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Varicocele: Reversal of the Testicular Blood Flow and Temperature Effects by Varicocele Repair
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0022-534 7/84/1316-1208$02.00/0 Vol. 131, June
THE JOURNAL OF UROLOGY
Copyright © 1984 by The Williams & Wilkins Co.
Printed in U.S.A.
VARICOCELE: REVERSAL OF THE TESTICULAR BLOOD FLOW AND TEMPERATURE EFFECTS BY VARICOCELE REPAIR K. FINNIE GREEN,* TERRY T. TURNER
AND
STUARTS. HOWARDS
From the Department of Urology, University of Virginia School of Medicine, Charlottesville, Virginia
ABSTRACT
An experimental left varicocele was created in the adult rat by partial ligation of the left renal vein. A varicocele repair was performed by high ligation of the internal spermatic vein. Testicular blood flow and temperature changes were measured in control and sham animals, animals 30 days after establishment of varicocele and animals 30 days after varicocele repair. There was a statistically significant (p :::: 0.01) bilateral elevation of testicular blood flow and temperature in the varicocele group compared to control and sham groups. Varicocele repair returned these blood flow and temperature values to normal. Average testicular blood flow for control, varicocele, sham varicocele and varicocele repair animals were 29.6 ± 1, 39.8 ± 2, 30. 7 ± 1 and 29.8 ± 1 ml. per min. per 100 gm. tissue, respectively. Testicular temperatures averaged 34.4 ± 0.1, 35.3 ± 0.2, 34.4 ± 0.1 and 34.5 ± 0.1 degrees C, respectively. It is possible that the elevation in blood flow is associated with the elevation of intratesticular temperature, which is known to impair spermatogenesis. The data support a relationship between the varicocele and potential testicular damage. The frequent association of varicocele with diminished male fertility has stimulated much clinical and scientific interest. This varicocele-infertility relationship has been supported in the literature by 3 recurrent observations. 1) The occurrence of varicocele in populations of infertile men is higher than in the general population. Compilation of data from several large clinical studies reveals the incidence of varicocele in men attending infertility clinics to be approximately 37 per cent (range 21 to 39 per cent), while the incidence of varicocele in the general population averages only 15 per cent (range 8 to 23 per cent). 1 2) A large percentage of men with varicoceles (50 to 80 per cent) have documented abnormal semen specimens. 2- 6 •14 These men demonstrate one or more features of the seminal stress pattern as described by McLeod. 7 Further evidence of impaired spermatogenesis in patients with varicoceles has been documented by the occurrence of bilateral changes seen in testicular biopsies. s-u 3) Varicocele repair is stated to improve semen quality in a large percentage of men and to improve male fertility as manifested by an increase in pregnancy rates of spouses. In a review of several clinical studies with a total of 1541 patients an average of 66 per cent of patients experienced an improvement in semen quality and 47 per cent impregnated their partner after varicocele repair. 1 •7• 12• 13- 16 Despite the therapeutic success of varicocele repair in the treatment of selected infertile men, several enigmas remain. For instance, the mechanism by which a varicocele adversely affects spermatogenesis is still not well understood. While multiple theories regarding the pathophysiology have been proposed, it is important to realize that the varicocele itself may not be responsible for the actual impairment of spermatogenesis. Instead, a varicocele may be a clinically detectable sign of a more complex underlying pathophysiologic process. Another paradox regarding the varicocele is that it is not uniformly detrimental to spermatogenesis. Many men with a varicocele have either a normal semen analysis or only modest impairment of spermatogenesis. While inconsistencies exist,
varicocele repair does seem to ameliorate the underlying pathophysiologic process in some men. Recent data by Saypol et al. 17 using an animal model show a bilateral elevation in testicular blood flow and temperature in response to a unilateral varicocele. The present study was undertaken to determine the effects of a conventional varicocele repair on these induced pathophysiologic changes. MATERIALS AND METHODS
Mature male Sprague-Dawley rats, weighing 350 to 500 gm., were obtained from the medical center vivarium. They were acclimated for 1 week prior to experimental use and were subsequently maintained in the vivarium for the duration of the experiment. Control and experimental groups were matched for approximate age and weight. Group I: Control (CJ. Five normal rats were weighed and anesthetized with 1.3 per cent halothane mixed with 1.5 liters per min. of room air. Continuous anesthesia was delivered through a face cone and maintained by a Flurotec 3 Vaporizer. Blood pressure in the left ventricle and right femoral artery was measured continuously by using 2 Gould Statham P23ID pressure transducers and a Sanborn Model 321 Dual Channel Pressure Recorder. Catheterization of the left ventricle through the right carotid artery was performed using PE50 pressure monitoring tubing which was connected to the 1st transducer. Localization of the catheter tip in the left ventricle was confirmed by the change in the pressure wave form, noted on the pressure recorder, as the catheter entered the left ventricle. The 2nd transducer was attached to PE50 tubing which was used to catheterize the right femoral artery. This line was used for blood pressure monitoring throughout each experiment. Rectal temperature was continuously monitored by a Bailey Thermometer Model BAT-4 and a Bailey Rectal Probe RET2. Rectal temperature was maintained at approximately 37C by a heat lamp. The technique of measuring organ blood flow and cardiac output by radioactive microsphere tracers was similar to that first described by Rudolph and Heyman. 18 Strontium 85 (SR85) labelled microspheres (15 ± 3 µ.), with a main gamma for publication January 26, 1984. f 100 514 k d 'fj f ·t f 15 1 *Accepted Requests for reprints: Dept. of Urology, Box 422, University of energy O per cent at ev an a speci lC ac lVl YO • Virginia School of Medicine, Charlottesville, VA 22908. mCi per gm., were suspended in a normal saline solution Supported by grant HD18252 from the National Institutes of Health. containing 0.05 per cent Tween-80 and 10 per cent dextran. 1208
REVERSAL OF EFI'ECTS OF VARICOCELE
1209
