Significance of Seminal Fructose Studies in Male Infertility

Vol. 24, No. 11, November 1973 Printed in U.S.A.

FERTILITY AND STERILITY

Copyright © 1973 by The Williams & Wilkins Co.

SIGNIFICANCE OF SEMINAL FRUCTOSE STUDIES IN MALE INFERTILITY A. M. PHADKE, M.B.B.S., N. R. SAMANT, M.B.B.S.,

AND

SHUBHADA D. DEVAL, B.Sc.

Family Welfare Bureau, Bombay 4, India

It has been almost half a century since McCarthy et al. 1 first noted the presence of a reducing sugar in semen, which they believed was glucose. Subsequent observers have confirmed their findings. 2 ' 4 However, it was Mann 5, 6 who proved that the seminal sugar was fructose and not glucose and that it was elaborated by the seminal vesicles under the influence of testosterone. Since then, numerous investigators have estimated the fructose content of semen and have tried to correlate it either with the sperm count or with the metabolic activity of spermatozoa, with conflicting results. 7 - 18 Surprisingly, so far nobody has studied the relationship between the seminal fructose levels and the spermatogenic activity in man, which the present study seeks to analyze precisely. MATERIALS AND METHODS

The patients for this study were selected from among those who had attended the Family Welfare Bureau in the past 7 years for investigation and treatment of infertility. They were healthy adult males between the second and fourth decades of life and did not show any evidence of endocrine disorder. For each patient, routine semen analyses and quantitative estimations of fructose by the resorcinol method were carried out two or three times, at intervals of 1 month, to assess the normal variations occurring spontaneously in semen. The mean fructose value was taken to represent the fructose concentration in the semen of each patient. In every case, abstinence from intercourse for 3-5 days was insisted Received March 20, 1973.

upon prior to collection of the semen specimen. The fructose estimation was usually carried out within half an hour after collection of the specimen. The present study is based on 3324 fructose estimations carried out in 1662 patients. In addition, it incorporates the study and analyses of 305 testicular biopSIes. The values for seminal fructose were found to vary; both high and low values were noted in each group. For the detailed statistical analysis, 51 patients from each group were chosen according to the principle of random selection. Care was taken that investigator bias did not creep into the selection of cases. Testicular biopsies were carried out in all cases of azoospermia and in all cases of severe oligospermia where the sperm count was less than 10 million/cu. cm. Every patient in whom testicular biopsy was carried out was included in this study. The only exceptions were cases which showed mixed lesions in the testicular pathology. In the past 7 years we had come across 19 cases of congenital bilateral absence of vas deferens and 9 cases of ejaculatory duct obstruction. Semen examinations and testicular biopsies were carried out in these cases but they were not included in the subsequent statistical analysis. The cases of obstructive azoospermia referred to in this paper were cases of azoospermia where testicular biopsies showed normal spermatogenesis. They were explored surgically for vaso-epididymal anastomosis. In these cases numerous motile spermatozoa were present in the head of the epididymis, and the site of

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SEMINAL FRUCTOSE STUDIES IN INFERTILITY

proven fertility, the value for seminal fructose was 253 mg ./100 mi. However, the difference in mean fructose levels in these two groups was statistically not significant. 2. The mean seminal fructose concentrations in cases of moderate oligospermia, severe oligospermia, and non obstructive azoospermia were 293, 309, and 326 mg./100 mi., respectively. These cases differed significantly from normospermic patients with sperm counts of more than 40 million/cu. cm. (at the 0.05% confidence level). Evidently, the fructose level was inversely proportional to the sperm count. 3. The cases of obstructive azoospermia differed markedly from the cases of nonobstructive azoospermia. The seminal fructose concentration in the former group was 254 mg./100 mi., and in the latter group it was 326 mg./100 mi. The difference in mean fructose levels in these two groups

obstruction was either at the lower pole of the epididymis or in the proximal portion of the vas deferens. RESULTS

Correlation between Seminal Fructose Levels and the Sperm Counts. The mean seminal fructose values in various groups are summarized in Table 1. The F ratio from Table 1 shows that the differences in mean fructose concentrations between the groups were not due only to chance. The fructose content in semen varied inversely with the sperm count: the higher the sperm count the lower the value for seminal fructose noted. 1. The mean value for seminal fructose in normospermic men with a sperm count of more than 40 million/cu. cm. was 231 mg./100 mi. In normospermic cases with

TABLE 1. Correlation between Sperm Counts and Fructose Concentrations No.

