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Fertility in Cryptorchidism: Further Development of an Experimental Model
0022-534 7/87 /1371-0128$02.00/0 THE JOURNAL OF UROLOGY
Vol. 137, January
Copyright© 1987 by The Williams & Wilkins Co.
Printed in U.S.A.
FERTILITY IN CRYPTORCHIDISM: FURTHER DEVELOPMENT OF AN EXPERIMENTAL MODEL BARRY A. KOGAN,* RAVI GUPTA
AND
KLAUS-PETER JUENEMANN
From the Department of Urology, University of California School of Medicine, San Francisco, California
ABSTRACT
Further development of an experimental model for evaluating fertility in cryptorchidism led to studies of unilateral cryptorchidism, endocrinological cryptorchidism, and the effects of treatment. The results demonstrate that rats with unilateral mechanical cryptorchidism have a significant diminution in the ability to impregnate females (impregnation rate, 45%) when compared with sham-operated controls (84%) and rats undergoing unilateral orchiectomy (88%). In addition, we demonstrated that lower doses of estradiol caused cryptorchidism and resulted in infertility of approximately the same degree as higher doses (impregnation rates, 18% and 0%, respectively), but avoided obvious side effects. Treatment of estradiol-induced cryptorchidism with human chorionic gonadotropin resulted in testicular descent, but did not significantly improve the ability to bear offspring (10% with hCG vs. 0% without). Surgical orchiopexy after surgically induced mechanical cryptorchidism resulted in improved fertility (30% vs. 0% ); however, the improvement was still significantly less than the control rate. In summary, this experimental model demonstrates the effects of various aspects of cryptorchidism and its treatment on fertility and can easily be adapted to evaluate important clinical problems. The fertility status of patients with cryptorchidism remains a controversial subject. 1- 3 The optimal form of treatment and the effects of treatment remain unclear. The reasons for this relate not only to problems with human experimentation, but also to factors unique to the disease, including its heterogeneity, the variability of treatment, and the lengthy followup required to evaluate fertility. Until recently, no experimental model was available to evaluate fertility in cryptorchidism or the effects of treatment. Previous work in our laboratory has shown that SpragueDawley rats with bilateral cryptorchidism (either mechanical or endocrinological) were unable to father offspring, a finding that was significantly different from controls. 4 In this limited study, rats with unilateral cryptorchidism were less able to impregnate females than were controls, but the difference was not statistically significant. Further studies were carried out to evaluate the following: 1) Is there a significant difference in fertility among rats with unilateral cryptorchidism, unilateral orchiectomy, and controls? 2) Could more subtle endocrinological abnormalities cause cryptorchidism and infertility? 3) Does surgical treatment of bilateral mechanical cryptorchidism improve fertility? 4) Does hormonal treatment of bilateral endocrinological cryptorchidism improve fertility? MATERIALS AND METHODS
Newborn Sprague-Dawley rats (Bantin and Kingman, Fremont CA) were treated to create cryptorchidism as described below. At 64 to 66 days, when sexually mature, each male rat was mated with two females that had previously produced at least one litter. After exactly 24 days, the females were sacrificed and the number and mean weight of all fetuses were determined. We have previously described the actual surgical and hormonal techniques used to establish cryptorchidism4 and, by creating the following groups, have varied the technique to test the effects of unilateral cryptorchidism. Accepted for publication July 3, 1986. * Requests for reprints: Dept. of Urology, U-518, University of California, San Francisco, CA 94143. Supported in part by grants from the Academic Senate and the Research Evaluation and Allocation Committee, University of California, San Francisco.
1. Surgical controls-16 newborn rats were given anesthesia and underwent sham surgery. 2. Unilateral mechanical cryptorchidism-19 newborn rats underwent exploration and fixation of one testis in its intraabdominal position. 3. Unilateral orchiectomy-13 newborn rats underwent unilateral orchiectomy. In creating endocrinological cryptorchidism, the following manipulations were performed to simulate the effect of hormonal abnormalities on testicular maturation and descent. The technique was based on that reported by Rajfer and Walsh (daily subcutaneous injections of 17-beta-estradiol [estradiol]), 6 but was modified for this study by the use of lower doses and shorter treatment intervals. Subgroups were as follows: 4. Controls-13 newborn male rats were injected for 30 days with sesame oil alone, the dosage increasing from 0.1 to 0.3 ml. 5. Full-dose estradiol-nine rats were injected for 30 days with estradiol, starting with 0.01 mg. in 0.1 ml. sesame oil and increasing to 0.03 mg. in 0.3 ml. as described. 6. Low-dose estradiol, short early course-five rats were injected from day 2 th:roug1i8with 0:001 mg. estradiol in 0.1 ml. sesame oil. 7. Low-dose estradiol, short delayed course-three rats were injected from day 11 through 17 with 0.001 mg. estradiol in 0.1 ml. sesame oil. 8. One-dose estradiol-three rats were injected on day two with 0.005 mg. estradiol in 0.1 ml. sesame oil. In studying the effects of hormonal treatment, the following group was formed: 9. Five rats were treated as in Group 5 above, but in addition were given human chorionic gonadotrophin (hCG) 1 IU subcutaneously daily after the first day of estradiol. To evaluate the success of surgical treatment for mechanical cryptorchidism two groups were created: 10. Bilateral mechanical cryptorchidism-16 newborn rats underwent suturing of both testes in their intraabdominal position at two to three days of life. 11. Bilateral orchiopexy-20 newborn rats had surgical fixation of the testes as in Group 2, then underwent surgical orchiopexy at 21 to 28 days of age. Differences among groups in number of females impregnated, litter size and mean fetal weight were compared with a oneway analysis of variance using Dunnett's method.
