Sperm morphology assessment using strict criteria and male fertility under in-vivo conditions of conception*

Transcription

1 Huma Reproductio vol.11 o.l pp , 1996 Sperm morphology assessmet usig strict criteria ad male fertility uder i-vivo coditios of coceptio* Waltraud Eggert-Kruse 1 ' 4, Heike Schwarz 1, Gerhard Rohr 2, Traute Demirakca 1, Wolfgag Tilge 3 ad Beo Ruebaum 1 'Departmet of Gyaecological Edocriology ad Fertility Disorders, Wome's Hospital, Uiversity of Heidelberg, VoPstrasse 9, Heidelberg, 2 Departmet of Iteral Medicie FV, Kliikum Maheim ad 3 Adrology Uit, Departmet of Dermatology, Uiversity of Heidelberg, Heidelberg, Germay ''To whom correspodece should be addressed The cliical sigificace of sperm morphology assessmet accordig to very strict criteria was determied usig seme samples of radomly chose males from couples ot submitted to assisted procreatio techiques, with a media duratio of ifertility of 4 years (rage 1-17; = 89). The relatioships of sperm morphological properties to the results of stadard sperm aalysis, icludig the differetiatio of roud cells i seme by moocloal atibodies ad seme cultures, the testig of sperm fuctioal capacity i vitro with the stadardized sperm-cervical mucus peetratio test (SCMPT) ad the subsequet pregacy rate uder i-vivo coditios of coceptio, were evaluated i a prospective study. The quick staiig method (DiffQuick stai) for sperm morphology proved to be practical ad suitable for routie use. The percetage of ormal forms accordig to strict criteria raged from 1 to 36, with a media of 12. Morphological fidigs were ot markedly related to the medical history, but sigificat relatioships betwee stadard parameters of sperm aalysis, i particular the sperm cout, the progressive motility ad stadard sperm morphology, were foud. The percetage of ormal forms was ot sigificatly associated with the microbial coloizatio of seme samples but was egatively related to high leukocyte rates. Seme samples with a higher percetage of ormal spermatozoa (show, for example, for >4, >7 or >14 ormal) were sigificatly more frequet i cases of a adequate SCMPT. The subsequet pregacy rate withi a observatio period of 12 moths was 20J.. The pregacy rate uder i-vivo coditios was sigificatly higher whe seme samples had a better sperm morphology, with sigificat differeces for thresholds at 4,7 ad 14 of strictly ormal forms- Although sperm morphology is oly oe amog a multiplicity of factors determiig fertility, the results suggest that the evaluatio of sperm morphology usig strict criteria provides valuable iformatio durig basic ifertility ivestigatios. This work was preseted i part at the Iteratioal Meetig o 'Adrology i the Nieties', Gek, Belgium, April, Europea Society for Huma Reproductio ad Embryology Key words: male fertility/sperm fuctio/sperm morphology/ sperm mucus iteractio/strict criteria Itroductio Sice the early observatios of va Leeuwehoeck ad Ham (1678) who described the 'aimalcules', sperm morphology has bee a matter of cosiderable debate. Although the assessmet of the morphological properties of spermatozoa is cosidered part of the stadard sperm aalysis of the World Health Orgaizatio (WHO), its cliical sigificace is cotroversial (e.g. MacLeod ad Gold, 1953; Eliasso, 1971; Fredricsso, 1979; Aitkcetal, 1982b; Rogers et al, 1983; Mahadeva ad Trouso, 1984; Dadoue, 1988; Polaski ad Lamb, 1988; Ama, 1989; Duphy et al, 1989; Bostofte et al, 1990, 1992; Davis ad Gravace, 1994). Recetly, very strict criteria for selectig morphologically 'ormal' spermatozoa have bee itroduced, referred to by some authors as 'Tygerberg' or 'Norfolk' criteria, ad correlated with the success of i-vitro fertilizatio (TVF; Kruger et al, 1986, 1988a; Mekveld et al., 1990). However, there is o iformatio about the cliical sigificace of this classificatio of sperm morphology for male fertility uder ivivo coditios of coceptio. I this prospective study, these strict criteria were used for a sperm morphology assessmet i a subfertile populatio ot submitted to ay meuiod of assisted reproductio such as IVF, gamete itra-fallopia trasfer or itrauterie isemiatio. I parallel, seme samples were evaluated usig the stadard procedure of microscopical evaluatio, as recommeded by WHO. A sperm aalysis also icluded seme cultures coverig a broad spectrum of micro-orgaisms, as well as a roud cell differetiatio i leukocytes ad cells of the early stages of spermatogeesis. Furthermore, aliquots of the same seme samples were used i a i-vitro sperm cervical mucus peetratio test (SCMPT) to determie the sperm fuctioal capacity. The female parters of the patiets were submitted to a comprehesive ifertility ivestigatio to detect female factors of ifertility. The relatioship of the sperm morphological assessmet with the cliical ad laboratory fidigs ad the couples' subsequet fertility uder i-vivo coditios of coceptio withi a observatio period of 1 year were determied. Materials ad methods Patiets The study populatio cosisted of 89 radomly chose couples presetig for ifertility ivestigatios at the Ifertility Uit of the Wome's Uiversity Hospital i Heidelberg, Germay. The age of 139 Dowloaded from by guest o 15 November 2018

