SEMEN ZINC LEVELS IN INFERTILE AND POSTVASECTOMY PATIENTS AND PATIENTS WITH PROSTATITIS*

Transcription

1 FERTILITY AND STERILITY Copyright c 1975 The American Fertility Society Vol. 26, No. 11, November 1975 Printed in U.S.A. SEMEN ZINC LEVELS IN INFERTILE AND POSTVASECTOMY PATIENTS AND PATIENTS WITH PROSTATITIS* JOEL L. MARMAR, M.D., SIDNEY KATZ, PH.D., DONALD E. PRAISS, M.D., AND THOMAS J. DEBENEDICTIS, M.D. South Jersey Urologic Associates, Cherry Hill, New Jersey 08034, and Department of Chemistry, Rutgers University, Camden, New Jersey The concentration of zinc in human seminal plasma and in sperm is extremely high compared with that of other body fluids and tissue. For example, the zinc concentration of blood plasma ranges between 75 and 115 JLg/100 ml, 1 whereas the zinc concentration of semen is 15 to 30 mg/100 ml,2 The value for zinc in sperm is even greater than that in semen, or 2.5 JLg/10 8 cells. 3 As a result of the extraordinarily high values for zinc in semen and sperm, semenologists have searched for the relationship among zinc metabolism, prostatic function, and reproduction. We have measured the semen zinc levels in volunteers and postvasectomy patients. These data were compared with the values in infertile patients and patients with prostatitis. In a selected number of patients, supplementary zinc sulfate was given. We have examined the responses to zinc therapy of patients with prostatitis. Finally, we examined the responses to zinc sulfate of patients with oligospermia and low semen zinc levels. MATERIALS AND METHODS Semen specimens were obtained by masturbation from volunteers and patients after 2 days of sexual abstinance. The volunteers were 18 apparently healthy medical students who denied any Received April 7, *Presented at the Thirty-First Annual Meeting ofthe American Fertility Society, April3 to 5, 1975, Los Angeles, Calif history of prostatic or venereal disease. There were 132 postvasectomy patients who were men of proven fertility but who were now azoospermic as a result of elective surgery. Patients with a history of prostatitis were eliminated from this group. There were 19 patients with abacterial prostatitis. These patients had recurrent symptoms referable to the prostate gland, including a positive finding of prostatic enlargement or tenderness and at least 20 to 25 white blood cells/high-power field on prostatic smear. Any patient with a positive urine culture following massage was eliminated from the study group, but no pure semen cultures were obtained. As part of an infertility evaluation, 153 semen specimens from 72 men who had not fathered a child were studied. Those eliminated from the study were patients with varicoceles, testicular atrophy, undescended testes, postpubertal mumps, or proven tubular fibrosis on biopsy. Not all of the remaining patients were subfertile. For the purpose of this study, the specimens were classified into four groups of differing fertility potential. The groups were organized by sperm count (less than 20 million sperm/ml, 20 to 60 million sperm/ml, 60 to 100 million sperm/ ml, and greater than 100 million sperm/ ml). Each specimen was collected in a separate, acid-washed, 25-ml polyethylene screw-cap vial. The semen analyses of the specimens were completed within 1.! hours after ejaculation. The speci-

2 1058 MARMAR ET AL. November 1975 mens were then refrigerated until the time of the zinc analysis. Each semen analysis included determination of volume, percentage motility, motility quality (0 to 4, with a grade of 4 representing excellent forward progression), motility index (the product of percentage motility and motility quality), and sperm density (expressed as millions of sperm per milliliter). The specimen was examined for morphology by Papanicolaou staining. The categories included normal, spindle forms (tapered), amorphous (bizarre heads), immature (cells with retained cystoplasm), and tail-less or double-headed. When patients were treated with supplementary zinc, they were given zinc sulfate, 80 mg by mouth two or three times/day (available as Vicon-C). Followup semen analyses were not done more frequently than every 8 weeks. Zinc analyses were done by atomic absorption spectrometry. A 0.1-ml aliquot of the liquefied semen was transferred to a graduate test tube, treated with 1 ml of 1% sodium chloride solution, and diluted to a volume of 10 ml with de-ionized water: Standards containing 0.1 to 5 mg of zindml were similarly prepared. A Perkin-Elmer model 214 atomic absorption spectrometer equipped with a zinc hollow cathode lamp and a specially fabricated aluminum burner was used for the measurements. Liquefied petroleum gas served as fuel, with compressed air as the oxidant. Measurements were made with a wavelength of nm, and the spectrometer