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The Effect of Graded Unilateral Testicular Biopsy on the Reproductive Capacity of Male Rats
0022-5347 /86/1351-0155$02.00 /0 Vol. 135, January Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1986 by The Williams & Wilkins Co.
THE EFFECT OF GRADED UNILATERAL TESTICULAR BIOPSY ON THE REPRODUCTIVE CAPACITY OF MALE RATS M. JAMES COSENTINO,* JOEL SHEINFELD, ERDAL ERTURK, AND ABRAHAM T. K COCKETT From the Department of Urology, University of Rochester School of Medicine and Dentistry, Rochester, New York
ABSTRACT
Fifty adult rats were subjected to unilateral testicular biopsy removing either 0.01, 0.1, 0.2 or 0.3 cc of testicular parenchyma. In addition, 20 rats underwent either hemicastration or sham surgery. After a 30-day recovery period each male was housed with two cycling females for 20 days. At the end of this breeding trial the percentage of fertile males, percentage of pregnant females and resulting embryo scores (no. of embryos x size of embryos) were determined for each group of male rats. After an additional 30 days (60 days post-biopsy) a second breeding trial was performed so as to note any long-term changes in fertility. In addition to the fertility parameters, mean seminiferous tubule diameters and serum testosterone levels were noted. After the first breeding trial the percentage of fertile males and percentage of pregnant females were inversely proportional to the amount of biopsy material removed (p < 0.05). However, the hemicastrate and sham-operated groups did not differ from the O cc control animals. The results of the second breeding trial showed a significant improvement in the percentage of females becoming pregnant (p < 0.05) and a tendency for improvement in the percentage of fertile males when compared to data of the first breeding trial. In addition, we found the mean seminiferous tubular diameter of the biopsied testes to be inversely proportional to the size of the biopsy (p < 0.01) with no apparent effect on the contralateral testes. We conclude that removing relatively large amounts of testicular parenchyma during unilateral testicular biopsy transiently affects male reproductive capacity, at least in the healthy animal model studied here. Ever since its introduction by Charny in 1940,1 the procedure of testicular biopsy has been established as an important diagnostic technique in male infertility. It has been particularly valuable in the assessment of such pathological conditions as Sertoli-cell-only syndrome, Klinefelter's syndrome, carcinoma in situ of the testes and obstructive azoospermia. 2 Although the need for evaluating biopsies from men with azoospermia is generally accepted, its role in evaluating oligospermic men has recently become less certain. Indeed, with the development of rapid FSH assays 3 in combination with accurate testicular size measurements4 even azoospermic patients with high FSH levels and reduced testicular size need not be biopsied since the spermatogonial population is usually irreversibly depleted. 5 However, if severe oligospermia is accompanied by normal FSH levels and normal testicular size, a biopsy showing normal spermatogenesis would indicate the presence of a partial duct obstruction. Thus, testicular biopsy may be indicated in a variety of conditions found in the infertile male. The literature has repeatedly demonstrated the fragility of the testis after various insults such as spermatic cord torsion 6 • 7 and cryptorchidism. 8 These studies showed that even after relatively mild insults to one testis, both testes degenerate as does subsequent fertility. 9 Thus, the question arises whether a biopsy could render a subfertile male infertile and if the size of the biopsy might play a role in the extent of the damage. To shed some light on these questions, the present study was designed to note the effect of unilateral testicular biopsies of various sizes on the fertility, fecundity and histology of rat testes. MATERIALS AND METHODS
Animals. Seventy adult male and 280 adult female SpragueDawley rats were used in this study. All animals were accliAccepted for publication July 26, 1985. *Requests for reprints: Dept. of Biology, University of Scranton, Scranton, PA 18510. Read at annual meeting of the American Urological Association, Atlanta, Georgia, 1985.
