DISTRIBUTION AND PROPERTIES OF SPERMATOZOA IN DIFFERENT FRACTIONS OF SPLIT EJACULATES*

FERTILITY AND STERILITY Copyright 1972 by The Williams & Wilkis Co. Vol. 23, No.4, April 1972 Prited i U.S.A. DISTRIBUTION AND PROPERTIES OF SPERMATOZOA IN DIFFERENT FRACTIONS OF SPLIT EJACULATES* R. ELIASSON, M.D., AND CHR. LINDHOLMER, M.D. Reproductive Physiology Uit, Departmet of Physiology, Faculty of Medicie, Karoliska Istitutet, Stockholm, Swede It is kow that the various glads cotributig to the huma seme discharge their products successively i a wellcotrolled patter. The first portio cotais maily prostate secretio, the midportio the highest cocetratio of spermatozoa, ad the last portio maily secretio from the semial vesicles. Durig the ejaculatio there is a partial mixture of the various secretios, the degree of which will ifluece the relative proportio of the various costituets i the fractios of a split ejaculate. If the ejaculate is collected i oly two fractios the first half will ormally cotai the mai part of the spermatozoa. 1 5 This observatio has led to a recommedatio to use the first part of the ejaculate for isemiatio (AIR) whe the husbad's seme was oligozoospermic or had a high volume. A additioal reaso for such a recommedatio was the observatio that the motility ad morphology of the spermatozoa were better i the first portio tha i the secod. 5 - The preset study was udertake to aalyze statistically how the desity ad some properties of the spermatozoa differed from each other i successive parts of the ejaculate ad from that i the whole seme. MATERIALS AND METHODS Seme samples were obtaied from 4 medical studets ad 7 ifertile patiets Received August 6, 1971. * Supported by the Swedish Medical Research Coucil (l4x-53) ad The Populatio Coucil. 252 with seme volumes over 6 ml. ad sperm desities below 55 millio/ml. Each subject delivered 1-3 (mea 2) seme samples for routie aalyses. I additio, each of them delivered 1-6 split ejaculates accordig to the guide lies give by Ludquist 4 ad Eliasso. I The umber of fractios obtaied varied betwee 4 ad 6. Each subject delivered his specimes withi a 3-moths' period. The specimes were collected by masturbatio after 3-5 days of cotiece. The aalyses icluded sperm desity, sperm motility (progressive ad quatitative), sperm viability, sperm morphology, as well as seme volume, acid phosphatase activity, fructose, ad zic cocetratios. The methods have bee preseted i detail elsewhere. 9, 10 For some of the split-ejaculate fractios the small volumes did ot allow for all aalyses, ad they were the give priority i the order give. The techical assistats, who performed the aalyses, were ot aware of the purpose of the study, or did they kow from whom the specimes origiated. This preveted all forms of bias i the aalytical work. The precislo of the various methods have bee preseted i the origial publicatios. The precisio of the morphologic aalyses, however, was agai checked i this study by radomly selectig 5 specimes. These were remarked ad a moth later evaluated by the techicia i charge of the laboratory. To obtai a basis for compariso the fractio i each experimet that had the highest sperm desity was used as refer-

April 1972 PROPERTIES OF SPERMATOZOA 253 ece (Fractio 0). The fractio before this was desiged -1, the fractios followig the referece +1, +2, etc. Noe of the subjects delivered the ejaculates i more tha 6 fractios. Ordiary statistical methods were used (N = umber of specimes or fractios; = degrees of freedom). 11 RESULTS Methodologic Aspects. Five radomly selected seme smears were examied twice. Paired aalysis demostrated that there was o sigificat differece betwee the results, e.g., the mea differece for "ormal spermatozoa" was 0.