After U/'_,,ewe,vu of the 0,1 to 0.15 mL of the suspension (60 to 95,000 spheres) was drawn up into a plastic syringe, placed into a counting vial and analyzed between 425 and 598 kev in a LKB 1280 Ultragamma II Counter. This suspension was then slowly injected into the left ventricular catheter over 10 sec. and flushed from the catheter by 0.3 ml. of heparinized saline over 15 sec. Prior to injection of the microsphere suspension, the right femoral artery catheter was attached to a Harvard Continuous Withdrawal Pump Model 600-950. Blood was withdrawn through the femoral artery catheter and collected into a glass syringe for 10 sec. before the injection of the microspheres, during the microsphere injection, and subsequently thereafter for a total withdrawal time of 70 sec. (;:,;L5 ml). The blood was then placed into a preweighed gamma counting vial. Testicular temperatures were obtained after the microsphere injection was complete and before the animal was sacrificed. Right and left intratesticular temperatures were obtained by making a small skin incision in each hemiscrotum and passing a 29 gauge needle microprobe into the parenchyma of each testis (Bailey Model MT-29/2, 0.25 sec. time constant). The animals were then sacrificed by injection of a saturated potassium chloride solution. Determination of organ blood flow was made by removal of the right and left testes, epididymides and kidneys, along with the prostate and a wedge section of liver. These organs were weighed and together with the blood sample and empty microsphere syringe, were placed in the LKB gamma counter for analysis, Total count time for each tissue sample was 3 minutes and was expressed as counts per min. Cardiac output was calculated by using the following equation:
Ix R C.O. = RBS C.O. = Cardiac Output (ml. per min, or ml. per min. per 100 gm. tissue). I = total injected count (CPM). R = withdrawal rate of blood (ml. per min.), RBS = counts in reference blood sample from femoral artery (CPM). Blood flow to each selected tissue was calculated by using the following equation: 100 T x R BF = - - - x - - - - - - RBS Tissue Wt. (gm.) BF= Blood flow (ml. per min, per 100 gm. tissue). T = tissue counts (CPM). R = withdrawal rate (ml. per min.). RBS = counts in reference blood sample (CPM). Preparation of the testes for included initial perfusion fixation in Bouins solution for approximately 5 minutes followed immersion fixation in Bouins solution for a minimum of 24 The fixed testes were embedded in paraffin and stained with and eosin. The specan imens were coded separately and scored subjectively independent histologist in a single blind procedure. The grading was made to appraise the tissue as being normal or abnormal (disorganization of germinal epithelium). Group II Varicocele (V). Eight rats were weighed, anesthetized and maintained at 37C as in Group I (C). A midline laparotomy was performed. The left renal vein was identified and partially ligated to an external diameter of 0.85 mm. A consistent stenosis was achieved by using a 4-0 silk suture which was ligated around both the renal vein and a metal probe. The probe was carefully removed and the vein allowed to expand against the loop of suture. The suture was positioned medially to both the adrenal and internal spermatic veins (fig. 1). After recovery from anesthesia the rats were returned and maintained in the vivarium. Thirty-one days after their initial operation the rats underwent blood flow and temperature measurements and histologic preparation of both testes using the same protocol as in Group I (C). In addition, dilatation of the left spermatic vein was quantitated at necropsy by measuring
FIG. 1. A, schematic of normal left kidney and normal left renal and spermatic veins. B, schematic of partial ligation of left renal vein with subsequent dilation of left renal, spermatic and meteral veins. C, arrows indicate left spermatic vein prior to partial ligation of left renal vein (20X). D, arrows indicate dilated spermatic vein (varicoce!e) 30 days after partial ligation of left renal vein (20X).
the external diameter of the varicocele as it crossed anteriorly to the iliolumbar veins. Group III. Sham varicocele repair (SVR). Six rats were weighed and anesthetized as in Group I (C). After the standard laparotomy, the left renal vein was identified and a 4-0 silk suture was passed posteriorly to the veiR No ligation was performed. After adequate recovery from anesthesia the rats were returned and maintained in the vivarium. Thirty days after this initial operation, these 6 rats were through a midline laparotomy, The left testicular vein was identified at the level of the left renal vein and sharp dissection around the testicular vein was performed. After adequate recovery from anesthesia the rats were returned and maintained in the vivarium. Group JV. Varicocele Repair (VR). Eleven rats were weighed and anesthetized as in Group I (C). The left renal vein was partially ligated as in Group II (V). After adequate recovery from anesthesia the rats were returned and maintained in the vivarium. Thirty days after the initial operation, these 11 rats were subjected to a 2nd midline laparotomy. A dilated left testicular vein was identified in all 11 animals and a varicocele repair, by a high ligation of the spermatic vein at its junction with the left renal vein, was performed. After adequate recovery from anesthesia the rats were returned to the vivarium. Thirty days after this 2nd operation, the rats in Group III (SVR) and IV (VR) underwent testicular blood flow and temperature measurements and histologic preparation as in Group I (C). The size of the repaired left spermatic vein was quantitated as in Group II (V).
1210
GREEN, TURNER AND HOWARDS TABLE 1.
Whole body weight, testicular weight, cardiac output, and systemic blood pressure* Whole Body Weight (gm.)
(R) Testicular Weight
(gm.)
(L) Testicular Weight (gm.)
Cardiac Output (ml./min./100 gm.)
Systemic Systolic Blood Pressure (mm.Hg)
419 ± 20 497 ± 17 473 ± 25
1.80 ± .20 1.88 ± .15 1.96 ± .10
1.80 ± .15 1.89 ± .15 1.95 ± 1.0
21.4 ± 1.0 20.9 ± 1.0 19.0 ± 1.0
115 ± 5.0 116 ± 4.2 116 ± 2.0
451 ± 10 None
1.84 ± .15 None
1.85 ± .15 None
19.4 ± 1.0 None
113 ± 3.5 None
Group I (control) no. = 5 Group II (varicocele) no.= 8 Group III (sham varicocele sham repair) no.= 6 Group IV (varicocele repair) no. = 11 Level of significant difference
* Values expressed as mean ± SEM. TABLE 2.
Group I (control) no. = 5 Group II (varicocele) no. = 8 Level of significant difference
Organ blood flow*
(R) Testis
(L) Testis
(R) Epididymis
(L) Epididymis
(R) Kidney
(L) Kidney
Liver
Prostate
29.9 ± 1.0 39.8 ± 2.0 p < 0.01
29.4 ± 1.0 39.8 ± 2.5 p < 0.01
21.8 ± 2.0 21.9 ± 2.0 None
21.0 ± 2.0 20.9 ± 1.5 None
462 ± 39 453 ± 62 None
477 ± 51 448 ± 76 None
20.3 ± 3.0 20.3 ± 2.5 None
35.3 ± 3.0 31.2 ± 2.0 None
* Values expressed as mean ± SEM in ml./min./100 gm. tissue.
