An experimental study of the effect of two distinct surgical techniques of orchiopexy on spermatogenesis and testicular damage in cryptorchid testes

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1 An experimental study of the effect of two distinct surgical techniques of orchiopexy on spermatogenesis and testicular damage in cryptorchid testes Gad Lotan, M.D., a Rachel Golan, Ph.D., b Yigal Efrati, M.D., a Margarita Vigodner, Ph.D., b Lawrence M. Lewin, Ph.D., b Lea Shochat, M.Sc., b and Baruch Klin, M.D. a a Department of Pediatric Surgery, Assaf Harofeh Medical Center, Zerifin; and b Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Objective: To compare the effect of two different techniques of testicular fixation on testicular function. Design: Experimental study. Setting: Surgical animal laboratory at an academic medical center. Patient(s): Sixteen mature golden hamsters underwent classic transfixation orchiopexy and true dartos pouch orchiopexy. Intervention(s): Classic transfixation orchiopexy (CTO) involved transfixation of the testicular wall at two different points and fixation of the dartos fascia. True dartos pouch orchiopexy (TDPO) involved creating a window in the dartos fascia, passage of the testicle, and closure of the window from both sides of the testicle. Main Outcome Measure(s): Flow cytometric separation of testicular cells into haploid, diploid, and tetraploid fractions for histogram analysis. Result(s): A significant decrease in testicular weight was observed in 6 out of 16 animals undergoing CTO. Diploid cells comprised the main cell fraction, and almost no haploid or tetraploid cells were observed, while in the 16 animals undergoing TDPO no change from the control pattern was observed. Conclusion(s): This experimental work supports our clinical impression that TDPO should replace CTO as the method of choice for the treatment of an undescended testicle in children. (Fertil Steril 2005;84: by American Society for Reproductive Medicine.) Key Words: Cryptorchidism, undescended testis, orchiopexy, dartos pouch, testicular function, flow cytometry Adults with a history of cryptorchidism are more likely to be infertile than normal men (1). The placement of the undescended testis into the scrotum was first advocated in A variety of surgical techniques of orchiopexy have been reported since then, from the various forms of dartos pouch to the commonly performed transparenchymal suture fixation. The deleterious effect of this latter technique on testicular histology and the phenomenon known as sympathetic orchiopathy (damage to the contralateral testis in cases of unilateral testicular injury or ischemia) raised questions concerning the ideal technique of orchiopexy. Although the advantages of early surgical intervention are rarely disputed (2), few studies have addressed the impact of different techniques of testicular fixation on testicular function the subject of the present study. MATERIALS AND METHODS Selection of Animals and Preparation of Samples for Analysis Sixteen mature golden hamsters were obtained from our surgical laboratory and maintained in conformance with Received June 15, 2004; revised and accepted February 24, Reprint requests: Gad Lotan, M.D., Department of Pediatric Surgery, Assaf Harofeh Medical Center, Zerifin 70300, Israel (FAX: ; lotan@asaf.health.gov.il). Israeli requirements, that is, they were fed standard pellets and given free access to water. Anesthesia was administered with halothane and maintained throughout the surgical procedure. The animals scrotums were shaved and cleaned with povidone iodine. A 1-cm transverse incision was made at the base of the scrotum. All the scrotal layers were systematically opened, avoiding injury to the testis. The first part of the experiment involved the creation of artificial unilateral cryptorchidism, induced by pushing the testis up to the inguinal area and closing the processus vaginalis from behind with a purse string absorbable suture, preventing the testis from descending lower than the level of the external inguinal ring. The unoperated side was used as a control. Four to 15 days later, the animals were sacrificed and their testicles surgically removed. After removal of fat and connective tissue, a portion of each testis was prepared for flow cytometry by mincing with surgical scissors to liberate individual cells. The cell suspensions and sperm samples were prepared in TNE buffer (0.01 M Tris buffer, 0.15 M NaCl, 0.01 M EDTA, ph 7.4), supplemented with glycerol 10% (v/v). The cell suspensions were frozen at 20 C for storage until analysis. The results showed significant damage to different types of germ cells on the operated side. The second phase of the experiment involved 18 mature golden hamsters 16 of which underwent operation, while /05/$30.00 Fertility and Sterility Vol. 84, No. 3, September 2005 doi: /j.fertnstert Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. 749