mated for 10 days, housed at 21C with a 12 hour light:12 hour dark cycle and fed standard pellets and water ad libitum. Testicular biopsy. The male rats were randomly divided into seven groups of ten animals each. The animals were anesthetized with sodium pentobarbital and the tunica albuginea of one testis was exposed. A 5 mm. incision was made in the anterior border of the testicular capsule and the appropriate amount of testicular parenchyma removed using iris scissors. The tunica albuginea was then closed with 6-0 chromic gut suture using a single figure-eight stitch. The scrotum was then closed using small animal wound clips and the animals were allowed to recover for one month. Four groups underwent unilateral testicular biopsy having either 0.01, 0.1, 0.2 or 0.3 cc of testicular parenchyma removed. The tissue volumes were determined by water displacement. This represents tissue masses having approximate diameters of 2, 5, 7 or 10 mm. respectively. A control group of ten animals had no parenchyma removed, but underwent a figure-eight stitch in the tunica albuginea using identical suture material. Another group of rats underwent hemicastration instead of unilateral testicular biopsy while yet another group only had the testis surgically exposed for 10 minutes while under general anesthesia. Fertility and fecundity tests. The assessment of fertility and fecundity of the male rats was done using a technique previously described by us. 6 That is, one month after surgery each male rat was housed with two adult female rats for 18 days, thus exposing each male rat to approximately eight female reproductive cycles. At the end of this period the female rats were sacrificed by an overdose of sodium pentobarbital and examined for gravidity. If either mate of a male rat became pregnant, that male was considered fertile. The number and mean length of embryos was determined for each pregnant female rat. The product of these two parameters (embryo score) was used as an index of fecundity. Two months after surgery a second breeding trial was conducted in the same manner described above to note any change in fertility status over time. Histological procedures. Three months after surgery both the
156
COSENTINO AND ASSOCIATES
ipsilateral and contralateral testes of each male rat were removed, immediately placed in Bouin's fixative and embedded in paraffin. The tissue sections were stained with hematoxylin and eosin and examined under 100 x magnification. The mean seminiferous tubular diameter was calculated to within 5 µ for each testis. This was done by averaging the diameters of ten round seminiferous tubules randomly selected in each tissue section. A tubule was considered round (cut in cross section) if the height was within 20 µ of the width. The measurements were done using a calibrated transparent overlay on a video monitor displaying the stained tissue sections at a final magnification of 400 X. Testosterone. At the time of orchiectomy, blood was obtained from the tail vein of each animal. The serum was assayed for testosterone concentration using a commercially available solid-phase radioimmunoassay (lmmuchem Corp.). This assay has an ED 50 of 3 to 5 mg./ml. and a per cent cross-reactivity of <8 per cent with dihydrotestosterone. Both the intra- and interassay variation has been established as less than 10 per cent (no.= 20). Statistical analyses. Data were analyzed for differences among groups using binomial Chi-square analyses or analysis of variance followed by Fisher's test for least significant difference. Differences between ipsilateral and contralateral seminiferous tubule diameters and differences between data obtained for the first and second fertility tests were done using paired t tests. The linearity of each parameter with respect to the amount of testicular parenchyma removed was calculated using Pearson's product-moment correlation. The significance of the number of animals showing ipsilateral tubule degeneration was calculated using binomial Chi-square analyses.
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RESULTS
Fertility of male rats. If a male rat impregnated either or both of the female rats with which it was housed, that male was considered fertile. The percentage of fertile male rats in each group one month after unilateral testicular biopsy was inversely proportional (r = -0.897, p < 0.05) to the amount of testicular parenchyma removed at the time of biopsy (fig. 1). Removal of the largest amount of parenchyma (0.3 cc) resulted in significantly (p < 0.01) depressed fertility, when compared to the control animals. In contrast, when these same animals were tested for fertility 2 months after the biopsies, no dependence on the size of the biopsy material was noted (fig. 1). Furthermore, the percentage of fertile males in each group was similar to that of the controls. In comparing all of the data obtained in this parameter one month after the biopsy with that obtained two months after the surgery, no significant difference was noted using a paired t test. However, the group of animals that underwent a 0.3 cc biopsy and demonstrated a depressed percentage of fertile males one month after surgery, showed significant (p < 0.05) improvement in this parameter to near control values when tested two months after the biopsies. Pregnancies. The percentage of female rats impregnated by male rats undergoing testicular biopsy one month before breeding was inversely proportional (r = -0.926, p < 0.05) to the size of the biopsy (fig. 2). The groups of female rats housed with males having 0.2 and 0.3 cc of testicular parenchyma removed one month earlier showed significantly (p < 0.05 and p < 0.01, respectively) fewer pregnancies when compared to control animals. The female rats housed with the same males two months after the biopsies showed significantly (p < 0.05) more pregnancies than those housed with the males one month after the biopsies (fig. 2). However, it was noted that those females housed with males having 0.3 cc of testicular parenchyma removed still exhibited significantly (p < 0.05) fewer pregnancies two months post-biopsy when compared to the control animals (fig. 2). Fecundity. The mean number of embryos per pregnant female (total of 2,583 embryos from 217 females) was not correlated
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to the size of the testicular biopsy nor did analysis of variance testing reveal any significant differences amongst the number of embryos produced by the male rats. This was the case for both the one-month and two-month fertility trials. Similarly, no changes were seen in the size of resulting embryos with respect to the amount of parenchyma removed from the male rats. These data were collectively represented using an overall index of fecundity which was obtained by multiplying the
157
EFFECT OF TESTICULAil ElCPSY OI\T REPRODlJCTIVE CAJ?ACITY
numbers of their size for each impregnated female and expressing the results as an embryo score (fig. 3). Seminiferous tubu/,e diameters. Examination of the ipsilateral testes revealed an inverse linear relationship (r = -0.982, p < 0.01) between the seminiferous tubular diameter and the biopsy size (fig. 4). Furthermore, the mean ipsilateral seminiferous tubular diameter resulting after biopsies of 0.1 cc or more were found to be significantly smaller (p < 0.01 to p < 0.05) than those of the control animals. Histological examination of the
TABLE 1. Percentage of testes with all seminiferous tubules degenerated to nonfunctional state three months after testicular biopsy
Testicular Biopsy Size (cc) Testes 0 Ipsilateral0 0 (0/10) Contralateral O (0/10)
0.01
0.10
0.20
0.30
11.1 (1/9) 0 (0/9)
30.0 (3/10) 0 (0/10)
33.3 (3/9)* 0 (0/9)
66.6 (6/9)** 0 (0/9)
0 Values obtained for the ipsi!ateral testes are significantly correlated to testicular biopsy size (r = 0.879; p <0.05). * Significantly larger than control values (p <0.05). ** Significantly larger than control values (p <0.01).