RESULTS
All blood flow and temperature data for paired organs within each of the 4 experimental groups were analyzed by Student's t test. No significant differences (p < 0.01) between paired organs within each group were noted. All
Sham varicocele repair animals, Group III (SVR), had mean testicular blood flows which were the same as in Group I (C) (table 3). Nine of 11 animals with varicocele repair (Group IV a) had mean testicular blood flows which were not significantly different from those in control or sham varicocele repair animals (Groups I & III). However, 2 of 11 animals with varicocele repair (Group IVb) had mean testicular blood flows which were elevated and were not significantly different from blood flow in animals with varicoceles (Group II). The mean testicular blood flows in controls (Group I), sham varicocele repair (Group III), and varicocele repair (Group IVa) were significantly lower (p < 0.01) when compared to corresponding blood flow values in animals with a varicocele (Group II) and the nonresponders in the varicocele repairs (Group IVb). Blood flow to other selected organs, including the epididymis, kidney, prostate and liver, was not significantly different between any of the 4 experimental groups. All 11 animals that underwent varicocele repair (Group IV) had a visible left varicocele, with a mean dilation of 0.85 ± 0.13 mm. before varicocele repair. The 2 animals in Group IVb (VR) with blood flow values that were the same as in Group II (V) had a persistent dilation of the internal spermatic vein after repair. The mean external diameter of these 2 persistent varicoceles was 0. 7 ± 0.15 mm. 3) Temperature. In control animals, mean right and left testicular temperatures were 34.4 ± 0.10 and 34.4 ± O.lOC,
respectively, when rectal temperature was maintained at 37C (table 4). In contrast, the testicular temperatures in Group II (V) were bilaterally elevated and statistically greater (p < 0.01) than the testicular temperatures in the control animals. In Group III (SVR) and in the 9 animals of Group IV a (VR) with normal blood flows, the mean testicular temperatures were statistically the same (p < 0.01) as in Group I (C) and significantly less (p < 0.01) than in Group II (V) (table 4). The 2 animals of Group IVb (VR) with elevated blood flows had mean testicular temperatures of an intermediate value. 4) Histology. All of Group I (C) demonstrated normal testicular histology. Of the 8 animals in Group II (V) with a grossly dilated testicular vein, only 1 showed bilateral germinal epithelial disorganization while 3 animals revealed unilateral disorganization. As a result of this minimal response to a 30 day varicocele no further histologic examination of the testes m Groups III (SVR) and IV (VR) was undertaken. DISCUSSION
The frequent association between varicocele and diminished male fertility has been a recurrent theme in the literature. 1- 13 An important recent advance in this field was the development of an adequate varicocele animal model. Al-Juburi et al., 19 Kay and Alexander, 2° Cockett and Caldamone 21 all have surgically created a unilateral varicocele in the animal model and have TABLE 3.
Testicular blood flow*
Group I (control) no. = 5 Group III (sham varicocele sham repair) no.= 6 Group !Va (varicocele repair) no. = 9 Group !Vb (varicocele repair) no. = 2 Group II (varicocele) no.= 8 Level of significant difference I, III, !Va vs. II, !Vb
(R) Testis
(L) Testis
29.9 ± 1.0 30.7 ± 1.0
29.4 ± 1.0 30.6 ± 1.0
29.8 ± 1.0 42.3 ± 1.5 39.8 ± 2.0 p < 0.01
30.0 ± 1.0 42.3 ± 1.0 39.8 ± 2.5 p < 0.01
* Values expressed as mean± SEM in ml./min./100 gm. tissue. TABLE 4.
Intratesticular temperature*
Group I (control) no. = 5 Group III (sham varicocele sham repair) no.= 6 Group !Va (varicocele repair) no. = 9 Group II (varicocele) no. = 8 Level of significant difference I, III, IV a vs. II Group !Vb (varicocele repair) no. = 2
(R) Testis
(L) Testis
34.4 ± .10 34.4 ± .10
34.4 ± .10 34.4 ± .10
34.5 ± .10 35.3 ± .20 p < 0.01
34.5 ± .10 35.3 ± .20 p < 0.01
34.7 ± .05
34.7 ± .05
* Values expressed as mean ± SEM in degrees centigrade. Rectal temperature
maintained at 37C.
1211.