128
129
FERTILITY IN CRYPTORCHIDISM RESULTS
TABLE 3.
Unilateral cryptorchidism. The testes descended spontaneously by 21 days of age in all 16 rats in the surgical control group (as assessed by visualization and palpation). Twenty-six of the 31 females mated with them became pregnant, with a mean litter size of 12.3 and a mean fetal weight of 3.13 gm. In Group 2 (unilateral cryptorchidism), only 17 of 38 females became pregnant, but the litter size and weights were comparable. This difference in pregnancy rate was statistically significant (p <0.02). In contrast, of the females mated with males that had a unilateral orchiectomy (Group 3) 23 of 26 were impregnated, significantly more than in the unilateral cryptorchidism group (p <0.02). Litter size and fetal weight were lower than with controls, but these differences were not statistically significant. These data are displayed in table 1. Endocrinological cryptorchidism. In Group 4 (controls), the testes descended spontaneously by 21 days of age. Twenty-five of the 26 females mated with these rats became pregnant, with a mean litter size of 11.9 and a mean fetal weight of 2.67 gm. No rat given full-dose estradiol (Group 5) had testicular descent and all were infertile. In Groups 6, 7 and 8, testicular descent occurred spontaneously, but was delayed from 21 to approximately 30 days. These rats were able to father offspring but with lesser rates (1/10, 2/B and 1/B, respectively). Results in all the experimental groups were significantly different from controls (p <0.01 to p <0.001). Table 2 shows a comparison of these data. Hormonal treatment of endocrinological cryptorchidism. Those rats treated with estradiol and hCG (Group 9) had spontaneous testicular descent at 16 days, but at 50 days the testes were grossly smaller than controls. Only one of ten females mated with these rats became pregnant and there was only one fetus in the litter. In this limited study, there was no statistically significant difference between those treated with hCG and those that were not. Table 3 compares these data with other groups. Surgical treatment of mechanical cryptorchidism. In those rats with bilateral mechanical cryptorchidism (Group 10), there
Group 4 Sesame oil Group 5 Full-dose estradiol Group 9 Estradiol + hCG
Numbr of Females Pregnant
Litter Size
21
25/26 (96%)
11.9
None
0/20 (0%)
16
1/10 (10%)
TABLE 4.
Group 1 Sham surgery Group 10 Bilateral mechanical cryptorchidism Group 11 Surgical orchiopexy
Hormonal treatment
Time of Testicular Descent (Days)
Mean Fetal Weight (gm.)
2.7
0.1
Surgical treatment
Time of Testicular Descent (Days)
Number of Females Pregnant
Litter Size
21
26/31 (84%)
12.3
3.1
None
0/32 (0%)
21-28
12/40 (30%)
4.3
0.1
Mean Fetal Weight (gm.)
was no spontaneous testicular descent and no evidence of fertility. Of those undergoing orchiopexy (Group 11), evaluation at a later date revealed testes in the scrotum in all; however, four were noted to be atrophic. These four occurred early in the series. Twelve of the forty females mated to these rats were impregnated, but the litter size was small and the fetuses were tiny. The pregnancy rate of those undergoing orchiopexy was significantly better than that of the bilateral cryptorchidism group (p <0.05), but remained significantly less than controls' (p <0.01) (see table 4). DISCUSSION
TABLE 1.
Unilateral mechanical cryptorchidism Time of Testicular Descent (Days)
Group 1 Sham surgery Group 2* Unilateral mechanical cryptorchidism Group 3* Unilateral orchiectomy
Number of Females Pregnant
Litter Size
Mean Fetal Weight (gm.)
21
26/31 (84%)
12.3
3.1
-/21
17/38 (45%)
11.8
2.9
-/21
23/26 (88%)
11.2
2.5
* In these groups only the contralateral testis descended normally.
TABLE
Group 4 Sesame oil only Group 5 Full-dose estradiol Group 6 Low-dose, short course Group 7 Low-dose, delayed course Group 8 One dose
2. Endocrinological cryptorchidism Time of Testicular Descent (Days)
Number of Females Pregnant
Litter Size
Mean Fetal Weight {gm.)
