Prepubertal testis hücre süspansiyonun dondurulmasında seeding etkisi

Gülnaz Kervancıoğlu; Gülnaz Kervancıoğlu; Şule Çetinel; Şule Çetinel; Elif Kervancıoğlu Demirci; Elif Kervancıoğlu Demirci; Gülçin Ekter Kanter; Gülçin Ekter Kanter; Ertan Kervancıoğlu; M. Ertan Kervancıoğlu

doi:10.5472/MMJ.2015.03894.1

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Year 2015, Volume: 28 Issue: 1, 13 - 20, 02.07.2015

Gülnaz Kervancıoğlu Gülnaz Kervancıoğlu Şule Çetinel Şule Çetinel Elif Kervancıoğlu Demirci Elif Kervancıoğlu Demirci Gülçin Ekter Kanter Gülçin Ekter Kanter Ertan Kervancıoğlu M. Ertan Kervancıoğlu

https://doi.org/10.5472/MMJ.2015.03894.1

Abstract

Objective: The cryopreservation of prepubertal testicular tissue promises to preserve fertility in boys under cancer treatment. The effects of a seeding process was investigated using different carriers for prepubertal testicular cell suspensions in order to develop an appropriate method for cryopreservation. Materials and Methods: Testicular cell suspensions of 7-8 day old male Wistar rats were frozen by a programmed slow freezing method. The straw and cryotube selected as carriers underwent a seeding process. The morphological effects on spermatogonial stem cells and Sertoli cells were evaluated semi-quantitatively by light microscopy and qualitatively with a transmission electron microscope. Results: The rate of vitality with or without seeding in straw was respectively, 90.6% (±3.0), 89.4 (±1.3) and in cryotube 77.6 (±4.2) and 85.4 (±2.7). The seeding process, showed no significant difference between the straw groups, but a significant difference was seen between the cryotube groups (p<0.0001). The nuclei, cytoplasmic structures and membranes of Sertoli and Conclusion: In this study, we compared two carriers to observe the effect of seeding during cryopreservation of prepubertal testicular cell suspensions and found that seeding in straw was the preferable

Keywords

Prepubertal testis, Testicular cell suspension, Programmed slow freezing, Spermatogonial stem cell, Seeding process