Group

No. of cases·

Mean fructose

1.

N ormospermic (with sperm count over 40 million/cu. cm.) Normospermic with proven fertility Moderate oligospermia (with sperm count between 10 and 40 million/cu. cm.) Severe oligospermia (with sperm count below 10 million/cu. em.) N onobstructive azoospermia Obstructive azoospermia

51 (651)

231

±18.03

51 (222) 51(208)

253 293

±17.35 ±22.48

51 (276)

309

±20.00

51 (144) 51 (i61)

326 254

±21.63 ±19.63

S.E.

mg./l00ml.

2. 3.

4.

5. 6.

306 (1662)

Total Comparison between Groups 1 and 2: t = Groups 1 and 3: t = Groups 1 and 4: t = Groups 1 and 5: t = Groups 1 and 6: t =

0.882 2.149 2.897 2.63 0.0763

For DF IOO • t at 0.05 level F = 3.794 For DF = 5/300 F at 0.01 level = 3.08 F at 0.05 level = 2.25

=

1.98; t at 0.01 level

=

2.63

* Numbers in parentheses indicate the total number of cases in that group.

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PHADKE ET AL.

was statistically highly significant (t = = 1.98). 4. The cardinal finding of our study was the fructose level in semen in cases of obstructive azoospermia. It was identical with the level of seminal fructose, 253 mg./100 mI., observed in normospermic cases with proven fertility. By contrast, the highest values for seminal fructose were noted in cases of non obstructive azoospermia. Relationship between Seminal Fructose Levels and Spermatogenesis. Table 2 summarizes mean seminal fructose concentrations in different types of spermatogenic lesions. The differences in the mean seminal fructose levels in these groups were statistically highly significant at the 0.01 % confidence level. It is obvious from this table that the seminal fructose content varied inversely with the germinal cell activity. The fructose content in the semen was highest (357.3 mg./100 ml.) in cases of germinal cell aplasia in which the germinal 2.431; t 100 at 0.05

epithelium was absent and the seminiferous tubules were lined by Sertoli cells only. The lowest values for seminal fructose (255 mg./lOO ml.) were encountered in cases of obstructive azoospermia in which the spermatogenesis was normal. The cases of spermatogenic arrest at various levels, hyalinization or atrophy of the tubules, and germinal cell aplasia, differed significantly at the 0.02% confidence level from cases of obstructive azoospermia in which spermatogenesis was normal. Absence of Fructose in Semen. In all of the 19 cases of congenital bilateral absence of vas deferens, seminal fructose was absent and the freshly obtained semen specimens failed to coagulate normally. In 9 cases of ejaculatory duct obstruction, the volume of semen was reduced to one or two drops. The material was diluted with 0.5 ml. of distilled water and was subsequently tested for the presence of fructose. In all such cases seminal fructose was absent.

TABLE 2. Mean Seminal Fructose Values in Different Lesions in Testicular Biopsies Mean seminal fructose

S.E.

Group

Testicular lesion

No. of cases

1.

Normal spermatogenesis (obstructive azoospermia) Normal spermatogenesis (with severe oligospermia) Spermatogenic arrest at various levels Severe tubular atrophy and/or hyalinization of tubules Germinal cell aplasia

51

255.1

±17.54

56

283.0

±17.96

67

312.0

±14.15

46

342.6

±13.95

85

357.3

±13.45

mg./l00mi.

2. 3. 4. 5.

