e inherite d paternall y (Sathanantha n et al., 1991; Schatten , 1994)
. Sper m centrosome s pla y a centra l rol e i n th e creatio n o f th e firs t an d subsequen t embryoni c mitoti c spindle s (Va n
Blerko
m an d Davis , 1995). Afte r sper m penetration , th e centrosom e nucleate s microtubule s t o for m a sperm-derive d aster , an d microtubule s appea r t o pla y a n importan t rol e i n bringin g th e mal e an d femal e pronucle i int o clos e appositio n (Navar a et al., 1994)
, an d the y ma y als o serv e t o establis h th e specifi c alignmen t o f th e pronuclea r membrane s require d fo r syngam y (Va n Blerko m et al., 1995)
.
Becaus
e th e microtubule s forme d fro m th e sper m centrosom e functio n bot h i n th e earl y stage s o f huma n fertilizatio n an d i n th e establishmen t o f th e first mitoti c spindl e (Palerm o et al., 1994; Va n Blerko m et al., 1995)
, centrosoma l defect s hav e bee n indicate d a s potentia l cause s o f fertilizatio n failur e an d developmenta l arres t a t th e pronuclea r stage , an d hav e bee n relate d t o subfertilit y i n human s (Schatten , 1994
; Va n Blerko m an d Davis , 1995)
.
Centrosom
e dysfunctio n ma y represen t a ne w clas s o f sper m defect s associate d wit h huma n earl y developmenta l failur e (Va n Blerkom , 1996). Suc h centrosoma l defect s probabl y accoun t fo r a smal l proportio n o f mal e infertilit y cases . Th e exten t t o whic h centrosoma l defect s contribut e t o idiopathi c mal e infertilit y wil l requir e th e analysi s o f centrosoma l functio n an d biochemistr y i n bot h norma l an d infertil e men , an d i n particular , thos e whos e spermatozo a fai l t o functio n eve n afte r intracytoplasmi c depositio n int o a n otherwis e developmentall y competen t ooplas m (Va n Blerkom , 1996)
. I f centrosoma l defect s ar e a caus e o f mal e infertility , the n thi s typ e o f infertilit y canno t b e overcom e simpl y b y th e insertio n o f a spermatozoo n int o th e ooplas m ( As h et al., 1995
; Simerl y et al, 1995
; Va n
Blerkom
, 1996).
Ultrastructural
abnormalities of the tail A variet y o f axonema l defect s hav e bee n documente d i n patient s wit h absen t o r impaire d sper m motility . Axonema l anomalie s consis t o f numerica l o r positiona l anomalie s o f th e microtubule s and/o r absenc e o f th e inne r o r oute r dynei n arms .Complet
e absenc e o f th e dynei n arm s i s associate d wit h immotil e spermatozo a 52Downloaded from https://academic.oup.com/humrep/article/14/suppl_1/47/635758 by guest on 15 August 2023
Fertilization potential of spermatozoa with abnormal morphology (Eliasso n et al., 1977), wherea s absenc e o f onl y th e oute r dynei n arm s i s no t accompanie d b y tota l immotilit y (Escalie r an d David , 1984)
. Th e subpopulatio n o f patient s wit h immotile-cili a syndrom e i s ver y hetero - geneou s an d occasionall y th e defec t doe s no t affec t 100
% o f th e spermatozo a (Johnso n et al., 1982)
. Afzeliu s an d Eliasso n (1979 ) reporte d o n patient s wit h variou s sper m cili a disturbance s an d suggeste d a geneti c caus e o f th e immotil e cili a syndrome . Tai l stum p syndrom e i s extremel y rar e an d i s cause d b y a dysfunctio n o f th e lates t stage s o f spermiogenesis , an d affect s spermatid s an d spermatozo a equall y (Barthelem y et al., 1989)
. Tai l stum p syndrom e i s characterize d b y flagella organize d a s uniflagellate s an d wit h extremel y shor t axoneme s wit h a 9+ 2 o r a 9+ 0 arrangement , generall y wit h dynei n arm s (Baccett i et al., 1993)
. I n shor t tai l syndrom e ther e i s a biflagellat e arrangement , wit h 9+ 0 o r 9+ 1 axoneme s an d a lac k o f dynei n arms . I n bot h syndromes , spermatozo a usuall y sho w unaffecte d sper m head s bu t unassemble d mitochondri a (Stal f et al., 1995)
. Th e pures t clinica l syndrom e i s tha t describe d b y Kartagene r (1933 ) wher e patient s presen t wit h 100
% immotil e bu t vita l spermatozo a i n norma l numbe r an d wit h norma l morpholog y accordin g t o ligh t microscopy , wit h reccurren t sinusiti s an d bronchiectasi s an d situ s inveru s (Kartagener , 1933)
. Th e lac k o f dynei n arm s o r nexi n link s i n Kartagener' s syndrom e impair s slidin g movement s betwee n tubule s alon g th e tai l s o tha t spermatozo a ar e immobile .
