{Received 24th May 1960)

Transcription

1 SEMEN PROSTAGLANDIN LEVELS IN FIFTY PATIENTS ATTENDING A FERTILITY CLINIC D. F. HAWKINS and A. H. LABRUM Obstetric Unit, University College Hospital, London, W.C.I {Received 24th May 1960) Summary. The levels of the smooth muscle stimulating substance prostaglandin in the semen of patients attending a fertility clinic were estimated. The data obtained were analysed in relation to the clinical and laboratory findings on these patients. While the results of this preliminary survey were not conclusive, they did suggest that the semen prostaglandin level is related in some positive way to the processes leading to conception. Further investigation of a larger group of subfertile couples where the wife is normal and the husband oligospermic is particularly indicated. INTRODUCTION That human prostatic secretion contains a smooth-muscle-contracting substance has been known for many years. Goldblatt (1935) described the pharmacological properties of this substance, and von Euler (1935, 1936) gave a further account of its activity and named it prostaglandin. Vogt (1958) has reviewed what is known of the chemical and physical properties of prosta glandin. He suggests that it is a hydroxy acid with an unsaturated long carbon chain. Prostaglandin appears to be a true secretion of the prostate, since its con centration in prostatic fluid is two to seven times greater than that in the gland itself (von Euler, 1934). In man, the substance is present in such high concentration in semen that, when the latter is diluted as much as 1 : 10,000, it will still cause spasm of isolated smooth muscle. In vitro, prostaglandin has been reported to stimulate contraction of isolated seminal vesicles (Goldblatt, 1935) and of isolated preparations of uterus from various species. Von Euler (1936) found that the substance caused isolated strips of human uterus to contract, but the majority of the specimens of human semen examined by Cockrill, Miller & Kurzrok (1935) caused relaxation of human uterine strips. The observations of Kurzrok & Lieb (1930) suggest that prostaglandin usually has a stimulant action on human uterus in vivo, since human semen applied by intra-cervical injection was rejected by the uterus whilst saline injections were retained. Karlson (1949), working with human subjects, found the variability of the changes in uterine motility produced by semen to be related to the 1

2 2 D. F. Hawkins and A. H. Labrum menstrual cycle. He reported that, in the few days prior to ovulation, semen caused stimulation of the corpus uteri, while the isthmus and cervix relaxed. During the secretory semen phase, caused a generalized contraction of the uterus. Karlson's (1959) later studies on infertile patients did not entirely confirm the earlier results. Asplund (1947a) has shown in animals that prosta glandin can cause relaxation of the tubai ostia. Both Asplund (1947a) and Karlson (1949, 1959) observed that effects on the uterus could be produced by the introduction of the active substance into the vagina. Asplund used rabbits in which the upper end of the vagina had been closed, and showed that prostaglandin introduced into the vaginal cavity caused systemic effects. This suggests that can prostaglandin be absorbed from the vagina and, by virtue of its effects on uterine motility, could be concerned in the process of insemination. Asplund (1947b, 1949) examined semen specimens from a series of patients attending a fertility clinic. He assayed the content of smooth-muscle-contracting substances on isolated preparations of rabbit ileum, but was unable to detect any relation to the results of seminal analysis. However, he believed that in some specimens the assay results were rendered inaccurate by interference from choline and another unidentified substance, and therefore did not strictly represent the prostaglandin content. In the present work, we employed isolated guinea-pig ileum for assaying prostaglandin in semen specimens. This preparation is more sensitive than rabbit ileum and, at the dilutions of semen used, the choline present in semen is quite inactive and does not interfere. In addition, we used an assay technique (Schild, 1942) that enabled us to detect the presence of an unknown interfering substance and to employ a measure of response that apparently eliminated the interference. The present report comprises the results of estimations of the concentration of prostaglandin in specimens of semen from a series of patients attending a fertility clinic. This work was intended as a preliminary survey of the levels to be found in normal and infertile patients and to suggest lines along which further investigations could profitably be carried out. Certain of our findings have already been mentioned briefly (Hawkins & Labrum, 1956). We are grateful to Dr G. I. M. Swyer for access to specimens of semen, the results of semen analyses, and the case records of the patients concerned. METHODS Semen specimens, produced by masturbation into dry sterile were jars, received at the fertility clinic within an hour or two of production. Measured volumes (0-5 to 5 ml) were added to 5-ml aliquots of phthalate buffer, ph 4-0, in sterile screw-capped bottles, and preserved at 4 C. The ph of the diluted specimens then lay between 5 and 7. It was found that no change in prostaglandin content occurred in specimens kept in this way for up to 6 weeks. In general, specimens were assayed within a day or two of receipt. Occasionally, they were preserved for longer periods than this but no specimens kept for longer than 21 days were included in the series.