signal was recorded on a strip chart recorder. Samples and standards were aspirated into the flame, and the zinc content of the samples was calculated from their recorded signals by reference to a calibration curve obtained from the signals from the standards. RESULTS Semen specimens from 18 volunteers were analyzed for zinc content by both TABLE 1. Mean Semen Zinc Values in 18 Healthy Volunteers by Different Techniques Technique Atomic absorption Neutron activation Signficance Mean zinc level 15.0 mg/100 ml 18.9 mg/100 ml p < 0.02 atomic absorption spectrometry and neutron activation. The results are compared in Table 1. The mean values obtained by neutron activation analysis were significantly higher than those obtained by atomic absorption spectrometry <R < 0.02). It is possible that this difference was due to a zinc impurity in the polyethylene irradiation container or to a suppression of the absorption by some component of the sample. The remainder of the zinc analyses in this study were done by atomic absorption spectrometry. Precision of the results obtained by atomic absorption spectrometry was evaluated by replicate analysis of the same samples. These data showed precision and are presented in Table 2. TABLE 2. Precision of Zinc Analyses by Atomic Absorption Spectrometry on Replicate Samples of Same Specimens Analysis J. c. Subject J. F. J. I. mg/iooml Mean SD To evaluate the possibility of zinc loss or contamination during storage, 12 semen specimens were analyzed weekly for 6 weeks. Three such samples are listed in Table 3. Although variations as high as 10% occurred, the zinc concentration remained approximately the same, indicating no loss or contamination. The variations may have been due to pipetting or dilutional errors.

3 Vol. 26, No. 11 SEMEN ZINC LEVELS IN MAN 1059 TABLE 3. Stability of Zinc Levels in Three Patients Whose Specimens Were Examined Weekly for 7 Weeks Patient Zinc level Day Day Day Day Day Day Day mg/100 ml M.B J. c c. c In Table 4, the mean semen zinc concentrations are reported for 18 volunteers, 136 postvasectomy patients, and 19 patients with abacterial prostatitis. From among the patients with prostatitis, 12 men were given zinc sulfate, 80 mg three times/day. The mean values for semen zinc were essentially the same for the volunteers and the postvasectomy patients. There was a statistically significant difference <P < 0.01) between the mean values for semen zinc in the volunteers and postvasectomy patients on the one hand and the patients with prostatitis on the other hand. In 12 patients with prostatitis who had been given zinc sulfate, the mean values for semen zinc were significantly greater than the values for patients with prostatitis before treatment <P < 0.01). As part of an infertility evaluation, 153 semen specimens from 72 men were examined. There were no positive correlations among zinc concentration, sperm count, motility, or morphology when all of the specimens were considered as a single group. However, it was our contention TABLE 4. Mean Semen Zinc Levels in 18 Volunteers, 136 Patients Postvasectomy, and 19 Patients with Abacterial Prostatitis, before and after Supplement with ZnSO 4 Subjects Volunteers Post vasectomy Prostatitis Prostatitis + ZnS0 4 Mean No. of semen subjects zinc level Range Significance mg/100 ml p < p < 0.01 TABLE 5. Arbitrary Parametric Classifwation of 153 Semen Specimens Parameter" Sperm count (millionlml) < >100 Zinc (mg/100 ml) Volume (ml) Motility (%) Motility quality (0-4) Sperm count (million/ml) No. of specimens Values are means. that these patients represented a mixture of populations.of fertile, subfertile, and absolutely infertile men. The specimens were regrouped into four classes according to sperm count (Table 5). Each class represented a population with a different potential for fertility. The mean sperm counts in each group differed significantly from the other <P < 0.01). The mean zinc concentrations showed an increasing trend but differed significantly only between the group with less than 20 million sperm/ml and the group with greater than 100 million sperm/ml <P < 0.01). The mean values for normal morphology were higher in the three classes with the greatest fertility potential (Table 6). A separate group of 11 patients with oligospermia (sperm count less than 60 million/ml) and low semen zinc levels (less than 15 mg/100 ml) was studied. Each patient was given zinc sulfate, 80 mg three times/day for at least 6 months. During the period of therapy the mean values increased significantly for zinc concentration, sperm count, motility in- TABLE 6. Arbitrary Morphologic Classifwation of 153 Semen Specimens Morphology" Sperm count (million/mil < ">100 ~ Normal Spindle Amorphous Immature Tail-less No. of specimens "Values are means.