Periaxonema
l anomalie s involv e dens e fibr e (abnorma l positio n o r size ) an d mitochondria l sheat h anomalie s a s wel l a s cytoplasmi c remnant s fo r th e midpiece , an d dens e fibres , longitudina l column s (abnorma l positio n o r size) , an d fibrou s sheat h anomalie s fo r th e principa l piec e (Courtad e et al., 1998). Disorganizatio n o f th e mitochondria l sheat h o r it s complet e absenc e ma y impai r motilit y du e t o th e lac k o f AT P synthesis . Mitochondri a ar e essentia l t o cel l metabolis m an d lac k o f thei r AT P productio n ca n caus e sper m degeneratio n (Kiipke r et al., 1998)
.
Anomalie
s o f th e periaxonema l structure s hav e bee n associate d wit h defect s o f th e axonema l comple x i n patient s wit h sever e o r tota l asthenozoospermi a (Escalie r an d David , 1984; Cheme s et al., 1987)
. Isolate d anomalie s o f th e periaxonema l element s ar e relate d t o motilit y disturbance s throug h a flagellar dyskinesi a tha t recentl y ha s bee n terme d 'periaxonema l flagellar dyskinesia ' (Davi d et al., 1993)
. I n patient s wit h persisten t unexplaine d asthenozoospermia , th e frequen t associ - atio n o f periaxonema l anomalie s wit h axonema l deficiencie s strongl y suggest s tha t axonema l deficiencie s ar e no t th e onl y caus e o f decrease d motilit y (Courtad e et al., 1998)
. Suc h anomalie s ar e frequentl y associate d wit h defect s i n th e component s o f th e periaxonema l structure s an d th e sper m head . Thus , transmissio n electro n microscop y analysi s shoul d includ e th e entir e spermatozoo n an d no t onl y th e axonem e (Courtad e et al., 1998)
.
Anothe
r seriou s defec t i s th e separatio n o f th e flagellum fro m th e hea d (headles s spermatozoon) . Thi s 'pin-head ' defec t i s assume d t o b e geneticall y inherite d (Zanevel d an d Polakowski , 1977). Instea d o f th e nucleus , a globula r cytoplasmi c mas s surroundin g th e proxima l segment s o f th e decapitate d flagellum ma y b e misinterprete d a s microcephali c spermatozo a unde r ligh t microscopy . 5
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Morphologica
l classificatio n o f spermatozo a Huma n spermatozo a sho w a grea t variatio n i n siz e an d structur e compare d t o othe r mammals . Norma l fertil e ejaculate s contai n spermatozo a exhibitin g considerabl e variation s no t onl y i n th e siz e an d shap e o f th e hea d an d th e acrosome , bu t als o i n th e degre e o f nuclea r vacuolation , siz e o f persistin g cytoplasmi c droplets , mid-piec e disturbance s an d tai l abnormalitie s (Ombele t et al., 1995). Aroun d th e tur n o f th e las t centur y variou s author s (Jensen , 1879
; Ballowitz , 1886
; Retzius, 1902; Branca, 1924) and others described not only normal but als o abnorma l spermatozo a i n detaile d drawings . Ther e wer e a s man y differen t interpretation s o f sper m morpholog y a s ther e wer e author s t o describ e them . Al l th e author s agree d tha t ther e wa s a definit e relationshi p betwee n abnorma l sper m morpholog y an d mal e subfertilit y bu t ther e wa s n o agreemen t a s t o wha t constitute d a n abnorma l spermatozoon . I t i s difficul t t o establis h th e criteri a fo r normalit y an d abnormalit y o f a spermatozoon . Variou s importan t attempt s hav e bee n mad e t o standardiz e criteri a fo r sper m morpholog y (MacLeo d an d Gold , 1951
; Freud , 1966
; Eliasson , 1971
; Davi d et al, 1975)
. Th e firs t Worl d Healt h Organizatio n (WHO ) classificatio n (1980 ) wa s a n importan t breakthroug h o f sper m morpholog y assessment . Hofman n an d Haide r (1985 ) reporte d anothe r syste m fo r sper m morpholog y assesmen t know n a s th e Diisseldor f classificatio n (Hofman n an d Haider , 1985)
. I n thi s classificatio n system , mor e emphasi s i s place d o n elongatio n o f spermatozo a an d acrosom e defects . Th e secon d WH O manua l (1987 ) revise d th e guideline s fo r seme n analysis . Kruge r an d Menkveld (Kruge r et al., 1986
, 1988
; Menkveld , 1987
; Menkvel d et al., 1990
) introduce d th e stric t Tygerber g criteri a fo r th e assessmen t o f sper m morphology . I n thi s classificatio n th e whol e spermatozoo n i s take n int o accoun t fo r assessmen t an d al l borderlin e an d slight y abnorma l hea d form s ar e considere d a s abnormal . Accordin g t o th e stric t Tygerber g criteria , morphologica l assessmen t result s ca n b e divide d int o thre e predictiv e categories : norma l morpholog y grou p (>14 % normal forms), good prognosis group (4-14% normal forms) and the poo r prognosi s grou p (<4 % norma l forms ) (Kruge r et al., 1988)
. I n th e thir d WH O manua l (1992) , muc h mor e attentio n wa s give n t o sper m morpholog y assessment . Beside s th e morphologicall y norma l group , fou r classe s o f abnormalitie s wer e describe d an d a teratozoospermi a inde x wa s calculated .