3 Semen prostaglandin 3 PROSTAGLANDIN ASSAY Estimates of prostaglandin content were made by biological assay, dilutions of the unknown solution being compared with a stable standard solution of prostaglandin prepared as described later. Isolated preparations of guinea-pig terminal ileum were maintained in oxygenated Tyrode's solution at 37 C. Doses of drugs were injected into the 5-ml isolated organ bath at intervals of 2\ or 3 min, and left in contact for 1 min. Contractions of the muscle were recorded with a servo-regulated isotonic ink-recorder (Hawkins & Smyth, 1954). Fresh preparations ofileum usually gave regular responses for up to 5 hr, After roughly equivalent doses of dilutions of the semen specimen and of the standard preparation of prostaglandin were determined, the prostaglandin content of the specimen was estimated by the recording of repeated graded responses, with the randomized assay described by Schild (1942). In general, four groups of responses, each consisting of two doses of unknown and two doses of standard, gave a satisfactory answer. In a number of the assays where four groups of responses were used, a Latin Square design was employed instead of a randomized order of doses within groups. Response heights were measured on the tracings after the drug had been in contact for 1 min. In this way, the effects of an interfering substance present in a few specimens could be avoided. The presence of this substance caused an initial transient peak response of the guinea-pig ileum which wore off within a minute, the response height settling to a level that, judged by the absence of deviation from parallelism, represented the true prostaglandin effect. Using this measure of response, we obtained results that were statistically valid when analysed as described by Schild and significant deviation from parallelism of the log. dose-response curves was only very rarely encountered. The 5% error limits of assay were of the order of ±25%, a satisfactory degree of accuracy for a biological assay of this type. Text-fig. 1 (a) shows four consecutive responses to the same dilution of semen. In Text-fig. 1 (b), two groups of 2 x2 responses are shown and it will be seen that satisfactory graded responses were obtained. In Text-fig. 2 (a and b), the observations of Goldblatt (1935) and von Euler (1936) are confirmed, in that atropine in sufficient concentration to reduce the response of the ileum to acetylcholine does not affect the response to prostaglandin. Cockrill et al. (1935) found that prostaglandin was antagonized by atropine; we were able to explain this observation, since considerably higher doses of atropine than were required to antagonize acetylcholine did in fact abolish the response to prostaglandin. The effects of choline on the guinea-pig ileum preparations were examined. Under the experimental conditions, choline chloride caused contractions of the ileum in concentrations down to 10 µg/ml. The concentration of choline present in semen is in the range 1-3 to 4 mg/ml (Lundquist, 1947). The dilutions of semen we employed, 1 : 5000 to 1 : 50,000, corresponded to a concentration of choline in the isolated-organ bath within the range 0 1 to 1-0 Mg/ml, well below the level that might interfere with the assays. Other constituents of semen known to be pharmacologically active, such as vitamins, amino acids, adenosine and its derivatives, noradrenaline, and