4 1060 MARMAR ET AL. November 1975 dex, and normal morphology. Within this group, there have been three pregnancies-two births and one miscarriage (Table 7). There had been no pregnancies within this group for at least 1 year prior to treatment. TABLE 7. Results ofzns0 4 Therapy for 11 Patients with Oligospermia and Low Semen Zinc Levels Values 11 Zinc (mg/100 ml) Sperm count (millionlml) Motility index Normal morphology "Values are means. Before ZnSO, DISCUSSION After ZnSO, Significance 18.9 p < p < p < p < 0.02 The high concentration of zinc in human seminal plasma results largely from the secretory contributions of the prostate gland. Mackenzie et al. 4 studied the zinc content of expressed human prostatic fluid, and they concluded that on a dry weight basis the prostatic fluid contained more zinc than any other human secretion. Lindholmer and Eliasson 5 examined a series of split ejaculates. The mean zinc concentration in the first fraction, or prostatic fraction, was 294 J.Lg/ml, whereas the mean zinc concentration in the last fraction, or seminal vesicular fraction, was only 44 J.Lg/ml. Furthermore, Eliasson 6 demonstrated a positive correlation between seminal acid phosphatase activity and seminal zinc concentration. Low levels of semen zinc occur either because of dilution from the seminal vesicular fluid or as a result of poor secretory function by the prostate gland. Eliasson et al. 7 noted that most patients with chronic prostato-seminal vesiculitis had a marked decrease in the seminal acid phosphatase and seminal zinc concentrations. Bush 8 reported low levels of semen zinc in patients with abacterial prostatitis. Many of these patients responded to therapy with zinc sulfate, 80 mg three times/ day (available as Vicon-C). Our data agree in principle with those of Bush. In our small series of patients with prostatitis, the levels of semen zinc rose dramatically after administration of zinc sulfate. The results are encouraging, but additional data are still being collected. Although there are some data concerning the utilization of 65 Zn radioisotope injected intravenously, 9 there are no specific reports in the literature concerning absorption, distribution, and utilization of ingested zinc in patients with prostatitis. Until more specific data become available, our protocol for the dosage of supplementary zinc sulfate will remain empirical for patients with prostatitis (between 150 and 240 mg of zinc sulfate/day). There has been considereable interest in determining the nutritional requirements for zinc in the past few years. The average total zinc content in 138 diets used in various metabolic studies in the United States was 12.2 mg/day. 10 In general, meat, eggs, milk products, and shellfish (especially oysters) are the best sources of zinc. Fruits and vegetables are usually poor sources. Balance studies indicate that the adult male requires approximately 4.9 to 6.1 mg of zinc/day. 11 These requirements are higher in adolescents and lactating women. Greater attention has been given to zinc nutrition and metabolism because a syndrome related to zinc deficiency has been documented in malnourished adolescents in Egypt and Iran The findings included dwarfism, iron deficiency anemia, hypogonadism, and retardation of sexual maturation. Although prostatic function was not studied specifically, maturation of the external genitalia occurred sooner in those patients given 100 mg of supplementary zinc sulfate/day. In the United States, a patient with regional enteritis had malabsorption, short stature, and retarded sexual development. Zinc deficiency was documented, and linear growth and sexual development

