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Objective: To study the mosaic (focal) pattern of spermatogenesis in azoospermic patients.
Materials and Methods: We conducted a cross-sectional study between June 2003 and February 2007 that included 87 non-obstructed azoospermic patients who underwent testicular sperm extraction (TESE) mapping. The extraction of spermatozoa into a culture medium was compared with testicular histology results. Both histopathology and TESE results were reviewed independently and blindly. Positive mosaic (focal) spermatogenesis was considered in positive mapping or positive TESE with a negative histology.
Results: Mosaic pattern was identified in 26.44% (23 patients) of the study sample. There was no difference in mosaic distribution between testes. There was a mosaic pattern in 10 of the 22 (22.22%) right testes tested and in 13 of the 42 (30.95%) left testes (P = 0.071; CI = 0.10209-0.27669). Histopathological background showed a difference in mosaic distribution. Mosaic pattern was identified in association with maturation arrest in 12 (13.79%) patients, testicular atrophy in 3 (3.45%), atrophy and maturation arrest in 1 (1.14%), Sertoli cell in 3 (3.45%), hypospermatogenesis in 2 (2.3%), and mixed histopathology in 2 (3.45%), in which atrophy, maturation arrest, and hypospermatogenesis all were identified in the same histopathological sample (P = 0.055; CI = 0.050-0.059).
Conclusion: Mosaic pattern of spermatogenesis was found in 26.44% of men with non-obstructive azoospermia. Mosaic (focal) spermatogenesis is more often identified in the histological background of maturation arrest. The chance of sperm retrieval is improved by taking biopsies from multiple sites of the testes.
Keywords: TESE, Mosaic TESE, Azoospermic patients, Mosaic pattern of spermatogenesis in azoospermic patients, Mosaic pattern of spermatogenesis
Correspondence: Mai Ahmed Banakhar, King Abdul Aziz University Hospital, Surgical Department-Urology, P.O. Box 80215, Jeddah, 21589, Kingdom of Saudi Arabia (KSA),
To Cite this Article: Banakhar MA, Farsi HA, Jamil ST. Azoospermic Patients: Mosaic Pattern in Testicular Sperm Extraction. UIJ. In Press. doi:10.3834/uij.1944-5784.2008.12.08
Advanced techniques of assisted reproduction have revolutionized treatment for male infertility. In particular, the introduction of intracytoplasmic sperm injection (ICSI) in combination with in vitro fertilization (IVF) has offered, for the first time, therapy with a high chance of producing a pregnancy from an azoospermic patient [1,2,3,4]. In addition, a number of novel techniques for spermatozoa retrieval have been introduced, including microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), and testicular spermatozoa extraction (TESE) [3,5,6,7,8,9]. However, several reports have indicated that TESE can also be applied in cases of serious testicular damage (including multifocal atrophy of seminiferous tubules) or disturbance of spermatid differentiation [10,11,12,13,14,15,16,17,18].
Pathological spermatogenesis might be focal, which underlines the need for multiple TESE biopsies. Only a few studies have investigated this claim under controlled circumstances [19,20,21,22]. In our study, we looked for evidence to support the hypothesis of focal spermatogenesis in non-obstructive azoospermia.
Materials and Methodology
A cross-sectional study was conducted between June 2003 and February 2007 and included all azoospermic patients (confirmed by minimum of 2 semen analyses) presenting with primary infertility and undergoing TESE (testicular mapping). Inclusion criteria included non-obstructive azoospermic patients, while obstructive patients were excluded. The experimental protocol was approved by the local ethics committee.
TESE was performed under local anesthesia (both spermatic and scrotal). A transverse incision was done through the scrotum and dartous until the tunica was identified. A small incision was performed to obtain testicular tissue. The specimen was then sent to the embryologist for examination. If the embryologist did not identify any sperm, the surgeon proceeded with testicular mapping. The surgeon took 3-7 testicular biopsies (mean = 5) randomly before moving to the contralateral testis. If still no sperm were found, the TESE was considered negative. If sperm were identified, more specimens were taken from the positive site for cryopreservation for further ICSI. Concomitantly, 1 specimen was sent for histopathology evaluation for all patients.
Examination of the TESE by the embryologist
The testicular tissue was immediately immersed into 1.0 mL of Earle’s medium (gassed overnight; supplemented with 4 mM of sodium bicarbonate, 21 mM of HEPES, 0.47 mM of pyruvate, and 10% vol/vol synthetic serum substitute), placed in sterile tube, and transferred to the andrology laboratory of the IVF unit. It was then transferred to a sterile, 2.5-cm Petri dish (Falcon 3002, Becton Dickinson and Co., Inc., Franklin Lakes, NJ) and maintained at 37°C.