Specifi
c sterilizin g defect s suc h a s globozoospermi a syndrom e shoul d b e men - tioned . Th e cut-of f valu e fo r normalit y change d fro m 50% (WH O 1987
) t o 30
% morphologicall y norma l spermatozo a (WH O 1992
) (Tabl e I) .
Abnorma
l sper m morpholog y an d fertilizatio n in vivo Seme n analysi s continue s t o b e th e mos t importan t too l fo r th e evaluatio n o f mal e fertilit y potential . Th e assessmen t o f sper m morphology , on e o f th e thre e 54Downloaded from https://academic.oup.com/humrep/article/14/suppl_1/47/635758 by guest on 15 August 2023
Fertilization potential of spermatozoa with abnormal morphology Tabl e I . A n overvie w o f sper m abnormalitie s a s describe d b y th e Worl d Healt h Organizatio n (WHO ) 1992 an d stric t Tygerber g criteria . Hea d
Midpiec
e Tai lCut-of
f valu e fo r normalit yBorderlin
e form sCytoplasmi
c droplet s a s proportio n o f hea d siz eShapeAcrosom
e Lengt h (L ) Widt h (W ) W: L rati oStrict Tygerberg criteria smooth , ova l 40-70% o f hea d 3- 5 u m 2- 3 |i m
0.60-0.6
7 L=1.5 X hea d lengt h W = < 1 |i m slender , axiall y attache d L = -4 5 (i m uniform , uncoile d >14 % abnorma l i WH O (1992 ) ova l 40-70% o f hea d 4-5. 5 (i m
2.5-3.
5 urn0.57-0.6
7 regular , no t ben t o r distended , inserte d a t 90° t o lon g axi s o f hea d no t broken , uncoiled , regular , n o termina l droplet s >30 % norma l
. O f th e conventiona l seme n parameter s examined , th e mos t revealin g wa s th e morphologica l characte r o f th e spermatozoa, whic h wa s significantl y poore r i n th e grou p wit h unexplaine d infertilit y tha n i n th e norma l fertil e contro l group . Ther e seem s t o b e a significan t relationshi p betwee n a n increasin g numbe r o f abnorma l spermatozo a i n sper m analysi s an d bot h a decreasin g probabilit y o f producin g livin g childre n an d a n increasin g tim e interva l t o th e first pregnanc y (Bostoft e et al., 1982
) bu t n o relationshi p t o abortion s an d pathologica l pregnancies . Roger s et al. (1983) conducted routine semen analysis and histological staining fo r sper m morphology , o n seme n sample s fro m 9 5 me n wh o wer e classifie d int o fertil e an d infertil e group s (Roger s et al., 1983)
. Th e seme n analysi s parameter s o f th e fertil e an d infertil e group s wer e significantl y different . A s a singl e parameter , decrease d numbe r o f norma l form s appeare d t o b e a goo d indicato r fo r clinica l infertilit y i f IV F testin g wa s no t available . A stud y (Zain i et al., 1985
) di d no t find an y significan t difference s betwee n fertil e an d infertil e me n regardin g sper m morpholog y classificatio n an d sper m motilit y (WHO , 1980)
. Thei r result s indicate d tha t sper m morpholog y a s currentl y assesse d ma y no t b e importan t i n predictin g fertilit y i n subfertil e men . I n anothe r stud y (Va n Zy l et al., 1990
) in-viv o conceptio n rate s o f 11.5 % wit h <4 % morphologicall y norma l spermatozo a an d 21.5
% for a group of men with 4-9% normal spermatozoa wa s reported , wit h a n overal l mea n pregnanc y rat e o f 19.1% . Check et al. 5
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(1992 ) classifie d severel y impaire d mal e fertilit y potentia l b y a resul t o f =£4 % spermatozo a wit h norma l morphology , an d score s >14 % indicate d norma l fertilizatio n potentia l (Chec k et al, 1992). The y di d no t find an y predictiv e valu e regardin g th e in-viv o conceptio n rat e usin g th e stric t Tygerber g criteria . A furthe r stud y (Haid l an d Schill , 1993
) assesse d sper m morpholog y i n 7 8 fertil e me n accordin g t o th e Diisseldor f classification . Ther e wer e 30
% norma l form s o n average , whic h conform s wit h th e WH O (1992 ) guidelines . Thi s differ s fro m th e stric t criteri a o f th e Tygerber g group , probabl y becaus e thes e criteri a wer e establishe d unde r in-vitr o conditions . A prospectiv e follow-u p stud y o n 90
7 infertil e me n (Wichman n et al., 1994
) foun d sper m morpholog y usin g th e WH O (1980 ) classification , t o b e a n independen t predictor o f th e fertilizatio n outcome . O n th e othe r hand , other s coul d no t pu t forwar d an y sper m characteristic , includin g sper m morphology , a s a sol e predictor o f mal e fertilit y statu s (Bartoo v et al., 1993)
. Nevertheles s tw o recen t studie s hav e pointe d t o th e importanc e o f th e evaluatio n o f sper m morpholog y usin g stric t criteri a fo r th e assessmen t o f in-viv o conceptio n probabilit y an d o f a man' s fertilizin g potentia l durin g basi c infertilit y investi - gation s (Eggert-Krus e et al., 1996
; Ombele t et al., 1997a)
. O f note , however , wa s a naturall y differin g cu t of f valu e fo r normalit y fro m thos e propose d b y th e WH O (1992) . I n conclusion , mos t o f th e abov e studie s lea d t o th e conclusio n tha t sper m morpholog y i s th e mos t significan t predictor o f in-viv o fertilization .