2 the remaining two served as controls. All the operated animals underwent classic transfixation orchiopexy (CTO) in the right hemiscrotum and true dartos pouch orchiopexy (TDPO) in the left hemiscrotum. Fifteen days later, all the hamsters were weighed and sacrificed. Their testicles were surgically removed, and their weights recorded. They were prepared for examination in the same way as reported in the first part of the experiment. Flow cytometric analysis of testicular cells into haploid, diploid, and tetraploid fractions was performed. The flow cytometry histograms of the right and left testis of each animal (including the control animals) were analyzed and compared. The surgical orchiopexies were performed as a doubleblind study so that the laboratory personnel were unaware until the end of the experiment which technique had been used for each animal examined. Surgical Technique The first part of the surgical procedure opening the inguinal area, releasing the processus vaginalis and the cremaster muscle, and passing the undescended testicle in the scrotum was the same for both techniques. The major difference was in the mode of testicular fixation. Classic transfixation orchiopexy involved transfixation of the testicular wall at two different points and fixation to the dartos fascia. Our proposed technique, TDPO, is substantially different in that no transfixation sutures are used for testicular fixation. Instead, a window is made in the dartos fascia, and once the testicle is passed through it, two stitches close this window from both sides of the testicle, avoiding its passage back to the inguinal canal. No stitches are passed through the testicular wall at any stage of the surgical procedure (Fig. 1). Flow Cytometric Analysis Flow cytometric separation of testicular cells into haploid, diploid, and tetraploid fractions was performed as described by Janca et al. (3) in 1986 and Golan et al. (4) in 2000 as follows: single-cell suspensions obtained as described above were thawed and centrifuged for 10 minutes at 500 g, and the pellet was resuspended in TNE buffer (0.2 ml). One tenth of a milliliter of single-cell suspension was mixed with 0.5 ml propidium iodide (25 g/ml final concentration) and aspirated into a flow cytometer (Becton Dickinson FACSort Flow Cytometer, San Jose, CA). Red fluorescence (BP650 LP filter) emitted from individual cells was recorded from approximately 10,000 cells per sample after excitation with a 488-nm argon laser. Results were analyzed using the WINMDI data processing program (J. Trotter, Histograms showing haploid, diploid, and tetraploid cells were analyzed and compared between left and right testes in each animal and with histograms obtained from the control animals. RESULTS The 16 mature golden hamsters operated on had CTO performed on the right hemiscrotum (the technique used by the great majority of pediatric surgeons) and TDPO performed on the left hemiscrotum (the technique proposed by us). In the 16 TDPO operations that were performed, the mean testicular weight was g. In 10 of 16 CTO operations performed, the mean testicular weight was g (hamsters numbers 7 16), while in the remaining six (numbers 1 6), a significant decrease in testicular weight was noted ( g) (Table 1). Complete numeric information concerning the mean testicular weight of the hamsters testes after TDPO and CTO can be seen in Table 1. Flow cytometry was used to investigate changes in cell populations of single testicular cell suspensions, comparing the results of the left and right testes, in both the experimental and control animals (Tables 2 and 3). The flow cytometric analysis produced histograms that separated the cell population according to ploidy (Fig. 2). In the control animals, the haploid cells were the major population, but peaks representing tetraploid, diploid, and condensed haploid cells were also observed (Fig. 2A). In six of the 16 CTO operations performed, diploid cells comprised the main cell fraction and almost no haploid and tetraploid cells were observed (Fig. 2C), whereas in all 16 TDPO operations performed, no change from the control pattern was observed (Fig. 2B). Numeric hard data of the flow cytometric analysis are shown in Tables 2 and 3. The above results clearly indicate that the CTO caused significant damage to different types of germ cells, resulting in spermatogenetic arrest in the early stages of spermatogenesis. No damage was observed with the TDPO. DISCUSSION The goals of orchiopexy in humans are to provide adequate scrotal fixation, to prevent recurrent torsion of the testis and spermatic cord or ascent of the testis, and to achieve these goals with minimal trauma to the testis. The best method of achieving fixation remains controversial. The methods in common use include CTO, scarification, the window technique, eversion of the parietal tunica vaginalis, and TDPO our preferred technique. The outcome of the experimental study presented here would suggest that the use of the popular technique of surgical fixation of the undescended testicle, involving one or two stitches in the dartos fascia and through the testicular wall CTO should be reconsidered. In the past, it was difficult to prove the above postulation, but our association with the Department of Clinical Biochemistry of Tel Aviv University provided us with a model to test our hypothesis. The department developed an animal model in hamsters for the study of spermatogenesis (4). This model proved itself ideally suited to our purpose, which was 750 Lotan et al. Orchiopexy technique and testicular damage Vol. 84, No. 3, September 2005