4 4 D. F. Hawkins and A. H. Labrum ergothioneine, would have been present in the isolated-organ bath in concentra tions far below those in which they have any action on the test preparation. Text-fig. 1. Records of isolated guinea-pig ileum preparations maintained in oxygenated Tyrode's solution at 37 C. (a) Contractions produced by four successive doses of semen added to the bath containing the preparation. semen added to give a final concentration of 1:25,000, left in contact 1 min, and then washed out. (b) Two groups of responses from a four-point assay (see text). and P/ and units/ml, respectively, of standard prostaglandin solution. U and U/2 large and small doses of 'unknown' solution containing prostaglandin. Text-fig. 2. Isolated guinea-pig ileum. Contractions produced by prostaglandin, units/ml (P), or acetylcholine 0-02 pg/ml (A). In (é), atropine 0-01 pg/ml present in the bath fluid. It therefore seems likely that under our conditions prostaglandin was the only active substance measured in these assays. STANDARD SOLUTION OF PROSTAGLANDIN A stable standard solution of prostaglandin was prepared in order that the activity present in semen specimens might be estimated by comparison. Pooled human seminal specimens were used as a source of prostaglandin. The original extraction described by von Euler in 1936 resulted, in our hands, in very low yields, only 1 to 5% of the activity in the original specimens being present in the product. The following modified extraction was therefore employed. The pooled semen specimens were taken to ph 4 with dilute hydrochloric acid and five volumes of acetone added. After filtration twice at the pump to remove the precipitated proteins, the acetone was distilled off at reduced pressure. The acidic solution remaining was then extracted twice with ten volumes of ether for several hours. The great majority of the activity was then present in the ether phase; the aqueous phase was practically inactive and was discarded. The ether was distilled off and the residue dried over phosphorus

5 Semen prostaglandin 5 pentoxide. The dry residue was then extracted four times with 50-ml aliquots of dry ether, filtered by gravity, and the ether distilled off. The residue was taken up in 10 to 20 ml of distilled water and filtered by gravity. Aliquots were sealed in ampoules, preserved at 4 C, and as a employed standard preparation for assay purposes. The yield with this extraction was 20 to 50%. The major loss occurred during the distillation of the acetone extract. The yield could be improved by omitting this stage, and extracting the acidified pooled specimens with ether, without any preceding protein precipitation. When this was done, the product tended to be translucent, probably due to lipoprotein the translucency could be removed by precipitation with trichloracetic acid without loss of prostaglandin activity. Some samples of prostaglandin prepared by the full extraction with acetone followed by ether described above were freeze-dried for storage. This resulted in approximately 50% further loss in activity but the product was stable and was sometimes employed as a standard preparation for assay purposes. The extraction described frees the product from choline, which is insoluble in ether. No evidence was obtained of the presence in the final extracts of any substance causing deviation from parallelism in the assays. DEFINITION OF UNIT An aqueous preparation of prostaglandin, distributed in ampoules, was selected as a primary standard and attributed a potency of 1 unit/ml. The biological activity of this preparation was checked repeatedly by its on activity various smooth muscle preparations and by comparison with other purified extracts and showed no deterioration over a period of 12 months during which the work was performed. The standard preparation in concentrations of the order of units/ml would give a small contraction of isolated or guinea-pig rabbit ileum. The unit is of the same order of magnitude as those employed by von Euler (1934, 1939) and von Euler & Hammarström (1937). The barium salt of prostaglandin prepared by von Euler in 1939 was equivalent to 12 of his units per mg. From time to time, new samples of prostaglandin were prepared and their potency estimated biologically in terms of the primary standard, with a rela tively high degree of accuracy (P 0 5 error limits of the order of ±10%). These substandards were used for the estimation of the prostaglandin content of semen specimens. SEMINAL ANALYSES Seminal analyses were made by the techniques described by Emmens (1947). Semen specimens were regarded as normal in all respects if the following criteria were satisfied: appearance normal not watery or mucoid; volume >2 ml; testicular cells not seen; spermatozoa >20 millions/ml; abnormal forms <40%; motility > 1 Junits after 5 hr at room temperature; motility estimate after 8 hr at room temperature more than half the value after 2 hr.