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testes three months after the biopsies revealed no change in the contralateral seminiferous tubule diameters regardless of the amount of parenchyma removed (fig. 4). When the seminiferous tubular diameters were grouped according to biopsy size, the ipsilateral testes showed significantly smaller (p < 0.001) mean diameters than that of the contralateral testes (fig.
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With respect to the seminiferous tubular diameter changes observed in the ipsilateral testes, we noted an all-or-none phenomenon. That is, those testes exhibited either normal tubular diameters relative to control values or were so degenerated that the diameters could not be measured (diameter = 0). The percentage of animals showing severely degenerated ipsilateral tubules was proportional to the size of the biopsy (r = 0.879, p < 0.05) with the 0.20 and 0.30 cc3 biopsies having significantly more (p < 0.05 and p < 0.01 respectively) degenerated testes compared to the control group (table 1). Indeed, when the contralateral testes of only the animals showing ipsilateral damage were examined, no changes were noted in the seminiferous tubular diameters of those contralateral testes. Thus, the damage induced by testicular biopsy appears to be isolated within the ipsilateral testes. Testosterone. Serum testosterone levels ranged from 0.3 ng./ ml. to 10.53 ng./ml. and did not differ significantly with respect to the amount of testicular parenchyma removed from the testes. Hemicastrated animals. The fertility and fecundity of animals that underwent hemicastration (HEMI) was compared to those animals that received a figure eight stitch in the tunica albuginea (STIT) and those that underwent testicular exposure only (SHAM) (table 2). Among these three groups, no significant differences were noted with respect to the per cent of fertile males, per cent of pregnant females or embryo scores one month post-surgeryo Furthermore, these values did not significantly change at the second breeding trial (two months post-surgery). Therefore, the changes seen in the fertility of the biopsied animals were probably not due to loss of germinal epithilium mass but due to the damage incurred in the biopsies. It was observed that the seminiferous tubule diameters of the testes at the end of the experiment were not m~:mncan.try different among the HEMI, STIT and SHAM groups Similarly, serum testosterone levels were essentially equal in these three groups. DISCUSSION
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(cc) FIG. 4. Seminiferous tubular diameters of rats having undergone testicular biopsies of various sizes three months earlier. (e) ipsilateral testes; (1!11) contralateral testes (means ± S.E.M.). * = significantly different from control values (0cc) (p < 0.05) **=significantly different from control values (0cc) (p < 0.01).
To date, only two studies on the effects of testicular biopsy in men have been undertaken. The first 10 was done on 20 inmate volunteers living in a penitentiary. Particular care was taken by these researchers to establish what their sperm counts were prior to biopsy. This was done by examining from nine to 16 ejaculates prior to surgery. After the biopsies, 55 per cent of the subjects showed no effect on sperm concentration for the entire follow-up period while 45 per cent experienced a significant depression in this parameter. Furthermore, 26 per cent of the subjects exhibited a secondary drop in sperm count or a drop that persisted throughout the study. Clearly, for about one half of the subjects, sperm counts were adversely affected by the biopsies at least temporarily, while one half of those
158
COSENTINO AND ASSOCIATES TABLE 2.
Fertility parameters of male rats following three surgical treatments (control groups)
First Fertility Trial-1 Month After Surgery Treatment Hemicastration T. albuginea suture Sham surgery
% Fertile Males
90 (9/10) 100 (10/10) 90.9 (10/11)
% Pregnant Females
Embryo* Score
80 (16/20) 90 (18/20) 81.8 (18/20)
162.7 ± 27.5 119.9 ± 20.0 129.9 ± 18.7
Second Fertility Trial-2 Months After Surgery
3 Months After Surgery
(µm.)
Serum* Testosterone (ng./ml.)
265 ± 2 260 ± 2 255 ± 10
2.18 ± 0.28 2.55 ± 0.33 1.82 ± 0.21
Mean* Seminiferous Tubular Diameter Hemicastration T. albuginea suture Sham surgery
100 (10/10) 100 (10/10) 90.9 (10/11)
90 (18/20) 90 (18/20) 86.4 (19/23)
144.9 ± 20.9 157.9 ± 19.9 172.2 ± 22.8
* Mean ± S.E.M.