3 FIGURE 1 Diagram of the true dartos pouch orchiopexy procedure. [1] A tunnel between the inguinal and the scrotal areas is created, using a cotton ball attached to a hemostat. [2] Through the scrotal incision the dartos fascia is liberated, with two mosquitos holding both corners. A dartos window is created using diathermy over the cotton ball. [3] The testis is grasped and pulled down through the dartos window, which is closed around the cord at both sides by double x sutures. [4] The skin and scrotal incisions are closed with absorbable sutures and Steristrip skin dressing. A, Inguinal incision; B, scrotal incision. Fertility and Sterility 751

4 FIGURE 1 CONTINUED to evaluate the damage caused by experimental cryptorchidism, comparing two different surgical techniques of testicular fixation. Evaluation of damage to the testicular cells caused by experimental cryptorchidism was assessed by Vigodner et al. (5), using flow cytometry and confocal microscopy. Using fluorescence staining of the cell DNA by propidium iodide or acridine orange followed by flow cytometric analysis, a marked decrease in the haploid condensed cell fraction was detected at the early stages of the experiment, which correlated with the confocal microscopy results. Spermiogenic arrest was detected at stages IX XI of seminiferous epithelium, and no mature forms of the haploid cells were observed by confocal microscopy. In cases of more severe damage, no haploid fractions were detected in the seminiferous tubules of the cryptorchid ani- 752 Lotan et al. Orchiopexy technique and testicular damage Vol. 84, No. 3, September 2005

5 TABLE 1 Testicular weight of the hamster testis after true dartos pouch orchiopexy (TDPO) and classic transfixation orchiopexy (CTO). Hamsters Right testis (g), CTO Left testis (g), TDPO Control Control mals, either by flow cytometry or confocal microscopy. In addition, flow cytometry revealed a significant decrease in the tetraploid cell fraction and an increase in the S-phase fraction, possibly explained by arrest before cell entrance into meiosis. Furthermore, confocal microscopy showed apoptotic cells, destruction of tubule structure, and cellular arrangement within the tubule. Flow cytometric analysis of the epididymal contents of the hemiscrotum treated by CTO showed no mature sperms, but diploid and tetraploid cells were detected, indicating a possible release of immature germ cells from the cryptorchid testis into the epididymis. Rosenmerkel in 1820 was the first to advocate placement of the undescended testis in the scrotum (6). The dartos pouch technique, in various forms, has been described by several investigators over the years, from Petrivalsky (7) in 1931 to Koop and Minor (8) in 1957 and Benson and Lofti (9) in A modified dartos pouch orchiopexy was reported by Ritchey and Bloom (10) in 1995 as an alternative to transparenchymal suture fixation. The techniques most closely resembling the one described here are those reported by De Netto and Goldberg (11) in 1964, as well as that reported by Redman (12) in 1990 the difference being that in De Netto s description the dartos fascia is closed snugly around the cord and not beside the testicle, as in our case. Both techniques are anatomically sound, technically simple, achieve good results, and avoid trauma to the scrotum. TABLE 2 Flow cytometric ploidy determination in single-cell suspensions of hamster testicular samples. Hamsters Side T (%) D (%) NCH (%) CH (%) 1 L R Trace 90 Trace Trace 2 L R L R Trace 90 Trace Trace 4 L R L R L R Trace 90 Trace Trace 7 L R L R L R L R L R L R L R L R L R L R Control L R Control L R Note: CH condensed haploid cells; D diploid cells; L left testis of hamsters subjected to true dartos pouch orchiopexy; NCH noncondensed haploid cells; R right testis of hamsters subjected to classic transfixation orchiopexy; T tetraploid cells. Basic studies of intratesticular arterial anatomy have shown that placement of sutures in the testicle, or even into the tunica albuginea alone, may result in testicular parenchymal damage (13, 14). Arterial casts of the testicular blood supply were examined after temporary placement of a 3-0 silk traction suture in the lower pole of the testis. Significant Fertility and Sterility 753