6 D. F. Hawkins and A. H. Labrum STATISTICAL METHODS Standard methods were employed. As the distribution of concentrations of prostaglandin in different patients was significantly skew (normal semen, t 2-47, <0 02; all cases, t 3-51, <0 001), while the distribution in terms of log. units/ml did not differ significantly from normal, the log. trans formation of this variable was employed in all computations, and mean values quoted in units/ml are geometric means. Certain of the variables for which correlation coefficients were calculated had skew distributions; in these cases, the values were obtained by recalculation of the data after appropriate norma lizing transformations had been applied. In view of the pilot nature of the present survey, relations significant at the 0-1 level were considered of interest. PATIENTS STUDIED The patients studied were fifty unselected couples who attended the Fertility Clinic of the Obstetric Hospital at University College Hospital, London, com plaining of infertility of from 3 months to 8 years duration. Of these, twentythree became pregnant during the period of study; of the remainder, nineteen were followed up for periods between 6 months and 5 years. RESULTS CONCENTRATION OF PROSTAGLANDIN IN SEMEN The distribution of the values obtained for concentration of prostaglandin in semen specimens from these fifty unselected infertility patients is shown in Text-fig. 3. It will be seen that the values have a skew distribution which is Semen prostaglandin (units/m Semen prostaglandin (log. units/ml] Text-fig. 3. Distributions of semen prostaglandin levels (units/ml and log. units/ml) in fifty men attending a fertility clinic. Filled blocks represent fifteen men in whom no hitherto known infertility factor was found. corrected by considering the logarithms of the values. The (geometric) mean concentration of prostaglandin in the semen specimens was 6 units/ml, with a standard deviation equivalent to 3-2 to 11-9 units/ml, and a range of 1-7 to 22-4 units/ml. The series included fifteen men who, as a result of clinical and laboratory investigation, were assessed as normal. These cases are indicated in the frequency diagrams in Text-fig. 3. The mean value attained for these normal

7 Semen prostaglandin men was 5-9, with a standard deviation corresponding to 2-9 to 11-8 units/ml, and a range of 1-9 to 20-8 units/ml. This observation that the values in men assessed as normal by the usual criteria do not differ from the values for the whole sub-fertile group, suggests that if the prostaglandin concentration has any influence on fertility, then this influence is not closely related to any of the known clinical or laboratory male infertility factors studied. Prostaglandin levels in specimens obtained from the same patient at different times varied somewhat, but this variation between repeated specimens from the same individual was significantly smaller than the variation between different individuals in the group. RELATION TO PROSTATIC FUNCTION Von Euler (1936) states that the highest concentrations of prostaglandin are found in the prostates of men aged 20 to 50 years and that the content is low in children and old men. Swyer (1944) has shown that the volume of the prostate increases up to the age of 25 years, remains approximately constant from 20 to 45 years, then either gradually declines, or increases due to benign hypertrophy. The ages of the patients in the present study were 26 to 43 years, and within this range the concentration of prostaglandin in the specimens did not vary with age. (Correlation coefficient r +0-04, 47.) Prostatic secretion provides a considerable proportion of the volume of the semen specimen. It was therefore not surprising to find that the results suggested a positive correlation between two functions of prostatic activity, viz. concentra tion of prostaglandin and the volume of the semen specimen (r +0-26, 50, <0 1). RELATION TO TESTICULAR FUNCTION There was no correlation between the amount of prostaglandin in the semen and the sperm density (r +0 6, 50). The mean levels in the six cases of azoospermia, the eleven cases of oligospermia, and the thirty-three cases with normal sperm counts ( >20 millions/ml) did not differ significantly. Walker (1953) has concluded that a high incidence of abnormal forms of spermatozoa is associated with infertility. In the present study, some relation between a low concentration of prostaglandin and a high incidence of abnormal spermatozoa was found. The correlation coefficient between prostaglandin content and percentage abnormal sperms was 0-30 (n 38, P<0T). The concentration of prostaglandin in the semen specimens was not related to the occurrence of testicular cells in the specimens or to the macroscopic appearance of the semen (described as watery, normal or mucoid). RELATION TO SPERM MOTILITY Motility assessments were made at varying times after the specimens of semen were received. From these measurements, the motility 2, 5 and 8 hr after production of the specimens was estimated by interpolation. On correlating