Comparison between Groups 1 and 2: t = 1.113 Groups 1 and 3: t = 2.56 Groups 1 and 4: t = 3.04 Groups 1 and 5: t = 4.628 For DF 100, t at 0.02 level = 2.36 F = 6.303 For DF = 4/300 Fat 0.01 level = 2.41 Fat 0.05 level = 3.38

,....<

.-(

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SEMINAL FRUCTOSE STUDIES IN INFERTILITY

DISCUSSION

Tyler 14 had noted wide fluctuations in seminal fructose values in the same patient on repeated examinations. Similar fluctuations in the fructose content of semen were noted by us, also, and for that reason the mean fructose value was taken as the representative value in each case. Even within the same group, wide variations in the seminal fructose levels were encountered. Hence, the data were subjected to statistical analysis on the basis of randomly selected cases, to enable us to draw valid and unbiased conclusions. The normal value for seminal fructose in Indian males ranged between 150 and 300 mg./lOO ml., with a mean of 231 mg./l00 ml. This compares favorably with the mean value of 225 mg./lOO ml. in Indian men, obtained by Sheth and Rao 17 with a chromatographic method. Figure 1 is a histogram illustrating the cumulative frequency distribution of fructose concentrations in normospermic men. The mean seminal fructose value in cases of obstructive azoospermia was 253 mg./l00 ml. This group is comparable with, and the findings for it are applicable to, patients in whom vasoligation has been carried out. Steniach had at one time believed that after vasoligation there is eventual atrophy of the seminiferous epithelium and that Leydig cells undergo compensatory hyperplasia, leading to the so-called rejuvenation. According to the present study, the endogenous androgenic activity, as reflected by the level offructose in semen, was normal in patients having obstructive lesions. Nelson,19 after estimating urinary 17 -ketosteroids in cases of obstructive azoospermia, likewise concluded that the urinary 17-ketosteroid values were neither increased nor decreased but remained normal. Correlation between the Fructose Content in Semen and the Sperm Count. In the

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present series the fructose content in semen was found to vary inversely with the sperm count. Davis and McCune 9 and Schirren 18 had noted such an inverse relationship. However, other observers, such as Tyler 14 and Sheth and Rao,17 failed to observe such an inverse correlation. Three explanations have been offered to account for this inverse relationship. First, it was suggested that the low values for seminal fructose in normospermic men could result from the utilization of fructose by spermatozoa. This tempting suggestion was obviously based on knowledge gained from studies of the metabolic activities of spermatozoa. Several investigators had proved that, under anaerobic conditions, spermatozoa were capable of utilizing fructose for their energy requirements. The progressively higher values for seminal fructose in cases of oligospermia and azoospermia were explained by the same logic: namely, that in such cases "nonutilization" of fructose by the progressively declining number of spermatozoa

-

(,)

z ::> '"o '" II:

LL

FRUCTOSE IN SEMEN IN mg PER 100 ml.

FIG. 1. Histogram illustrating cumulative frequency distribution of fructose content in semen of normospermic men.

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PHADKE ET AL.

was responsible for the high(;I seminal fructose content. This "utilization" hypothesis can be restated to say that essentially there are no "real" differences in the seminal fructose content in infertile men but that the "apparent" lower values in the normospermic men are due to the utilization of fructose by the spermatozoa. The second suggestion was put forward by Tyler 14 and by MacLeod and Freund. 20 It was argued that, in specimens with high concentrations of spermatozoa, the spermatozoa themselves occupy an appreciable volume of the ejaculate and hence the fructose concentration per cubic centimeter is low. Third, Sheth and Rao 17 have suggested that the higher values for seminal fructose obtained by the resorcinol method in azoospermic men could be due to other reducing substances, namely tryptophan, present in the semen. According to all of the above-mentioned theories, the seminal fructose values in cases of obstructive azoospermia ought to have been the highest, as there were no spermatozoa in semen either to utilize the fructose or to occupy the volume of the ejaculate appreciably. Likewise, if tryptophan-like reducing substances were present in higher concentrations in the azoospermic semen samples, the seminal fructose values ought to have been the highest in this group. Surprisingly, the mean seminal fructose value in this group was identical with the mean fructose value obtained in fertile individuals. By contrast, the fructose values in cases of nonobstructive azoospermia were highest in the present series. Rationally, therefore, it was concluded that the fructose production itself varied inversely with the sperm count and that the seminal fructose content depended on the state of the germinal epithelium rather than on the number of spermatozoa present in the semen. Vaishwanar 15 noted the elevated seminal fructose values in azoospermic cases. How-

