Reduced prostaglandin levels in the semen of men with

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1 Reduced prostaglandin levels in the semen of men with very high sperm concentrations R. W. Kelly, I. Cooper and A. A. Templeton Medical Research Council, Unit ofreproductive Biology, 2 Forrest Road, Edinburgh EHI 2QW, and *Department of Obstetrics and Gynaecology, University of Edinburgh, 23 Chalmers Street, Edinburgh EH3 9ER, U.K. Summary. The prostaglandin levels have been measured in a group of men with sperm concentrations greater than 300 \m=x\106/ml and compared with the levels in men with sperm concentrations of 50 to 150 \m=x\106/ml. The distribution of the PG levels in all groups was highly skewed but the data could be transformed to a normal distribution by taking logarithms. Comparison of the PG levels showed a highly significant lowering of the PG levels in the polyzoospermic group when compared with either of the groups with normal sperm concentrations. Introduction Although prostaglandins (PGs) were first discovered in human seminal plasma (Goldblatt, 1933; von Euler, 1934, 1935) and although a relationship between PG levels in semen and fertility were apparently established in early investigations of seminal PG content (Bygdeman, Fredricsson, Svanborg & Samuelsson, 1970), the function of these compounds in semen is still not clear. The first PGs to be found in human semen were those of the E and F series (Samuelsson, 1963) which were followed by reports of the A, B, 19hydroxy A and 19hydroxy (Hamberg series & Samuelsson, 1966).The finding that the 19hydroxy PGEs are the major PGs of human semen (Taylor & Kelly, 1974; Jonsson, Middleditch & Desiderio, 1975) has led to the suggestion that the A, B, 19hydroxy A and 19hydroxy PGs in semen and elsewhere are artefacts (Middleditch, 1975). The average ejaculate of a fertile man will contain around 1 mg PG which consists mainly of the 4 E type PGs; PGE1, PGE2, 19OH PGE1 and 19OH PGE2 (Templeton, Cooper & Kelly, 1978). Early measurements of seminal PGs showed that there was no apparent correlation between the PG content of the semen and any common measure of semen quality (Hawkins, 1968; Bygdeman et al, 1970; Brummer & Gillespie, 1972) although several studies have reported an apparently significant difference in the PG content of semen from couples with otherwise unexplained infertility when compared to levels in fertile men (Bygdemann et al, 1970; Ito & Katayama, 1971; Brummer & Gillespie, 1972; Collier, Flower & Stanton, 1975). All these comparisons have been complicated, and sometimes perhaps invalidated, by the skewed distribution of most seminal variables, including PG content (Hawkins, 1968) and the wide range of PG levels in normal fertile men (Templeton et al, 1978). Now that the qualitative nature of the seminal PGs is more clearly understood, we have reexamined the possibility of a connection between sperm count and seminal PG levels. In this study we have measured the PG content of the semen from a group of men with sperm counts greater than /ml who are all polyzoospermic (Eliasson, 1975) and compared this with the PG content of semen samples in which the count was in the range of lovml.

2 Materials and Methods Single semen samples were obtained from fertile men between 20 and 40 years of age (Templeton et al, 1978) and from men attending an infertility clinic. All samples were obtained by masturbation between 07:00 and 10:00 h. Routine measurements of sperm count, motility and seminal volume were carried out on samples as soon as they were received, which was usually within 3 h of ejaculation. All semen samples had motilities greater than 35%. Aliquots of the semen samples were placed into methoxyamine hydrochloride solution within 6 h of ejaculation and were then stored at 4 C until analysed for PG levels using a gas chromatography method (Cooper & Kelly, 1975). The precision of this method was 6% (s.d.) for PGE measurements and 12% (s.d.) for 19OH PGE measurements. The variation of PG levels from day to day for one person has been estimated at 23% for PGEs and 25% for 19OH PGEs (Templeton et al, 1978). The sensitivity of the method was 02 pg PG/ml semen. Semen samples were categorized according to their sperm count. Group A consisted of 32 men attending an infertility clinic who had sperm counts in the range /ml. Group included 15 men who had fathered children within the last year with sperm counts in the same range. Group C comprised 9 men from the infertility clinic and one man who had fathered a child within the last year with sperm counts greater than 300 x 106/ml. Data were analysed for significance using the Wilcoxon Rank Sum test (Wilcoxon, 1945) or were transformed by taking logarithms and then analysed by Student's t test. Results The widest range of PG levels (Table 1) was found in Group B. The total PGE + 19OH PGE content of the ejaculate in this group ranged from 191 to 3717 pg with a mean content of 1049 pg. The PG levels and seminal volumes in Group A did not differ significantly from those of Group but Group A was without the few very high levels of PG found in Group B. The total PGE + 19OH PGE content of Group A ranged from 211 to 3783 pg with a mean of 1077 pg. The range of sperm concentrations in Group C was from 300 to /ml and the range of total sperm content of the ejaculates was from 213 to 2750 IO6. The total PGE + 19OH PGE content of the ejaculates in Group C was from 71 to 2285 pg with a mean of 464 pg. The concentrations of 19OH PGE and of PGE + 19OH PGE in Group C were highly significantly different from those of Group A and (Table 1). The differences in PGE levels were less marked with a significant difference clearly demonstrated between Groups C and A only. When the PG content of the ejaculates was examined the total E + 19OH PGE content in ta) io Groups A + H P 5 10 I (b) Groups A + _D_ Group C 5 Group C Concentration (^ug/ml) Rr^r Content fag) Textfig. 1. The distribution of (a) concentration and (b) content of PGE + 19OH PGE in ejaculates from Groups A + and Group C. 4000