Abnorma
l sper m morpholog y an d intrauterin e inseminatio n (IUI ) Th e result s afte r IU I treatmen t fo r mal e infertilit y diffe r tremendousl y betwee n th e variou s centres . Poo r result s hav e bee n describe d whe n IU I wa s performe d i n natura l cycle s fo r mal e subfertilit y indication s (Glas s an d Ericsonn , 1978; Marr s et al., 1983
; Confin o et al, 1986
; Kirb y et al., 1991
) althoug h tw o studie s hav e show n pregnanc y rate s pe r cycl e o f 1 6 an d 21
% afte r IU I i n patient s wit h poo r seme n qualit y (Keri n et al., 1984
; Byr d et al., 1987)
. Fo r a bette r cycl e outcom e IU I shoul d b e couple d wit h ovaria n stimulatio n (She r et al., 1984
;
Blumenfel
d an d Nahhas , 1989; Tarlatzi s et al., 1991
; Iriann i et al, 1993)
. I n a revie w o f 110
0 IU I treatmen t cycles , Ombele t et al. (1996 ) foun d a significan t differenc e i n cycl e fecundit y comparin g th e 'poor-prognosis ' (<5 % norma l form s usin g th e Tygerber g stric t criteria ) wit h th e 'good-prognosis ' grou p (5-8 % normal forms) (Ombelet et al, 1996). Various studies emphasize the valu e o f sper m morphology fo r th e predictio n o f th e outcom e o f IU I (Comhair e et al, 1994
; Tone r et al, 1994
; Bur r et al, 1996)
. Wit h a tota l motil e sper m coun t o f <1X1O 6 fo r insemination , sper m morpholog y become s a ver y usefu l predictiv e too l fo r th e outcom e o f IU I (Ombele t et al, 1997b)
. 5
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Fertilization potential of spermatozoa with abnormal morphologyInfluenc
e o f sper m preparatio n technique s o n sper m morpholog yMethod
s o f sper m preparatio n whic h improv e th e proportio n o f spermatozo a wit h norma l morphology , norma l intac t acrosomes , velocit y an d linearit y shoul d achiev e bette r result s i n assiste d reproductiv e technology . Whil e variou s method s o f sper m preparatio n hav e bee n develope d ove r th e years , th e mos t popula r ar e th e swim-u p procedure , th e discontinuou s Percol l gradien t metho d an d th e mini -Percol
l gradien t technique .Spermatozo
a ar e sensitiv e t o oxidativ e damage , an d i t ha s bee n demonstrate d tha t centrifuga l pelletin g o f unselecte d sper m population s fro m huma n ejaculate s cause s th e productio n o f reactiv e oxyge n specie s (ROS ) withi n th e pelle t whic h induc e irreversibl e damag e i n th e spermatozo a (Aitki n an d Clarkson , 1987). Th e effec t o f RO S o n th e pathophysiolog y o f huma n sper m functio n ha s bee n emphasize d i n recen t years . Oxidativ e stres s i s though t t o b e associate d wit h mal e infertliti y (Alvare z et al, 1987
; Aitke n et al., 1989
, 1991
, 1993
; D e
Lamirand
e an d Gagnon , 1992). Th e tw o majo r source s o f RO S i n seme n preparation s ar e leukocyte s an d spermatozo a (Aitke n an d Clarkson , 1987
; Aitke n an d West , 1990
; Iwasak i an d Gagnon , 1992
; Kessopoulo u et al., 1992
; Zin i et al., 1993)
. Oligozoospermi c patient s ten d t o hav e hig h RO S productio n b y spermatozo a (Aitke n et al., 1989
) an d oligozoospermi c sample s shoul d b e spu n gentl y durin g th e swim-u p procedure , sinc e abnorma l spermatozo a ma y otherwis e releas e RO S tha t ca n damag e norma l spermatozo a (Edward s an d Brody , 1995)
. Sper m preparatio n wit h a discontinuou s Percol l gradien t reduce s th e productio n o f RO S cause d b y lipi d peroxidatio n (Edward s an d Brody , 1995)
.
Regardin
g recover y o f spermatozo a wit h norma l morphology , mos t studie s comparin g th e swim-u p metho d an d Percol l gradien t centrifugatio n favoure d th e swim-u p technique , whic h remain s th e metho d mos t commonl y use d b y IV F laboratories . Althoug h Percol l an d mini-Percol l resul t i n greate r yield s o f motil e spermatozo a tha n th e swim-u p preparation , th e swim-u p procedur e select s highe r proportion s o f spermatozo a wit h improve d characteristic s (velocity , intac t acrosome s an d norma l morphology ) whic h relat e t o fertilizatio n rate s in vitro (N g et al., 1992), Th e swim-u p metho d shoul d remai n th e standar d choice , particularl y fo r a sampl e whic h i s likel y t o yiel d a sufficien t numbe r o f motil e spermatozo a fo r insemination , becaus e o f it s simplicit y an d als o becaus e i t yield s spermatozo a o f hig h quality . Anothe r optio n i s th e sequentia l mini-Percol l an d swim-u p preparatio n whic h improve s yield s o f hig h qualit y spermatozo a fro m som e abnorma l seme n sample s an d therefor e ha s potentia l fo r improvin g fertilizatio n rate s (Ng et al., 1992).