21
25/26 (96%)
11.9
2.7
None
0/20 (0%)
30
1/10 (10%)
6
1.9
30
2/6 (33%)
10
0.1
30
1/6 (17%)
9
0.1
Cryptorchidism is a clinical condition that has been recognized for thousands of years. Despite this, many facets of this disease process remain poorly understood-particularly the associated infertility. The reasons for this are many, but are in large part due to the inability to study the disease scientifically in humans. Investigations are hampered by the difficulty of treatment and the lengthy followup necessary. Even the best examiner can confuse retractile, incompletely descended, and ectopic testes. Obviously, the ramifications of each diagnosis are quite different. Similarly, the group with true cryptorchidism includes those patients with both unilateral and bilateral undescended testes, as well as those whose testes descended spontaneously after birth. Further, in each group the cause may be different, including mechanical, endocrinological, and inherent testicular abnormalities, among others. Finally, treatment can be hormonal, surgical or both, and the age at treatment may vary considerably. Even the evaluation of fertility is difficult, as semen analysis alone does not take into account the numerous other factors involved in fathering offspring (antisperm antibodies, sperm penetration, etc.). Paternity is probably the best measure of fertility for these purposes, but proof of paternity is complex and expensive, requiring lengthy and individual followup as well as HLA typing of patients and children. Hence the validity of conclusions in any study of fertility reached 20 to 30 years after the diagnosis of cryptorchidism, and including widely disparate patients, must be questioned. For all the above reasons, it remains, even in 1986, nearly impossible to counsel parents accurately about the chances for fertility in their newborns with cryptorchidism. It is with this in mind that a new approach for an animal model was developed. Other models have been reported, but
130
KOGAN AND ASSOCIATES
these have generally used adult animals, a choice that does not really simulate human cryptorchidism. 6 - 8 In models which did use newborn animals, the endpoints evaluated included testicular histology, enzyme analysis, evaluation of serum antisperm antibody levels, and sperm count on semen analysis, all of which have significant deficiencies. 9 - 12 Further, previous models have only evaluated fertility in mechanical, not endocrinological, cryptorchidism. 6 - 12 Equally important, no other models have simulated different treatments to permit observation of their effects on fertility. The particular model developed uses Sprague-Dawley rats since they are readily available, have a short lifespan, and are relatively inexpensive. Further, later phases of sexual differentiation, including penile growth and testicular descent, occur after birth and have been extensively studied. 5• 13 Consequently it is possible, both hormonally and mechanically, to prevent testicular descent in newborn rats and thereby simulate cryptorchidism in the human fetus. Male rats are highly fertile and females have multiple pregnancies; thus it appears that mating studies involving rats can detect subtle changes in fertility. 14• 15 Our early study demonstrated marked and statistically significant differences between bilaterally cryptorchid rats and controls, whether the etiology was mechanical or endocrinological. In our preliminary work, there were differences between unilaterally cryptorchid animals and controls, but these were not statistically significant. With the collection of further data, there are now clear-cut and statistically significant differences between controls, unilateral cryptorchid animals, and those with bilateral cryptorchidism. This is in accordance with data in the literature on human cryptorchidism and further supports the validity of this model. Even more interesting is the significant difference between the unilateral orchiectomy and unilateral cryptorchidism groups. This suggests that the cryptorchid testis is in some way inhibiting the contralateral "normal" testis, a phenomenon that many clinicians feel affects fertility in human cryptorchidism and has been noted in testicular torsion. 12 • 13 Also in our preliminary research, the dosage of estradiol was based on that reported previously. 5 The animals did indeed have bilateral cryptorchidism, but they were also small and drank large amounts of water, suggesting that the estradiol caused other pituitary effects, possibly a deficiency in growth hormone and antidiuretic hormone. We therefore attempted to achieve similar results with lower doses of estradiol. Using several different regimens, we were able to achieve a model of delayed testicular descent, from the norm of 21 days to 30. Interestingly, this was associated with significant infertility when compared with controls. Since it is questionable whether the slight delay could be the sole causative factor, the implication is that the estradiol, or perhaps decreased testosterone associated with suppression of luteinizing hormone by the estradiol, may contribute to the infertility. The human male fetus is subjected to large amounts of estrogen, and reports in the literature have shown decreased serum testosterone and luteinizing hormone in children with cryptorchidism and also in those with delayed spontaneous descent. 16• 17 Although we have yet to measure serum testosterone and luteinizing hormone in these rats, the physiology of estradiol's effects suggests that our model of endocrinological cryptorchidism may be very similar to the human disease process and that it provides further evidence that hormonal abnormalities may be clinically important in the development of infertility in these patients. 18 We are currently investigating hormone levels in newborns with undescended testes. The question of the effects of hCG is very suited to an animal model. Clinical studies of its effectiveness show widely divergent results, 19- 21 again related to the difficulties inherent in human experimentation. In our particular model, hCG did bring about testicular descent, but either contributed to the infertility or failed to prevent it completely. Timing of treatment and