2 W.Eggert-Kruse et al the male patiets varied betwee 22 ad 45 years (media 33.3); the age of the female parters was betwee 22 ad 40 years (media 30.5). The media duratio of ifertility was 4 years (rage 1-17). Primary ifertility was foud i 70.4 ad secodary ifertility i 29.6 of patiets. Patiets with cliical symptoms or sigs of ifectio of the lower geital tract were excluded from our study, as well as couples with azoospermia of the male parter or a severe female factor of ifertility (e.g. wome with tuba] occlusio, severe adhesios i both Fallopia tubes, severe edometriosis, uterus myomatosus, itrauterie adhesios or uterus duplex). All female parters were ivestigated thoroughly for potetial ifertility factors. Tubal patecy was proved by hysterosalpigography ad/or laparoscopy. Some pelvic pathologies (mostly discrete adhesios of oe Fallopia tube but patecy of the other side, or a mior uterie factor such as a slight hypoplasia) were foud i 32 of the wome, but all had at least oe free patet tube ad thus a reasoable chace of pregacy. A comprehesive, cycle-related edocrial screeig was performed o all the wome, icludig several tests for ovaria ad pituitary fuctio, as well as adreal (e.g. adreocorticotrophic hormoe test) ad thyroid (e.g. thyrotrophi-releasig hormoe test) fuctio, i the early follicular phase, ad multiple determiatios of progesteroe, 17f3-oestradiol ad prolacti cocetratio i the luteal phase, which were completed by further hormoal assays if ecessary. Luteal isufficiecy was defied as a mea progesteroe cocetratio of <10 g/ml, determied o days 5, 7 ad 9 of the hyperthermic phase. I all, 23.0 of the wome had regular ovulatio without ay hormoal disorders ad icludig a adequate luteal phase. Usig this differetiated edocrial examiatio, mior edocrial aomalies (mostly discrete luteal isufficiecy or precliical disorders of thyroid or adreal fuctio) were detected i 64.0 of the wome despite spotaeous ovulatory cycles. Oligoameorrhoea was foud i 12.5 of the wome. If ecessary, specific treatmet was give accordig to the results of the edocrial examiatio, which resulted i regular ovulatory cycles with a adequate luteal phase i all wome i our study. A detailed medical history was obtaied ad a adrological examiatio was performed i all males. All males were apparetly healthy, apart from their complait of beig ivolutarily childless. A routie seme aalysis gave sperm couts of <40X10 6 /ml or a reduced progressive motility (<40) i early half of the me (50.6). I some patiets, adrological treatmet had bee recommeded by colleagues from the Adrology Uit, Uiversity of Heidelberg, Heidelberg, Germay, based o the results of stadard sperm aalyses. The curret medicatio (kallikrei, = 16; petoxifyllie, = 2; testosteroe udecaoate, = 1; goadotrophis, 1) was ot discotiued prior to the study ad the strict morphological assessmet. Durig the time of the study, oe of the couples were submitted to assisted procreatio techiques such as i-vitro fertilizatio (IVF), gamete itra-fallopia trasfer or itrauterie isemiatio. Seme aalysis Ejaculates were obtaied by masturbatio ito sterile glass jars i hospital after a period of sexual abstiece of at least 5 days. Samples were used for all tests directly after liquefactio. A seme aalysis, icludig the determiatio of sperm volume, ph, sperm cout, progressive motility, morphology ad fructose cocetratio, was performed usig routie criteria (WHO, 1987). I parallel, two slides were prepared from each seme sample for a assessmet of the sperm morphology usig the strict criteria, as described i detail by Kruger et al (1986, 1988a). Liquefied seme (5 uj) was pipetted oto carefully precleaed stadard microscope 140 slides ad a thi smear was prepared. Staiig was performed usig the DiffQuick stai set (America Hospital Supply dell Caribe Ic., Aguada, Puerto Rico) usig the solutio for fixatio ad solutios 1 ad 2 for staiig, as recommeded by the maufacturer. After air dryig, slides were rised with distilled water, mouted with Glycergel (Dako, Hamburg, Germay) ad covered with a coverslip. Assessmet was carried out o the same day. A total of 100 spermatozoa were evaluated o each slide usig the high power magificatio (X1000, i oil) of a light microscope. The percetages of ormal ad pathological forms, icludig the morphological idex (combiatio of ormal forms ad mior abormalities) accordig to Kruger et al. (1988a), were determied based o the strict criteria. Throughout the whole study, the strict sperm morphology evaluatio was performed by the same observer. Whe the results of the sperm morphology evaluatio usig the strict criteria o the two slides prepared from each seme specime were correlated, the Spearma rak testig offered a correlatio coefficiet (r) of 0.88 for the percetage of ormal forms ad 0.89 for the morphological idex (P < ). The results of the first slide were used for further comparisos. The percetage of ormal forms was chose for further statistical aalyses as the mai parameter of sperm morphological quality based o the suggestios of Kruger etal. (1986, 1988a). Examiatio ad differetiatio of roud cells Seme smears were prepared ad the percetage of leukocytes of roud cells was determied as described previously (Eggert-Kruse et al., 1992a). Briefly, the umber of roud cells was first couted usig a Neubauer chamber (LDH, Heidelberg, Germay). For the differetiatio of roud cells, moocloal atibodies of high specificity for commo leukocyte atige were used (Dako LC ; Dakopatts, Glostrup, Demark). Staiig was performed with a streptavidibioti system (HistoStai, SP kit; Zymed Laboratories, South Sa Fracisco, CA, USA). Slides were icubated i a blockig solutio to elimiate the o-specific backgroud. Primary atibody, biotiylated secod atibody, ezyme cojugate ad substrate chromogee mixture were applied accordig to the maufacturer's istructios. Smears were the couterstaied with Meyer's haematoxyli ad mouted i a aqueous moutig solutio (Glycergel ; Dako). Positive (peripheral leukocytes) ad egative cotrols were icluded i each of the test series. Usig the high power field of a stadard light microscope, 100 roud cells were examied ad the percetage of leukocytes was determied. All readigs were performed i duplicate, ad the mea value was used i the aalysis. Microbial screeig Aliquots from each ejaculate take for a sperm morphological assessmet were submitted to seme cultures, icludig mycoplasmas {Mycoplasma homiis ad Ureaplasma urealyticum), potetially pathogeic aerobic ad aaerobic bacteria, ad species of the physiological flora. I additio, immuoglobuli (Ig) G atibodies to Chlamydia trachomatis were determied i serum samples of all patiets as a marker for previous ifectio by these micro-orgaisms (idirect fluorescece atibody test; Virgo ; Schiapparelli Biosystems Ic., Columbia). Stadard methods were used for the idetificatio of micro-orgaisms, as described i detail previously (Eggert-Kruse et al., 1992b; Departmet of Microbiology ad Hygiee ad Microbiological Laboratories, Departmet of Dermatology, Uiversity of Heidelberg, Heidelberg, Germay). Simultaeously, a microbial screeig was performed i the female parters of patiets for the determiatio of potetially sexually trasmitted orgaisms. Microbial cultures i female patiets also icluded a evaluatio of the edocerv- Dowloaded from by guest o 15 November 2018

3 Sperm morphology assessmet usig strict criteria ical material for C.trachomatis (McCoy cell culture) ad herpes simplex virus, ad of yeasts i the forix posterior vagialis. Sperm fuctio testig To evaluate sperm fuctioal capacity, the i-vitro SCMPT was performed i parallel with a morphological evaluatio of the spermatozoa with aliquots of the same ejaculates. Cervical mucus from the wives of the patiets, ad additioally cervical mucus from fertile doors i the crossed SCMPT, was used as the peetratio medium. The cervical mucus was take from the edocervix with a special device (Aspiglaire ; IMV, L" Aigle, Frace). The quality of the cervical mucus was classified accordig to Isler et al. (1972), ad the ph was determied with paper strips (ph-idikatorpapier, Merck, Darmstadt, Germay). To obtai the most reproducible results, freshly obtaied cervical mucus from all the wome was used, after stadardized oral treatmet with oestroges (80 ig/day ethiyl oestradiol) for 1 week precedig the test The SCMPT usig this stadardized hormoal approach has bee described i detail elsewhere (Eggert-Kruse et al, 1989a,b). Briefly, capillary tubes were carefully filled with cervical mucus avoidig air bubbles ad sealed at oe ed with modellig clay. With oe drop of cervical mucus protrudig at the other ed, the tubes were placed i the reservoirs of a peetratio meter (Kremer, 1968) filled with fresh seme directly after liquefactio. After icubatio periods of 0.5, 2 ad 6 h i a moist chamber at 37 C, the peetratio distace, sperm desity ad sperm motility grade were determied. Results were summarized i a cumulative SCMPT score. Based o the SCMPT score after a icubatio period of 6 h, samples were selected i those offerig a adequate or iadequate ability to peetrate the cervical mucus i vitro. Statistical methods The pregacy rate was determied after 12 moths. Data were processed usig the statistical aalysis systems SAS ad SPSS. x 2. Fisher's two-tailed exact tests, Wilcoxo's rak-sum tests, Spearma rak correlatio ad logistic regressio aalysis were used. Statistical sigificace was cosidered to be achieved at P < Results Microscopical seme aalysis Stadard criteria Seme aalyses showed oligozoospermia (<20X 10 6 spermatozoa/ml) i 15.7, a progressive motility of = 40 i 49.4 (44/89) ad cosiderable astheozoospermia (<20) i 14.6 of patiets. Some.3 (24/88) of seme samples showed <60 of ormal spermatozoa accordig to stadard WHO criteria, with a media percetage of ormal forms of 63 (rage 24 77). Media values (rages) of the other parameters of stadard sperm aalysis were for ejaculate volume 3.8 ml ( ml), ph 7.3 ( ), viability 65 (45-80) ad fructose cocetratio 1580 g/ml ( ). Sperm cocetratio, progressive motility ad stadard morphology (WHO) were sigificatly iterrelated. I samples offerig 2*60 ormal forms based o WHO criteria, a progressive motility of >40 was sigificatly more frequet (foud i 67.2 of these samples compared with 8.3 of specimes with <60 ormal forms; P <0.0001), ad a sperm cout of 5*20X 10 6 spermatozoa/ml was foud i 92.2 of samples compared with 62.5 i samples with a reduced stadard morphology (P < 0.001; 2 aalysis). Spearma rak testig offered correlatio coefficiets (r) of 0.73 for the percetage of ormal forms based o WHO criteria ad sperm