Under a dissecting microscope, the seminiferous tubules were teased apart using 21-gauge needles. Using sterile slides, the contents were squeezed into the surrounding media and examined under a dissecting microscope for the presence or absence of mature sperm.
Processing for cryopreservation of testicular tissue
The tubules were transferred to a 15-mL conical tube containing 1 mL of fresh media, and the cell suspension was transferred to a separate centrifuge tube. Both tubes were incubated at 37°C for 15–30 minutes, and the supernatant of the first tube (containing tubules) was combined with the cell suspension in the second tube. The suspension was centrifuged at 500× g for 5 minutes, and the pellet was resuspended in 1 mL of media with 0.3% BSA. A cell count was performed, and the suspension was diluted or concentrated to 0.5–1.0 million sperm/mL. Before freezing, an aliquot was removed to assess sperm quality.
At cryopreservation, multiple aliquots of sperm were frozen whenever possible. The cell suspension was slowly diluted 1:1 with sperm freezing media supplemented with glycerol. The samples were slow cooled at a rate of −0.5°C/min to 4°C and then packaged in 1-mL cryovials (Nunc Cryotubes, Roskilde, Denmark). The vials were frozen at a rate of −10°C/min to −90°C and were stored in liquid nitrogen at −196°C.
Pieces of testicular biopsy were fixed immediately in Bouin’s solution. Semi-thin paraffin wax sections (4 micron) were stained and examined by light microscopy at x400 magnification using standard techniques. The slides were read by 3 assessors unaware of the results of the TESE. Testicular histology was classified into hypospermatogenesis (reduction in the degree of normal spermatogenic cells), maturation arrest (an absence of the later stages of spermatogenesis), Sertoli cell only (the absence of germ cells in the seminiferous tubules), atrophy, obstructed (normal spermatogenesis), or mixed (more than 1 histopathology type).
Both histopathology and TESE results were reviewed independently and blindly for the presence of spermatozoa. Next, results were recorded and reviewed by the investigator for the constructed mosaic (focal) spermatogenesis criteria.Positive mosaic spermatogenesis was considered if there was positive mapping (i.e. moving from a negative to a positive spot in the testis) or positive TESE with a negative histology.
We used the SPSS computer program (v.15 for Windows) for data analysis. Values are presented as percentages and P values with 95% confidence intervals. A P value of < 0.05 was judged as statistically significant.
A total of 87 patients with mean age of 31 years and a history of primary infertility for an average of 2-13 years were included in the study. Mosaic pattern of spermatogenesis was identified in 26.44% (23) of patients. There was no difference in mosaic distribution between testes. Mosaic pattern was present in 10 of the 45 (22.22%) right testes tested and in 13 of the 42 (30.95%) left testes (P = 0.071; CI = 0.10209-0.27669). Histopathological background showed a difference in mosaic distribution. Mosaic pattern was identified in association with maturation arrest in 12 (13.79%) patients, testicular atrophy in 3 (3.45%), atrophy and maturation arrest in 1 (1.14%), Sertoli cell in 3 (3.45%), hypospermatogenesis in 2 (2.30%), and 2 (3.45%) patients had mixed histopathology in which atrophy, maturation arrest, and hypospermatogenesis all were identified in the same histopathological sample (P = 0.055; CI = 0.050-0.059). The results are shown in Table 1, Table 2, Table 3, and Figure 1.
It is well established that mature testicular spermatozoa can be found in cases of non-obstructive azoospermic men [23,24]. Moreover, it has also been proven that such sperm has fertilization ability after the intracytoplasmic sperm injection (ICSI) procedure [12,25,26,27,28]. These findings made the achievement of genetic offspring possible for a population of men who were previously advised to use donor spermatozoa. As a result, testicular sperm retrieval procedures in non-obstructive azoospermic men are becoming increasingly popular.
At present, there are no means of predicting the presence of mature spermatozoa in non-obstructed azoospermic men except by performing testicular biopsy [23,24,29].
In fertile men, sperm cells are produced throughout the testis. Steinberger and Tjioe  and Roosen-Range  stated that a single large sample is representative of the entire testis. Levin  examined the testes of infertile men and found mixed histological patterns of germinal cell aplasia and focal spermatogenesis in 6% of patients. A similar histology of side-by-side presence of different patterns was observed in non-obstructed azoospermic men [25,26,33,34,35,36,37,38,39]. The incidence of such mosaic pattern of spermatogenesis has not been determined previously. However, our study showed an incidence of 26.4% for the mosaic pattern of spermatogenesis in azoospermic patients.