dosage of hCG could certainly be varied in our study, although the present regimen was adequate to affect testicular descent. The fact that the testes were later noted to be small suggests that the hormonal imbalances are important contributing factors to the infertility. A deleterious effect of hCG itself has been suggested by other investigators and may also be clinically relevant. 22 The limited success of orchiopexy is difficult to explain. The obviously beneficial effects as compared with bilateral cryptorchidism are encouraging, but these animals were still significantly less fertile than controls. This may be attributable to problems with this model, in that merely suturing the testis may damage it or initiate an autoimmune process. Further, the orchiopexy in these animals was performed at 21 to 28 days. Since rats attain sexual maturity within a relatively short time (approximately 65 days), this seven-day difference, which seems minimal, may well be significant. In humans, the failure of surgery to prevent infertility or neoplasia is often attributed to the delay in treatment, hence the recommendations for earlier surgery. Studies are currently underway to see if this delay is likewise relevant in the rat model. Some clinicians have also suggested that either hormonal factors or inherent testicular abnormalities limit the success of surgery, no matter how early it is performed. In our model these factors were controlled and did not play a role in the infertility. A major benefit of this particular model is its ability to separate different variables, something not possible in human studies. In this instance, the results demonstrate that the abnormal position of the testis is the direct cause of infertility. The inability of human studies clearly to define the etiology, pathophysiology and optimal treatment of cryptorchidism is a source of considerable frustration to clinicians. It was because of this that an animal model was developed to elucidate the causes and treatment of the infertility associated with cryptorchidism. Our preliminary work demonstrated that the model was feasible. In this report, we have shown that the model can simulate different types of both mechanical and endocrinological cryptorchidism and can be used to study treatment of these conditions. Further studies are now possible evaluating numerous other treatment options. Although the results of research in rats cannot be immediately extrapolated to humans, this model does provide a means, previously unavailable, to study the disease. REFERENCES
1. Gilhooly, P. E., Meyers, F. and Lattimer, J. K.: Fertility prospects for children with cryptorchidism. Am. J. Dis. Child., 138: 940, 1984. 2. Fallon, B. and Kennedy, T. J.: Long-term follow-up of fertility in cryptorchid patients. Urology, 25: 502, 1985. 3. Kogan, S. J.: Fertility in cryptorchidism. In: Cryptorchidism: Management and Implications. Edited by F. Hadziselimovic. Berlin: Springer Verlag, pp. 71-82, 1983. 4. Juenemann, K.-P., Kogan, B. A. and Abozeid, M. H.: Fertility in cryptorchidism: an experimental model. J. Urol., 136: 214, 1986. 5. Rajfer, J., and Walsh, P. C.: Hormonal regulation of testicular descent: experimental and clinical observations. J. Urol., 118: 985, 1977. 6. Nelson, W. 0.: Mammalian spermatogenesis: effect of experimental cryptorchidism in the rat and non-descent of the testis in man. Rec. Prog. Horm. Res., 6: 29, 1951. 7. Clegg, E. J.: Studies on artificial cryptorchidism: compensatory changes in the scrotal testes of unilaterally cryptorchid rats. J. Endocrinol., 33: 259, 1965. 8. Keel, B. A. and Abney, T. 0.: Alterations of testicular function in the unilaterally cryptorchid rat. Proc. Soc. Exp. Biol. Med., 166: 489, 1981. 9. Atkinson, P. M.: The effects of early experimental cryptorchidism and subsequent orchidopexy on the maturation of the guineapig testicle. Br. J. Surg., 60: 253, 1973. 10. Kiesewetter, W. B., Kalayoglu, M. and Sachs, B.: The effect of
FERTILITY rN :CRYPTORCHIDISM
11.
12.
13.
14.
15.
abnormal position, scrotal repositioning, and human gonadotropic hormone on the developing puppy testis. J. Pediatr. Surg., 8: 739, 1973. Urry, R. L. and Middleton, R. G.: Evidence for a contralateral effect of unilateral cryptorchidism on spermatogenesis, enzyme systems, and sperm transport of the scrotal testicle. Surg. Forum, 34: 649, 1983. Kogan, S. J., Vatrandaslar, F., Gondos, B., Bennett, B., Nehlsen, S., Sun, E., Fleckman, A., Smey, P. and Levitt, S.: Experimental unilateral cryptorchidism: is there a contralateral effect on the descended testis? Presented at the 52nd Annual Meeting of the American Academy of Pediatrics, October 22-27, 1983, San Francisco, CA. (Program for Scientific Sessions [Section on Urology], p. 18). Hadziselimovic, F.: Embryology of testicular descent and maldescent. In: Cryptorchidism: Management and Implications. Edited by F. Hadziselimovic. Berlin: Springer-Verlag, pp. 11-34, 1983. Nagler, H. M. and White, R. deV.: Long-term effects of testicular torsion on reproductive capacity in the adult male rat. Surg. Forum, 33: 619, 1982. Cosentino, M. J., Rabinowitz, R., Valvo, J. R. and Cockett, A. T. K.: The effect of prepubertal spermatic cord torsion on subse-
131
quent fertility in rats. J. Androl., 5: 93, 1984. 16. Gendrel, D., Job, J.-C. and Roger, M.: Reduced post-natal rise of testosterone in plasma of cryptorchid infants. Acta Endocrinol., 89: 372, 1978. 17. Padovani, E., Pizzo, P., Chiaffoni, P. and Tato, L.: Retarded testicular descent and hypophyso-gonadal axis in premature newborns. Pediatr. Res., 16; 905, 1982 (Abstr.). 18. Hadziselimovic, F., Thommen, L., Girard, J. and Herzog, B.: The significance of post-natal gonadotrophin surge for testicular development in normal and cryptorchid testis. J. Urol., 136: 274, 1986. 19. Ehrlich, R. M., Dougherty, L. J., Tomashefsky, P. and Lattimer, J. K.: Effect of gonadotropin in cryptorchism. J. Urol., 102: 793, 1969. 20. Garagorri, J.-M., Job, J.-C., Canlorbe, P. and Chaussain, J.-L.: Results of early treatment of cryptorchidism with human chorionie gonadotropin. J. Pediatr., 101: 923, 1982. 21. Bierich, J. R.: Gonadotropin therapy for the undescended testis. In: Pediatric Andrology. Edited by S. J. Kogan and E. S. E. Hafez. Hague/Boston/London: Martinus Nijhoff, pp. 163-171, 1981. 22. Heine, J.P., Novick, H.P. and Hinman, F. Jr.: Effects of premature puberty on cryptorchid testes in rats. Surg. Forum, 24: 552, 1973.