8 8 D. F. Hawkins and A. H. Labrum the values with the concentration of prostaglandin in the specimens, it was found that a high prostaglandin concentration was associated with a low 2-hr motility (r 0-32, 31, <0 1), but that after 8 hr the motility was no longer related to the concentration of this substance. Closer examination of the data suggested that this relation arose primarily from a small group of cases where subnormal 2-hr motility was associated with a higher than usual prostaglandin level. No relation between prostaglandin concentration and the rate of decline of motility could be detected. RELATION TO COITUS The patient's libido did not appear to bear any relation to his prostaglandin level. Frequency of intercourse varied from two to twenty-six times per month, and was unrelated to the results of the assays. There was one case of partial impotence in the series. The patient's prostaglandin level was average. No relation to whether or not the wife was satisfied at intercourse, or to the occurrence of unexplained dyspareunia or pain after coitus, was detected. FEMALE INFERTILITY FACTORS No relation of prostaglandin activity to cervical hostility (normal semen, but only dead spermatozoa in the cervical mucus), incompetence of the internal os, or disease of the cervix (chronic cervicitis, cervical erosions, etc.) could be demonstrated. Ten patients had tubai insufflations within 12 hr of coitus. The occurrence of unexplained high pressures or initial spasm did not appear to be related to the concentration of prostaglandin in the husband's semen. RELATION TO PAST HISTORY OF CONCEPTION The wives were divided into three groups: those who had never conceived, those who had conceived in the past but with a frequency of one or less per 2 years of marital relations, and those who had conceived more often than this. The mean semen prostaglandin levels in the three corresponding groups of husbands were 5-5 (s.d. 3-1 to 9-8, 23), 6-3 (s.d. 3-0 to 13-2, 19) and 9-3 (s.d. 4-9 to 17-8, 7), respectively. While these measurements have no diagnostic value, the value for the difference between the first and last groups is approximately 0 5. These observations are therefore compatible with the view that there is a positive relation between the concentration of prosta glandin in the husband's semen and the ability ofthe couple to effect conception. DISCUSSION Our findings on the concentrations of prostaglandin in the semen of patients attending a fertility clinic are in close agreement with those of Asplund (1947b). This author found no relation between prostaglandin concentration and the results of full seminal analyses. Our results suggest that there is an inverse relation between prostaglandin concentration and the percentage of abnormal spermatozoa, and that there is a group of infertility patients in which subnormal

9 Semen prostaglandin 9 motility is associated with a high prostaglandin concentration. Neither of these associations is, of course, necessarily causal ; they may be the common products of some underlying process. RELATION OF PROSTAGLANDIN TO CONCEPTION Although in a preliminary survey of a moderate number of unselected cases we did not expect to obtain any definite evidence that prostaglandin is a fertility factor, we were of course curious about this point. With the justification that the results would indicate which type of case was most suitable for further in relation to the outcome of the study, our results were therefore analysed period of infertility. In the group of patients where the husband's sperm count was greater than 20 millions/ml, no relation between prostaglandin concentra tion and conception was detected. In the group of eight cases where the husband was oligospermic (sperm count 0-5 to 19 millions/ml), and no bar to conception had been detected in the wife, we found the values for prostaglandin in semen shown in Table 1. Table 1 prostaglandin levels in the semen of eight oligospermic patients where no bar to conception was found in the wife Case Duration of infertility {years) Spermatozoa (millionslml) 11 < <1 <1 15 Semen analysis Motility Subnormal Normal Normal Normal Subnormal Prostaglandin (unitsjml) Outcome Pregnant Pregnant It will be seen that in this small group of cases a high semen prostaglandin level appeared to be associated with a favourable outcome. We therefore feel it is in this group of subfertile patients, where improvement in any one of a number offertility factors might achieve conception, that evidence of a function of prostaglandin is most likely to be found. ACKNOWLEDGMENTS We are grateful to Professor W. C. W. Nixon and Dr G. I. M. Swyer for their encouragement and advice, and to the Nuffield Foundation for financial support. REFERENCES Asplund, J. (1947a) Some preliminary experiments in connection with the effect of prostaglandin on the uterus and tubae in vivo. Acta physiol. scand. 13, 109. Asplund, J. (1947b) A quantitative determination of the content of contractive substances in human sperm and their significance for the motility and vitality of the spermatozoa. Acta physiol. scand. 13, 103.

