Sperm retrieval techniques in rats with suppressed spermatogenesis by experimental cryptorchidism

Lia Mara Rossi1,6, Luís A.V. Pereira2, Luciana de Santis2, Fábio F. Pasqualotto3, Assumpto Iaconelli, Jr4, Valdemar Ortiz5 and Edson Borges, Jr4

1 Medical School at Jundiaí, and Fertility Center, 2 Universidade Estadual de Campinas, 4 Fertility Center and 5 Department of Urology, Universidade Federal São Paulo, SP and 3 Department of Embriology, Universidade de Caxias do Sul, RS, Brazil

6 To whom correspondence should be adressed at: Fertility Center for Assisted Fertilization, Avenida Brigadeiro Luís Antônio, 4.251, São Paulo, SP, Brazil. Email: lia{at}fertility.com.br


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
OBJECTIVE: Our aim was to assess the suppression of spermatogenesis and sperm retrieval rate after testicular sperm extraction (TESE) or testicular sperm aspiration (TESA) in adult rats with surgically induced cryptorchidism. METHODS: Adult rats were submitted to TESE and TESA procedures after 15 days of induced cryptorchidism. After spermatozoa retrieval, the testicles were extracted, weighed and a morphological analysis by conventional light microscopy was done. The numbers of spermatozoa retrieved in both TESA and TESE were rated and compared. RESULTS: Histological analysis of the testicles revealed Sertoli cell-only syndrome in 60% of the testicles, and maturation arrest in the remaining cryptorchid testicles. Significant differences were seen in the number of spermatozoa retrieved (P < 0.05) between cryptorchidic and control rats. When sperm retrieval techniques were compared, no differences were detected in the number of spermatozoa obtained (P > 0.05). CONCLUSIONS: It seems that a 15 day period of cryptorchidism is enough to induce spermatogenesis disorders. No differences were detected in the number of spermatozoa retrieved in the right or left testicles, irrespective of the testicular pole. Furthermore, and even more importantly, no differences in the retrieval rate were seen between the two techniques.

Key words: aspiration/cryptorchidism/extraction/sperm/testicle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Since the first report using the ICSI procedure in patients with severe oligoasthenoteratozoospermia and the first pregnancies obtained by this method, the management of the azoospermic infertile patient has changed. (Palermo et al., 1992Go; Van Steirteghem et al., 1993Go). In fact, this new therapy which can involve surgical retrieval of spermatozoa combined with assisted reproduction has given new hope to those patients previously considered infertile (Silber et al., 1988Go; Schoysman et al., 1993Go; Devroey et al., 1995Go; Mansour et al., 1997Go; Gil-Salom et al., 1998Go; Madgar et al., 1998Go; Palermo et al., 1999Go; Tournaye, 1999Go; Pasqualotto et al., 2002Go).

The utilization of spermatozoa extracted from the testis for the ICSI procedure in men with obstructive azoospermia allowed the achievement of high pregnancy rates following ICSI (Craft et al., 1993Go). Additionally, within a short period of time, testicular sperm extraction (TESE) was expanded to include patients with non-obstructive azoospermia (Turek et al., 1997Go; Ezeh et al., 1998Go; Schlegel, 1999Go; De Croo et al., 2000Go; Silber, 2000Go). Since the size of the testis bears a direct correlation to testicular function, TESE involves one or typically more biopsies of the testis, especially in these patients (Takihara et al., 1987Go; Ezeh et al., 1998Go; Hauser et al., 1998Go; Ostad et al., 1998Go; Schulze et al., 1999Go). However, testicular open biopsies may have considerable side effects, including haematomas, inflammation and even permanent devascularization of the testis resulting in testicular atrophy (Schlegel et al., 1997; Amer et al., 2000Go). Despite potential complications, sperm retrieval is successful in 30–70% of non-obstructive azoospermic patients (Ezeh et al., 1999Go; Schulze et al., 1999Go). The theoretical basis for attempting the retrieval of spermatozoa from the testis of men with apparent absence of spermatogenesis was based on quantitative histological studies from testicular biopsies in fertile and infertile men (Silber and Rodriguez-Rigau, 1981Go). Some men with non-obstructive azoospermia have focal areas of spermatogenesis within the testis despite the fact that global spermatogenic function of the testis is severely impaired. It thus appears that a certain minimum threshold of spermatogenesis is needed in order for spermatozoa to be able to reach the ejaculate.