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ever, the conclusion at which he arrived was erroneous. He suggested that the "fructose test" would be helpful in differentiating between the cases of obstructive and nonobstructive azoospermia. According to Vaishwanar, high seminal fructose values in azoospermic men were indicative of obstructive lesions, and in nonobstructive azoospermia the fructose values would be low. The basis for his contention is not clear, as testicular biopsies were not performed in his series. Our findings are exactly contrary to this speculative hypothesis. The "utilization" hypothesis had built-in objections and fallacies from the beginning. In normal, healthy men, spermatozoa are not stored in the seminal vesicles prior to ejaculation but are stored in the ampullae of the vas deferens. Second, increased fructolysis inevitably should result in increased lactic acid production. Schirren 18 rightly pointed out that, in the semen specimens of normospermic men, the lactic acid content is not increased. Again, during normal coitus, the transient period during which spermatozoa come into contact with seminal plasma during ejaculation is hardly sufficient for appreciable fructolysis to occur. Last, the amount of fructose which is available to the spermatozoa is far in excess of their utilization needs for energy requirements. Relationship between Seminal Fructose and Spermatogenesis. It is obvious from Table 2 that the fructose content of semen varies inversely with the germinal cell activity. This finding is of much endoMann and crinologic significance. Parsons 21 , 22 demonstrated that the fructose level in semen depends on endogenous testosterone activity. Our finding accordingly means that there is progressively increased Leydig cell activity with progressive inactivity of the germinal epithelium. However, Leydig cells require stimulation from pituitary interstitial cell-stimulating hormone (ICSH). Can it then be said

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SEMINAL FRUCTOSE STUDIES IN INFERTILITY

logically that there is progressively increased production of ICSH with decreasing germinal cell activity? The crucial question is whether there is increased activity of Leydig cells in lesions of spermatogenic arrest, atrophy, or hyalinization of the seminiferous tubules and germinal cell aplasia. We have frequently noted hyperplasia of Leydig cells associated with these lesions. Typical Leydig cell hyperplasia in such conditions is illustrated in Figs. 2-4. Figure 5 shows normal spermatogenesis and is shown for comparison. The histologic evidence of Leydig cell hyperplasia corroborates the high seminal fructose content observed in such cases. The classical work of Howard et al. 23 has proved that there is a progressive increase in follicle-stimulating hormone (FSH) in urine in cases of spermatogenic arrest, hyalinization of tubules, and germinal cell aplasia. They revived the theory of "in-

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hibin," initially suggested by McCullagh 24 in 1932. The germinal epithelium, according to them, elaborates a water-soluble, nonhormonal substance (inhibin) which has a suppressive effect on the anterior hypophysis. In cases of decreasing germinal cell activity, the production of inhibin accordingly is less. In turn, the suppressive effect on the anterior hypophysis is less, which results in the increased production and release of gonadotropins. Heller et al. 25 confirmed the findings of Howard et al. 23 They conceded that, with progressive germinal cell inactivity, there are increasing amounts of FSH in urine . The point on which they differed was in interpretation. Heller et al. 25, 26 postulated that there was no real increased production of gonadotropins by the pituitary but that there was progressive nonutilization of gonadotropins by the germinal epithelium. Their utilization hypothesis was based on

FIG. 2. Testicular biopsy showing a typical case of complete spermatogenic arrest after the formation of stray primary spermatocytes which are exfoliated in the lumina of the tubules. The seminiferous tubules are small and round and lack tortuosity. The interstitial tissue shows marked hyperplasia of Leydig cells. Hematoxylin and eosin, x 350.