Vol. 137, January
Copyright© 1987 by The Williams & Wilkins Co.
Printed in U.S.A.
FERTILITY IN CRYPTORCHIDISM: FURTHER DEVELOPMENT OF AN EXPERIMENTAL MODEL BARRY A. KOGAN,* RAVI GUPTA
AND
KLAUS-PETER JUENEMANN
From the Department of Urology, University of California School of Medicine, San Francisco, California
ABSTRACT
Further development of an experimental model for evaluating fertility in cryptorchidism led to studies of unilateral cryptorchidism, endocrinological cryptorchidism, and the effects of treatment. The results demonstrate that rats with unilateral mechanical cryptorchidism have a significant diminution in the ability to impregnate females (impregnation rate, 45%) when compared with sham-operated controls (84%) and rats undergoing unilateral orchiectomy (88%). In addition, we demonstrated that lower doses of estradiol caused cryptorchidism and resulted in infertility of approximately the same degree as higher doses (impregnation rates, 18% and 0%, respectively), but avoided obvious side effects. Treatment of estradiol-induced cryptorchidism with human chorionic gonadotropin resulted in testicular descent, but did not significantly improve the ability to bear offspring (10% with hCG vs. 0% without). Surgical orchiopexy after surgically induced mechanical cryptorchidism resulted in improved fertility (30% vs. 0% ); however, the improvement was still significantly less than the control rate. In summary, this experimental model demonstrates the effects of various aspects of cryptorchidism and its treatment on fertility and can easily be adapted to evaluate important clinical problems. The fertility status of patients with cryptorchidism remains a controversial subject. 1- 3 The optimal form of treatment and the effects of treatment remain unclear. The reasons for this relate not only to problems with human experimentation, but also to factors unique to the disease, including its heterogeneity, the variability of treatment, and the lengthy followup required to evaluate fertility. Until recently, no experimental model was available to evaluate fertility in cryptorchidism or the effects of treatment. Previous work in our laboratory has shown that SpragueDawley rats with bilateral cryptorchidism (either mechanical or endocrinological) were unable to father offspring, a finding that was significantly different from controls. 4 In this limited study, rats with unilateral cryptorchidism were less able to impregnate females than were controls, but the difference was not statistically significant. Further studies were carried out to evaluate the following: 1) Is there a significant difference in fertility among rats with unilateral cryptorchidism, unilateral orchiectomy, and controls? 2) Could more subtle endocrinological abnormalities cause cryptorchidism and infertility? 3) Does surgical treatment of bilateral mechanical cryptorchidism improve fertility? 4) Does hormonal treatment of bilateral endocrinological cryptorchidism improve fertility? MATERIALS AND METHODS
Newborn Sprague-Dawley rats (Bantin and Kingman, Fremont CA) were treated to create cryptorchidism as described below. At 64 to 66 days, when sexually mature, each male rat was mated with two females that had previously produced at least one litter. After exactly 24 days, the females were sacrificed and the number and mean weight of all fetuses were determined. We have previously described the actual surgical and hormonal techniques used to establish cryptorchidism4 and, by creating the following groups, have varied the technique to test the effects of unilateral cryptorchidism. Accepted for publication July 3, 1986. * Requests for reprints: Dept. of Urology, U-518, University of California, San Francisco, CA 94143. Supported in part by grants from the Academic Senate and the Research Evaluation and Allocation Committee, University of California, San Francisco.
1. Surgical controls-16 newborn rats were given anesthesia and underwent sham surgery. 2. Unilateral mechanical cryptorchidism-19 newborn rats underwent exploration and fixation of one testis in its intraabdominal position. 3. Unilateral orchiectomy-13 newborn rats underwent unilateral orchiectomy. In creating endocrinological cryptorchidism, the following manipulations were performed to simulate the effect of hormonal abnormalities on testicular maturation and descent. The technique was based on that reported by Rajfer and Walsh (daily subcutaneous injections of 17-beta-estradiol [estradiol]), 6 but was modified for this study by the use of lower doses and shorter treatment intervals. Subgroups were as follows: 4. Controls-13 newborn male rats were injected for 30 days with sesame oil alone, the dosage increasing from 0.1 to 0.3 ml. 5. Full-dose estradiol-nine rats were injected for 30 days with estradiol, starting with 0.01 mg. in 0.1 ml. sesame oil and increasing to 0.03 mg. in 0.3 ml. as described. 6. Low-dose estradiol, short early course-five rats were injected from day 2 th:roug1i8with 0:001 mg. estradiol in 0.1 ml. sesame oil. 7. Low-dose estradiol, short delayed course-three rats were injected from day 11 through 17 with 0.001 mg. estradiol in 0.1 ml. sesame oil. 8. One-dose estradiol-three rats were injected on day two with 0.005 mg. estradiol in 0.1 ml. sesame oil. In studying the effects of hormonal treatment, the following group was formed: 9. Five rats were treated as in Group 5 above, but in addition were given human chorionic gonadotrophin (hCG) 1 IU subcutaneously daily after the first day of estradiol. To evaluate the success of surgical treatment for mechanical cryptorchidism two groups were created: 10. Bilateral mechanical cryptorchidism-16 newborn rats underwent suturing of both testes in their intraabdominal position at two to three days of life. 11. Bilateral orchiopexy-20 newborn rats had surgical fixation of the testes as in Group 2, then underwent surgical orchiopexy at 21 to 28 days of age. Differences among groups in number of females impregnated, litter size and mean fetal weight were compared with a oneway analysis of variance using Dunnett's method.