Substantial data have been published on fine-needle aspiration techniques in patients with non-obstructive azoospermia. These authors suggest that there is spermatogenesis heterogeneity within the failing atrophic testis. Indeed, ‘patches’ of sperm production may be localized within the testis with this technique. Therefore, the testicular sperm aspiration (TESA) approach may enable one to reach more testicular sites, without causing extensive testicular damage and consequently having fewer side effects (Turek et al., 1997Go).

On the other hand, percutaneous biopsy with a 19 gauge butterfly needle is a quick and reliable method for obtaining spermatozoa for ICSI. However, for a detailed histopathological diagnosis, the needle biopsies gave poor results, whereas the material from the open testicular biopsies was assessable (Rosenlund et al., 1998Go). In addition, testicular needle biopsy using a 14 gauge biopsy needle gave a sufficient amount of tissue and spermatozoa for ICSI, cryopreservation and histology, even in non-obstructive azoospermia (Tuuri et al., 1998Go; Rosenlund et al., 2001Go). This technique is simpler and cheaper than open biopsy, it can be done in the office setting without general anaesthesia and, hence, it can be considered the optimal method for the retrieval of testicular spermatozoa. Ultrasound-guided TESA is also a safe and accurate method for sperm retrieval in azoospermic patients (Belenky et al., 2001Go).

Several investigators have described degenerative changes in the undescended testis in cryptorchid rats, implying that this kind of animal model might be valuable to study the degenerative changes (Agarwala and Mitra, 1996Go; Kogan et al., 1987Go; Srinivas et al., 1998Go; Zakaria et al., 1998Go; Altay et al., 2000Go). In order to assess histological features and to compare sperm retrieval results obtained through TESA or TESE, we surgically induced cryptorchidism in rats to promote spermatogenic damage.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
The study was approved by the Universidade Estadual de Campinas, according to the Declaration of Helsinki (protocol number 333/2, CEEA-IB-UNICAMP). Forty male adult rats (Wistar) were operated on according to the group assigned.

All rats received similar amounts of commercial food at a scheduled time every day. Water was available as necessary throughout the study period. The rats were housed in cages in the animal laboratory at the Universidade Estadual de Campinas, which has central air-conditioning. To simulate natural conditions, 12 h light and dark cycles were maintained in the laboratory.

The rats were subjected to anaesthesia with ether at the time of surgery. All instruments were sterilized in 2% glutaraldehyde. After positioning the rat on the board, the ventral surface was prepped with povidone iodine and a transverse lower abdominal incision was made. The incision was then closed in layers with 6-0 polyglactin and the wound cleaned with povidone iodine.

In a pilot study, eight adult male rats underwent a bilateral induced cryptorchidism to induce spermatogenesis failure. Testicular evaluation was done at different time intervals (two rats evaluated after 7 days of cryptorchidism; two rats, 10 days; two rats, 15 days; and two rats, 30 days). Gubernaculectomy was done and the gubernaculum was anchored to the anterior abdominal wall with 6-0 polypropylene. Testes from all animals at each time interval were extracted, weighed and a morphological analysis was done to show the efficacy of the induced cryptorchidism technique. Biopsy tissue was placed in Bouin's solution and submitted to histological analysis. Histological analysis revealed a normal pattern in the 7 and 10 day period of cryptorchidism. On the other hand, histological features of testicular damage were noted in testes submitted to cryptorchidism for the 15 day period as well as the 30 day period. "All of the 50 testis from the experimental group submitted to 15-day period of cryptorchidism reported impairment in the spermatogenesis process [60%-30 testicles—showing only Sertoli cell and 40%-20 testicles—showing maturation arrest (arrest at the primary spermatocytes stage)] (Figure 3B). Normal histology was noted in all testicles of the control group (Figure 3A). From the remaining animals 25 underwent bilateral induced cryptorchidism and 7 were used as controls (sham operated). Tissue samples were otained from both testis in the control and experimental group, 15 days after surgical intervention. All thirty-two right cryptorchid testicles (experimental and control groups) underwent TESA (performed with a 16G needle) as the first procedure, with only two points of needle entrance, but searching for spermatozoa in all testis extension. Afterwards, TESE was done in the 3 different poles of the testicles (superior, middle, and inferior) (Figure 1). Regarding all 32 left testicles, the procedure of sperm aspiration was different. We performed the aspiration in the three different poles (superior, middle, and inferior) of the testicles. After the sperm aspiration procedure was done, TESE was performed in the same way that was described for the right testis (Figure 2)."