REVEiliSAL OF EFFECTS OF \lARICJCELE
demonstrated its adverse effect on both testes. These studies reported the presence of a seminal stress pattern, a bilateral impairment of spermatogenesis on testicular biopsy and a bilateral increase in testicular temperaturern-zi Saypol et a!. 17 using this animal model studied the effects of varicocele on testicular blood flow and temperature. They observed a significant bilateral increase in both testicular blood flow and temperature as the result of a unilateral varicocele. The present investigation corroborates the effect of the experimental varicocele as described by Saypol et al.1 7 The varicocele effect of a statistically significant, bilateral elevation of both mean testicular blood flow and temperature was observed when comparing data from Groups I (C) and II (V) (tables 2, 3 and 4). These data support the hypothesis that deleterious bilateral testicular effects result from unilateral varicocele. The exact mechanism by which the elevation in testicular blood flow produces an elevation in temperature might speculatively be related to a relative inefficiency of the counter-current heat exchange system in the pampiniform plexus. The effectiveness of this heat exchange system is diminished as the testicular arterial blood flow is increased, 22 • 23 Despite the therapeutic success ofvaricocele repair, the exact mechanism by which it corrects the effects of a varicocele remains unknown. If it is true that increased testicular blood flow and temperature are important to the underlying pathophysiology of varicocele, then any procedure which reverses the clinical effects of varicocele should also reverse these blood flow and temperature effects. We performed our varicocele repair experiment to determine if, indeed, the repair altered the testicular changes associated with an experimental varicocele, A total of 11 animals underwent varicocele repair and they were divided into 2 groups according to their blood flow values after repair. Nine of 11 animals, Group IVa (tables 3 and 4), had mean testicular blood flow and temperature values that were the same as in controls. All 9 animals of Group IV a had visible varicoceles at the time of the varicocele repair. The response to varicocele repair, therefore, was a dramatic normalization of both testicular blood flow and testicular temperature. This observation is important for several reasons. First, it supports our initial hypothesis as to the underlying pathophysiology of the varicocele, Through the creation of a varicocele in the animal model we have observed consistent and quantifiable pathophysiologic responses which may be detrimental to spermatogenesis in the long term. Secondly, by a high ligation of the dilated internal spermatic vein we have applied the standard clinical treatment for a varicocele to our experimental animal modeL The response was a reversal of the underlying pathophysiology. To our knowledge this is the 1st 'Nell-documented report detailing the actual physiologic events The exact manner by which which occur after varicocele a total obstruction of the internal c,p,cuuaw., vein leads to a decrease in the testicular arterial flow and to a decrease in the testicular temperature has not yet been elucidated. The data from the 2 animals in Group IVb (VR) were also intriguing. The testicular blood flow in these animals was not significantly different from the testicular blood flow in animals with a varicocele (tables 3 and 4). In addition, a persistent dilatation of the internal spermatic vein was seen in both animals. These 2 animals responded as if the varicocele was still present. The etiology of this persistent varicocele effect after high ligation of the internal spermatic vein could result from what has been described in the clinical literature as the recurrent varicocele phenomena. This has been reported to occur in approximately 5 per cent of men treated with varicocele repair. 24 Collateral venous channels which establish a connection between the left renal vein and the internal spermatic vein below the level of the ligation have been observed in these recurrences. In Group IVb a dilated collateral venous channel between the left renal vein and the left spermatic vein was
!TI l animal, The cause of noted at varicocele in other animal remains unknown. His to logic evaluation of the effect of a varicocele on testicular morphology revealed a minimal negative influence in a small number of animals with the experimental varicocele. This may have been due to the short exposure of the testis to the varicocele, relative to the duration of the rat spermatogenic cycle. Further investigation of the hemodynamic and thermal changes associated with varicocele and varicocele repair are critical to a more precise understanding of the relationship between varicocele and male infertility. 'AQC'Wmcu
REFERENCES L Greenberg, S. M.: Varicocele and male fertility. Fertil. Steril., 28: 699, 1977. 2. Greenberg, S. M., Lipshultz, L. Land Wein, A. J.: Experience with 425 subfertile male patients. J. Urol., IHI: 507, 1978. 3. Cockett, A. T. K., Urry, R. L. and Dougherty, K. A.: The varicocele and semen characteristics. J. Urol., 121: 435, 1979. 4. Cockett, A. T. K., Netto, I. C. V., Dougherty, K. A. and Urry, R. L.: Semen analysis: a review of samples from 225 men seen at an infertility clinic. J. Urol., 114: 560, 1975. 5. Rodriguiz-Rigau, L. J., Smith, K D. and Steinberger, E.: Varicocele and the morphology of spermatozoa. Fertil. Steril., 35: 54, 1981. 6. Fariss, B. L., Fenner, D. K., Plymate, S. R., Brannen, G. E., Jacob, W. M. and Thomason, A. M.: Seminal characteristics in the presence of a varicocele as compared with those of expectant fathers and prevasectomy men. FertiL Steril., 35: 325, 1981. 7, MacLeod, J .: Seminal cytology in the presence of varicocele. Fertil. Steril., 16: 735, 1965. 8. Etriby, A., Girgis, S. M., Hefnawy, H. and Ibrahim, A. A.: Testicular changes in subfertile males with varicocele. Fertil. Steril., 18: 666, 1967. 9. Dubin, L. and Hotchkiss, R. S.: Testis biopsy in subfertile men with varicocele. Fertil. Steril., 20: 50, 1969. 10. Ibrahim, A. A., Awad, ,H. A., El-Haggar, S. and Mitawi, B. A.: Bilateral testicular biopsy in men with varicocele. Fertil. Steril., 28: 663, 1977. 11. McFadden, M. R. and Mehan, D. J.: Testicular biopsies in 101 case.s of varicocele. J. Urol., 119: 372, 1978. 12. Charny, C. W. and Baum, S.: Varicocele and infertility. JAMA, 204: 75, 1968. 13. Stewart, B. H.: Varicoce!e in infertility: incidence and results of surgical therapy. J. Urol., 112: 222, 1974. 14. Brown, J. S.: Varicocele repair in the subfertile male: a ten-year experience with 295 cases. Fertil. Steri!., 27: 1046, 1976. 15. Glezerman, M., Rakowszczyk, M., Lunenfeld, B., Beer, R. and Goldman, B.: Varicocele in oligospermic patients: pathophysiology and results after ligation and division of the internal spermatic vein. J. UroL, 115: 562, 1976. 16. K. and Amelar, R. D.: Varicocelectomy: 986 cases in a study. l 0: 446, 1977. 17. Saypol, C., Howards, Turner, T. T. and Iviiller, E. D.: Influence of surgically induced varicocele on testicular biood flow, temperature, and histology in adult rats and dogs. J. Clin. 68: 39, 1981. 18. Rudolph, IVL and Heymann, M. A.: The circulation of the fetus in utero, Methods for studying distribution of blood flow, cardiac output and organ blood flow. Circ. Res., 21: 163, 1967. 19. Cockett, A. T. K., A!-Juburi, A., Altebarmakian, V., Vergamini, R. F. and Caldamone, A. A.: The varicocele: new experimental and clinical data. Urology, 15: 492, 1980, 20. Kay, R., Alexander, N. J. and Baugham, W. L.: Induced varicoceles in Rhesus monkeys. Fertil. Steril., 31: 195, 1979. 21. Caldamone, A. A. and Cockett, A. T. K.: Recent advances in male infertility research. Urol. Clin. N. Am., 8: 63, 1981. 22. Dahl, E. V. and Herrick, J. F.: A vascular mechanism for maintaining testicular temperature by counter-current exchange. Surg. Gynecol. Obstet., 108: 697, 1959. 23. Waites, G. M. H. and Moule, G. R.: Relation of vascular heat exchange to temperature regulation in the testis of the ram. J. Reprod. Fertil., 2: 213, 1961. 24. Sayfan, J., Adam, Y. G. and Soffer, Y.: A natural "venous bypass" causing postoperative recurrence of a varicocele. J. Androl., 2: 108, 1981.