128
129
FERTILITY IN CRYPTORCHIDISM RESULTS
TABLE 3.
Unilateral cryptorchidism. The testes descended spontaneously by 21 days of age in all 16 rats in the surgical control group (as assessed by visualization and palpation). Twenty-six of the 31 females mated with them became pregnant, with a mean litter size of 12.3 and a mean fetal weight of 3.13 gm. In Group 2 (unilateral cryptorchidism), only 17 of 38 females became pregnant, but the litter size and weights were comparable. This difference in pregnancy rate was statistically significant (p <0.02). In contrast, of the females mated with males that had a unilateral orchiectomy (Group 3) 23 of 26 were impregnated, significantly more than in the unilateral cryptorchidism group (p <0.02). Litter size and fetal weight were lower than with controls, but these differences were not statistically significant. These data are displayed in table 1. Endocrinological cryptorchidism. In Group 4 (controls), the testes descended spontaneously by 21 days of age. Twenty-five of the 26 females mated with these rats became pregnant, with a mean litter size of 11.9 and a mean fetal weight of 2.67 gm. No rat given full-dose estradiol (Group 5) had testicular descent and all were infertile. In Groups 6, 7 and 8, testicular descent occurred spontaneously, but was delayed from 21 to approximately 30 days. These rats were able to father offspring but with lesser rates (1/10, 2/B and 1/B, respectively). Results in all the experimental groups were significantly different from controls (p <0.01 to p <0.001). Table 2 shows a comparison of these data. Hormonal treatment of endocrinological cryptorchidism. Those rats treated with estradiol and hCG (Group 9) had spontaneous testicular descent at 16 days, but at 50 days the testes were grossly smaller than controls. Only one of ten females mated with these rats became pregnant and there was only one fetus in the litter. In this limited study, there was no statistically significant difference between those treated with hCG and those that were not. Table 3 compares these data with other groups. Surgical treatment of mechanical cryptorchidism. In those rats with bilateral mechanical cryptorchidism (Group 10), there
Group 4 Sesame oil Group 5 Full-dose estradiol Group 9 Estradiol + hCG
Numbr of Females Pregnant
Litter Size
21
25/26 (96%)
11.9
None
0/20 (0%)
16
1/10 (10%)
TABLE 4.
Group 1 Sham surgery Group 10 Bilateral mechanical cryptorchidism Group 11 Surgical orchiopexy
Hormonal treatment
Time of Testicular Descent (Days)
Mean Fetal Weight (gm.)
2.7
0.1
Surgical treatment
Time of Testicular Descent (Days)
Number of Females Pregnant
Litter Size
21
26/31 (84%)
12.3
3.1
None
0/32 (0%)
21-28
12/40 (30%)
4.3
0.1
Mean Fetal Weight (gm.)
was no spontaneous testicular descent and no evidence of fertility. Of those undergoing orchiopexy (Group 11), evaluation at a later date revealed testes in the scrotum in all; however, four were noted to be atrophic. These four occurred early in the series. Twelve of the forty females mated to these rats were impregnated, but the litter size was small and the fetuses were tiny. The pregnancy rate of those undergoing orchiopexy was significantly better than that of the bilateral cryptorchidism group (p <0.05), but remained significantly less than controls' (p <0.01) (see table 4). DISCUSSION
TABLE 1.
Unilateral mechanical cryptorchidism Time of Testicular Descent (Days)
Group 1 Sham surgery Group 2* Unilateral mechanical cryptorchidism Group 3* Unilateral orchiectomy
Number of Females Pregnant
Litter Size
Mean Fetal Weight (gm.)
21
26/31 (84%)
12.3
3.1
-/21
17/38 (45%)
11.8
2.9
-/21
23/26 (88%)
11.2
2.5
* In these groups only the contralateral testis descended normally.
TABLE
Group 4 Sesame oil only Group 5 Full-dose estradiol Group 6 Low-dose, short course Group 7 Low-dose, delayed course Group 8 One dose
2. Endocrinological cryptorchidism Time of Testicular Descent (Days)
Number of Females Pregnant
Litter Size
Mean Fetal Weight {gm.)