References

1. Orwig KE, Schlatt S. Cryopreservation and transplantation of spermatogonia and testicular tissue for preservation of male fertility. J Natl Cancer Inst Monogr 2005;34:51-6. doi:10.1093/jncimonographs/lgi029
2. Schlatt S, Ehmcke J, Jahnukainen K. Testicular stem cells for fertility preservation: Preclinical studies on male germ cell transplantation and testicular grafting. Pediatr Blood Cancer 2009;53:274-80. doi:10.1002/pbc.22002
3. Goossens E, Tournaye H. Is there a clinical future for spermatogonial stem cells? Curr Stem Cell Res Ther 2007;2:189-95. doi:10.2174/157488807781696258
4. Wyns C, Curaba M, Petit S, et al. Management of fertility preservation in prepubertal patients: 5 years’ experience at the Catholic University of Louvain. Hum Reprod 2011;26:737- 47. doi: 10.1093/humrep/deq387.
5. Wyns C, Langendonckt AV,Wese F-X, Donnez J, Curaba M. Long-term spermatogonial survival cryopreserved and xenografted immature human testicular tissue. Hum Reprod 2008;23:2402-14. doi:10.1093/humrep/den272.
6. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Ogura A, Toyokuni S, Shinohara T. Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stemcells. Hum Reprod 2003;18:2660-67. doi:10.1093/ humrep/deg483
7. Avarbock MR, Brinster CJ, Brinster RL. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat Med 1996;2:693-6. doi:10.1038/nm0696-693
8. Wyns C, Curaba M, Martinez-Madrid B, Langendonckt AV, François-Xavier W, Donnez J. Spermatogonial survival after cryopresrvation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice. Hum Reprod 2007;22:1603-11. doi: 10.1093/humrep/dem062
9. Jezek D, Schulze W, Kalanj-Bognar S, Vukelic Z, MilavecPuretic. V, Krhen I. Effects of various cryopreservation media and freezing thawing on the morphology of rat testicular biopsies. Andrologia 2001;33:368-78. doi: 10.1046/j.1439- 0272.2001.00459.x
10. Keros V, Rosenlund B, Hultenby K, Aghajanova L, Levkov L, Hovatta O. Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants. Hum Reprod 2005;20:1676-87. doi: 10.1093/humrep/deh797
11. Keros V, Hultenby K, Borgström B, Fridström M, Jahnukainen K, Hovatta O. Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys under-going gonadotoxic cancer treatment. Hum Reprod 2007;22:1384-95. doi: 10.1093/humrep/del508
12. Milazzo JP, Vaudreuil L, Cauliez B, et al. Comparison of conditions for cryopreservation of testicular tissue from immature mice. Hum Reprod 2008;23:17-28. doi: 10.1093/ humrep/dem355
13. Izadyar F, Matthijs-Rijsenbilt JJ, den Ouden K, Creemers LB, Woelders H, de Rooij DG. Development of a cryopreservation protocol for type a spermatogonia. J Androl 2002;23:537-45. doi: 10.1002/j.1939-4640.2002.tb02276.x
14. Schlatt S, von Schonfeldt V, Schepers AG. Male germ cell transplantation: an experimen-tal approach with a clinical perspective. Br Med Bull 2000;56:824-36. doi: 10.1258/0007142001903409
15. Brockbank KGM, Song YC, Khirabadi BS, Lightfoot FG, Boggs JM, Taylor MJ. Storage of tissues by vitrification. Transpl Proc 2000;32:3-4. doi: 10.1016/S0041- 1345(99)00851-9
16. Mazur P, Leibo SP, Chu EHY. A two-factor hypothesis of freezing injury evidence from Chinese hamster tissue culture cells. Exp Cell Res 1972;71:345-55. doi: 10.1016/0014- 4827(72)90303-5
17. Mazur P, Rall WF, Leibo SP. Kinetics of water loss and the likelihood of intracellular freezing in mouse ova. Influence of the method of calculating the temperature dependence of water permeability. Cell Biophys 1984;6:197-213. doi: 10.1007/BF02788619
18. Delilbaşı L. In Vitro Fertilizasyon (IVF) Laboratuvar Yöntemleri (Yeni uygulamalar ve güncel yaklaşımlar). Ankara: Güneş Kitabevleri, 2008:61-83. ISBN: 9789752771697
19. Morris GJ, Acton E. Controlled ice nucleation in cryopreservation. Cryobiology 2013; 66:85-92. doi: 10.1016/j. cryobiol.2012.11.007
20. Russell LD, Ettlin RA, Hikim APS, Clegg ED, (editors). Histopathological evaluation of the testis. 1st Edition. Clearwater: Cache River Press, 1990:1-58.
21. Gouk S S, Loh YFJ, Kumar S D, Watson PF, Kuleshova LL. Cryopreservation of mouse testicular tissue: prospect for harvesting spermatogonial stem cells for fertility preservation. Fertil Steril 2011; 95: 2399-2403. doi: 10.1016/j.fertnstert. 2011.03.035.
22. Trump BF, Laiho KA, Mergner WJ, Arstila AU. Studies on the subcellular pathophysiology of acute lethal cell injury. Beitr Path Bd 1974;125:243–71. doi:10.1016/S0005-8165(74)80177-0
23. Baert Y, Goossens E , Saen D V, Ning L, Veld P, Tournaye H. Orthotopic grafting of cryopreserved prepubertal testicular tissue: in search of a simple yet effective cryopreservation protocol. Fertil Steril 2012; 97:1152-7. doi: 10.1016/j. fertnstert.2012.02.010.
24. Baert Y, Saen DV, Haentjens P, Veld PI, Tournaye H, Goossens E. What is the best cryopreservation protocol for human testicular tissue banking? Hum Reprod 2013;28:1816- 26. doi: 10.1093/humrep/det100
25. Milazzo JP, Travers A, Bironneau AA, et al. Rapid screening of cryopreservation protocols for murine prepubertal testicular tissue by histology and PCNA immunostaining. J Androl 2010;31: 617-30. doi:10.2164/jandrol.109.009324.
26. Griswold MD. The central role of Sertoli cells in spermatogenesis. Cell Dev Biol 1998;9:411-16. doi: 10.1006/ scdb.1998.0203
27. Nugent D, Meirow D, Brook PF, Y, Gosden RG. Transplantation in reproductive medicine: previous experience, present knowledge and future prospects. Hum Reprod Update 1997;3:267-80. doi: 10.1093/humupd/3.3.267
28. Hossain A, Nagamani M. Erkek üreme hücrelerinin dondurularak saklanması (Çev: Kervancıoğlu G, Kervancıoğlu E). İnfertilite ve Yardımla Üreme Teknikleri. Ankara: Güneş Kitabevi, 2012:466-77. ISBN: 9789752773899
29. Crabbe E, Verheyen G, Tournaye H, Van Steırteghem A. Freezing of testicular tissue as a minced suspension preserves sperm quality better than whole-biopsy freezing when glycerol is used as cryoprotectant. Int J Androl 1999;22:43-8. doi: 10.1046/j.1365-2605.1999.00149.x
30. Nagano R,Tabata S, Nakanishi Y, Ohsako S, Kurohmaru M, Hayashi Y. Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis. Anat Rec 2000;258:210-20. doi: 10.1002/(SICI)1097- 0185(20000201)258:2<210::AID-AR10>3.0.CO;2-X
31. Robbins H, Dores C, Coyle K, Dobrinski I. Germ cells and testicular somatic cells have different sensitivity to cryopreservation. Reprod Fertil Dev 2012;25:184-184. doi: 10.1071/RDv25n1Ab74
32. Bloom W, Fawcett DW. A Textbook of Histology. 11th Edition. Philadelphia: Saunders Company, 1986:796-848. ISBN-10: 0721617298
33. Ross MH, Pawlina W. Histology: A Text and Atlas. 6th edition. Philadelphia PA: Lippincott, Williams and Wilkins, 2011:784-818.
34. Travers A, Milazzo JP, Perdrix A, A, et al. Assessment of freezing procedures for rat immature testicular tissue. Theriogenology 2011;76:981-990. doi: 10.1016/j. theriogenology.2011.04.025
35. Unni S, Kasiviswanathan S, D’Souza S, S, et al. Efficient cryopreservation of testicular tissue: effect of age, sample state and concentration of cryoprotectant. Fertil Steril 2011;97:200- 8. doi: 10.1016/j.fertnstert. 2011.10.018