affected had more long-term affects. A larger study (100 subjects) by Rowley et al.11 similarly showed a decrease in sperm count in about 40 per cent of the subjects after testicular biopsy. However, all of these men recovered to pre-biopsy sperm counts within 18 weeks. The men who exhibited a decrease in sperm count were classified into two groups: 1) those that had an abrupt decrease followed by an immediate return to normal and 2) those having an immediate decrease that persisted for 10 weeks after the biopsies. This suggests that the spermatogenic process of the quick recovery group was not affected since it takes from 60 to 90 days for new sperm to show up in the ejaculate of man. However, the second group did not begin recovery until 70 days after the biopsies, suggesting some upset of spermatogenesis occurred at the time of biopsy. Unfortunately it is not possible to determine from those studies if the effect noted on sperm count was due to the actual biopsy or the surgical trauma to the scrotum. Indeed, the study by Gordon et al. 10 incorporated the use of post-surgical ice packs for 12 to 18 hours. This alone may have had some effect on sperm concentration. 12 The present study also noted transient changes of rat fertility following testicular biopsy. That is, even though the fertility of the male rats and the number of pregnancies they produced were inversely proportional to the size of the testicular biopsy one month after surgery (figs. 1 and 2) assessment of these parameters two months after surgery showed fertility returning to control levels. Indeed, the quality of the pregnancies that did occur (embryo score, fig. 3) was unaffected regardless of the time after surgery. Of particular interest to us was the observation that testicular biopsy reduced the seminiferous tubular diameter in an all-ornone fashion (table 1). These data suggest that some structure (perhaps vascular) critical to the maintenance of the rest of the organ was distributed more often with the larger biopsies. The mechanism of this phenomenon, however, remains to be elucidated. The amount of tissue removed during the biopsies, 0.01, 0.1, 0.2 or 0.3 cc, represents approximately 1 per cent, 10 per cent, 20 per cent and 30 per cent respectively of a rat testis volume. However, extrapolation to the human situation where approximately 1 per cent or less of testicular parenchyma is removed, should be cautioned against since it is not known if the percentage of the testis volume removed is important or if the absolute amount of parenchyma (for example, 0.2 cc) is the critical factor. Since we noted that hemicastration in the adult male rat did not affect fertility (table 2) it is apparent that the changes seen in fertility of the biopsied animals was not due to loss of germinal epithelium mass from the organism, but to some damage incurred to the tissue remaining in the organism. That is, perhaps some critical structure (such as a blood vessel) was damaged during biopsy. Certainly, the larger the biopsy the more likely one would be to damage such a structure. This, however, remains to be determined. Hemicastration of 65-day-old rats 13 or 6 to 10-month-old
rats 14 did not result in contralateral compensatory hypertrophy of the remaining testes and is in agreement with the data of the present study (table 2). However, rats aged 22 to 24 months did show striking hemicastration-induced testicular enlargement.14 A similar response was also noted in adult rams. 15 Thus, the response of the remaining testis to hemicastration is variable depending on the age and species of the organism. The data of the present study indicate that fertility in the healthy adult male rat undergoing unilateral testicular biopsy is not adversely affected unless removal of relatively large amounts of parenchyma occurs. However, whether or not this procedure adversely affects fertility of an already subfertile man remains speculative. REFERENCES
1. Charny, C. W.: Testicular biopsy: its value in male sterility. JAMA, 115: 1429, 1940. 2. Leong, A. S. Y. and Matthews, C. D.: The role of testicular biopsy in the investigation of male infertility. Pathol., 14: 205, 1982. 3. de Kretser, D. M., Burger, H. G. and Hudson, B.: The relationship between germinal cells and serum FSH levels in males with infertility. J. Clin. Endocrinol. Metabol., 38: 787, 1974. 4. Takihara, H., Sakatoku, J., Fujii, M., Nasu, T., Cosentino, M. J. and Cockett, A. T. K.: Significance of testicular size measurement in andrology. I. A new orchiometer and its clinical application. Fertil. Steril., 39: 836, 1983. 5. de Kretser, D. M.: Testicular biopsy in the management of male infertility. Int. J. Androl., 5: 449, 1982. 6. Cosentino, M. J., Rabinowitz, R., Valvo, J. R. and Cockett, A. T. K.: The effect of prepubertal spermatic cord torsion on subsequent fertility in rats. J. Androl., 5: 93, 1984. 7. Cosentino, M. J., Nishida, M., Rabinowitz, R. and Cockett, A. T. K.: Histological changes occurring in the contralateral testes of prepubertal rats subjected to various durations of unilateral spermatic cord torsion. J. Urol., 133: 906, 1985. 8. Urry, R. L.: Evidence for a contralateral testicular effect in unilaterally cryptorchid animals. (abstract). J. Androl., 1: 64, 1980. 9. Wallace, D. M.A., Gunter, P.A., Landon, G. V., Pugh, R. C. B. and Hendry, W. F.: Sympathetic orchiopathia: an experimental and clinical study. Br. J. Urol., 54: 765, 1982. 10. Gordon, D. L., Barr, A. B., Herrigel, J. E. and Paulsen, C. A.: Testicular biopsy in man. I. The effect upon sperm concentration. Fertil. Steril., 16: 522, 1965. 11. Rowley, M. J., O'Keefe, K. B. and Heller, C. G.: Decreases in sperm concentration due to testicular biopsy procedure in man. J. Urol., 101: 347, 1969. 12. Mulrahy, J. J.: Scrotal hypothermia and the infertile man. J. Urol., 132: 469, 1984. 13. Lindgren, S., Danber, J. E. and Carstensen, H.: Compensatory testosterone secretion in unilaterally orchidectomized rats. Life Sci., 18: 1203, 1976. 14. Johnson, L. and Neaves, W. B.: Enhanced daily sperm production in the remaining testes of aged rats following hemicastration. J. Androl., 4: 162, 1983. 15. Voglmayr, J. K. and Matner, P. E.: Compensatory hypertrophy in the remaining testis following unilateral orchidectomy in the adult ram. J. Reprod. Fertil., 17: 179, 1968.
THE JOURNAL OF UROLOGY
Copyright© 1986 by The Williams & Wilkins Co.