=--
0022-534 7/84/1316-1208$02.00/0 Vol. 131, June
THE JOURNAL OF UROLOGY
Copyright © 1984 by The Williams & Wilkins Co.
Printed in U.S.A.
VARICOCELE: REVERSAL OF THE TESTICULAR BLOOD FLOW AND TEMPERATURE EFFECTS BY VARICOCELE REPAIR K. FINNIE GREEN,* TERRY T. TURNER
AND
STUARTS. HOWARDS
From the Department of Urology, University of Virginia School of Medicine, Charlottesville, Virginia
ABSTRACT
An experimental left varicocele was created in the adult rat by partial ligation of the left renal vein. A varicocele repair was performed by high ligation of the internal spermatic vein. Testicular blood flow and temperature changes were measured in control and sham animals, animals 30 days after establishment of varicocele and animals 30 days after varicocele repair. There was a statistically significant (p :::: 0.01) bilateral elevation of testicular blood flow and temperature in the varicocele group compared to control and sham groups. Varicocele repair returned these blood flow and temperature values to normal. Average testicular blood flow for control, varicocele, sham varicocele and varicocele repair animals were 29.6 ± 1, 39.8 ± 2, 30. 7 ± 1 and 29.8 ± 1 ml. per min. per 100 gm. tissue, respectively. Testicular temperatures averaged 34.4 ± 0.1, 35.3 ± 0.2, 34.4 ± 0.1 and 34.5 ± 0.1 degrees C, respectively. It is possible that the elevation in blood flow is associated with the elevation of intratesticular temperature, which is known to impair spermatogenesis. The data support a relationship between the varicocele and potential testicular damage. The frequent association of varicocele with diminished male fertility has stimulated much clinical and scientific interest. This varicocele-infertility relationship has been supported in the literature by 3 recurrent observations. 1) The occurrence of varicocele in populations of infertile men is higher than in the general population. Compilation of data from several large clinical studies reveals the incidence of varicocele in men attending infertility clinics to be approximately 37 per cent (range 21 to 39 per cent), while the incidence of varicocele in the general population averages only 15 per cent (range 8 to 23 per cent). 1 2) A large percentage of men with varicoceles (50 to 80 per cent) have documented abnormal semen specimens. 2- 6 •14 These men demonstrate one or more features of the seminal stress pattern as described by McLeod. 7 Further evidence of impaired spermatogenesis in patients with varicoceles has been documented by the occurrence of bilateral changes seen in testicular biopsies. s-u 3) Varicocele repair is stated to improve semen quality in a large percentage of men and to improve male fertility as manifested by an increase in pregnancy rates of spouses. In a review of several clinical studies with a total of 1541 patients an average of 66 per cent of patients experienced an improvement in semen quality and 47 per cent impregnated their partner after varicocele repair. 1 •7• 12• 13- 16 Despite the therapeutic success of varicocele repair in the treatment of selected infertile men, several enigmas remain. For instance, the mechanism by which a varicocele adversely affects spermatogenesis is still not well understood. While multiple theories regarding the pathophysiology have been proposed, it is important to realize that the varicocele itself may not be responsible for the actual impairment of spermatogenesis. Instead, a varicocele may be a clinically detectable sign of a more complex underlying pathophysiologic process. Another paradox regarding the varicocele is that it is not uniformly detrimental to spermatogenesis. Many men with a varicocele have either a normal semen analysis or only modest impairment of spermatogenesis. While inconsistencies exist,
varicocele repair does seem to ameliorate the underlying pathophysiologic process in some men. Recent data by Saypol et al. 17 using an animal model show a bilateral elevation in testicular blood flow and temperature in response to a unilateral varicocele. The present study was undertaken to determine the effects of a conventional varicocele repair on these induced pathophysiologic changes. MATERIALS AND METHODS
Mature male Sprague-Dawley rats, weighing 350 to 500 gm., were obtained from the medical center vivarium. They were acclimated for 1 week prior to experimental use and were subsequently maintained in the vivarium for the duration of the experiment. Control and experimental groups were matched for approximate age and weight. Group I: Control (CJ. Five normal rats were weighed and anesthetized with 1.3 per cent halothane mixed with 1.5 liters per min. of room air. Continuous anesthesia was delivered through a face cone and maintained by a Flurotec 3 Vaporizer. Blood pressure in the left ventricle and right femoral artery was measured continuously by using 2 Gould Statham P23ID pressure transducers and a Sanborn Model 321 Dual Channel Pressure Recorder. Catheterization of the left ventricle through the right carotid artery was performed using PE50 pressure monitoring tubing which was connected to the 1st transducer. Localization of the catheter tip in the left ventricle was confirmed by the change in the pressure wave form, noted on the pressure recorder, as the catheter entered the left ventricle. The 2nd transducer was attached to PE50 tubing which was used to catheterize the right femoral artery. This line was used for blood pressure monitoring throughout each experiment. Rectal temperature was continuously monitored by a Bailey Thermometer Model BAT-4 and a Bailey Rectal Probe RET2. Rectal temperature was maintained at approximately 37C by a heat lamp. The technique of measuring organ blood flow and cardiac output by radioactive microsphere tracers was similar to that first described by Rudolph and Heyman. 18 Strontium 85 (SR85) labelled microspheres (15 ± 3 µ.), with a main gamma for publication January 26, 1984. f 100 514 k d 'fj f ·t f 15 1 *Accepted Requests for reprints: Dept. of Urology, Box 422, University of energy O per cent at ev an a speci lC ac lVl YO • Virginia School of Medicine, Charlottesville, VA 22908. mCi per gm., were suspended in a normal saline solution Supported by grant HD18252 from the National Institutes of Health. containing 0.05 per cent Tween-80 and 10 per cent dextran. 1208
REVERSAL OF EFI'ECTS OF VARICOCELE
1209