3 Table 1. PGE (Mg/ml) (range) log10of data Prostaglandin concentrations in a group of polyzoospermic men (Group C, >300 x lovml) and two groups of men with normal sperm counts (Groups A and B, lovml) Group (sperm count) Difference between groups* A (N 32) = ( =15) ( 10) = C from A C from B A from 55 (5139) (2262) OH PGE fag/ml) (range) (460670) (641094) (31310), log10of data PGE + 19OH PGE (jig/ml) 302 (range) (72783) 257 log10of I 241 data I 239 Volume (ml) (range) 37 (1678) (563) < < 0005 < < 001 P<001 < 0001 < (911239) (45341) < 001 < < 0001 < (1346) (0667) 245 * Student's t test was used on log10 data and the Wilcoxon Rank Sum test was used on untransformed data. t Not significant at the 01 level using Student's t test (see text). t Not significant at the 01 level using Wilcoxon Rank Sum test. Groups A and B, combined together as shown in Textfig. 1 and independently, differed significantly (P < 001 using the Wilcoxon Rank Sum test) from the PG content in Group C. The distributions of PG concentrations and content were all skewed (see Textfig. 1). No significant differences in motility were observed between the three groups. Discussion Previous studies of the PG content of semen have not revealed a correlation of PG levels with other commonly measured parameters such as sperm count, motility, longevity of the spermatozoa or seminal volume (Asplund, 1947; Hawkins, 1968; Bygdeman et al, 1970; Brummer & Gillespie, 1972; Collier et al, 1975) although several reports have noted an apparently significant increase in seminal PG levels in a group of azoospermic men when compared with a group of oligozoospermic men (Sturde, 1968; Perry & Desiderio, 1977). The difference between these groups, however, took account neither of the aetiology of the azoospermia nor of the skewed distribution of the seminal PG levels which has previously been described (Hawkins, 1968) and is confirmed in this study. The results given here show a highly significant difference of seminal PG levels in men with sperm counts greater than 300x 106/ml and in two populations of men with counts of /ml. All three groups exhibited a highly skewed distribution of PG levels as is evident from

4 the difference between the mean and the median levels (Table 1) and is shown for Groups A + and Group C in Textfig. 1. This skewed distribution was transformed to a near normal distribution by taking logarithms of the data, thus bringing the mean and the median almost into coincidence for the 19OH PGE and the PGE + 19OH PGE levels. Although there appears to be a possible significant difference between the PGE levels in Group A and Group B, nonparametric statistics fail to demonstrate significance; this is because of the few very high levels of PGE and 19OH PGE in Group (fertile men) which were absent from the group attending the infertility clinic. This confirms the findings of Brummer & Gillespie (1972) although there is no obvious explanation. The possibility that the difference in the PG levels between the polyzoospermic men and the other groups is due to a difference in semen volumes is discounted because there was no significant difference between semen volumes of the groups when investigated by parametric or nonparametric statistics. The absence of a difference in semen volumes between the two groups indicates that the polyzoospermia is not a result of the decreased secretion by the seminal vesicles and this cannot therefore explain the lowered PG levels. Moreover, significant differences can be demonstrated between the PG (E + 19OHE) content of semen in Group C and the content in either of Groups A and or the content of Groups A and together (Textfig. 1). These lowered PG levels in polyzoospermic men could be explained in two ways; first, some common unidentified factor could be responsible both for low PG levels and high sperm counts, or secondly, the spermatozoa could be binding (or metabolizing) the prostaglandins and rendering them unavailable for measurement. The first possibility cannot easily be pursued because it is difficult to suggest the common factor which might be responsible; although androgens are known to stimulate PG production by the seminal vesicles (Skakkebaek, Kelly & Corker, 1976), and therefore poor androgenization might account for the low PG levels, there is no evidence that low androgen levels are a cause or a result of polyzoospermia. However, it has been reported by Joel (1968) that polyzoospermia is "more than rarely" associated with impotence. The second possibility gains some support from the findings that although human spermatozoa are reported to bind prostaglandins of the E and F series (Mercado, Villalobos, Domínguez & Rosado, 1978) very little PG can be recovered from centrifuged spermatozoa (Cooper & Kelly, 1975). Another possibility is that the spermatozoa bind the endoperoxide precursors to the PGs which might be sufficiently stable to interact with the spermatozoa immediately before ejaculation. If binding by the spermatozoa is responsible for the reduced PG levels in polyzoospermia it would lend further support to the idea that the function of seminal PGs is to modify sperm metabolism at the time of ejaculation (Kelly, 1977). We thank Professor R. V. Short, frs, for advice and encouragement, Mr K. Donachie for routine analysis of the semen, and the staff of the SubFertility Clinic, Royal Infirmary of Edinburgh, for their help. References Asplund, J. (1947) A quantitative determination of the content of contractive substances in human semen and their significance for the motility and vitality of the spermatozoa. Ada physiol. scand. 13, Brummer, H.C. & Gillespie, A. (1972) Seminal prosta glandins and fertility. Clin. Endocr. 1, Bygdeman, M., Fredricsson, B., Svanborg, K. & Samuelsson, B. (1970) The relation between fertility and prostaglandin content of seminal fluid in man. Fert. Steril. 21, Collier, J.C, Flower, R.L. & Stanton, S.L. (1975) Seminal prostaglandins in infertile men. Fert. Steril. 26, Cooper, I. & Kelly, R.W. (1975) The measurement of E and 19hydroxy E prostaglandins in human seminal fluid. Prostaglandins 10, Eliasson, R. (1975) Analysis of semen. In Progress in Infertility, pp Eds S. J. Behrman & R. W. Kistner. Little, Brown & Co., Boston. Goldblatt, M.W. (1933) A depressor substance in seminal fluid. Chem. & Ind. 52,