6 TABLE 3 Flow cytometric analysis of mean percentages of cell population distribution of control hamsters and hamsters treated by true dartos pouch orchiopexy (TDPO) and classic transfixation orchiopexy (CTO). Hamsters T (%) D (%) NCH (%) CH (%) Control TDPO (1 16) CTO (1 6) Trace 90 Trace Trace CTO (7 16) Note: CH condensed haploid cells; D diploid cells; NCH noncondensed haploid cells; T tetraploid cells. FIGURE 2 Representative histograms displaying flow cytometric separation of single-cell suspension from control hamster testes (A), testes after TDPO (B), and testes after CTO (C), displaying red fluorescence intensity versus events (number of cells). CTO classic transfixation orchiopexy; D diploid; FL2 Height red fluorescence; IMH immature haploid; MH mature haploid; T tetraploid; TDPO true dartos pouch orchiopexy. arterial blockage was found in 14% of casts from internal spermatic artery injection and 100% of the casts from deferential artery injection, showing clearly that segmental or complete infarction of the testis can result from sutures placed through the tunica albuginea. Bellinger et al. (15) in 1989, studying the effects of surgical technique on testicular histology, emphasized the risk of testicular parenchymal injury after suture fixation, particularly with absorbable suture material. At the same time, they found complete circumferential adherence and normal spermatogenesis in 94% and minimal focal tubular atrophy in 23% using dartos-fixed testes. Dixon et al. (16) also evaluated the effect of transparenchymal suture fixation of the testis on testicular histology. Significant inflammatory reactions were observed in all groups of animals with suture fixation, regardless of suture size and material. However, only 5% of the animals in the dartos pouch control group had an inflammatory response. These findings raise concerns about the effect of surgical technique on the future reproductive capabilities of the testis when the transfixation technique is used. Another argument against transfixation orchiopexy is the phenomenon known as sympathetic orchiopathy, which refers to damage to the contralateral testis in cases of unilateral testicular injury or ischemia. The mechanism for this process is autoimmunization, which occurs when breakdown of the blood-testis barrier exposes tubular antigens to the immune system (17). In addition to contralateral testicular damage, autoimmunization can produce antisperm antibodies (18). Since autoimmunization occurs when breakdown of the blood-testis barrier allows for exposure of tubular antigens to the immune system, it does not occur with the TDPO technique. In conclusion, this experimental work has convinced us that dartos pouch orchiopexy, as described here, is the 754 Lotan et al. Orchiopexy technique and testicular damage Vol. 84, No. 3, September 2005

7 method of choice for orchiopexy. Anatomically sound, technically simple, very effective, and avoiding the placement of traumatizing anchoring sutures through the testicle itself, it should replace CTO in the treatment of the undescended testis in children. REFERENCES 1. Lipshultz LI, Caminos-Torres R, Greenspan CS, Snyder PJ. Testicular function after orchiopexy for unilaterally undescended testis. N Engl J Med 1976;295: Puri P, O Donnell B. Semen analysis of patients who had orchidopexy at or after seven years of age. Lancet 1988;2: Janca FC, Jost LK, Evenson DP. Mouse testicular and sperm cell development characterized from birth to adulthood by dual parameter flow cytometry. Biol Reprod 1986;34: Golan R, Weissenberg R, Oschry Y, Shochat L, Lewin LM. Spermatogenesis in the golden hamster during the first spermatogenetic wave: a flow cytometric analysis. Mol Reprod Dev 2000;55: Vigodner M, Lewin LM, Shochat L, Oschry I, Lotan G, Klin B, et al. Evaluation of damage to the testicular cells of golden hamsters caused by experimental cryptorchidism using flow cytometry and confocal microscopy. Int J Androl 2003;26: Cabot H, Nesbit RM. Undescended testis principles and methods of treatment. Arch Surg 1931;22: Petrivalsky J. Zur Behandlung des Leistenhodens. Zentralbl f Chir 1931;58: Koop CE, Minor CL. Observations on undescended testis: the technique of surgical management. Arch Surg 1957;75: Benson CD, Lofti MW. The pouch technique in the surgical correction of cryptorchidism in infants and children. Surgery 1967;62: Ritchey ML, Bloom DA. Modified dartos pouch orchiopexy. Urology 1995;45: De Netto NF, Goldberg HM. A method of orchiopexy. Surg Gynecol Obstet 1964;118: Redman JF. Simplified technique for scrotal pouch orchiopexy. Urol Clin North Am 1990;17: Jarow JP. Intratesticular arterial anatomy. J Androl 1990;11: Smith JA. Biopsy and the testicular artery of the horse. Equine Vet J 1974;6: Bellinger MF, Abromowitz H, Brantley S, Marshall G. Orchiopexy: an experimental study of the effect of surgical technique on testicular histology. J Urol 1989;142: Dixon TK, Ritchey ML, Boykin W, Harper B, Zeidman E, Thompson IM. Transparenchymal suture fixation and testicular histology in a prepubertal rat model. J Urol 1993;149: Wallace DM, Gunter PA, Landon GV, Pugh RC, Hendry WF. Sympathetic orchiopathia an experimental and clinical study. Br J Urol 1982;54: Peters AJ, Coulam CB. Review: sperm antibodies. Am J Reprod Immunol 1991;27: Fertility and Sterility 755