There was no difference in the pattern of spermatogenesis between the right and left testes (P = 0.071), but different testis histology showed an effect on the pattern of spermatogenesis. We found the maturation arrest to have the highest incidence (13.79%) of mosaic pattern in comparison to the other histology (Table 3, Figure 1).
Mosaic pattern of spermatogenesis was found in 26.44% of men with non-obstructive azoospermia. Mosaic (focal) spermatogenesis is most often identified in the histological background of maturation arrest. The chance of sperm retrieval is improved by taking biopsies from multiple sites of the testes.
- Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992 Jul 4;340(8810):17-8.
PubMed - CrossRef
- Redgment CJ, Yang D, Tsirigotis M, Yazdani N, al Shawaf T, Craft IL. Experience with assisted fertilization in severe male factor infertility and unexplained failed fertilization in vitro. Hum Reprod. 1994 Apr;9(4):680-3.
- Tsirigotis M, Pelekanos M, Yazdani N, Boulos A, Foster C, Craft IL. Simplified sperm retrieval and intracytoplasmic sperm injection in patients with azoospermia. Br J Urol. 1995 Dec;76(6):765-8.
- Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, Wisanto A, Devroey P. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod. 1993 Jul;8(7):1061-6.
- Craft I, Tsirigotis M, Bennett V, Taranissi M, Khalifa Y, Hogewind G, Nicholson N. Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia. Fertil Steril. 1995 May;63(5):1038-42. PubMed
- Schoysman R, Vanderzwalmen P, Nijs M, Segal L, Segal-Bertin G, Geerts L, van Roosendaal E, Schoysman D. Pregnancy after fertilisation with human testicular spermatozoa. Lancet. 1993 Nov 13;342(8881):1237.
PubMed - CrossRef
- Schoysman R, Vanderzwalmen P, Nijs M, Segal-Bertin G, van de Casseye M. Successful fertilization by testicular spermatozoa in an in-vitro fertilization programme. Hum Reprod. 1993 Aug;8(8):1339-40.
- Silber SJ, Asch RH. Epididymal surgery. Infertility. 1992;133-53.
- Silber SJ, Nagy ZP, Liu J, Godoy H, Devroey P, Van Steirteghem AC. Conventional in-vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration. Hum Reprod. 1994 Sep;9(9):1705-9.
- Fishel S, Green S, Bishop M, Thornton S, Hunter A, Fleming S, al-Hassan S. Pregnancy after intracytoplasmic injection of spermatid. Lancet. 1995 Jun 24;345(8965):1641-2.
PubMed - CrossRef
- Gil-Salom M, Minguez Y, Rubio C, Remohi J, Pellicer A. Intracytoplasmic testicular sperm injection: an effective treatment for otherwise intractable obstructive azoospermia. J Urol. 1995 Dec;154(6):2074-7.
PubMed - CrossRef
- Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum Reprod. 1995 Jan;10(1):148-52.
- Tournaye H, Liu J, Nagy PZ, Camus M, Goossens A, Silber S, Van Steirteghem AC, Devroey P. Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa. Hum Reprod. 1996 Jan;11(1):127-32.
- Yemini M, Vanderzwalmen P, Mukaida T, Schoengold S, Birkenfeld A. Intracytoplasmic sperm injection, fertilization, and embryo transfer after retrieval of spermatozoa by testicular biopsy from an azoospermic male with testicular tubular atrophy. Fertil Steril. 1995 May;63(5):1118-20.
- Craft I, Tsirigotis M. Simplified recovery, preparation and cryopreservation of testicular spermatozoa. Hum Reprod. 1995 Jul;10(7):1623-6.
- Haimov-Kochman R, Lossos F, Nefesh I, Zentner BS, Moz Y, Prus D, Bdolah Y, Hurwitz A. The value of repeat testicular sperm retrieval in azoospermic men. Fertil Steril. 2008 Aug 1.
- Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia? A systematic review. Hum Reprod Update. 2007 Nov-Dec;13(6):539-49. Epub 2007 Sep 24. Review.
PubMed - CrossRef
- Hauser R, Yogev L, Paz G, Yavetz H, Azem F, Lessing JB, Botchan A. Comparison of efficacy of two techniques for testicular sperm retrieval in nonobstructive azoospermia: multifocal testicular sperm extraction versus multifocal testicular sperm aspiration. J Androl. 2006 Jan-Feb;27(1):28-33.