5 Hamberg, M. & Samuelsson,. (1966) Prostaglandins in human seminal plasma. J. biol. Chem. 241, Hawkins, D.F. (1968) Relevance of prostaglandins to problems of human subfertility. In Prostaglandin Symposium of the Worcester Foundation, pp Eds P. W. Ramwell & J. E. Shaw. Interscience, New York. Ito, H. & Katayama, T. (1971) Male infertility and prostaglandins. Proc. 7th Wld Congr. Fert. Steril, pp Excerpta Medica (Int. Congr. Seri. No. 234a), Amsterdam. Joel, CA. (1968) Male impotence (with special reference to hyperspermia and therapy with Diazepine derivatives). In Progress in Infertility, pp Eds H. R. Behrman & R. W. Kistner. Little, Brown & Co., Boston. Jonsson, H.T., Middleditch, B.S. & Desiderio, D.M. (1975) Prostaglandins in human seminal fluid: two novel compounds. Science, N.Y. 187, Kelly, R.W. (1977) Effect of seminal prostaglandins on the metabolism of human spermatozoa. J. Reprod. Fert. 50, Mercado, E., Villalobos, M., Domínguez, R. & Rosado,. (1978). Differential binding of PGE, and PG F2<z to the human spermotozoa membrane. Life Sciences 22, Middleditch, B.S. (1975) PGA: fact or artifact? Prosta glandins 9, Perry, D.L. & Desiderio, D.M. (1977) Utility of d4pge2 as an internal standard to quantify endogenous levels of PGE PGE2, 19OH PGE, and 19OH PGE2 in human seminal fluid by GCMS. Prostaglandins 14, Samuelsson, B. (1963) Isolation and identification of prostaglandins from human seminal plasma. J. biol. Chem. 238, Skakkebaek, N.E., Kelly, R.W. & Corker, C.S. (1976) Prostaglandin concentrations in the semen of hypogonadal men during treatment with testosterone. J. Reprod. Fert. 47, Sturde, H.C (1968) Experimentelle Untersuchungen zur Frage der Prostaglandine und ihrer Beziehungen zur männlichen Fertilität. ArzneimittelForschung 18, , , Taylor, P.L. & Kelly, R.W. (1974) 19Hydroxylated prostaglandins as the major prostaglandins of human semen. Nature, Lond. 250, Templeton, A.A., Cooper, I. & Kelly, R.W. (1978) Prostaglandin concentrations in the semen of fertile men. J. Reprod. Fert. 52, Von Euler, U.S. (1934) Zur Kenntnis der pharmakologischen Wirkungen von Nativesekreten und extrac ten männlicher accessorischer Geschlectsdrüsen. Archiv, exp. Path. Pharmak. 175, Von Euler, U.S. (1935) A depressor substance in the vesicular gland. J. Physiol., Lond. 84, 21p22p. Wilcoxon, F. (1945) Individual comparison by ranking methods. Biometrics Bull. 1, Received 6 September 1978