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FIG. 3. Testicular biopsy showing hyalinization of seminiferous tubules. On the left is seen a group of nine seminiferous tubules which are represented by cordons of hyaline material surrounded by a single layer of fibroblasts. They lack lumina and even the Sertoli cells are not seen. The germinal epithelium is absent. On the right is a mass of Leydig cells. Hematoxylin and eosin, x 350.

two facts. They found normal or slightly lower values for urinary 17 -ketosteroids in cases of spermatogenic arrest, hyalinization of tubules, and germinal cell aplasia. They could not detect increased amounts of ICSH in urine. Logically, according to them, if inhibin production was less in such cases there ought to be increased production of FSH and ICSH, and both of these hormones should be present in increasing amounts in urine; but all the same, Heller et aI. 25 and Heller and Nelson 26 could not account for the Leydig cell hyperplasia in cases of atrophy and aplasia of germinal epithelium. Howard, Simmons, and Sniffen 27 have noted similar hyperplasia of Leydig cells in cases of sclerosing tubular degeneration and germinal cell aplasia, and so has Charny 28 in cases of spermatogenic arrest. They solved the riddle by assuming that the Leydig cell hyperplasia was not "real" but was "apparent." It was

simply the reduced diameter of seminiferous tubules which gave a false impression of relatively increased numbers of Leydig cells. When the histologic evidence was overwhelming and could not be ignored, it was argued that the Leydig cells were morphologically abundant but functionally failing. Our findings prove that the Leydig cell hyperplasia associated with these lesions is real and is reflected in the increased values for seminal fructose in such cases. Besides, how certain is the criterion of estimation of urinary 17 -ketosteroids in judging the activity of pituitary ICSH? Maddock, Epstein, and Nelson 29 believe that, urinary estrogens are better guides' to ICSH activity than are urinary 17 -ketosteroids. Previous investigators, such as Howard et aI.,23, 27 Heller et aI.,25 and Heller and Nelson,26 have established the reciprocal relationship between the excretion of FSH

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in urine and the germinal cell activity. deferens was detected in 19 cases; seminal From our observations, it may be deduced fructose was uniformly absent in these that there may be increased production of cases. Amelar and Hotchkiss 34 were the ICSH with decreasing germinal cell activ- first to demonstrate the absence of fructose ity. The chief function of seminal fructose in semen in cases of congenital bilateral is not to provide the spermatozoa with a absence of vas deferens. The seminal vesisuitable substrate for their metabolic ac- cles arise embryologically as an out-budtivity. Essentially, fructose should be ding of vasa deferentia, and in cases of looked upon as an index of Leydig cell congenital bilateral absence of vas deferens function. The capacity of spermatozoa to there is invariably the congenital absence utilize fructose is to be acknowledged as an of seminal vesicles also. Fructose in semen incidental finding. In the vast majority of is solely produced by the seminal vesicles. cases of male infertility there is no andro- Similarly, the protein substrate which is genic deficiency, as judged by the seminal essential for coagulation of semen is confructose content. Treatment of such cases tributed by the seminal vesicles, and its with testosterone is rationally unwarranted absence results in the failure of the semen and will inevitably end in failure if un- to coagulate. 32 wisely attempted. The absence of fructose in semen in cases Absence of Fructose in Semen. Congeni- of ejaculatory duct obstruction is selftal bilateral absence of vas deferens is by explanatory. This malady was noted in 9 no means rare. Various investigators 30' 34 cases, primarily of Koch's origin, in the have reported such cases. In the present series. Recently, Kaplan et al. 35 have demonseries, congenital bilateral absence of vas

FIG. 4. Testicular biopsy showing germinal cell aplasia. The seminiferous tubules (three are incompletely seen) are small in diameter and lack tortuosity. They are lined by Sertoli cells only. The germinal epithelium is absent. The interstitial tissue shows extensive hyperplasia of Leydig cells. Hematoxylin and eosin, x 350.