21
25/26 (96%)
11.9
2.7
None
0/20 (0%)
30
1/10 (10%)
6
1.9
30
2/6 (33%)
10
0.1
30
1/6 (17%)
9
0.1
Cryptorchidism is a clinical condition that has been recognized for thousands of years. Despite this, many facets of this disease process remain poorly understood-particularly the associated infertility. The reasons for this are many, but are in large part due to the inability to study the disease scientifically in humans. Investigations are hampered by the difficulty of treatment and the lengthy followup necessary. Even the best examiner can confuse retractile, incompletely descended, and ectopic testes. Obviously, the ramifications of each diagnosis are quite different. Similarly, the group with true cryptorchidism includes those patients with both unilateral and bilateral undescended testes, as well as those whose testes descended spontaneously after birth. Further, in each group the cause may be different, including mechanical, endocrinological, and inherent testicular abnormalities, among others. Finally, treatment can be hormonal, surgical or both, and the age at treatment may vary considerably. Even the evaluation of fertility is difficult, as semen analysis alone does not take into account the numerous other factors involved in fathering offspring (antisperm antibodies, sperm penetration, etc.). Paternity is probably the best measure of fertility for these purposes, but proof of paternity is complex and expensive, requiring lengthy and individual followup as well as HLA typing of patients and children. Hence the validity of conclusions in any study of fertility reached 20 to 30 years after the diagnosis of cryptorchidism, and including widely disparate patients, must be questioned. For all the above reasons, it remains, even in 1986, nearly impossible to counsel parents accurately about the chances for fertility in their newborns with cryptorchidism. It is with this in mind that a new approach for an animal model was developed. Other models have been reported, but
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these have generally used adult animals, a choice that does not really simulate human cryptorchidism. 6 - 8 In models which did use newborn animals, the endpoints evaluated included testicular histology, enzyme analysis, evaluation of serum antisperm antibody levels, and sperm count on semen analysis, all of which have significant deficiencies. 9 - 12 Further, previous models have only evaluated fertility in mechanical, not endocrinological, cryptorchidism. 6 - 12 Equally important, no other models have simulated different treatments to permit observation of their effects on fertility. The particular model developed uses Sprague-Dawley rats since they are readily available, have a short lifespan, and are relatively inexpensive. Further, later phases of sexual differentiation, including penile growth and testicular descent, occur after birth and have been extensively studied. 5• 13 Consequently it is possible, both hormonally and mechanically, to prevent testicular descent in newborn rats and thereby simulate cryptorchidism in the human fetus. Male rats are highly fertile and females have multiple pregnancies; thus it appears that mating studies involving rats can detect subtle changes in fertility. 14• 15 Our early study demonstrated marked and statistically significant differences between bilaterally cryptorchid rats and controls, whether the etiology was mechanical or endocrinological. In our preliminary work, there were differences between unilaterally cryptorchid animals and controls, but these were not statistically significant. With the collection of further data, there are now clear-cut and statistically significant differences between controls, unilateral cryptorchid animals, and those with bilateral cryptorchidism. This is in accordance with data in the literature on human cryptorchidism and further supports the validity of this model. Even more interesting is the significant difference between the unilateral orchiectomy and unilateral cryptorchidism groups. This suggests that the cryptorchid testis is in some way inhibiting the contralateral "normal" testis, a phenomenon that many clinicians feel affects fertility in human cryptorchidism and has been noted in testicular torsion. 12 • 13 Also in our preliminary research, the dosage of estradiol was based on that reported previously. 5 The animals did indeed have bilateral cryptorchidism, but they were also small and drank large amounts of water, suggesting that the estradiol caused other pituitary effects, possibly a deficiency in growth hormone and antidiuretic hormone. We therefore attempted to achieve similar results with lower doses of estradiol. Using several different regimens, we were able to achieve a model of delayed testicular descent, from the norm of 21 days to 30. Interestingly, this was associated with significant infertility when compared with controls. Since it is questionable whether the slight delay could be the sole causative factor, the implication is that the estradiol, or perhaps decreased testosterone associated with suppression of luteinizing hormone by the estradiol, may contribute to the infertility. The human male fetus is subjected to large amounts of estrogen, and reports in the literature have shown decreased serum testosterone and luteinizing hormone in children with cryptorchidism and also in those with delayed spontaneous descent. 16• 17 Although we have yet to measure serum testosterone and luteinizing hormone in these rats, the physiology of estradiol's effects suggests that our model of endocrinological cryptorchidism may be very similar to the human disease process and that it provides further evidence that hormonal abnormalities may be clinically important in the development of infertility in these patients. 18 We are currently investigating hormone levels in newborns with undescended testes. The question of the effects of hCG is very suited to an animal model. Clinical studies of its effectiveness show widely divergent results, 19- 21 again related to the difficulties inherent in human experimentation. In our particular model, hCG did bring about testicular descent, but either contributed to the infertility or failed to prevent it completely. Timing of treatment and