Prepubertal testis hücre süspansiyonun dondurulmasında seeding etkisi

Year 2015, Volume: 28 Issue: 1, 13 - 20, 02.07.2015

https://doi.org/10.5472/MMJ.2015.03894.1

Abstract

Amaç: Prepubertal testis kriyoprezervasyonu, kanser tedavisi
yapılacak erkek çocuklarda fertilitenin korunması için günümüz
teknolojisinde umut vadetmektedir. Kriyoprezervasyonda en
uygun yöntemin geliştirilmesi için prepubertal testis hücre
süspansiyonunda farklı taşıyıcılar kullanarak seeding etkisi
araştırıldı.
Gereç ve Yöntem: Yedi-sekiz günlük Wistar yavru erkek sıçan
testis hücre süspansiyonları, programlı yavaş dondurma yöntemiyle
donduruldu. Taşıyıcı olarak seçilen yüksek güvenlikli straw ve
kriyotüpe seeding uygulandı. Spermatogonyal kök hücreler ve
Sertoli hücreleri üzerindeki morfolojik etki, semi-kantitatif olarak
ışık, kalitatif olarak geçirimli elektron mikroskobunda incelendi.
Bulgular: Canlılık oranı, yüksek güvenlikli straw’da seeding
uygulanmayan ve uygulanan gruplarda sırasıyla %90,6±3,0,
%89,4±1,3, kriyotüpte %77,6±4,2 ve %85,4±2,7 bulundu. Seeding
uygulanmasıyla, canlılık oranında straw grupları arasında fark
görülmemesine rağmen, kriyotüp grupları arasında fark anlamlıydı
(p

Keywords

Prepubertal testis, Testis hücre süspansiyonu, Programlı yavaş dondurma, Spermatogonyal kök hücre, Seeding