After U/'_,,ewe,vu of the 0,1 to 0.15 mL of the suspension (60 to 95,000 spheres) was drawn up into a plastic syringe, placed into a counting vial and analyzed between 425 and 598 kev in a LKB 1280 Ultragamma II Counter. This suspension was then slowly injected into the left ventricular catheter over 10 sec. and flushed from the catheter by 0.3 ml. of heparinized saline over 15 sec. Prior to injection of the microsphere suspension, the right femoral artery catheter was attached to a Harvard Continuous Withdrawal Pump Model 600-950. Blood was withdrawn through the femoral artery catheter and collected into a glass syringe for 10 sec. before the injection of the microspheres, during the microsphere injection, and subsequently thereafter for a total withdrawal time of 70 sec. (;:,;L5 ml). The blood was then placed into a preweighed gamma counting vial. Testicular temperatures were obtained after the microsphere injection was complete and before the animal was sacrificed. Right and left intratesticular temperatures were obtained by making a small skin incision in each hemiscrotum and passing a 29 gauge needle microprobe into the parenchyma of each testis (Bailey Model MT-29/2, 0.25 sec. time constant). The animals were then sacrificed by injection of a saturated potassium chloride solution. Determination of organ blood flow was made by removal of the right and left testes, epididymides and kidneys, along with the prostate and a wedge section of liver. These organs were weighed and together with the blood sample and empty microsphere syringe, were placed in the LKB gamma counter for analysis, Total count time for each tissue sample was 3 minutes and was expressed as counts per min. Cardiac output was calculated by using the following equation:
Ix R C.O. = RBS C.O. = Cardiac Output (ml. per min, or ml. per min. per 100 gm. tissue). I = total injected count (CPM). R = withdrawal rate of blood (ml. per min.), RBS = counts in reference blood sample from femoral artery (CPM). Blood flow to each selected tissue was calculated by using the following equation: 100 T x R BF = - - - x - - - - - - RBS Tissue Wt. (gm.) BF= Blood flow (ml. per min, per 100 gm. tissue). T = tissue counts (CPM). R = withdrawal rate (ml. per min.). RBS = counts in reference blood sample (CPM). Preparation of the testes for included initial perfusion fixation in Bouins solution for approximately 5 minutes followed immersion fixation in Bouins solution for a minimum of 24 The fixed testes were embedded in paraffin and stained with and eosin. The specan imens were coded separately and scored subjectively independent histologist in a single blind procedure. The grading was made to appraise the tissue as being normal or abnormal (disorganization of germinal epithelium). Group II Varicocele (V). Eight rats were weighed, anesthetized and maintained at 37C as in Group I (C). A midline laparotomy was performed. The left renal vein was identified and partially ligated to an external diameter of 0.85 mm. A consistent stenosis was achieved by using a 4-0 silk suture which was ligated around both the renal vein and a metal probe. The probe was carefully removed and the vein allowed to expand against the loop of suture. The suture was positioned medially to both the adrenal and internal spermatic veins (fig. 1). After recovery from anesthesia the rats were returned and maintained in the vivarium. Thirty-one days after their initial operation the rats underwent blood flow and temperature measurements and histologic preparation of both testes using the same protocol as in Group I (C). In addition, dilatation of the left spermatic vein was quantitated at necropsy by measuring
FIG. 1. A, schematic of normal left kidney and normal left renal and spermatic veins. B, schematic of partial ligation of left renal vein with subsequent dilation of left renal, spermatic and meteral veins. C, arrows indicate left spermatic vein prior to partial ligation of left renal vein (20X). D, arrows indicate dilated spermatic vein (varicoce!e) 30 days after partial ligation of left renal vein (20X).
the external diameter of the varicocele as it crossed anteriorly to the iliolumbar veins. Group III. Sham varicocele repair (SVR). Six rats were weighed and anesthetized as in Group I (C). After the standard laparotomy, the left renal vein was identified and a 4-0 silk suture was passed posteriorly to the veiR No ligation was performed. After adequate recovery from anesthesia the rats were returned and maintained in the vivarium. Thirty days after this initial operation, these 6 rats were through a midline laparotomy, The left testicular vein was identified at the level of the left renal vein and sharp dissection around the testicular vein was performed. After adequate recovery from anesthesia the rats were returned and maintained in the vivarium. Group JV. Varicocele Repair (VR). Eleven rats were weighed and anesthetized as in Group I (C). The left renal vein was partially ligated as in Group II (V). After adequate recovery from anesthesia the rats were returned and maintained in the vivarium. Thirty days after the initial operation, these 11 rats were subjected to a 2nd midline laparotomy. A dilated left testicular vein was identified in all 11 animals and a varicocele repair, by a high ligation of the spermatic vein at its junction with the left renal vein, was performed. After adequate recovery from anesthesia the rats were returned to the vivarium. Thirty days after this 2nd operation, the rats in Group III (SVR) and IV (VR) underwent testicular blood flow and temperature measurements and histologic preparation as in Group I (C). The size of the repaired left spermatic vein was quantitated as in Group II (V).
1210
GREEN, TURNER AND HOWARDS TABLE 1.
Whole body weight, testicular weight, cardiac output, and systemic blood pressure* Whole Body Weight (gm.)
(R) Testicular Weight
(gm.)
(L) Testicular Weight (gm.)
Cardiac Output (ml./min./100 gm.)
Systemic Systolic Blood Pressure (mm.Hg)
419 ± 20 497 ± 17 473 ± 25
1.80 ± .20 1.88 ± .15 1.96 ± .10
1.80 ± .15 1.89 ± .15 1.95 ± 1.0
21.4 ± 1.0 20.9 ± 1.0 19.0 ± 1.0
115 ± 5.0 116 ± 4.2 116 ± 2.0
451 ± 10 None
1.84 ± .15 None
1.85 ± .15 None
19.4 ± 1.0 None
113 ± 3.5 None
Group I (control) no. = 5 Group II (varicocele) no.= 8 Group III (sham varicocele sham repair) no.= 6 Group IV (varicocele repair) no. = 11 Level of significant difference
* Values expressed as mean ± SEM. TABLE 2.
Group I (control) no. = 5 Group II (varicocele) no. = 8 Level of significant difference
Organ blood flow*
(R) Testis
(L) Testis
(R) Epididymis
(L) Epididymis
(R) Kidney
(L) Kidney
Liver
Prostate
29.9 ± 1.0 39.8 ± 2.0 p < 0.01
29.4 ± 1.0 39.8 ± 2.5 p < 0.01
21.8 ± 2.0 21.9 ± 2.0 None
21.0 ± 2.0 20.9 ± 1.5 None
462 ± 39 453 ± 62 None
477 ± 51 448 ± 76 None
20.3 ± 3.0 20.3 ± 2.5 None
35.3 ± 3.0 31.2 ± 2.0 None
* Values expressed as mean ± SEM in ml./min./100 gm. tissue.