5 Vol. 26, No. 11 SEMEN ZINC LEVELS IN MAN 1061 accelerated when the patient was given zinc sulfate, 100 to 400 mg/day.u These cases represent extreme examples of zinc deficiency and malnutrition, but recently in this country there have been reports of marginal zinc deficiency. Hambridge et al. 14 studied the zinc concentration of hair clippings from 338 apparently healthy Denver school children. Ten children in this group had hair zinc levels similar to those of malnourished Egyptian adolescents with zinc deficiency. These 10 children were in the lowest loth percentile on the growth charts, and they had altered taste acuity. Henkin et al,l 5 reported a group of patients with hypogeusia. The patients demonstrated dramatically improved taste acuity with supplemental zinc sulfate. In our opinion, more data are needed to evaluate the status of zinc nutrition in this country. These data would be particularly important for patients with prostatitis and low levels of semen zinc. Although the relationship of seminal zinc to prostatic secretory function seems to be well documented, the relationship of zinc to the metabolism and motility of sperm remains unclear. In lower mammalian s~ecies such as the rat, the zinc concentration of seminal plasma is low. 16 The sperm obtain zinc from the testis, and the zinc content of epididymal and ejaculated spermatozoa remains the same. The situation is different in the dog. Saito et al. 17 reported that canine seminal plasma is rich in zinc. As a result, the ejaculated sperm contain more zinc than the epididymal sperm. When the canine epididymal sperm were bathed in ZnCl2 at 0.02 or 0.2 mm concentration, the motility of these sperm was enhanced to the level of motility of ejaculated sperm. 18 In man, Janick et al. 19 reported levels of zinc in sperm obtained from the vas deferens significantly lower than those of ejaculated sperm. However, in contrast to observations in the dog, there was no difference in the motility of vasal sperm versus ejaculated sperm. In a series of experiments in man, Lindholmer and Eliasson20 studied the exchange of zinc between sperm and seminal plasma. They concluded that the prostatic fluid contained some factor which protected the motility of the sperm by regulating the exchange of zinc and other cations across cell membrane of the sperm. From our data on 153 semen specimens studied as part of a work-up for infertility in 72 men, it was demonstrated that the specimens with the greatest fertility potential had the highest mean zinc concentrations. For example, the group with greater than 100 million sperm/ml had a significantly higher mean zinc concentration than the group with less than 20 million sperm!ml. Our data agree with those of other reports on human ejaculates. When human ejaculates were grouped into arbitrary classes according to percentage motility and motility quality, the class with the highest scores had a significantly greater mean zinc concentration. 19 In a separate study in man, 72% of patients with evidence of decreased prostatic secretory function (i.e., low semen zinc concentration and reduced seminal acid phosphatase activity) demonstrated teratospermia and asthenospermia.21 In a small group of 11 patients with oligospermia and low semen zinc, we have prescribed supplementary zinc sulfate. While on therapy, these patients demonstrated significantly greater mean levels of semen zinc, mean sperm count, and mean motility index. Three pregnancies have occurred during the period of treatment. We suspect that the supplementary zinc sulfate was beneficial to over-all prostatic function, but we are unable to explain the apparent beneficial effect on the other parameters of the semen analyses. In conclusion, we believe that the analyses of zinc in human seminal plasma provide valuable information about the