THE EFFECT OF GRADED UNILATERAL TESTICULAR BIOPSY ON THE REPRODUCTIVE CAPACITY OF MALE RATS M. JAMES COSENTINO,* JOEL SHEINFELD, ERDAL ERTURK, AND ABRAHAM T. K COCKETT From the Department of Urology, University of Rochester School of Medicine and Dentistry, Rochester, New York
ABSTRACT
Fifty adult rats were subjected to unilateral testicular biopsy removing either 0.01, 0.1, 0.2 or 0.3 cc of testicular parenchyma. In addition, 20 rats underwent either hemicastration or sham surgery. After a 30-day recovery period each male was housed with two cycling females for 20 days. At the end of this breeding trial the percentage of fertile males, percentage of pregnant females and resulting embryo scores (no. of embryos x size of embryos) were determined for each group of male rats. After an additional 30 days (60 days post-biopsy) a second breeding trial was performed so as to note any long-term changes in fertility. In addition to the fertility parameters, mean seminiferous tubule diameters and serum testosterone levels were noted. After the first breeding trial the percentage of fertile males and percentage of pregnant females were inversely proportional to the amount of biopsy material removed (p < 0.05). However, the hemicastrate and sham-operated groups did not differ from the O cc control animals. The results of the second breeding trial showed a significant improvement in the percentage of females becoming pregnant (p < 0.05) and a tendency for improvement in the percentage of fertile males when compared to data of the first breeding trial. In addition, we found the mean seminiferous tubular diameter of the biopsied testes to be inversely proportional to the size of the biopsy (p < 0.01) with no apparent effect on the contralateral testes. We conclude that removing relatively large amounts of testicular parenchyma during unilateral testicular biopsy transiently affects male reproductive capacity, at least in the healthy animal model studied here. Ever since its introduction by Charny in 1940,1 the procedure of testicular biopsy has been established as an important diagnostic technique in male infertility. It has been particularly valuable in the assessment of such pathological conditions as Sertoli-cell-only syndrome, Klinefelter's syndrome, carcinoma in situ of the testes and obstructive azoospermia. 2 Although the need for evaluating biopsies from men with azoospermia is generally accepted, its role in evaluating oligospermic men has recently become less certain. Indeed, with the development of rapid FSH assays 3 in combination with accurate testicular size measurements4 even azoospermic patients with high FSH levels and reduced testicular size need not be biopsied since the spermatogonial population is usually irreversibly depleted. 5 However, if severe oligospermia is accompanied by normal FSH levels and normal testicular size, a biopsy showing normal spermatogenesis would indicate the presence of a partial duct obstruction. Thus, testicular biopsy may be indicated in a variety of conditions found in the infertile male. The literature has repeatedly demonstrated the fragility of the testis after various insults such as spermatic cord torsion 6 • 7 and cryptorchidism. 8 These studies showed that even after relatively mild insults to one testis, both testes degenerate as does subsequent fertility. 9 Thus, the question arises whether a biopsy could render a subfertile male infertile and if the size of the biopsy might play a role in the extent of the damage. To shed some light on these questions, the present study was designed to note the effect of unilateral testicular biopsies of various sizes on the fertility, fecundity and histology of rat testes. MATERIALS AND METHODS
Animals. Seventy adult male and 280 adult female SpragueDawley rats were used in this study. All animals were accliAccepted for publication July 26, 1985. *Requests for reprints: Dept. of Biology, University of Scranton, Scranton, PA 18510. Read at annual meeting of the American Urological Association, Atlanta, Georgia, 1985.
mated for 10 days, housed at 21C with a 12 hour light:12 hour dark cycle and fed standard pellets and water ad libitum. Testicular biopsy. The male rats were randomly divided into seven groups of ten animals each. The animals were anesthetized with sodium pentobarbital and the tunica albuginea of one testis was exposed. A 5 mm. incision was made in the anterior border of the testicular capsule and the appropriate amount of testicular parenchyma removed using iris scissors. The tunica albuginea was then closed with 6-0 chromic gut suture using a single figure-eight stitch. The scrotum was then closed using small animal wound clips and the animals were allowed to recover for one month. Four groups underwent unilateral testicular biopsy having either 0.01, 0.1, 0.2 or 0.3 cc of testicular parenchyma removed. The tissue volumes were determined by water displacement. This represents tissue masses having approximate diameters of 2, 5, 7 or 10 mm. respectively. A control group of ten animals had no parenchyma removed, but underwent a figure-eight stitch in the tunica albuginea using identical suture material. Another group of rats underwent hemicastration instead of unilateral testicular biopsy while yet another group only had the testis surgically exposed for 10 minutes while under general anesthesia. Fertility and fecundity tests. The assessment of fertility and fecundity of the male rats was done using a technique previously described by us. 6 That is, one month after surgery each male rat was housed with two adult female rats for 18 days, thus exposing each male rat to approximately eight female reproductive cycles. At the end of this period the female rats were sacrificed by an overdose of sodium pentobarbital and examined for gravidity. If either mate of a male rat became pregnant, that male was considered fertile. The number and mean length of embryos was determined for each pregnant female rat. The product of these two parameters (embryo score) was used as an index of fecundity. Two months after surgery a second breeding trial was conducted in the same manner described above to note any change in fertility status over time. Histological procedures. Three months after surgery both the
156
COSENTINO AND ASSOCIATES
ipsilateral and contralateral testes of each male rat were removed, immediately placed in Bouin's fixative and embedded in paraffin. The tissue sections were stained with hematoxylin and eosin and examined under 100 x magnification. The mean seminiferous tubular diameter was calculated to within 5 µ for each testis. This was done by averaging the diameters of ten round seminiferous tubules randomly selected in each tissue section. A tubule was considered round (cut in cross section) if the height was within 20 µ of the width. The measurements were done using a calibrated transparent overlay on a video monitor displaying the stained tissue sections at a final magnification of 400 X. Testosterone. At the time of orchiectomy, blood was obtained from the tail vein of each animal. The serum was assayed for testosterone concentration using a commercially available solid-phase radioimmunoassay (lmmuchem Corp.). This assay has an ED 50 of 3 to 5 mg./ml. and a per cent cross-reactivity of <8 per cent with dihydrotestosterone. Both the intra- and interassay variation has been established as less than 10 per cent (no.= 20). Statistical analyses. Data were analyzed for differences among groups using binomial Chi-square analyses or analysis of variance followed by Fisher's test for least significant difference. Differences between ipsilateral and contralateral seminiferous tubule diameters and differences between data obtained for the first and second fertility tests were done using paired t tests. The linearity of each parameter with respect to the amount of testicular parenchyma removed was calculated using Pearson's product-moment correlation. The significance of the number of animals showing ipsilateral tubule degeneration was calculated using binomial Chi-square analyses.