motility (P < ), ad of 0.49 for sperm cout ad progressive motility (/» < ). Strict criteria Markedly less morphologically ormal spermatozoa compared with the stadard classificatio were foud whe the strict criteria (Kruger et al, 1986, 1988a) were applied. The media percetage (rage) for ormal spermatozoa was 12 (1-36). For statistical aalyses, thresholds at 4 ad 14 ormal forms, based o previous reports (Kruger et al., 1986, 1988a), were used, with a additioal cut-off at 7 ormal forms. Sperm morphology accordig to strict criteria was sigificatly improved whe routie parameters showed adequate progressive motility (>40), a sperm cout of >20XlO 6 /ml or 5*60 ormal forms based o the WHO classificatio, as ca be see i Table I. Spearma rak testig offered a sigificat correlatio of these parameters with strictly determied sperm morphology. Microbial screeig The majority of ejaculates were coloized with bacteria. Potetially pathogeic aerobic species (Escherichia coli, B- streptococci, eterococci, Proteus mirabilis, Staphylococcus aureus ad others with a lower prevalece) were foud i 53.4 (47/88), potetially pathogeic aaerobic microorgaisms i 6.8 ad mycoplasmas (M.homiis ad/or U. urealyticum) were cultured i 12.5 of seme samples. Additioally, species of the physiological flora were foud i 85.2 oif ejaculates. Microbial fidigs were ot sigificatly related to the morphological properties of the spermatozoa with regard to cut-offs at 4, 7 ad 14 strictly ormal forms, as well as to medias ad rages. I additio, o sigificat relatioship was foud with respect to IgG atibodies to C.trachomatis, which were elevated (titre 3=1:256) i serum samples of 11 of me. Furthermore, the results of the morphological assessmet i seme samples did ot show a relatioship with the microbial fidigs i the lower geital tract of the female parters. Differetiatio of roud cells The outcome of roud cell differetiatio usig moocloal atibodies was related to the sperm morphological evaluatio usig strict criteria i 48 specimes (53.9). Roud cells were foud i all specimes ad leukocytes i 72.4 of samples. The percetage of leukocytes correlated sigificatly with the umber of leukocytes per ml, as well as the umber of leukocytes per ejaculate (r > 0.8, P < ). The media percetage of leukocytes of the roud cells was 3 (rage 0-53). For a statistical aalysis, cut-offs at leukocyte rates of 3 (media), 10 ad 15 were used. Ejaculates with 5*15 of leukocytes of the roud cells were cosidered 'leukocyte positive', based o previous observatios (Eggert- Kruse et al., 1992a). The umber of roud cells was ot sigificatly related to strictly determied sperm morphological properties. The associatio betwee leukocyte rates ad sperm morphology 141 Dowloaded from by guest o 15 November 2018

4 W.Eggert-Krus* et al Tab\e L Relatioship betwee strict morphological criteria ad stadard parameters of sperm aalysis Stadard parameter Sperm morphology (strict criteria) ormal forms* ormal forms* Total Correlatio coefficiet (r) b =s7 >7 <14 5»14 c " * Sperm cout»20 X \()P/ml Progressive motility *40 Stadard morphology *60 ormal Total ' 33.3' 44.4 f e 58.1' 85.3 f 69.7 *X 2 aalysis, compared with correspodig group below the threshold idicated. b Spearma rak correlatio. dumber of patiets per group. ''Percetage of patiets per group. 'P < 'P < STotal umber per group with >1 ormal forms = 61. 'Total umber per group with <14 ormal forms = 53. Table IL Relatioship of leukocyte rates after roud cell differetiatio ad sperm morphology usig strict criteria Normal forms (strict criteria) >4 >7»14 Total f Leukocyte rate* c " h ' 60.4' 60.7 NS = ot sigificat. Immuologically determied percetage of leukocytes of the roud cells i seme. "Fisher's two-tailed exact test, compared with correspodig groups below the threshold idicated. 'Number of patiets per group. d Percetage of patiets per group. 'Cosidered 'leukocyte positive', based o previous reports. 'Total umber ad percetage of patiets ' 68.6' 91.4' P value b s»15 e P value b Total NS NS based o the strict criteria is show i Table II. I 'leukocyte positive' samples, ormal sperm morphology was sigificatly less frequet (P < 0.05). Noe of these samples showed >14 ormal forms evaluated with strict criteria. Sigificat differeces were also see whe a threshold at 10 leukocytes was used (P < 0.05 for cut-offs at 4, 7 ad 14 ormal forms, Fisher's two-tailed exact tests), e.g. 5*14 strictly ormal sperm forms were foud i 64.1 (25/39) of samples whe the leukocyte rate was <10 compared with 22.2 (2/ 9) i cases where roud cell differetiatio offered a higher leukocyte rate (3=10). Sperm fuctio testig The sperm mucus iteractio i vitro was evaluated usig the i-vitro SCMPT with cervical mucus from the female parters i 82 couples (92.1). The outcome of this migratio test, based o the cumulative SCMPT score after 6 h observatio, was iadequate i 39 ad adequate i 61 of cases. The relatioship betwee SCMPT results ad sperm morphology assessmet is show i Table m. Sperm morphology based o strict criteria was sigificatly better i the group with ^ 0.3 l r 0.26' adequate SCMPT outcome. This could be demostrated for thresholds at 4, 7 ad 14 ormal forms. The sigificace of sperm morphological properties for mucus migratio ability i vitro