RESULTS
All blood flow and temperature data for paired organs within each of the 4 experimental groups were analyzed by Student's t test. No significant differences (p < 0.01) between paired organs within each group were noted. All
Sham varicocele repair animals, Group III (SVR), had mean testicular blood flows which were the same as in Group I (C) (table 3). Nine of 11 animals with varicocele repair (Group IV a) had mean testicular blood flows which were not significantly different from those in control or sham varicocele repair animals (Groups I & III). However, 2 of 11 animals with varicocele repair (Group IVb) had mean testicular blood flows which were elevated and were not significantly different from blood flow in animals with varicoceles (Group II). The mean testicular blood flows in controls (Group I), sham varicocele repair (Group III), and varicocele repair (Group IVa) were significantly lower (p < 0.01) when compared to corresponding blood flow values in animals with a varicocele (Group II) and the nonresponders in the varicocele repairs (Group IVb). Blood flow to other selected organs, including the epididymis, kidney, prostate and liver, was not significantly different between any of the 4 experimental groups. All 11 animals that underwent varicocele repair (Group IV) had a visible left varicocele, with a mean dilation of 0.85 ± 0.13 mm. before varicocele repair. The 2 animals in Group IVb (VR) with blood flow values that were the same as in Group II (V) had a persistent dilation of the internal spermatic vein after repair. The mean external diameter of these 2 persistent varicoceles was 0. 7 ± 0.15 mm. 3) Temperature. In control animals, mean right and left testicular temperatures were 34.4 ± 0.10 and 34.4 ± O.lOC,
respectively, when rectal temperature was maintained at 37C (table 4). In contrast, the testicular temperatures in Group II (V) were bilaterally elevated and statistically greater (p < 0.01) than the testicular temperatures in the control animals. In Group III (SVR) and in the 9 animals of Group IV a (VR) with normal blood flows, the mean testicular temperatures were statistically the same (p < 0.01) as in Group I (C) and significantly less (p < 0.01) than in Group II (V) (table 4). The 2 animals of Group IVb (VR) with elevated blood flows had mean testicular temperatures of an intermediate value. 4) Histology. All of Group I (C) demonstrated normal testicular histology. Of the 8 animals in Group II (V) with a grossly dilated testicular vein, only 1 showed bilateral germinal epithelial disorganization while 3 animals revealed unilateral disorganization. As a result of this minimal response to a 30 day varicocele no further histologic examination of the testes m Groups III (SVR) and IV (VR) was undertaken. DISCUSSION
The frequent association between varicocele and diminished male fertility has been a recurrent theme in the literature. 1- 13 An important recent advance in this field was the development of an adequate varicocele animal model. Al-Juburi et al., 19 Kay and Alexander, 2° Cockett and Caldamone 21 all have surgically created a unilateral varicocele in the animal model and have TABLE 3.
Testicular blood flow*
Group I (control) no. = 5 Group III (sham varicocele sham repair) no.= 6 Group !Va (varicocele repair) no. = 9 Group !Vb (varicocele repair) no. = 2 Group II (varicocele) no.= 8 Level of significant difference I, III, !Va vs. II, !Vb
(R) Testis
(L) Testis
29.9 ± 1.0 30.7 ± 1.0
29.4 ± 1.0 30.6 ± 1.0
29.8 ± 1.0 42.3 ± 1.5 39.8 ± 2.0 p < 0.01
30.0 ± 1.0 42.3 ± 1.0 39.8 ± 2.5 p < 0.01
* Values expressed as mean± SEM in ml./min./100 gm. tissue. TABLE 4.
Intratesticular temperature*
Group I (control) no. = 5 Group III (sham varicocele sham repair) no.= 6 Group !Va (varicocele repair) no. = 9 Group II (varicocele) no. = 8 Level of significant difference I, III, IV a vs. II Group !Vb (varicocele repair) no. = 2
(R) Testis
(L) Testis
34.4 ± .10 34.4 ± .10
34.4 ± .10 34.4 ± .10
34.5 ± .10 35.3 ± .20 p < 0.01
34.5 ± .10 35.3 ± .20 p < 0.01
34.7 ± .05
34.7 ± .05
* Values expressed as mean ± SEM in degrees centigrade. Rectal temperature
maintained at 37C.
1211.