10 10 D. F. Hawkins and A. H. Labrum Asplund, J. (1949) Bestämningar av kontraherande substans i homosperma. Nord. med. 42, Cockrill, J. R., Miller, E. G. Jr. & Kurzrok, R. (1935) The substance in human seminal fluid affecting uterine muscle. Amer. J. Physiol. 112, 577. Emmens, G W. (1947) The motility and viability of rabbit spermatozoa at different hydrogen-ion concentrations. J. Physiol. 106, 471. Euler, U. S. von. (1934) Zur Kenntnis der pharmakologischen Wirkungen von Nativsekreten und Extrakten männlicher accessorischer Geschlectsdrüsen. Arch. exp. Path. Pharmak. 175, 78. Euler, U. S. von. (1935) Über die spezifische blutdrucksenkende Substanz des menschlichen Prostataund Samenblasensekretes. Klin. Wschr. 14, Euler, U. S. von. (1936) On the specific vaso-dilating and plain muscle stimulating substances from accessory genital glands in man and certain animals (prostaglandin and vesiglandin). J. Physiol. 88,213. Euler, U. S. von. (1939) Weitere Untersuchungen über Prostaglandin, die physiologisch aktive Substanz gewisser Genitaldrüsen. Skand. Arch. Physiol. 81, 65. Euler, U. S. von & Hammarström, S. (1937) Über das Vorkommen des Prostaglandins in Tierorganen. Skand. Arch. Physiol. 77, 96. Goldblatt, M. W. (1935) Properties of human seminal plasma. J. Physiol. 84, 208. Hawkins, D. F. & Labrum,. H. (1956) Function of the prostate. Brit. med. J. ii, Hawkins, D. F. & Smyth, C. N. (1954) A servo-regulated ink recorder for isolated smooth muscle preparations. J. Physiol. 124, 8P. Karlson, S. (1949) Spermas inverkan pâ motiliteten inom den icke gravida uterus hos människa. Nord. med. 42, Karlson, S. (1959) The influence of seminal fluid on the motility of the non-pregnant human uterus. Acta obstet, gynec. scand. 38, 503. Kurzrok, R. & Lieb, C. C. (1930) Biochemical studies of human semen. II. The action of semen on the human uterus. Proc. Soc. exp. Biol., N.T. 28, 268. Lundçhjist, F. (1947) Studies on the biochemistry of human semen. I. The natural substrate of prostatic phosphatase. Acta physiol. scand. 13, 322. Schild, H. O. (1942) A method of conducting a biological assay on a preparation giving repeated graded responses illustrated by the estimation of histamine. J. Physiol. 101, 115. Swyer, G. I. M. (1944) Post-natal growth changes in the human prostate. J. Anat., Lond. 78, 130. Vogt, W. (1958) Naturally occurring lipid-soluble acids of pharmacological interest. Pharmacol. Rev. 10, 407. Walker,. (1953) The investigation of male infertility. Problems of Fertility in General Practice, 2nd edn, p. 30. Cassell & Co, London.