was cofirmed whe the cervical mucus of fertile doors, also obtaied uder stadardized coditios, was used for the crossed SCMPT ( = 81; see Table m). Medical history The medical histories revealed geeral illesses i 11.2, mumps i 69.0 ad previous geital ifectios i 14.0 of the patiets. Of the me, 20.9 reported geital surgery ad 8.1 varicocoelectomy. Noe of the me remembered ay geital ijury. I all, 22.5 of the patiets ( = 20) received adrological medicatio, predomiatly kallikrei (16/20). I 4.5 of the me the duratio of adrological treatmet was 2=2 years. These variables, as well as the age of the patiets, smokig (>10 cigarettes/day i 33.3), alcohol cosumptio (55.7), stress due to workig coditios (28.4) or ifertility ivestigatio ad treatmet (11.3), ad ifertility factors i their Dowloaded from by guest o 15 November 2018

5 Sperm morphology assessmet usig strict criteria Table III. Relatioship of sperm morphology usig mucus peetratio test (SCMFIT strict criteria ad sperm fuctioal capacity, evaluated with the stadardized i-vitro sperm-cervical Normal forms (strict criteria) Adequate 6 Iadequate 5 Total P value c SCMPT I (with parters' cervical mucus) d >4 >7 3=14 Total f Crossed SCMPT (SCMPT II) (with doors' cervical >4 >7 *14 Total f mucus) c Sperm-mucus iteractio testig performed i vitro uder hormoally stadardized coditios. b Based o the total SCMPT score after 6 h of icubatio. X 2 aalysis or Fisher's two-tailed exact test, compared with the correspodig group below the threshold value idicated. d SCMPT performed with fresh cervical mucus from the female parters of the patiets. ^Cervical mucus used for crossed SCMPT obtaied from fertile female doors. f Total umber ad percetage of patiets Table IV. Sperm morphology usig strict criteria ad subsequet fertility uder i-vivo coditios of coceptio* Normal forms Pregacy rate No. pregat/total o. P value b (strict criteria) () of couples per group =S4 >4 *7 > 7 < 14 s=14 Total NS = ot sigificat. "Determied after 12 moths. b Fisher's two-tailed exact test 1/10 17/79 1/ 17/62 6/54 1ZG5 18/89 NS <0.02 <0.01 female parters, did ot offer a sigificat relatioship with the outcome of the sperm morphological assessmet usig strict criteria. Pregacy rate The pregacy rate after 12 moths i this prospective study was 20.2 (18/89). The relatioship of sperm morphology usig strict criteria ad subsequet fertility is show i Table IV. Statistical aalysis revealed marked differeces for the percetage of ormal forms based o strict criteria whe 4, 7 or 14 ormal forms were take as cut-off values. The pregacy rate was more tha three times higher whe seme samples had 5*14 ormal forms compared with those havig <14 ormal spermatozoa (34.3 versus 11.1; P < 0.01). For a threshold value of 7 ormal forms, pregacy rates were.4 whe seme samples had 3=7 ormal forms compared with 3.7 for those havig <7 ormal spermatozoa. Sigificat differeces were also foud whe 20 strictly ormal was take as a additioal cut-off value. The subsequet pregacy rate was 14.1 (10/71) whe morphological assessmet based o strict criteria offered «20 ormal forms, compared with 44.4 (8/18) whe samples showed >20 ormal spermatozoa (P < 0.01, <0.04 <0.04 <0.03 <0.04 <0.04 <0.03 Fisher's two-tailed exact test). However, poor sperm morphology (= 4 ormal) did ot exclude a subsequet pregacy. Sigificat differeces i the sperm morphological properties of those patiets who did ad those who did ot achieve a pregacy withi the observatio period were also foud whe media ad rage values were compared with a media percetage of strictly ormal spermatozoa of 16.5 (rage 3-33) i the fertile group compared with 10.0 (rage 1-36) i the o-fertile group of me (P < 0.01, Wilcoxo's raksum test). A logistic regressio aalysis icludig, i additio to strictly determied sperm morphology, the basic parameters of routie sperm aalysis (sperm cout, motility ad stadard morphology) offered a P value of 0.02 for the percetage of ormal forms accordig to strict criteria; the other parameters did ot achieve statistical sigificace. Whe stadard sperm morphology was excluded from the logistic regressio model, sperm motility also achieved sigificace (P < 0.02), whereas the impact of the other parameters remaied uchaged (sigificat with P < 0.02 for strictly determied sperm morphology, ot sigificat for sperm cout). The cliical relevace of the morphological properties for subsequet sperm fertilizig capacity was cofirmed i further statistical aalyses, after exclusio of, for example, those couples offerig potetially ifluecig female factors of ifertility (e.g. mior tubal pathology or hormoal disorders such as oligoameorrhoea prior to treatmet). The pregacy rate i this subgroup was slightly higher (14/52, 26.9). Agai, the percetage of strictly ormal forms i seme samples (with regard to the differet threshold values ad the medias ad rages) was sigificatly related to the subsequet fertility of the couples uder i-vivo coditios of coceptio, e.g. the pregacy rate was 47.6 i cases whe 3=14 ormal forms were foud compared with 12.9 whe a morphological seme evaluatio usig the strict criteria proved <14 ormal spermatozoa (P < 0.01), as see i Table V. Dowloaded from by guest o 15 November