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Figure 3. (a) Photomicrograph showing seminiferous tubules from control group testes. The small cells at the base of the seminiferous epithelium are spermatogonia. The cells with the largest nuclei are pachytene spermatocytes. There are spermatids (round and elongating) about to be released into the lumen as well as spermatozoa. Haematoxylin and eosin, 700x. (b) Photomicrograph showing seminiferous tubules from experimental group testes. Histological analysis of testes to confirm the cryptorchidic condition. Seminiferous tubules are shown only as small cells at the base of the epithelium. Haematoxylin and eosin, 350x.

 


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Figure 1. All right testes underwent first a TESA procedure, with only two points of needle entrance but searching for spermatozoa in all testis extensions, and afterwards TESE in three different poles (superior, middle and inferior).

 


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Figure 2. All left testes underwent first a TESA searching for spermatozoa in three different poles (superior, middle and inferior) and afterwards, TESE in a similar way as described for the right testis.

 
The testicular parenchyma (~2 mm in diameter) was excised with sharp, curved iris scissors and placed in human tube fluid (HTF) culture medium. Individual tubules are isolated with two sterile glass slides. Additional mechanical dispersal was performed by mincing the tissue with sterile scissors in the HTF medium and sequential passes of tissue suspension through a 24 gauge angiocatheter were performed. Wet preparation of this suspension is immediately examined under the phase contrast microscope at 100x and 400x power.

Once the procedures of sperm retrieval were finished, both testes from all animals were extracted, weighed and a morphological analysis was done to demonstrate the induced histological features. Biopsy tissue was placed in Bouin's solution and all material obtained was processed for histological analysis.

Comparisons between TESA and TESE were performed according to the number of spermatozoa retrieved from the testes. For this, we created four different scores according to the number of sperm observed under the light microscope: 0 (no sperm), 1 (few sperm, 1–3 x106 sperm/ml), 2 (fairly good, 3–15 x 106 sperm/ml) and 3 (good, >15 x 106 sperm/ml).

Statistical analysis
Statistical analysis of variance (ANOVA), Student's t-test and {chi}2 analysis were used to analyse the differences between groups. Values were considered significantly different when P<0.05 with the use of a two-tailed test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
There was a decrease in testis weight up to 15 days following the induction of cryptorchidism. The left and right testis of the cryptorchid rats demonstrated significantly lower weight compared with the controls (Table I).


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Table I. Weight and comparison between the testis after 15 days of cryptorchidism

 
We found significant differences in the number of sperm retreived between the right cryptorchid testis and its controls. The same was found in the left cryptorchid testis when compared with controls. No differences were seen in scores between the cryptorchidic groups, as well as between the control groups, irrespective of the sperm retrieval technique employed (Table II).


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Table II. Scores and comparison between the testis after 15 days of cryptorchidism irrespective of sperm retrieval technique

 
When we compared the scores found in the left and right testes considering the TESA technique only, no differences were seen. Also, there were no differences in the scores obtained from the left and right testes with the TESE technique (Tables III and IV).