References

1. Orwig KE, Schlatt S. Cryopreservation and transplantation of spermatogonia and testicular tissue for preservation of male fertility. J Natl Cancer Inst Monogr 2005;34:51-6. doi:10.1093/jncimonographs/lgi029
2. Schlatt S, Ehmcke J, Jahnukainen K. Testicular stem cells for fertility preservation: Preclinical studies on male germ cell transplantation and testicular grafting. Pediatr Blood Cancer 2009;53:274-80. doi:10.1002/pbc.22002
3. Goossens E, Tournaye H. Is there a clinical future for spermatogonial stem cells? Curr Stem Cell Res Ther 2007;2:189-95. doi:10.2174/157488807781696258
4. Wyns C, Curaba M, Petit S, et al. Management of fertility preservation in prepubertal patients: 5 years’ experience at the Catholic University of Louvain. Hum Reprod 2011;26:737- 47. doi: 10.1093/humrep/deq387.
5. Wyns C, Langendonckt AV,Wese F-X, Donnez J, Curaba M. Long-term spermatogonial survival cryopreserved and xenografted immature human testicular tissue. Hum Reprod 2008;23:2402-14. doi:10.1093/humrep/den272.
6. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Ogura A, Toyokuni S, Shinohara T. Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stemcells. Hum Reprod 2003;18:2660-67. doi:10.1093/ humrep/deg483
7. Avarbock MR, Brinster CJ, Brinster RL. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat Med 1996;2:693-6. doi:10.1038/nm0696-693
8. Wyns C, Curaba M, Martinez-Madrid B, Langendonckt AV, François-Xavier W, Donnez J. Spermatogonial survival after cryopresrvation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice. Hum Reprod 2007;22:1603-11. doi: 10.1093/humrep/dem062
9. Jezek D, Schulze W, Kalanj-Bognar S, Vukelic Z, MilavecPuretic. V, Krhen I. Effects of various cryopreservation media and freezing thawing on the morphology of rat testicular biopsies. Andrologia 2001;33:368-78. doi: 10.1046/j.1439- 0272.2001.00459.x
10. Keros V, Rosenlund B, Hultenby K, Aghajanova L, Levkov L, Hovatta O. Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants. Hum Reprod 2005;20:1676-87. doi: 10.1093/humrep/deh797
11. Keros V, Hultenby K, Borgström B, Fridström M, Jahnukainen K, Hovatta O. Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys under-going gonadotoxic cancer treatment. Hum Reprod 2007;22:1384-95. doi: 10.1093/humrep/del508
12. Milazzo JP, Vaudreuil L, Cauliez B, et al. Comparison of conditions for cryopreservation of testicular tissue from immature mice. Hum Reprod 2008;23:17-28. doi: 10.1093/ humrep/dem355
13. Izadyar F, Matthijs-Rijsenbilt JJ, den Ouden K, Creemers LB, Woelders H, de Rooij DG. Development of a cryopreservation protocol for type a spermatogonia. J Androl 2002;23:537-45. doi: 10.1002/j.1939-4640.2002.tb02276.x
14. Schlatt S, von Schonfeldt V, Schepers AG. Male germ cell transplantation: an experimen-tal approach with a clinical perspective. Br Med Bull 2000;56:824-36. doi: 10.1258/0007142001903409
15. Brockbank KGM, Song YC, Khirabadi BS, Lightfoot FG, Boggs JM, Taylor MJ. Storage of tissues by vitrification. Transpl Proc 2000;32:3-4. doi: 10.1016/S0041- 1345(99)00851-9
16. Mazur P, Leibo SP, Chu EHY. A two-factor hypothesis of freezing injury evidence from Chinese hamster tissue culture cells. Exp Cell Res 1972;71:345-55. doi: 10.1016/0014- 4827(72)90303-5
17. Mazur P, Rall WF, Leibo SP. Kinetics of water loss and the likelihood of intracellular freezing in mouse ova. Influence of the method of calculating the temperature dependence of water permeability. Cell Biophys 1984;6:197-213. doi: 10.1007/BF02788619
18. Delilbaşı L. In Vitro Fertilizasyon (IVF) Laboratuvar Yöntemleri (Yeni uygulamalar ve güncel yaklaşımlar). Ankara: Güneş Kitabevleri, 2008:61-83. ISBN: 9789752771697