6 W.Eggert-Kruse et al Table V. Sperm morphology usig strict criteria ad subsequet fertility uder i-vivo coditios of coceptio (evaluated after the exclusio of couples with mior factors of female ifertility)* Normal forms Pregacy rate No. pregataotal o. P value 6 (strict criteria) () of couples per group <H > «7 6.3 > < * Total 26.9 NS = ot sigificat. Determied after 12 moths. Viser's two-tailed exact test. Discussio 1/7 13/45 1/16 13/36 4/31 10/21 14/52 NS <0.01 The role of the traditioal seme parameters, icludig sperm morphology as a progostic idicator of sperm fertilizig capacity, is a matter of costat debate (e.g. Falk ad Kaufma, 1950; Page ad Houldig, 1951; Hitig et al., 1980; Aitke et al., 1982b; Hargreave ad Elto, 1983; Bostofte et al, 1985, 1992; Jeuli et al., 1986; Liu ad Baker, 1988, 1992; Polaski ad Lamb, 1988; Ama, 1989). Some authors have idicated that the morphological evaluatio of spermatozoa usig a very strict classificatio is of value i IVF protocols (Kruger et al, 1986, 1987a, 1988a,b), but there is a lack of iformatio for these criteria to be useful uder i-vivo coditios. Usig this strict classificatio, markedly less 'ormal' spermatozoa are foud tha with the stadard procedure employed i most laboratories. The quick staiig method used i our study proved to be easy ad practical, ad allowed the clear differetiatio of spermatozoa. Advatages of the DiffQuick stai techique over the classic Papaicolaou stai are a complete staiig to readig time of <7 mi, commercially prepared reagets ad a easy staiig procedure. Care has to be take that slides are cleaed thoroughly ad that a very thi slide is prepared to avoid backgroud stais that could egatively ifluece the results. A sperm morphological evaluatio is cosidered to be a highly subjective procedure. Ulike the haematopoietic cells for example, the difficulty i classifyig huma sperm morphology is compouded by the large variety of abormal forms ecoutered i the seme of ifertile me. Oly certai types of abormality ca be quatitated objectively. Comparative studies carried out >20 years ago cocluded that the assessmet of sperm morphology was persoality orietated, qualitative, o-repeatable ad difficult to teach to studets ad techicias (Freud, 1966). O the other had, a low iter- ad itratechicia variability usig strict criteria has bee reported (Kruger et al, 1987b). I our study, the laboratory error i determiig the sperm morphological properties (by strict criteria) was miimized by usig oe observer for all assessmets. The percetage of ormal forms was take as a parameter for morphological quality, ad offered a media percetage of ormal forms of 12 (rage 1-36). This correspods well with the 12 (rage 0-29) reported by Kruger et al (1987b) but is lower tha i other studies of the Tygerberg group 144 (Kruger et al, 1988a,b). Ulike the selected study populatio of these authors (Kruger et al, 1986, 1987a,b, 1988a,b), samples with <30 motility or <2OX1O 6 spermatozoa/ml were ot excluded. Our fidigs cofirm previous reports, some of which were published may years ago, which demostrated that the differet characteristics of seme quality are sigificatly iterrelated (Falk ad Kaufma, 1950; Page ad Houldig, 1951; MacLeod ad Gold, 1951, 1953; Overstreet et al, 1981; Bostofte et al, 1984). I the preset study, this correlatio could be show particularly for stadard morphology ad other seme parameters, but also with regard to strictly determied sperm morphological properties ad, for example, sperm cout ad motility. These factors must be take ito cosideratio whe iterpretig the morphological fidigs. Sperm morphology is regarded as a relatively stable parameter. Abormal sperm morphology may be a reflectio of poor testicular physiology. No sigificat relatioship was foud betwee the medical history ad the results from the cliical examiatio. As the impairmet of testicular fuctio might be caused by iflammatory processes, all of the ejaculates were screeed for microbial coloizatio ad leukocytospermia, although all patiets were asymptomatic i terms of geital tract ifectio. The majority of ejaculates were coloized by bacteria, i accordace with the fidigs of a much larger subfertile populatio (Eggert-Kruse et al, 1992b). However, this did ot iterfere with the outcome of the strict morphological assessmet. Nevertheless, these observatios do ot exclude the fact that the results might be quite differet i me with curret ifectios of the lower geital tract. Elevated rates of leukocytes i seme are a potetial marker for a subcliical ifectio, which may have a effect o sperm fuctioal capacity ad subsequet fertility. The relatioship betwee leukocyte rates ad sperm morphology usig strict criteria has ot bee reported previously. It was show that roud cells maily represeted early spermatogeic cells, with a media percetage of leukocytes of oly 3. Because the relevace ad upper limit of ormality of white blood cells i seme is cotroversial (Wolff, 1995), differet cut-off values were used to relate the leukocyte rates to the morphological fidigs. I 'leukocyte positive' samples, defied accordig to previous reports (Eggert-Kruse et al, 1992a), a poor sperm morphology accordig to strict criteria was sigificatly more frequet, possibly reflectig impaired spermatogeesis. Our study idicates that sperm morphological