Th e smal l volum e (mini-Percoll ) gradien t i s though t t o b e th e bes t techniqu e fo r sper m preparatio n i n IV F fo r oligozoospermi c sample s b y som e investigator s (Or d et al., 1990
; Caldero n et al., 1991)
. I n term s o f th e clinica l result s fo r IVF , swim-u p an d Percol l preparatio n o f fres h seme n d o no t differ . Som e studie s repor t a greate r improvemen t i n morpholog y afte r swim-u p (Engler t et al., 1992
) whil e other s reporte d a bette r morpholog y afte r Percol l preparatio n (Va n de r Zwalme n et al., 1991)
. Thes e difference s ca n b e explaine d eithe r b y th e lac k o f uniformit y i n th e Percol l gradien t 5
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Tabl e II . Comparativ e recover y o f morphologica l norma l spermatozo a usin g glas s woo l filtratio n (GWF ) o r GW F an d swim-u p preparatio n i n patien t grou p wit h >10 % an d <10 % morphologicall y norma l spermatozo a i n nativ e semen . Norma l sper m morpholog y >10 % (n = <10 % (n ---43) = 17 )Native 29.8 ± 4. 1 ±Semen 19. 7 1. 9GWF 35.
3 ± 6. 5 ±: 21.2 : 4.
2GWF-SU
41.9 ± 21.6
* 8. 5 ± 7. 0 * P < 0.05 , i n compariso n wit h nativ e seme n usin g th e paire d ?-tes t
Adapte
d fro m Al-Hasan i et al. (1996) . preparatio n technique , o r b y th e classificatio n use d t o describ e morphology . I t i s probabl e tha t ther e ar e als o difference s i n th e population s o f seme n anomalie s selecte d fo r IVF . Th e double-was h swim-u p preparation fo r IV F substantiall y improve s th e recover y o f spermatozo a wit h norma l morpholog y accordin g t o th e stric tTygerber
g criteria , an d th e benefi t i s mos t substantia l i n patient s wit h abnorma l sper m sample s (Scot t et al., 1989), whil e other s (Cha n et al., 1991
) suggeste d tha t th e multiple-tub e swim-u p metho d i s effectiv e fo r th e separatio n o f spermato - zo a fro m normozoospermi c seme n sample s fo r us e i n infertilit y treatment .
Al-Hasan
i et al. (1996) , i n a prospectiv e study , examine d th e sper m quality , especiall y sper m morphology , an d th e outcom e o f IV F results , whe n glas s woo l filtratio n (GWF ) an d swim-u p wer e use d i n combinatio n fo r th e preparatio n o f spermatozoa . GW F wa s compare d wit h GWF/swim-u p b y usin g aliquot s fro m th e sam e seme n samples . The y foun d tha t GW F alon e di d no t produc e significantl y bette r result s but , i n combinatio n wit h swim-up , i t resulte d i n significantl y bette r recover y o f spermatozo a wit h norma l sper m morpholog y (Tabl e II ) an d i n a n improve d outcom e o f IV F -embry o transfe r fo r fres h seme n sample s wit h >10 % morphologicall y norma l spermatozoa . T o conclude , despit e som e contradictor y results , mos t studie s favoure d th e swim-u p techniqu e fo r recover y o f spermatozo a wit h norma l morphology .Abnorma
l sper m morpholog y an d fertilizatio n in vitro (IVF )Regardin
g IVF , numerou s studie s hav e bee n publishe d emphasizin g th e impor - tanc e o f sper m morpholog y a s a predictor fo r men' s fertilizin g potentia l in vitro. Th e first studie s use d a variet y o f criteri a fo r sper m morphology , includin g thos e o f th e WH O (1980 , 1987). Th e mos t recen t studie s almos t al l use d th e stric t
Tygerber
g criteria . Th e first descriptio n o f a n associatio n betwee n sper m morpholog y abnormalitie s an d poo r reproductiv e potentia l in vitro wa s mad e i n 1984 (Yovic h an d Stanger , 1984)
. They reported a normal fertilization rate but a significant delay in reaching th e pronuclea r stag e fo r th e abnorma l morpholog y grou p (WHO , 1980)
. Th e stud y grou p wa s small , however , includin g onl y 1 0 patients . I n som e studie s sper m morpholog y wa s reporte d t o b e o f n o valu e i n predictin g 5
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Fertilization potential of spermatozoa with abnormal morphology fertilizatio n i n IVF , o r t o b e o f minima l valu e i n predictin g IV F succes s (Alpe r et al, 1985; Jeyendra n et al, 1986
; Hirsc h et al, 1986
; Talber t et al, 1987
;
Rosenbor
g et al, 1990). I n othe r studies , sper m morpholog y wa s reporte d t o b e th e bes t sol e predicto r regardin g fertilizin g capacit y o f a seme n sampl e an d th e outcom e o f IV F (Kruge r et al, 1986
, 1988
, 1990
; Acost a et al, 1988
; Cha n et al, 1988
; Sco t et al, 1989
; Hintin g et al, 1990
; Engins u et al, 1991
;
Kobayash
i et al, 1991; D e Geyte r et al, 1992
; Dunca n et al, 1993)
. Possibl y th e mai n reaso n fo r th e conflictin g result s describe d i n th e abov e mentione d studie s i s a hig h leve l o f variatio n i n th e estimatio n o f sper m morpholog y usin g a variet y o f morphologica l criteria , eithe r simila r t o thos e recommende d b y th e WH O (WH O 1980
, 1987
) o r not . Th e us e o f ver y stric t Tygerber g criteria , a s introduce d b y Kruge r an d Menkvel d (Kruge r et al, 1986
, 1987
, 1988
; Menkveld et al, 1990
; WHO , 1992
) ma y resul t i n a reliabl e an d reproducibl e metho d fo r assessin g sper m morphology , wit h a goo d predictiv e valu e i n assiste d reproductio n (Engins u et al, 1991
, 1992)
.