% (stadard error of mea of differeces 2.9, p = 0.). Voluteers. Oe of the voluteers preseted seme specimes with low sperm desity (10-20 millio/mi.). The specimes from the others were regarded ormal accordig to the criteria give. The distributio of the spermatozoa i the various fractios ad some of their properties are preseted i Fig. 1 ad Table 1. The mai part of the cells were foud i the first half of the ejaculate. The motility (both progressive ad quatitative) de- 150-2 -1 0 1 2 3 FRACTION NUMBER 100 ~... o <: :::j "''''.,,, ",,,, "''''" " l:~...... ~~ 00 so ~ ~ FIG. 1. Distributio ad properties of spermatozoa i various fractios of 17 split ejaculates from 4 healthy voluteers. The meas ad S.D. for the percetage motile ad live spermatozoa are preseted. I each experimet the fractio havig the highest sperm desity was used as a referece (Fractio 0). TABLE 1. Values for Sperm Motility. Viability. ad Morphology i the Various Fractios of Split Ejaculates from 4 Voluteers t* t t -1 0 47 1.11 2. 0.46 64 1.23 55 67 62 6 N 19 3.0 Fractio Per cet motile spermatozoa 43 35 27 1 2.61 5.0t 4. lot 1O.22t 17 17 17 10 Progressive motility 2.7 2.6 2.4 2.4 2.65 3.63t 4.2lt 4.42t 1 16 15 Per cet live spermatozoa 62 56 4 46 1. 75 2.05 4.22t 2.57 1 15 14 6 69 19 Per cet ormal spermatozoa 6 1 65 16 74 15 6.25t 2 '" t Values refer to paired aalyses betwee a give fractio ad Fractio O. tp< 0.001. + p< 0.005. creased with icreasig fractio umbers. A correspodig impairmet of the viability was oted, but oly i Fractio 3 was the differece statistically sigificat (p < 0.005) from that i the referece fractio (Fractio 0). A iterestig observatio is that motility was more impaired i the later fractios tha correspoded to the decrease i per cet live spermatozoa (Fig. 1). The per cet spermatozoa with ormal cofiguratio was the same i all fractios. (The differece betwee Nad is due to the fact that i some fractios the volume was too small to allow for all aalyses-see "Methods"). The properties of the spermatozoa i Fractio 0 was compared with those i the whole ejaculates from each idividual. No differece was oted betwee the mea values for morphology, motility, or viability. Part of the results is preseted i Table 2. Patiets. Seve patiets with seme volumes betwee 7 ad 10 ml. ad sperm 2.0 49 75

254 ELIASSON AND LINDHOLMER Vol. 23 TABLE 2. Values for the Per Cet Motile ad Dead Spermatozoa i the Fractios with the Highest Sperm Desity (Fractio 0) ad the Whole Ejaculates from 4 Voluteers Subject Fractio 0 Motile spermatozoa % Dead spermatozoa % Sperm desity (l0'/ml.) Whole t Value Fractio 0 Whole t Value Whole ejaculates ejaculates ejaculates A 62.0 55.0 2.3 25.0 134 (N = 3) (N = 1) (N = 3) (N = 1) (N = 1) B 4.3 47.5 0.04 51.3 50 0.15 12 (N = 3) (N = 2) (N = 3) (N = 2) (N = 2) C 51.7 4.3 1. 76 33.2 2 0.0 4 (N = 6) (N = 3) (N = 6) (N = 3) (N = 3) D 61.0 62.5 0.44 27.0 3.5 1.97 10 (N = 5) (N = 2) (N = 5) (N = 2) (N = 2) values for sperm desity i the whole ejaculates are also preseted. t Values calculated for meas of upaired data. No sigificat differece was oted. desity below 55 millio/ml. (i most cases below 40 millio/ml.) were asked to preset split ejaculates i the same way as the voluteers. The properties of the spermatozoa i the most sperm rich fractio were compared with those of whole specimes from the same idividuals. Part of the results is preseted i Table 3. The oly differece betwee the results i Tables 3 ad 2 cocers the per cet spermatozoa with ormal cofiguratio. It is i specimes from the patiets probably higher i the sperm rich fractio tha i the whole ejaculate (p < 0.01). No differece could be oted with regard to motility ad viability of the spermatozoa. DISCUSSION Earlier observatios showig that the mai part of the spermatozoa is preset i the first half of the huma ejaculate have bee further cofirmed i this study. We could also