properties accordig to strict criteria are related to sperm fuctioal capacity. A poor sperm morphology based o strict criteria was sigificatly more frequet whe the results of the SCMPT with cervical mucus from the female parters were iadequate. The sigificace of morphological properties for the ability of spermatozoa to migrate could be cofirmed whe door cervical mucus was used i the crossed SCMPT. This highlights the marked filterig capacity of huma cervical mucus for abormally cofigured spermatozoa (Fredricsso ad Bjork, 1977; Perry et al., 1977; Jeuli et al., 1985), particularly for spermatozoa with abormal heads (Kremer, 1968; Haso ad Overstreet, 1981). O the other had, the sigificat correlatio of sperm morphology with progressive motility (Overstreet Dowloaded from by guest o 15 November 2018

7 Sperm morphology assessmet usig strict criteria et al., 1981; Katz et al., 1982), also kow to be importat for cervical mucus peetratio (Kremer, 1968; Aitke et al., 1985; Eggert-Kruse et al., 1989b), has to be cosidered. The relevace of a more differetiated sperm pathology (with regard to the differet types of aomaly of the sperm head, eck ad tail accordig to strict criteria) for the sperm-mucus iteractio i vitro ad i vivo is evaluated i aother study (Eggert-Kruse et al., 1995). The associatio of seme quality ad male ifertility has bee recogized for >40 years (MacLeod ad Gold, 1953). However, there is o agreemet as to what characteristics are uique to a fertile spermatozoo. The defiitio of morphological 'ormality' is a matter of debate, as are the cliically relevat limits for the rate of pathological forms. The barrier fuctio of the cervix is a importat factor for fertility uder i-vivo coditios of coceptio. Ulike reports o morphology ad IVF outcome (Hitig et al., 1980; Mahadeva ad Trouso, 1984; Jeuli etal, 1986; Krugerefa/., 1986, 1987a, 1988a,b; Liu ad Baker, 1992), this factor has ot bee overcome with assisted reproductive techologies i our study. Uder i-vivo coditios of coceptio, it ca also be demostrated that the pregacy rate was sigificatly higher i cases of better sperm morphology based o strict criteria whe threshold values for the rate of ormal forms, defied i FVF studies (e.g. Kruger et al., 1988a,b), were used; this was also the case whe the media rate of ormal spermatozoa was used. For example, the pregacy rate withi 12 moths of a sperm morphological assessmet was 34.3 whe 5=14 ormal forms were foud i the seme samples, ad 11.1 whe <14 were foud (P < 0.01); with regard to a cut-off of 7, cumulative pregacy rates were.4 versus 3.7 (P < 0.02) whe 5=7 versus <7 ormal forms were foud i the seme samples. Sigificat differeces were also foud whe 20 ormal forms was take as a additioal limit (P < 0.01). However, it could also be demostrated that very poor sperm morphology (<4 ormal forms) did ot exclude subsequet fertility uder i-vivo coditios. Particularly uder the usual i-vivo coditios of coceptio, pregacy rates are iflueced by a multiplicity of differet parameters. Subfertility has to be cosidered as a problem of the couple. Therefore, both parters were submitted to a comprehesive ifertility ivestigatio i our study. Couples with a cliically cosiderable female factor of ifertility, e.g. severe tubal pathology, were excluded. With regard to their female parters, all patiets had a reasoable chace of achievig a pregacy. The cliical relevace of discrete hormoal disorders, e.g. some luteal isufficiecy or borderlie results of fuctio tests, i cases of spotaeous ovulatory cycles is cotroversial. However, because mior disorders i female parters could also potetially ifluece the pregacy rate, additioal statistical aalyses were performed i this study payig particular attetio to these variables, which cofirmed the sigificat relatioship of strictly determied sperm morphology ad subsequet fertility. O the other had, with regard to the male factor, strictly assessed sperm morphology must be cosidered as oly oe of may cliically relevat variables. The medical history ad the results of a cliical adrological examiatio of the patiet have to be carefully aalysed, as well as psychosexual factors ad potetial evirometal iflueces. The iterrelatioships of the differet parameters of microscopical seme aalysis should also be cosidered, as well as biochemical factors ad the ability of the spermatozoo to peetrate the cervical mucus ad reach the site of fertilizatio [o pregacy was achieved i our study whe SCMPT offered a iadequate result (P < 0.01), cofirmig previous fidigs (Eggert-Kruse et al., 1989a,b). I additio, the duratio of the patiets' ifertility is also importat (e.g. MacLeod ad Gold, 1953; Aafjes et al., 1978), as well as may other adrological factors. Therefore, durig ifertility follow-up, it is ecessary to base the estimatio of fertility progosis ad therapeutic decisios ot oly o oe but o several differet determiats. Although the evaluatio of strict morphology aloe is ot sufficiet for a exact defiitio of sperm fertilizig capacity, a sperm morphological assessmet accordig to strict criteria provides valuable iformatio about sperm quality ad is therefore useful for basic ifertility ivestigatios. Refereces Aafjes, J.H., va der Vijver, CM. ad Scheck, P. (1978) The duratio of