Abnorma
l sper m morpholog y a t th e tim e o f IV F impair s bot h th e fertilizatio n rat e an d th e overal l reproductiv e potential . Eve n thoug h fertilizatio n ma y b e achieve d b y correctiv e measure s (increasin g inseminatio n concentration) , sever e teratozoospermi a als o predispose s t o a highe r abortio n rat e (Oehinge r et al, 1988). Severe sperm head abnormalities have a lower ability to establish successfu l pregnancie s followin g IV F (Oehninge r et al, 1988)
. Engins u et al. (1991 ) showe d tha t evaluatio n o f sper m morpholog y accordin g t o stric t Tygerber g criteri a i s mor e effectiv e i n predictin g fertilizatio n tha n evaluatio n accordin g t o WH O criteria . The y demonstrate d tha t th e metho d o f evaluatin g sper m morpho - log y base d o n ver y stric t criteri a allow s a mor e accurat e predictio n o f th e chanc e o f fertilizatio n in vitro. Wit h spermatozo a havin g morphologica l impairmen t bu t o f sufficien t qualit y tha t the y ca n sometime s penetrat e th e zon a an d achiev e fertilization , a proble m o f delaye d fertilizatio n an d poo r embryoni c developmen t i s eviden t (Ron-E l et al, 1991)
. Th e stric t criteri a fo r evaluatio n o f huma n sper m morpholog y play s a n importan t rol e i n predictin g IV F results , an d concentratio n o f progressivel y motil e spermatozo a ca n b e a n optiona l metho d (Engins u et al, 1992)
. Earlie r studie s i n IV F patient s suggeste d a n associatio n betwee n abnorma l sper m morphology ; especiall y sper m hea d abnormalitie s o f th e postacrosoma l region , an d poo r embry o morpholog y (Perinau d et al, 1993)
. Simila r association s wer e show n fo r lowe r implantatio n an d ongoin g pregnanc y rate s afte r increasin g th e sper m concentration s fo r IV F i n severel y teratozoospermi c patient s (Gro w et al, 1994)
. It is evident that a morphological evaluation using very strict criteria not onl y predict s th e rat e o f fertilizatio n in vitro bu t als o th e conceptio n rat e i n individua l couple s accepte d int o a n IV F programm e (Ombele t et al, 1994)
. Bot h sper m motilit y parameter s an d percentag e norma l morpholog y determine d wit h th e stric t criteri a ar e significan t factor s i n predictin g fertilizatio n an d pregnanc y rate s i n IV F (Donnell y et al, 1998)
, whil e sper m morpholog y ca n b e use d a s a determinin g facto r i n performin g IV F o r ICS I i n a give n borderlin e seme n sampl e (Salla m et al, 1998)
. 5
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Th e deleteriou s effec t o f teratozoospermi a o n bot h fertilizatio n rat e an d pregnanc y rat e pe r cycl e afte r IVF-embry o transfe r ha s bee n establishe d i n th e abov e studies . However , Robinson et al. (1994) , i n a retrospectiv e analysi s o f 2144 consecutiv e IVF/embry o transfe r treatmen t cycles , examine d th e effec t o f th e stric t classificatio n o f sper m morpholog y o n th e outcom e o f IV F an d embry o transfer . The y foun d n o statisticall y significan t differenc e betwee n th e patien t wit h >14 % norma l form s an d patient s wit h ^4 % an d =^14 % norma l form s wit h regar d t o th e percentag e o f patient s achievin g th e normalize d media n fertilizatio n rat e o r higher . Ther e wa s a statisticall y significantl y lowe r chanc e o f achievin g thi s rat e i n patient s wit h <4 % norma l forms , bu t stil l 68.6
% di d achiev e tha t fertilizatio n rat e o r higher . Th e majorit y o f patient s achieve d th e normalize d media n fertilizatio n rat e eve n wit h <4 % morphology , a s lon g a s sper m concentra - tio n an d motilit y wer e withi n th e norma l range . However , thes e differen t conclusion s ma y resul t fro m difference s i n stainin g techniques , sper m preparatio n protocol s an d morphologica l classificatio n systems . T o conclude , sper m morpholog y i s th e mos t significan t determinin g facto r i n predictin g th e outcom e o f a n IV F cycl e i n case s o f teratozoospermia , an d a cut - of f valu e o f 4 % norma l morphology , usin g th e stric t criteria , ha s bee n suggeste d fo r IV F cycle s t o maximiz e th e chance s o f achievin g a hig h fertilizatio n an d pregnanc y rate .