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RESULTS
Fertility of male rats. If a male rat impregnated either or both of the female rats with which it was housed, that male was considered fertile. The percentage of fertile male rats in each group one month after unilateral testicular biopsy was inversely proportional (r = -0.897, p < 0.05) to the amount of testicular parenchyma removed at the time of biopsy (fig. 1). Removal of the largest amount of parenchyma (0.3 cc) resulted in significantly (p < 0.01) depressed fertility, when compared to the control animals. In contrast, when these same animals were tested for fertility 2 months after the biopsies, no dependence on the size of the biopsy material was noted (fig. 1). Furthermore, the percentage of fertile males in each group was similar to that of the controls. In comparing all of the data obtained in this parameter one month after the biopsy with that obtained two months after the surgery, no significant difference was noted using a paired t test. However, the group of animals that underwent a 0.3 cc biopsy and demonstrated a depressed percentage of fertile males one month after surgery, showed significant (p < 0.05) improvement in this parameter to near control values when tested two months after the biopsies. Pregnancies. The percentage of female rats impregnated by male rats undergoing testicular biopsy one month before breeding was inversely proportional (r = -0.926, p < 0.05) to the size of the biopsy (fig. 2). The groups of female rats housed with males having 0.2 and 0.3 cc of testicular parenchyma removed one month earlier showed significantly (p < 0.05 and p < 0.01, respectively) fewer pregnancies when compared to control animals. The female rats housed with the same males two months after the biopsies showed significantly (p < 0.05) more pregnancies than those housed with the males one month after the biopsies (fig. 2). However, it was noted that those females housed with males having 0.3 cc of testicular parenchyma removed still exhibited significantly (p < 0.05) fewer pregnancies two months post-biopsy when compared to the control animals (fig. 2). Fecundity. The mean number of embryos per pregnant female (total of 2,583 embryos from 217 females) was not correlated
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Biopsy Size
(cc) FIG. 2. Percentages of female rats becoming pregnant following two breeding trials with male rats having undergone testicular biopsies of various sizes. (e) 30 days after surgery: (B) 60 days after surgery. * = significantly different from control values (0cc) (p < 0.05). ** significantly different from control values (0cc) (p < 0.01).
to the size of the testicular biopsy nor did analysis of variance testing reveal any significant differences amongst the number of embryos produced by the male rats. This was the case for both the one-month and two-month fertility trials. Similarly, no changes were seen in the size of resulting embryos with respect to the amount of parenchyma removed from the male rats. These data were collectively represented using an overall index of fecundity which was obtained by multiplying the
157
EFFECT OF TESTICULAil ElCPSY OI\T REPRODlJCTIVE CAJ?ACITY
numbers of their size for each impregnated female and expressing the results as an embryo score (fig. 3). Seminiferous tubu/,e diameters. Examination of the ipsilateral testes revealed an inverse linear relationship (r = -0.982, p < 0.01) between the seminiferous tubular diameter and the biopsy size (fig. 4). Furthermore, the mean ipsilateral seminiferous tubular diameter resulting after biopsies of 0.1 cc or more were found to be significantly smaller (p < 0.01 to p < 0.05) than those of the control animals. Histological examination of the
TABLE 1. Percentage of testes with all seminiferous tubules degenerated to nonfunctional state three months after testicular biopsy
Testicular Biopsy Size (cc) Testes 0 Ipsilateral0 0 (0/10) Contralateral O (0/10)
0.01
0.10
0.20
0.30
11.1 (1/9) 0 (0/9)
30.0 (3/10) 0 (0/10)
33.3 (3/9)* 0 (0/9)
66.6 (6/9)** 0 (0/9)
0 Values obtained for the ipsi!ateral testes are significantly correlated to testicular biopsy size (r = 0.879; p <0.05). * Significantly larger than control values (p <0.05). ** Significantly larger than control values (p <0.01).