ifertility: a importat datum for the fertility progosis of me with seme abormalities. Fertil SteriL, 30, Aitke, RJ., Best, F.S.M., Richardso, D.W., Djahabakhch, 0. ad Les, M.M. (1982a) The correlates of fertilizig capacity i ormal fertile me. Fertil SteriL, 38, Aitke, RJ., Best, F.S.M., Richardso, D.W., Djahabakhch, O., Mortimer, D., Templeto, AA ad Les, M.M. (1982b) Aalysis of sperm fuctio i case of uexplaied ifertility: covetioal criteria, movemet characteristics ad fertilizig capacity. FertiL SteriL, 38, Aitke, RJ., Sutto, M., Warer, P. ad Richardso, D.W. (1985) Relatioship betwee the movemet characteristics of huma spermatozoa ad their ability to peetrate cervical mucus ad zoa-free hamster oocytes. J. Reprod., FertiL, 73, 441^t49. Ama, R.P. (1989) Ca the fertility potetial of a semial sample be predicted accurately? J. AdroL, 10, Bostofte, E., Serup, J. ad Rebbe, H. (1984) Iterrelatios amog the characteristics of huma seme, ad a ew system for classificatio of male fertility. FertiL SteriL, 41, Bostofte, E., Serup, J. ad Rebbe, H. (1985) The cliical value of morphological ratig of huma spermatozoa. It. J. FertiL, 30, Bostofte, E., Bagger, P., Michael, A. ad Stakema, G. (1990) Fertility progosis for ifertile me: results of follow-up study of seme aalyses i ifertile me from two differet populatios evaluated by the Cox regressio model. FertiL SteriL, 54, Bostofte, E., Bagger, P., Michael, A. ad Stakema, G. (1992) The sperm peetratio test (P-test) ca predict fecudability i the male parter from ifertile couples. Adrologia, 24, Dadoue, J.P. (1988) Ultrastructural abormalities of huma spermatozoa. Hum. Reprod., 3, Davis, R.O. ad Gravace, C.G. (1994) Cosistecy of sperm morphology classificatio methods. J. AdroL, 15, Duphy, B.C., Neal, LJvt. ad Cooke, I.D. (1989) The cliical value of covetioal seme aalysis. 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8 W.Eggert-Kruse et al Eggert-Kruse, W., Reima-Aderse, J., Rohr, G., Poh], S., Tilge, W. ad Ruebaum, B. (1995) Cliical relevace of sperm morphology assessmet usig strict criteria ad relatioship with sperm-mucus iteractio i vivo ad i vitro. Fertil. SteriL, 63, Eliasso, R. (1971) Stadards for ivestigatio of huma seme. Adrologia, 3, Falk, H.C. ad Kaufma, S.A. (1950) What costitutes a ormal seme? Feil. Steril.. 1, Fredricsso, B. (1979) Morphologic evaluatio of spermatozoa i differet laboratories. Adrologia, 11, Fredricsso, B. ad BjOrk, G. (1977) Morphology of postcoital spermatozoa ad the cervical secretio ad its cliical sigificace. FertiL Steril., 28, Freud, M. (1966) Stadards for the ratig of huma sperm morphology: a cooperative study. It. J. Fertil., 11, Haso, F.W. ad Overstreet, J.W. (1981) The iteractio of huma spermatozoa with cervical mucus i vivo. Am. J. Obstet. GvecoL, 140, Hargreave, T.B. ad Elto, R.A. (1983) Is covetioal sperm aalysis of ay use? Br. J. 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Thesis, Uiversity of Groige, The Netherlads. Kruger, T.F., Mekveld, R., Stader, F.S.H., Lombard, C.V., va der Merve, J.P. et al., (1986) Sperm morphologic features as a progostic factor i i vitro fertilizatio. Fertil. SteriL, 46, Kruger, T.F., Acosta, A.A., Simmos, K.F., Swaso, RJ., Matta, J.F. et al., (1987a) New method of evaluatig sperm morphology with predictive value for huma i vitro fertilizatio. Urology, 30, Kruger, T.F., Ackerma, S.B., Simmos, K.F., Swaso, RJ., Brugo, S.S. ad Acosta, A.A. (1987b) A quick, reliable staiig techique for huma sperm morphology. Arch. Adrol, 18, 5-7. Kruger, T.F., Acosta, A.A., Simmos, K.F., Swaso, RJ., Matta, J.F. ad Oehiger, S. (1988a) Predictive value of abormal sperm morphology i i vitro fertilizatio. Fertil. Steril, 59, Kruger, T.F., Haque, D., Acosta, A.A., Pleba, P., Swaso, RJ., Simmos, K.F., Matta, J.F., Morshedi, M. ad Oehiger, S. (1988b) Correlatio betwee sperm morphology, acrosi, ad fertilizatio i a IVF program. Arch. Adrol, 20, Liu, DJ. ad Baker, H.W.G. (1988) The proportio of huma sperm with poor morphology but ormal itact acrosomes detected with Pisum sativum agglutii correlates with fertilizatio i vitro. FertiL Steril,'S0, Liu, DJ. ad Baker, H.W.G. (1992) Tests of huma sperm fuctio ad fertilizatio i vitro. Fertil. SteriL, 58, MacLeod, J. ad Gold, RZ (1951) The male factor i fertility ad ifertility. IV. Sperm morphology i fertile ad ifertile marriage. FertiL SteriL, 2, 394-^14. MacLeod, J. ad Gold, R.Z. (1953) The male factor i fertility ad ifertility. IV. Seme quality ad certai other factors i relatio to ease of coceptio. FertiL SteriL, 4, Mahadeva, M.M. ad Trouso, A.O. (1984) The ifluece of semial characteristics o the success rate of huma i vitro fertilizatio. FertiL Steril, 42, Mekveld, R., Stader, F.S.H., Kotze, TJ.W, Kruger, T.F. ad va Zyl, J.A. (1990) The evaluatio of morphologic characteristics of huma spermatozoa accordig to stricter criteria. Hum. 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(1995) The cliical sigificace of white blood cells i seme. Feil. SteriL, 63, World Health Orgaizatio (1987) WHO Laboratory Maual for the Examiatio of Huma Seme ad Seme-Cervical Mucus Iteractio. 2d ed, Cambridge Uiversity Press, Cambridge, UK. Received o August 8, 1994; accepted o September 22, 1995 Dowloaded from by guest o 15 November 2018