Abnorma
l sper m morpholog y an d intracytoplasmi c sper m injectio n (ICSI ) Th e majo r breakthroug h i n th e treatmen t o f mal e infertilit y wa s th e introductio n o f ICS I becaus e i t enable d patient s wit h previou s IV F failur e an d patient s wit h extremel y impaire d seme n parameter s t o achiev e fertilizatio n an d pregnanc y (Palerm o et al., 1992). Thus , wit h ICSI , th e presence o f onl y a fe w barel y motil e spermatozo a i n a seme n sampl e i s al l tha t i s require d fo r fertilizatio n becaus e ICS I bypasse s th e natura l selectio n proces s whe n sever e sper m abnormalitie s ar e presen t (Va n Steirteghe m et al., 1993)
. Palerm o et al. (1993 ) reporte d tha t non e o f th e individua l sper m parameters , suc h a s concentration , progressiv e motilit y o r morphology , wa s associate d wit h th e outcom e o f ICSI . I t seem s tha t n o significan t differenc e i n fertilizatio n an d pregnanc y rate s afte r ICS I i s presen t i n patient s wit h sever e teratozoospermi a a s compare d wit h les s sever e form s o f mal e infertilit y (Cohe n et al., 1994
; Mansou r et al., 1995)
.
Spermatozo
a fro m th e mos t sever e case s o f oligoasthenoteratozoospermi a (OAT ) produc e th e sam e pregnanc y rate s a s thos e fro m mil d case s o f mal e facto r infertilit y whic h wer e n o differen t fro m thos e produce d b y spermatozo a fro m me n wit h norma l sper m undergoin g conventiona l IV F (Nag y et al., 1995). I t appear s tha t neithe r th e mos t sever e morphologica l defect , no r th e mos t sever e motilit y defect , no r th e tinies t numbe r o f spermatozo a i n th e ejaculat e hav e an y negativ e effec t o n th e pregnanc y rat e wit h ICSI . Fertilizatio n failur e wit h ICS I result s no t fro m th e mos t sever e morphologica l defec t o f th e spermatozo a bu t 6
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Fertilization potential of spermatozoa with abnormal morphology rathe r fro m eithe r poo r oocyt e qualit y o r sper m non-viabilit y (Li u et al,1995b)
. I t i s clea r tha t th e outcom e o f IVF-embry o transfe r i s no t relate d t o teratozoospermi a whe n fertilizatio n i s achieve d usin g ICSI , whic h bypasse s bot h zon a bindin g an d fusio n t o th e oolemm a (Oehninge r et al, 1995; Sherin s et al, 1995
; Svalander et al., 1996). Although the implantation and ongoing pregnancy rate s ar e ver y low , wit h a hig h incidenc e o f earl y pregnanc y loss , ICS I seem s t o b e th e onl y availabl e treatmen t modalit y i n case s wit h totall y morphologicall y abnorma l spermatozo a (Tasdemi r et al., 1997)
.
Accordin
g t o Kiipke r et al (1998) , sever e hea d defect s o f th e spermatozo a ar e mainl y associate d wit h long-ter m infertilit y an d failur e o f fertilizatio n followin g classica l IV F treatment . However , th e severit y o f sper m defect s appear s t o hav e n o predictiv e valu e wit h regar d t o th e succes s o f ICSI . I n a comprehensiv e analysi s o f 683 microinjection cycles, no difference was found in the fertilization,
embry o development , transfe r o r pregnanc y rate s i n relatio n t o sper m morpholog y usin g ejaculate d spermatozo a (Nag y et al., 1998). Thi s i s especiall y importan t whe n on e consider s th e standar d IV F technique , wher e accordin g t o th e majorit y o f report s (se e above) , th e fertilizatio n rat e fall s i f th e proportio n o f spermatozo a wit h norma l morpholog y a s determine d b y stric t criteria , i s <1 4 % , an d especiall y whe n i t i s < 4 % . A n explanatio n o f th e unimpaire d fertilizatio n rate s wit h ICS I ma y b e th e fac t tha t generall y th e mos t normal-lookin g spermatozoo n i s selecte d fo r microinjection . ICS I offer s a mean s t o bypas s th e barrier s o f th e oocyte , whic h woul d otherwis e b e a n obstacl e fo r th e morphologicall y abnorma l spermatozoon . I t wa s noteworth y i n th e sam e analysi s (Nag y et al., 1998
) tha t th e embry o qualit y wa s no t affecte d adversel y b y th e injectio n o f spermatozo a wit h abnorma l morphology , no r th e initia l pregnanc y los s rate . S o th e author s conclude d tha t th e morphologica l abnormalitie s o f spermatozo a hinderin g thei r abilit y t o penetrat e th e oocyt e ma y no t reflec t an y othe r geneti c abnormalit y o f thes e gamete s apar t fro m thei r morpholog y (Rosenbuc h et al., 1992
; Nag y et al., 1998)
.