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Table III. Scores obtained with TESA in the left and right testicles in the experimental and control groups

 

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Table IV. Scores obtained with TESE in the left and right testicles in the experimental and control groups

 
No difference in number of sperm retrieved was found when left and right testes of cryptorchid rats were submitted to TESA or TESE techniques. The same applied to the control groups (Table V).


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Table V. Scores between different methods of sperm retrieval in right and left experimental testicles

 
No differences were found when employing TESA across the three poles of cryptorchid testes in left testicles and TESE in left and right testicles (Table VI). Furthermore, no differences were found across the three poles of both testicles of control rats when employing TESE bilaterally and TESA in the left testis (Table VII).


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Table VI. Mean scores between the different poles in the cryptorchid testicles in the experimental group

 

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Table VII. Mean scores between the different poles in the cryptorchid testicles in the control group

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
IVF was developed originally for patients with tubal infertility (Palermo et al., 1992Go). However, ICSI has allowed some of these couples to have genetic offspring using a small number of spermatozoa in the testicles (Mansour et al., 1997Go; Gil-Salom et al., 1998Go; Ezeh et al., 1999Go; Lewin et al., 1999Go; Palermo et al., 1999Go; Tournaye, 1999Go; Ubaldi et al., 1999Go). In order to assess the suppression of spermatogenesis and to compare sperm retrieval results obtained by fine-needle TESA or TESE, cryptorchidism was surgically performed in rats to induce spermatogenesis failure, and the retrieval procedures were performed afterwards. We believe that, despite the differences between humans and rodents, we could extrapolate our findings to our clinical practice.

In our study, this model for cryptorchidism induction resulted in Sertoli cell-only syndrome or maturation arrest after a 15 day period. Therefore, histological study of the testis revealed that cryptorchidism induced by surgery in adult rats could cause failure in the spermatogenesis process. Also, there was a decrease in the weight of the testis following cryptorchidism. The weight of the left testis in the cryptorchid rats was significantly lower compared with both testes of control rats.

There are publications in the literature discussing the best way to retrieve sperm in patients with obstructive and non-obstructive azoospermia (Friedler et al., 1997; Tournaye et al., 1998Go; Mercan et al., 2000Go). To assess whether TESE results in larger amounts of sperm retrieved than TESA techniques in testicles with failure of spermatogenesis, we created four different scores based on the number of sperm retrieved. The results of the present study showed no differences in the scores when sperm retrieval techniques were compared. Since the amounts of sperm retrieved in both procedures were comparable in our study and, based on the fact that TESA is an office-based technique with few associated side effects and complications, this procedure might be elected as the first choice instead of TESE, which is not an office-based procedure and has possible complications such as bleeding and infection. This study therefore supports TESA as a sperm retrieval technique to be used in patients with spermatogenesis failure. However, further studies with a larger number of testicles analysed are needed to confirm our results.

It is important to emphasize that no differences were found in the number of spermatozoa retrieved across the three different regions of the left testicles, irrespective of the type of sperm retrieval technique. The evaluation of the three different regions has concluded that induction of defective spermatogenesis through cryptorchidism, at least in a rat model, had effects in the entire testicle. This result suggests that incomplete testicular failure, at least surgically induced cryptorchidism, may involve a sparse multi-focal distribution of spermatogenesis throughout the entire testicle, rather than a regional distribution. Our results are in accordance with others that show that patients with non-obstructive azoospermia have small foci of spermatogenesis in the testis although remaining azoospermic over all (Silber et al., 1997Go).


    Conclusion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
We conclude from our study that 15 days of cryptorchidism are enough to induce spermatogenesis disorders as seen in 60% of the testicles presenting Sertoli cell-only syndrome and 40% presenting maturation arrest (early maturation arrest). No differences were seen in the scores obtained in the right and left testis, irrespective of testicular pole. Furthermore, and even more importantly, no differences in the retrieval rate were seen between TESA and TESE.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
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Submitted on April 21, 2004; resubmitted on July 13, 2004; accepted on September 10, 2004.





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