250
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200
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testes three months after the biopsies revealed no change in the contralateral seminiferous tubule diameters regardless of the amount of parenchyma removed (fig. 4). When the seminiferous tubular diameters were grouped according to biopsy size, the ipsilateral testes showed significantly smaller (p < 0.001) mean diameters than that of the contralateral testes (fig.
150
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0
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0
0.1
0.2
0.3
0.4
Biopsy Size (cc) FIG. 3. Embryo scores (embryo size X number of embryos) of female rats following two breeding trials with male rats having undergone testicular biopsies of various sizes (means ± S.E.M.). (0) 30 days after surgery: (Ill) 60 days after surgery.
-.__ -f--- __ --f--------I
Contraiatera!
4).
With respect to the seminiferous tubular diameter changes observed in the ipsilateral testes, we noted an all-or-none phenomenon. That is, those testes exhibited either normal tubular diameters relative to control values or were so degenerated that the diameters could not be measured (diameter = 0). The percentage of animals showing severely degenerated ipsilateral tubules was proportional to the size of the biopsy (r = 0.879, p < 0.05) with the 0.20 and 0.30 cc3 biopsies having significantly more (p < 0.05 and p < 0.01 respectively) degenerated testes compared to the control group (table 1). Indeed, when the contralateral testes of only the animals showing ipsilateral damage were examined, no changes were noted in the seminiferous tubular diameters of those contralateral testes. Thus, the damage induced by testicular biopsy appears to be isolated within the ipsilateral testes. Testosterone. Serum testosterone levels ranged from 0.3 ng./ ml. to 10.53 ng./ml. and did not differ significantly with respect to the amount of testicular parenchyma removed from the testes. Hemicastrated animals. The fertility and fecundity of animals that underwent hemicastration (HEMI) was compared to those animals that received a figure eight stitch in the tunica albuginea (STIT) and those that underwent testicular exposure only (SHAM) (table 2). Among these three groups, no significant differences were noted with respect to the per cent of fertile males, per cent of pregnant females or embryo scores one month post-surgeryo Furthermore, these values did not significantly change at the second breeding trial (two months post-surgery). Therefore, the changes seen in the fertility of the biopsied animals were probably not due to loss of germinal epithilium mass but due to the damage incurred in the biopsies. It was observed that the seminiferous tubule diameters of the testes at the end of the experiment were not m~:mncan.try different among the HEMI, STIT and SHAM groups Similarly, serum testosterone levels were essentially equal in these three groups. DISCUSSION
0
0.1
0.2
0.3
0.4
Biopsy Size
(cc) FIG. 4. Seminiferous tubular diameters of rats having undergone testicular biopsies of various sizes three months earlier. (e) ipsilateral testes; (1!11) contralateral testes (means ± S.E.M.). * = significantly different from control values (0cc) (p < 0.05) **=significantly different from control values (0cc) (p < 0.01).
To date, only two studies on the effects of testicular biopsy in men have been undertaken. The first 10 was done on 20 inmate volunteers living in a penitentiary. Particular care was taken by these researchers to establish what their sperm counts were prior to biopsy. This was done by examining from nine to 16 ejaculates prior to surgery. After the biopsies, 55 per cent of the subjects showed no effect on sperm concentration for the entire follow-up period while 45 per cent experienced a significant depression in this parameter. Furthermore, 26 per cent of the subjects exhibited a secondary drop in sperm count or a drop that persisted throughout the study. Clearly, for about one half of the subjects, sperm counts were adversely affected by the biopsies at least temporarily, while one half of those
158
COSENTINO AND ASSOCIATES TABLE 2.
Fertility parameters of male rats following three surgical treatments (control groups)
First Fertility Trial-1 Month After Surgery Treatment Hemicastration T. albuginea suture Sham surgery
% Fertile Males
90 (9/10) 100 (10/10) 90.9 (10/11)
% Pregnant Females
Embryo* Score
80 (16/20) 90 (18/20) 81.8 (18/20)
162.7 ± 27.5 119.9 ± 20.0 129.9 ± 18.7
Second Fertility Trial-2 Months After Surgery
3 Months After Surgery
(µm.)
Serum* Testosterone (ng./ml.)
265 ± 2 260 ± 2 255 ± 10
2.18 ± 0.28 2.55 ± 0.33 1.82 ± 0.21
Mean* Seminiferous Tubular Diameter Hemicastration T. albuginea suture Sham surgery
100 (10/10) 100 (10/10) 90.9 (10/11)
90 (18/20) 90 (18/20) 86.4 (19/23)
144.9 ± 20.9 157.9 ± 19.9 172.2 ± 22.8
* Mean ± S.E.M.