PubMed - CrossRef
- Friedler S, Raziel A, Strassburger D, Soffer Y, Komarovsky D, Ron-El R. Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum Reprod. 1997 Jul;12(7):1488-93.
PubMed - CrossRef
- Turek PJ, Cha I, Ljung BM. Systematic fine-needle aspiration of the testis: correlation to biopsy and results of organ “mapping” for mature sperm in azoospermic men. Urology. 1997 May;49(5):743-8.
PubMed - CrossRef
- Schulze W, Thoms F, Knuth UA. Testicular sperm extraction: comprehensive analysis with simultaneously performed histology in 1418 biopsies from 766 subfertile men. Hum Reprod. 1999 Sep;14 Suppl 1:82-96.
- Tsujimura A, Miyagawa Y, Takao T, Takada S, Koga M, Takeyama M, Matsumiya K, Fujioka H, Okuyama A. Salvage microdissection testicular sperm extraction after failed conventional testicular sperm extraction in patients with nonobstructive azoospermia. J Urol. 2006 Apr;175(4):1446-9; discussion 1449.
PubMed - CrossRef
- Hauser R, Temple-Smith PD, Southwick GJ, de Kretser D. Fertility in cases of hypergonadotropic azoospermia. Fertil Steril. 1995 Mar;63(3):631-6.
- Tournaye H, Camus M, Goossens A, Liu J, Nagy P, Silber S, Van Steirteghem AC, Devroey P. Recent concepts in the management of infertility because of non-obstructive azoospermia. Hum Reprod. 1995 Oct;10 Suppl 1:115-9.
- Devroey P, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, Van Steirteghem A, Silber S. Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod. 1995 Jun;10(6):1457-60.
- Gil-Salom M, Mínguez Y, Rubio C, De los Santos MJ, Remohí J, Pellicer A. Efficacy of intracytoplasmic sperm injection using testicular spermatozoa. Hum Reprod. 1995 Dec;10(12):3166-70.
- Kahraman S, Ozgür S, Alatas C, Aksoy S, Balaban B, Evrenkaya T, Nuhoglu A, Tasdemir M, Biberoglu K, Schoysman R, Vanderzwalmen P, Nijs M. High implantation and pregnancy rates with testicular sperm extraction and intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia. Hum Reprod. 1996 Mar;11(3):673-6.
- Mansour RT, Aboulghar MA, Serour GI, Fahmi I, Ramzy AM, Amin Y. Intracytoplasmic sperm injection using microsurgically retrieved epididymal and testicular sperm. Fertil Steril. 1996 Mar;65(3):566-72.
- Oates RD, Mulhall J, Burgess C, Cunningham D, Carson R. Fertilization and pregnancy using intentionally cryopreserved testicular tissue as the sperm source for intracytoplasmic sperm injection in 10 men with non-obstructive azoospermia. Hum Reprod. 1997 Apr;12(4):734-9.
PubMed - CrossRef
- Steinberger E, Tjioe DY. A method for quantitative analysis of human seminiferous epithelium. Fertil Steril. 1968 Nov-Dec;19(6):959-61.
- Roosen-Runge EC. Quantitative investigations on human testicular biopsies. I. Normal testes. Fertil Steril. 1956 May-Jun;7(3):251-61.
- Levin HS. Testicular biopsy in the study of male infertility: its current usefulness, histologic techniques, and prospects for the future. Hum Pathol. 1979 Sep;10(5):569-84.
- Giwercman A, Skakkebaek NE. The role of testicular biopsy in the evaluation of male infertility. In: Burger HG, Oshima H, eds. An Approach to Clinical Andrology. Rome, Italy: Serono Symposia Review; 1993:67-75.
- Haimov-Kochman R, Imbar T, Lossos F, Nefesh I, Zentner BS, Moz Y, Prus D, Bdolah Y, Hurwitz A. Technical modification of testicular sperm extraction expedites testicular sperm retrieval. Fertil Steril. 2008 Feb 2.
- Silber SJ, Nagy Z, Devroey P, Tournaye H, Van Steirteghem AC. Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure. Hum Reprod. 1997 Nov;12(11):2422-8. Erratum in: Hum Reprod 1998 Mar;13(3):780.
PubMed - CrossRef
- Vernaeve V, Verheyen G, Goossens A, Van Steirteghem A, Devroey P, Tournaye H. How successful is repeat testicular sperm extraction in patients with azoospermia? Hum Reprod. 2006 Jun;21(6):1551-4. Epub 2006 Feb 10.
PubMed - CrossRef