cofirm previous reports 5, 7, that the motility of the spermatozoa (qualitative ad quatitative) is sigificatly impaired i the later fractios compared to that i the first fractios. However, with regard to morphology of the spermatozoa there was o differece betwee the various fractios. Eliasso ad TreichPo have demostrated a high correlatio betwee motil- TABLE 3. Values for Differet Parameters Measured for Whole Seme ad the Most Sperm Rich Fractio of Split Ejaculates from 7 Patiets with High Seme Volumes ad Low Sperm Desities Whole seme Fractio with highest sperm desity Sperm desity, 27 165 106/ml. Motile sperma- 56 56 tozoa, per cet Live spermatozoa, 65 59 per cet Normal sperma- 59 66 tozoa, per cet Seme volume, 7.2 1.1 milliliter t Values calculated as for Table 2. tp <0.001. :j: p <0.01. t Value* 5.t 0.00 1.21 3.3:j: ity ad viability of the spermatozoa whe the latter is determied with the preset supravital staiig method. The discrepacy oted i this study betwee motility ad viability i the later fractios (Table 1) may therefore idicate the presece of a factor i the secretio from the semial vesicles that specifically suppresses sperm motility or a stimulatig factor i the prostate fluid. Evidece for a stimulatig effect of the prostate secretio o sperm motility has, however, ot bee reported. 12

April 1972 PROPERTIES OF SPERMATOZOA 255 It is usually accepted that sperm motility ad morphology are more importat for male fertility tha sperm desity. If, therefore, the recommedatio to use the first part of the ejaculate for isemiatio should have a sigificat basis oe would require that the spermatozoa i the fractio with the highest sperm desity had better motility ad morphology tha i the whole ejaculate. Such a differece could, however, ot be oted i our experimets except for the probably higher per cet "ormal" spermatozoa i the sperm rich fractios from the patiets. The real sigificace of a differece betwee 66 ad 59% spermatozoa with ormal cofiguratio must, however, be ope for doubt. Our results are at some variace with these reported by Amelar ad Hotchkiss. Oe reaso could be that we have aalyzed several samples from each subject withi a short period of time ad refer to differeces i mea values. O the other had, the other authors base their opiio o aalyses of specimes from a much larger umber of subjects. The oly previously published study cocerig this subject i which the data has bee preseted i such a way that a statistical aalysis has bee possible is that by Amelar ad Hotchkiss. Whe the t test for paired aalysis was applied to the data for patiets whose wives coceived followig isemiatio with the first portio of a split ejaculate it was sigificat that the motility was better (p < 0.005) i split tha i whole ejaculates obtaied from patiets whose wives were sigificatly differet for sperm morphology. The per cet motile cells ad per cet ormal spermatozoa were probably higher (p < 0.005) i split tha i whole ejaculates obtaied from patiets whose wives were isemiated but did ot coceive. Five patiets reported pregacy without isemiatio or ay other therapy. For this group the sperm motility ad morphology were the same i split ad whole ejaculates. It thus appears that successful isemiatio could be related to the relative quality of the first portio of the ejaculate. O the other had, there were differeces betwee the groups that call for a reservatio. t Test was also used for the differeces betwee meas of upaired data, for seme volume, sperm desity, motility, ad morphology. The patiets were classified i 3 groups: those who reported pregacy after isemiatio (Group, those whose wives did ot become pregat (Group 11), ad those who reported coceptio without ay help (Group III). The oly differeces oted betwee these groups were: (a) the per cet ormal spermatozoa