affected had more long-term affects. A larger study (100 subjects) by Rowley et al.11 similarly showed a decrease in sperm count in about 40 per cent of the subjects after testicular biopsy. However, all of these men recovered to pre-biopsy sperm counts within 18 weeks. The men who exhibited a decrease in sperm count were classified into two groups: 1) those that had an abrupt decrease followed by an immediate return to normal and 2) those having an immediate decrease that persisted for 10 weeks after the biopsies. This suggests that the spermatogenic process of the quick recovery group was not affected since it takes from 60 to 90 days for new sperm to show up in the ejaculate of man. However, the second group did not begin recovery until 70 days after the biopsies, suggesting some upset of spermatogenesis occurred at the time of biopsy. Unfortunately it is not possible to determine from those studies if the effect noted on sperm count was due to the actual biopsy or the surgical trauma to the scrotum. Indeed, the study by Gordon et al. 10 incorporated the use of post-surgical ice packs for 12 to 18 hours. This alone may have had some effect on sperm concentration. 12 The present study also noted transient changes of rat fertility following testicular biopsy. That is, even though the fertility of the male rats and the number of pregnancies they produced were inversely proportional to the size of the testicular biopsy one month after surgery (figs. 1 and 2) assessment of these parameters two months after surgery showed fertility returning to control levels. Indeed, the quality of the pregnancies that did occur (embryo score, fig. 3) was unaffected regardless of the time after surgery. Of particular interest to us was the observation that testicular biopsy reduced the seminiferous tubular diameter in an all-ornone fashion (table 1). These data suggest that some structure (perhaps vascular) critical to the maintenance of the rest of the organ was distributed more often with the larger biopsies. The mechanism of this phenomenon, however, remains to be elucidated. The amount of tissue removed during the biopsies, 0.01, 0.1, 0.2 or 0.3 cc, represents approximately 1 per cent, 10 per cent, 20 per cent and 30 per cent respectively of a rat testis volume. However, extrapolation to the human situation where approximately 1 per cent or less of testicular parenchyma is removed, should be cautioned against since it is not known if the percentage of the testis volume removed is important or if the absolute amount of parenchyma (for example, 0.2 cc) is the critical factor. Since we noted that hemicastration in the adult male rat did not affect fertility (table 2) it is apparent that the changes seen in fertility of the biopsied animals was not due to loss of germinal epithelium mass from the organism, but to some damage incurred to the tissue remaining in the organism. That is, perhaps some critical structure (such as a blood vessel) was damaged during biopsy. Certainly, the larger the biopsy the more likely one would be to damage such a structure. This, however, remains to be determined. Hemicastration of 65-day-old rats 13 or 6 to 10-month-old
rats 14 did not result in contralateral compensatory hypertrophy of the remaining testes and is in agreement with the data of the present study (table 2). However, rats aged 22 to 24 months did show striking hemicastration-induced testicular enlargement.14 A similar response was also noted in adult rams. 15 Thus, the response of the remaining testis to hemicastration is variable depending on the age and species of the organism. The data of the present study indicate that fertility in the healthy adult male rat undergoing unilateral testicular biopsy is not adversely affected unless removal of relatively large amounts of parenchyma occurs. However, whether or not this procedure adversely affects fertility of an already subfertile man remains speculative. REFERENCES
1. Charny, C. W.: Testicular biopsy: its value in male sterility. JAMA, 115: 1429, 1940. 2. Leong, A. S. Y. and Matthews, C. D.: The role of testicular biopsy in the investigation of male infertility. Pathol., 14: 205, 1982. 3. de Kretser, D. M., Burger, H. G. and Hudson, B.: The relationship between germinal cells and serum FSH levels in males with infertility. J. Clin. Endocrinol. Metabol., 38: 787, 1974. 4. Takihara, H., Sakatoku, J., Fujii, M., Nasu, T., Cosentino, M. J. and Cockett, A. T. K.: Significance of testicular size measurement in andrology. I. A new orchiometer and its clinical application. Fertil. Steril., 39: 836, 1983. 5. de Kretser, D. M.: Testicular biopsy in the management of male infertility. Int. J. Androl., 5: 449, 1982. 6. Cosentino, M. J., Rabinowitz, R., Valvo, J. R. and Cockett, A. T. K.: The effect of prepubertal spermatic cord torsion on subsequent fertility in rats. J. Androl., 5: 93, 1984. 7. Cosentino, M. J., Nishida, M., Rabinowitz, R. and Cockett, A. T. K.: Histological changes occurring in the contralateral testes of prepubertal rats subjected to various durations of unilateral spermatic cord torsion. J. Urol., 133: 906, 1985. 8. Urry, R. L.: Evidence for a contralateral testicular effect in unilaterally cryptorchid animals. (abstract). J. Androl., 1: 64, 1980. 9. Wallace, D. M.A., Gunter, P.A., Landon, G. V., Pugh, R. C. B. and Hendry, W. F.: Sympathetic orchiopathia: an experimental and clinical study. Br. J. Urol., 54: 765, 1982. 10. Gordon, D. L., Barr, A. B., Herrigel, J. E. and Paulsen, C. A.: Testicular biopsy in man. I. The effect upon sperm concentration. Fertil. Steril., 16: 522, 1965. 11. Rowley, M. J., O'Keefe, K. B. and Heller, C. G.: Decreases in sperm concentration due to testicular biopsy procedure in man. J. Urol., 101: 347, 1969. 12. Mulrahy, J. J.: Scrotal hypothermia and the infertile man. J. Urol., 132: 469, 1984. 13. Lindgren, S., Danber, J. E. and Carstensen, H.: Compensatory testosterone secretion in unilaterally orchidectomized rats. Life Sci., 18: 1203, 1976. 14. Johnson, L. and Neaves, W. B.: Enhanced daily sperm production in the remaining testes of aged rats following hemicastration. J. Androl., 4: 162, 1983. 15. Voglmayr, J. K. and Matner, P. E.: Compensatory hypertrophy in the remaining testis following unilateral orchidectomy in the adult ram. J. Reprod. Fertil., 17: 179, 1968.