dosage of hCG could certainly be varied in our study, although the present regimen was adequate to affect testicular descent. The fact that the testes were later noted to be small suggests that the hormonal imbalances are important contributing factors to the infertility. A deleterious effect of hCG itself has been suggested by other investigators and may also be clinically relevant. 22 The limited success of orchiopexy is difficult to explain. The obviously beneficial effects as compared with bilateral cryptorchidism are encouraging, but these animals were still significantly less fertile than controls. This may be attributable to problems with this model, in that merely suturing the testis may damage it or initiate an autoimmune process. Further, the orchiopexy in these animals was performed at 21 to 28 days. Since rats attain sexual maturity within a relatively short time (approximately 65 days), this seven-day difference, which seems minimal, may well be significant. In humans, the failure of surgery to prevent infertility or neoplasia is often attributed to the delay in treatment, hence the recommendations for earlier surgery. Studies are currently underway to see if this delay is likewise relevant in the rat model. Some clinicians have also suggested that either hormonal factors or inherent testicular abnormalities limit the success of surgery, no matter how early it is performed. In our model these factors were controlled and did not play a role in the infertility. A major benefit of this particular model is its ability to separate different variables, something not possible in human studies. In this instance, the results demonstrate that the abnormal position of the testis is the direct cause of infertility. The inability of human studies clearly to define the etiology, pathophysiology and optimal treatment of cryptorchidism is a source of considerable frustration to clinicians. It was because of this that an animal model was developed to elucidate the causes and treatment of the infertility associated with cryptorchidism. Our preliminary work demonstrated that the model was feasible. In this report, we have shown that the model can simulate different types of both mechanical and endocrinological cryptorchidism and can be used to study treatment of these conditions. Further studies are now possible evaluating numerous other treatment options. Although the results of research in rats cannot be immediately extrapolated to humans, this model does provide a means, previously unavailable, to study the disease. REFERENCES
1. Gilhooly, P. E., Meyers, F. and Lattimer, J. K.: Fertility prospects for children with cryptorchidism. Am. J. Dis. Child., 138: 940, 1984. 2. Fallon, B. and Kennedy, T. J.: Long-term follow-up of fertility in cryptorchid patients. Urology, 25: 502, 1985. 3. Kogan, S. J.: Fertility in cryptorchidism. In: Cryptorchidism: Management and Implications. Edited by F. Hadziselimovic. Berlin: Springer Verlag, pp. 71-82, 1983. 4. Juenemann, K.-P., Kogan, B. A. and Abozeid, M. H.: Fertility in cryptorchidism: an experimental model. J. Urol., 136: 214, 1986. 5. Rajfer, J., and Walsh, P. C.: Hormonal regulation of testicular descent: experimental and clinical observations. J. Urol., 118: 985, 1977. 6. Nelson, W. 0.: Mammalian spermatogenesis: effect of experimental cryptorchidism in the rat and non-descent of the testis in man. Rec. Prog. Horm. Res., 6: 29, 1951. 7. Clegg, E. J.: Studies on artificial cryptorchidism: compensatory changes in the scrotal testes of unilaterally cryptorchid rats. J. Endocrinol., 33: 259, 1965. 8. Keel, B. A. and Abney, T. 0.: Alterations of testicular function in the unilaterally cryptorchid rat. Proc. Soc. Exp. Biol. Med., 166: 489, 1981. 9. Atkinson, P. M.: The effects of early experimental cryptorchidism and subsequent orchidopexy on the maturation of the guineapig testicle. Br. J. Surg., 60: 253, 1973. 10. Kiesewetter, W. B., Kalayoglu, M. and Sachs, B.: The effect of
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abnormal position, scrotal repositioning, and human gonadotropic hormone on the developing puppy testis. J. Pediatr. Surg., 8: 739, 1973. Urry, R. L. and Middleton, R. G.: Evidence for a contralateral effect of unilateral cryptorchidism on spermatogenesis, enzyme systems, and sperm transport of the scrotal testicle. Surg. Forum, 34: 649, 1983. Kogan, S. J., Vatrandaslar, F., Gondos, B., Bennett, B., Nehlsen, S., Sun, E., Fleckman, A., Smey, P. and Levitt, S.: Experimental unilateral cryptorchidism: is there a contralateral effect on the descended testis? Presented at the 52nd Annual Meeting of the American Academy of Pediatrics, October 22-27, 1983, San Francisco, CA. (Program for Scientific Sessions [Section on Urology], p. 18). Hadziselimovic, F.: Embryology of testicular descent and maldescent. In: Cryptorchidism: Management and Implications. Edited by F. Hadziselimovic. Berlin: Springer-Verlag, pp. 11-34, 1983. Nagler, H. M. and White, R. deV.: Long-term effects of testicular torsion on reproductive capacity in the adult male rat. Surg. Forum, 33: 619, 1982. Cosentino, M. J., Rabinowitz, R., Valvo, J. R. and Cockett, A. T. K.: The effect of prepubertal spermatic cord torsion on subse-
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quent fertility in rats. J. Androl., 5: 93, 1984. 16. Gendrel, D., Job, J.-C. and Roger, M.: Reduced post-natal rise of testosterone in plasma of cryptorchid infants. Acta Endocrinol., 89: 372, 1978. 17. Padovani, E., Pizzo, P., Chiaffoni, P. and Tato, L.: Retarded testicular descent and hypophyso-gonadal axis in premature newborns. Pediatr. Res., 16; 905, 1982 (Abstr.). 18. Hadziselimovic, F., Thommen, L., Girard, J. and Herzog, B.: The significance of post-natal gonadotrophin surge for testicular development in normal and cryptorchid testis. J. Urol., 136: 274, 1986. 19. Ehrlich, R. M., Dougherty, L. J., Tomashefsky, P. and Lattimer, J. K.: Effect of gonadotropin in cryptorchism. J. Urol., 102: 793, 1969. 20. Garagorri, J.-M., Job, J.-C., Canlorbe, P. and Chaussain, J.-L.: Results of early treatment of cryptorchidism with human chorionie gonadotropin. J. Pediatr., 101: 923, 1982. 21. Bierich, J. R.: Gonadotropin therapy for the undescended testis. In: Pediatric Andrology. Edited by S. J. Kogan and E. S. E. Hafez. Hague/Boston/London: Martinus Nijhoff, pp. 163-171, 1981. 22. Heine, J.P., Novick, H.P. and Hinman, F. Jr.: Effects of premature puberty on cryptorchid testes in rats. Surg. Forum, 24: 552, 1973.