Althoug
h ICS I i s th e onl y solutio n fo r fertilizatio n i n case s wit h tota l globozoospermia , thi s i s th e onl y abnorma l conditio n o f sper m morpholog y whic h give s ver y poo r fertilizatio n rate s an d poo r result s afte r ICSI . A fe w pregnancie s have.bee n reporte d afte r ICS I wit h round-heade d spermatozoa , bu t th e genera l result s afte r ICS I wit h acrosomeles s spermatozo a ar e agai n rathe r limite d (Lundi n et al., 1994; Li n et al., 1995
; Trokoude s et al., 1995
; Tournay e et al, 1998
; Li u et al, 1995a)
.
Fertilizatio
n rate s wit h ICS I ar e generall y poo r fo r patient s wit h onl y immotil e spermatozo a availabl e i n th e ejaculat e (Nij s et al, 1996). Pregnancie s hav e als o bee n reporte d afte r subzona l inseminatio n (Wol f et al, 1993)
. Th e reaso n wh y ICS I result s ar e s o poo r i n patient s sufferin g fro m immotile-cili a syndrom e i s no t clear . Man y o f th e ultrastructura l defect s ma y hav e concomitan t defect s affectin g othe r microtubula r structure s suc h a s th e sper m centrosome , an d deficiencie s a t thi s leve l ma y b e involve d i n fertilizatio n failur e an d poo r embryoni c developmen t afte r ICS I (Asc h et al, 1995
; Tournay e et al, 1998)
. Tai l stum p syndrome , whic h ma y b e associate d wit h primar y ciliar y dyskinesia , i s als o associate d wit h morphologica l defect s o f th e flagellum resultin g i n sever e 6
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N.Nikolettos et al.
Tabl e III . Influenc e o f sper m morpholog y o n embry o qualit y i n th e swim-u p contro l grou p an d i n th e grou p wit h combinatio n o f glas s woo l filtratio n (GWF ) an d swim-u p techniqu e fo r seme n preparatio nPreparatio
n metho d Norma l sper m Embryo s o f excellen t morpholog y (% ) qualit y (% )Swim-u
p <1 0 8 1 >1 0 7 0GWF-swim-u
p <1 0 7 1 6 9Adapte
d fro m Al-Hasan i et al. (1996 ) asthenozoospermia . Stal f et al. (1995) reported a pregnancy after ICSI of immotile spermatozo a fro m a patien t wit h tai l stum p syndrom e (Stal f et al., 1995). I n conclusion , wit h th e exceptio n o f globozoospermi a an d immotil e cili a syndrome , neithe r th e typ e no r th e exten t o f sper m impairmen t seem s t o hav e a n importan t rol e o n th e outcom e o f ICSI , whic h seem s t o b e th e bes t metho d o f treatmen t i n case s o f sever e male-facto r subfertility .
Relationshi
p betwee n seme n morpholog y an d embry o qualit y i n IV F an d ICS I Som e studie s hav e compare d th e fertilizatio n rate , implantatio n rat e an d embry o qualit y o f patient s wit h sever e teratozoospermi a wh o wer e treate d b y IVF , modifie d IV F usin g hig h inseminatio n concentratio n (HIC ) o r ICSI . I n a stud y (Al-Hasan i et al., 1996), th e impac t o f sper m morpholog y an d th e preparatio n techniqu e o n th e qualit y o f embryo s obtaine d b y IV F wa s foun d t o b e insignifican t (Tabl e III) . Hal l et al. (1995 ) compare d th e us e o f HIC-IV F an d ICS I fo r a simila r grou p o f teratozoospermi c patient s wh o wer e diagnose d usin g stric t criteria . Althoug h th e numbe r o f patient s receivin g a 'pure ' transfe r o f embryo s derive d eithe r fro m HIC-IV F o r ICS I an d th e tota l numbe r o f embryo s examine d wer e small , the y foun d simila r fertilization , implantatio n an d pregnanc y rate s fo r
HIC-IV
F an d ICS I (Hal l et al., 1995). The y conclude d tha t whe n concern s o f safet y ar e full y allaye d an d th e cos t o f ICS I ca n b e brough t int o lin e wit h IVF , the n ICS I ma y becom e th e firs t optio n fo r treatmen t o f teratozoospermi c patients . I n a retrospectiv e comparativ e analysi s o f embry o implantatio n i n patient s wit h sever e teratozoospermi a treate d wit h eithe r HIC-IV F o r ICSI , i t wa s reveale d tha t HIC-IV F resulte d i n a highe r fertilizatio n rat e tha n ICS I i n patient s wit h sever e teratozoospermia, bu t tha t thi s positiv e resul t wa s offse t b y a deleteriou s effec t o n embry o qualit y (Oehninge r et al., 1996)
. Thi s suggeste d a negativ e effec t o n earl y embryogenesi s o f putativ e sper m factor s release d i n th e vicinit y o f th e eggs . Embryo s produce d b y ICS I ha d a significantl y highe r morphologica l scor e and , afte r uterin e transfer , showe d a tendenc y towar d superio r implantatio n an d pregnanc y rate s tha n HIC-derive d embryos .