6 1062 MARMAR ET AL. November 1975 function of the prostate gland. This gland is a target organ of androgen activity and appears to be responsive to supplementary zinc sulfate. The prostate may hold the key to some yet unknown factors necessary for successful fertilization. SUMMARY The semen zinc levels were determined in 18 volunteers, 132 patients postvasectomy, and in 19 patients with prostatitis. The mean values for semen zinc were the same in the volunteers and postvasectomy patients. However, the mean zinc concentration in the volunteers and postvasectomy patients was significantly different from that of the patients with prostatitis. When a group of 12 patients with prostatitis was given supplementary zinc sulfate, the levels of semen zinc rose dramatically and were significantly different from those before treatment. As part of an infertility evaluation, 153 specimens from 72 men who had not fathered a child were examined. A complete semen analysis of each specimen was made, including the semen zinc concentration. The specimens were separated into four arbitrary groups with differing fertility potential. The groups were organized by sperm counts (less than 20 million sperm/ml, 20 to 60 million sperm/ ml, 60 to 100 million sperm/ml, and greater than 100 million sperm/ml). There was a trend toward increasing zinc concentrations in the groups with increasing fertility potential. Eleven patients with oligospermia and low semen zinc levels were given supplementary zinc sulfate, 80 mg three times/day for 6 months to 1 year. The parameters of the semen analysis improved in all categories. There were three pregnancies during the period of treatment. REFERENCES 1. Halsted JA, Smith JC Jr, Irwin Ml: A conspectus of research on zinc requirements of man. J Nutr 104:347, Eliasson R, Lindholmer Ch: Zinc in human seminal plasma. Andrologie 3:147, Huacuja L, Sosa A, Delago NM, Rosado A: A kinetic study of the participation of zinc in human spermatozoa metabolism. Life Sci 13: 1383, Mackenzie AR, Hall T, Whitmore WF Jr: Zinc content of expressed human prostatic fluid. Nature 193:72, Lindholmer Ch, Eliasson R: Zinc and magnesium in human spermatozoa. Int J Fertil 17: 153, Eliasson R: Biochemical analysis of human semen in the study of the physiology and pathophysiology of the male accessory genital glands. Fertil Steril 19:344, Eliasson R, Molin L, Rajka G: Involvement of the prostate and seminal vesicles in urethritis with special reference to semen analysis. Andrologie 2:179, Bush IM: Personal communication 9. Chisholm LD, Short MD, Ghanadian R, McRae CU, Glass HI: Radiozinc uptake and scintiscanning in prostatic disease. J Nucl Med 15:739, Osis D, Kramer L, Wiatrowski E, Spencer H: Dietary zinc intake in man. Am J Clin Nutr 25:582, Sandstead HH: Zinc nutrition in the United States. Am J Clin Nutr 26:1251, Prasad AS, Halsted JA, Nadimi M: Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism, and geophagia. Am J Med 31:532, Halsted JA, Ronaghy H, Abadi P, Haghshenass M, Amerhakemi GH, Barakat R, Reinhold JG: Zinc deficiency in man. Am J Med 53:277, Hambridge KM, Hambridge C, Jacobs M, Baum JD: Low levels of zinc in hair, anorexia, poor growth, and hypogeusia in children. Pediatr Res 6:868, Henkin Rl, Schecter PJ, HoyeR, Mattern CFT: Idiopathic hypogeusia with dysgeusia, hyposmia, and dysosmia: a new syndrome. JAMA 217:34, Birnbaum D, Hall Th, LeeR: The zinc content of rat sperm cells from ejaculate, vas, epididymis and testis. Proc Soc Exp Bioi Med 108:321, Saito S, Zeitz L, Busch IM: Zinc content of spermatozoa from various levels of canine and rat reproductive tract. Am J Physiol 213:749, 1967

7 Vol. 26, No. 11 SEMEN ZINC LEVELS IN MAN Saito S, Bush IM, Mackenzie AR, Whitmore WF Jr: The effects of certain metals and chelating agents on the motility of dog epididymal and ejaculated spermatozoa. Invest Urol4:546, Janick J, Zeitz L, Whitmore WF Jr: Seminal fluid and spermatozoon zinc levels and their relationship to human spermatozoon motility. Fertil Steril 22:573, Lindholmer Ch, Eliasson R: The effects of albumin, magnesium, and zinc on human sperm survival in different fractions of split ejaculates. Fertil Steril 25:424, Eliasson R: Correlation between the sperm density, morphology, and motility and the secretory function of the accessory genital glands. Andrologie 2:165, 1970