was sigificatly lower i specimes from me i Group II tha from me i Group I or III (p < 0.001); ad (b) specimes from me i Group III had higher sperm desity (p < 0.005) ad higher per cet motile cells (p < 0.001) tha those from me i Group I. If oe accepts that sperm desity is of less importace for male fertility tha the fuctioal properties of the spermatozoa the the mai differece betwee Group I (successful isemiatio with split ejaculate) ad Group III (pregacy without help) was the per cet motile cells. O the other had, the motility of the spermatozoa is susceptible to temperature ad decreases with time after ejaculatio. Careful cotrol of the experimetal variables is therefore ecessary if a real differece would be verified. I the light of our ow observatios ad after a careful aalysis of the results preseted by others we feel that more ad well-cotrolled studies are eeded to scietifically justify the use of AIH with split ejaculates. Iformatio about sex physiology ad istructios about coital positios to favor the depositio of the seme at the posterior forix may give

256 ELIASSON AND LINDHOLMER Vol. 23 equally good results as AIR with split ejaculates. Patiets with large seme volumes may beefit from istructios how to cocetrate the seme at the cervix by withdrawal after the expulsio of the first portio of the ejaculate. We have durig a year give this istructio to patiets with large seme volumes but without ay other otable defects i the seme. Our results are, however, ot at all as positive with regard to pregacies (zero) as reported by others. 6, SUMMARY Sperm desity, motility, viability, ad morphology were estimated i the various fractios of huma split ejaculates ad whole seme from healthy voluteers ad from ifertile patiets with oligozoospermia ad large seme volumes. The motility was sigificatly lower i the fractios represetig the secod half of the total volume tha i the first fractios. The per cet spermatozoa with ormal cofiguratio was the same i all fractios. The qualitative properties of the spermatozoa were the same i the most sperm rich fractio ad the whole ejaculate from the same idividuals. The implicatios of these fidigs with referece to isemiatio with the sperm rich first half of a ejaculate are discussed. Ackowledgmets. The skillful techical assistace by Mrs. Valborg Arbert ad Mrs. Moica Dahlfors is gratefully ackowledged. REFERENCES 1. ELIASSON, R Studies o prostagladi. Occur rece, formatio ad biological actios. Dissertatio Stockholm. Acta Physiol Scad 46(Suppl. 15): 1959. 2. ELIASSON, R Prostagladi-properties, actios ad sigificace. Biochem Pharmacol 12:405, 1963. 3. GUTMAN, E. B., AND GUTMAN, A. B. Quatitative relatios of a prostatic compoet (acid phosphatase) of huma semial fluid. Edocriology 2:115, 1941. 4. LUNDQUIST, F. Aspects of the biochemistry of huma seme. Acta Physiol Scad 19 (Suppl. 66): 1949. 5. MACLEOD, J., AND HOTCHKISS, R S. The distributio of spermatozoa ad of certai chemical costituets i the huma ejaculate. JUral 4: 225, 1942. 6. HARVEY, C., AND JACKSON, M. H. A method of cocetratig spermatozoa i huma seme. J Cli Path :341, 1955. 7. FARRIS, E. J., AND MURPHY, D. P. The characteristics of the two parts of the partitioed ejaculate ad the advatages of its use for itrauterie isemiatio. Fertil Steril 11:465, 1960.. AMELAR, R. D., AND HOTCHKISS, R S. The split ejaculate. Its use i the maagemet of male ifertility. Fertil Steril 16:46, 1965. 9. ELIASSON, R Stadards for ivestigatio of huma seme. Adrologie 3:49, 1971. 10. ELIASSON, R, AND TREICHL, L. Supravital staiig of huma spermatozoa. Fertil Steril 22:134, 1971. 11. RICHTERICH, R Cliical Chemistry Theory ad Practice. Karger, Basel, 1969. 12. HARVEY, C. The use of partitioed ejaculates i ivestigatig the role of accessory secretios i huma seme. Stud Fertil :3, 1956.