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FIG. 5. Testicular biopsy showing normal spermatogenesis in a typical case of obstructive azoospermia. Note occasional Leydig cells in the intertubular spaces. Hematoxylin and eosin, x 350.

strated that, in cases of congenital cystic fibrosis, invariably there is congenital bilateral absence of vas deferens and seminal vesicles. The seminal fructose in such cases was always missing. More males with cystic fibrosis are living into the reproductive age because of advances in medical care, and the recognition of this entity merits the special attention of the physician. SUMMARY

The present study is based on 3324 fructose estimations carried out in 1662 cases of male infertility. The "normal" values for seminal fructose in Indian males are reported. The correlation between the seminal fructose findings and sperm counts was investigated. It was observed that the fructose level in semen varied inversely with the sperm count. This inverse relationship was not due to the varying "utilization" of fructose by the spermatozoa.

Instead, it was established that the fructose production in the semen itself varies inversely with the sperm count. For the first time the relationship between the seminal fructose findings and the state of spermatogenesis was studied on the basis of 305 testicular biopsy findings. It is concluded that the level of fructose in semen varies inversely with the germinal cell activity. In testicular lesions associated with higher seminal fructose values there was evidence of Leydig cell hyperplasia. In obstructive azoospermia the mean value for seminal fructose was not raised and was similar to the mean fructose levels observed in fertile men. Evidence presented here indirectly supports the "inhibin" hypothesis. Seminal fructose was absent in 19 cases of congenital bilateral absence of vas deferens and in 9 cases of ejaculatory duct obstruction which were encountered in the past 7 years.

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Acknowledgment. The authors thank Dr. L. D. Sanghavi, M.Sc., Ph.D., Dean, Indian Cancer Research Centre, Parel, Bombay 12, for making arrangements for the photomicrographs published in this paper. REFERENCES 1. MCCARTHY, J. F., STEPTIA, C. T., JOHNSTON, M. B., AND KILLIAN, J. F. Biochemical studies of prostato-vesicular secretions. J Urol 19:43, 1928. 2. HUGGINS, C. B., AND JOHNSON, C. Chemical observations on fluids of the seminal tract. Amer J Physiol 103:574, 1933. 3. GOLDBLATT, M. Properties of human seminal plasma. J Physiol (London) 84:208, 1935. 4. MACLEOD, J. The metabolism of human spermatozoa. Proc Soc Exp Bioi Med 42:153, 1939. 5. MANN, T. The origin and function of seminal fructose. Biochem J 40:29, 1946. 6. MANN, T. Studies on the metabolism of semen. Fructose as a normal constituent of seminal plasma. Site of formation and function of fructose in semen. Biochem J 40:481,1946. 7. HUGGINS, C. "Role of Accessory Glands in Fertility." In Diagnosis in Sterility, Engle, E. T., Ed. Blackwell, Oxford, England, 1946, p. 67. 8. PRYDE, J. Sugar of human semen. Nature (London) 157:660, 1946. 9. DAVIS, M. E., AND MCCUNE, W. W. Fructolysis of human spermatozoa. Fertil Steril 1:362, 1950. 10. HARVEY, C. "Fructose and Citric Acid in Human Semen." In Proceedings of the Society for the Study of Fertility. Blackwell, Oxford, England, 1951, p. 56. 11. LANDAU, R. L., AND LOUGHEAD, R. Seminal fructose concentration as an index of androgenic activity in man. J Clin Endocr 11:1411, 1951. 12. MCCULLAGH, E. B., AND SCHAFFENBURG, C. A. Hormonal activity in semen. J Clin Endocr 11 :403, 1951. 13. RABOCH, J., AND HARDEC, J. Quantitative fructose Bestimmungen in menschichen Ejakulate. Endokrinologie 31:171, 1954. 14. TYLER, E. T. Seminal fructose studies in infertility. Fertil Steril 6:247, 1955. 15. VAISHWANAR, P. S. Fructolysis of human spermatozoa in semen. Amer J Obstet Gynec 75:139, 1958. 16. VASTERLING, H. W. Fructose and fructolysis in human seminal fluid. Z Geburtsh Gynaek 152:36, 1959. 17. SHETH, A. R., AND RAO, S. S. Fructose levels in human semen determined chromatographically. Indian J Med Sci 16:709, 1962. 18. SCHIRREN, C. Relation between fructose content of semen and fertility in man. J Reprod FertiI5:347, 1963. 19. NELSON, W. O. "Spermatogenesis in Testes of

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