REVEiliSAL OF EFFECTS OF \lARICJCELE
demonstrated its adverse effect on both testes. These studies reported the presence of a seminal stress pattern, a bilateral impairment of spermatogenesis on testicular biopsy and a bilateral increase in testicular temperaturern-zi Saypol et a!. 17 using this animal model studied the effects of varicocele on testicular blood flow and temperature. They observed a significant bilateral increase in both testicular blood flow and temperature as the result of a unilateral varicocele. The present investigation corroborates the effect of the experimental varicocele as described by Saypol et al.1 7 The varicocele effect of a statistically significant, bilateral elevation of both mean testicular blood flow and temperature was observed when comparing data from Groups I (C) and II (V) (tables 2, 3 and 4). These data support the hypothesis that deleterious bilateral testicular effects result from unilateral varicocele. The exact mechanism by which the elevation in testicular blood flow produces an elevation in temperature might speculatively be related to a relative inefficiency of the counter-current heat exchange system in the pampiniform plexus. The effectiveness of this heat exchange system is diminished as the testicular arterial blood flow is increased, 22 • 23 Despite the therapeutic success ofvaricocele repair, the exact mechanism by which it corrects the effects of a varicocele remains unknown. If it is true that increased testicular blood flow and temperature are important to the underlying pathophysiology of varicocele, then any procedure which reverses the clinical effects of varicocele should also reverse these blood flow and temperature effects. We performed our varicocele repair experiment to determine if, indeed, the repair altered the testicular changes associated with an experimental varicocele, A total of 11 animals underwent varicocele repair and they were divided into 2 groups according to their blood flow values after repair. Nine of 11 animals, Group IVa (tables 3 and 4), had mean testicular blood flow and temperature values that were the same as in controls. All 9 animals of Group IV a had visible varicoceles at the time of the varicocele repair. The response to varicocele repair, therefore, was a dramatic normalization of both testicular blood flow and testicular temperature. This observation is important for several reasons. First, it supports our initial hypothesis as to the underlying pathophysiology of the varicocele, Through the creation of a varicocele in the animal model we have observed consistent and quantifiable pathophysiologic responses which may be detrimental to spermatogenesis in the long term. Secondly, by a high ligation of the dilated internal spermatic vein we have applied the standard clinical treatment for a varicocele to our experimental animal modeL The response was a reversal of the underlying pathophysiology. To our knowledge this is the 1st 'Nell-documented report detailing the actual physiologic events The exact manner by which which occur after varicocele a total obstruction of the internal c,p,cuuaw., vein leads to a decrease in the testicular arterial flow and to a decrease in the testicular temperature has not yet been elucidated. The data from the 2 animals in Group IVb (VR) were also intriguing. The testicular blood flow in these animals was not significantly different from the testicular blood flow in animals with a varicocele (tables 3 and 4). In addition, a persistent dilatation of the internal spermatic vein was seen in both animals. These 2 animals responded as if the varicocele was still present. The etiology of this persistent varicocele effect after high ligation of the internal spermatic vein could result from what has been described in the clinical literature as the recurrent varicocele phenomena. This has been reported to occur in approximately 5 per cent of men treated with varicocele repair. 24 Collateral venous channels which establish a connection between the left renal vein and the internal spermatic vein below the level of the ligation have been observed in these recurrences. In Group IVb a dilated collateral venous channel between the left renal vein and the left spermatic vein was
!TI l animal, The cause of noted at varicocele in other animal remains unknown. His to logic evaluation of the effect of a varicocele on testicular morphology revealed a minimal negative influence in a small number of animals with the experimental varicocele. This may have been due to the short exposure of the testis to the varicocele, relative to the duration of the rat spermatogenic cycle. Further investigation of the hemodynamic and thermal changes associated with varicocele and varicocele repair are critical to a more precise understanding of the relationship between varicocele and male infertility. 'AQC'Wmcu
REFERENCES L Greenberg, S. M.: Varicocele and male fertility. Fertil. Steril., 28: 699, 1977. 2. Greenberg, S. M., Lipshultz, L. Land Wein, A. J.: Experience with 425 subfertile male patients. J. Urol., IHI: 507, 1978. 3. Cockett, A. T. K., Urry, R. L. and Dougherty, K. A.: The varicocele and semen characteristics. J. Urol., 121: 435, 1979. 4. Cockett, A. T. K., Netto, I. C. V., Dougherty, K. A. and Urry, R. L.: Semen analysis: a review of samples from 225 men seen at an infertility clinic. J. Urol., 114: 560, 1975. 5. Rodriguiz-Rigau, L. J., Smith, K D. and Steinberger, E.: Varicocele and the morphology of spermatozoa. Fertil. Steril., 35: 54, 1981. 6. Fariss, B. L., Fenner, D. K., Plymate, S. R., Brannen, G. E., Jacob, W. M. and Thomason, A. M.: Seminal characteristics in the presence of a varicocele as compared with those of expectant fathers and prevasectomy men. FertiL Steril., 35: 325, 1981. 7, MacLeod, J .: Seminal cytology in the presence of varicocele. Fertil. Steril., 16: 735, 1965. 8. Etriby, A., Girgis, S. M., Hefnawy, H. and Ibrahim, A. A.: Testicular changes in subfertile males with varicocele. Fertil. Steril., 18: 666, 1967. 9. Dubin, L. and Hotchkiss, R. S.: Testis biopsy in subfertile men with varicocele. Fertil. Steril., 20: 50, 1969. 10. Ibrahim, A. A., Awad, ,H. A., El-Haggar, S. and Mitawi, B. A.: Bilateral testicular biopsy in men with varicocele. Fertil. Steril., 28: 663, 1977. 11. McFadden, M. R. and Mehan, D. J.: Testicular biopsies in 101 case.s of varicocele. J. Urol., 119: 372, 1978. 12. Charny, C. W. and Baum, S.: Varicocele and infertility. JAMA, 204: 75, 1968. 13. Stewart, B. H.: Varicoce!e in infertility: incidence and results of surgical therapy. J. Urol., 112: 222, 1974. 14. Brown, J. S.: Varicocele repair in the subfertile male: a ten-year experience with 295 cases. Fertil. Steri!., 27: 1046, 1976. 15. Glezerman, M., Rakowszczyk, M., Lunenfeld, B., Beer, R. and Goldman, B.: Varicocele in oligospermic patients: pathophysiology and results after ligation and division of the internal spermatic vein. J. UroL, 115: 562, 1976. 16. K. and Amelar, R. D.: Varicocelectomy: 986 cases in a study. l 0: 446, 1977. 17. Saypol, C., Howards, Turner, T. T. and Iviiller, E. D.: Influence of surgically induced varicocele on testicular biood flow, temperature, and histology in adult rats and dogs. J. Clin. 68: 39, 1981. 18. Rudolph, IVL and Heymann, M. A.: The circulation of the fetus in utero, Methods for studying distribution of blood flow, cardiac output and organ blood flow. Circ. Res., 21: 163, 1967. 19. Cockett, A. T. K., A!-Juburi, A., Altebarmakian, V., Vergamini, R. F. and Caldamone, A. A.: The varicocele: new experimental and clinical data. Urology, 15: 492, 1980, 20. Kay, R., Alexander, N. J. and Baugham, W. L.: Induced varicoceles in Rhesus monkeys. Fertil. Steril., 31: 195, 1979. 21. Caldamone, A. A. and Cockett, A. T. K.: Recent advances in male infertility research. Urol. Clin. N. Am., 8: 63, 1981. 22. Dahl, E. V. and Herrick, J. F.: A vascular mechanism for maintaining testicular temperature by counter-current exchange. Surg. Gynecol. Obstet., 108: 697, 1959. 23. Waites, G. M. H. and Moule, G. R.: Relation of vascular heat exchange to temperature regulation in the testis of the ram. J. Reprod. Fertil., 2: 213, 1961. 24. Sayfan, J., Adam, Y. G. and Soffer, Y.: A natural "venous bypass" causing postoperative recurrence of a varicocele. J. Androl., 2: 108, 1981.