19. Morris GJ, Acton E. Controlled ice nucleation in cryopreservation. Cryobiology 2013; 66:85-92. doi: 10.1016/j. cryobiol.2012.11.007
20. Russell LD, Ettlin RA, Hikim APS, Clegg ED, (editors). Histopathological evaluation of the testis. 1st Edition. Clearwater: Cache River Press, 1990:1-58.
21. Gouk S S, Loh YFJ, Kumar S D, Watson PF, Kuleshova LL. Cryopreservation of mouse testicular tissue: prospect for harvesting spermatogonial stem cells for fertility preservation. Fertil Steril 2011; 95: 2399-2403. doi: 10.1016/j.fertnstert. 2011.03.035.
22. Trump BF, Laiho KA, Mergner WJ, Arstila AU. Studies on the subcellular pathophysiology of acute lethal cell injury. Beitr Path Bd 1974;125:243–71. doi:10.1016/S0005-8165(74)80177-0
23. Baert Y, Goossens E , Saen D V, Ning L, Veld P, Tournaye H. Orthotopic grafting of cryopreserved prepubertal testicular tissue: in search of a simple yet effective cryopreservation protocol. Fertil Steril 2012; 97:1152-7. doi: 10.1016/j. fertnstert.2012.02.010.
24. Baert Y, Saen DV, Haentjens P, Veld PI, Tournaye H, Goossens E. What is the best cryopreservation protocol for human testicular tissue banking? Hum Reprod 2013;28:1816- 26. doi: 10.1093/humrep/det100
25. Milazzo JP, Travers A, Bironneau AA, et al. Rapid screening of cryopreservation protocols for murine prepubertal testicular tissue by histology and PCNA immunostaining. J Androl 2010;31: 617-30. doi:10.2164/jandrol.109.009324.
26. Griswold MD. The central role of Sertoli cells in spermatogenesis. Cell Dev Biol 1998;9:411-16. doi: 10.1006/ scdb.1998.0203
27. Nugent D, Meirow D, Brook PF, Y, Gosden RG. Transplantation in reproductive medicine: previous experience, present knowledge and future prospects. Hum Reprod Update 1997;3:267-80. doi: 10.1093/humupd/3.3.267
28. Hossain A, Nagamani M. Erkek üreme hücrelerinin dondurularak saklanması (Çev: Kervancıoğlu G, Kervancıoğlu E). İnfertilite ve Yardımla Üreme Teknikleri. Ankara: Güneş Kitabevi, 2012:466-77. ISBN: 9789752773899
29. Crabbe E, Verheyen G, Tournaye H, Van Steırteghem A. Freezing of testicular tissue as a minced suspension preserves sperm quality better than whole-biopsy freezing when glycerol is used as cryoprotectant. Int J Androl 1999;22:43-8. doi: 10.1046/j.1365-2605.1999.00149.x
30. Nagano R,Tabata S, Nakanishi Y, Ohsako S, Kurohmaru M, Hayashi Y. Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis. Anat Rec 2000;258:210-20. doi: 10.1002/(SICI)1097- 0185(20000201)258:2<210::AID-AR10>3.0.CO;2-X
31. Robbins H, Dores C, Coyle K, Dobrinski I. Germ cells and testicular somatic cells have different sensitivity to cryopreservation. Reprod Fertil Dev 2012;25:184-184. doi: 10.1071/RDv25n1Ab74
32. Bloom W, Fawcett DW. A Textbook of Histology. 11th Edition. Philadelphia: Saunders Company, 1986:796-848. ISBN-10: 0721617298
33. Ross MH, Pawlina W. Histology: A Text and Atlas. 6th edition. Philadelphia PA: Lippincott, Williams and Wilkins, 2011:784-818.
34. Travers A, Milazzo JP, Perdrix A, A, et al. Assessment of freezing procedures for rat immature testicular tissue. Theriogenology 2011;76:981-990. doi: 10.1016/j. theriogenology.2011.04.025
35. Unni S, Kasiviswanathan S, D’Souza S, S, et al. Efficient cryopreservation of testicular tissue: effect of age, sample state and concentration of cryoprotectant. Fertil Steril 2011;97:200- 8. doi: 10.1016/j.fertnstert. 2011.10.018

There are 35 citations in total.

Details

Primary Language	Turkish
Journal Section	Articles
Authors	Gülnaz Kervancıoğlu This is me Gülnaz Kervancıoğlu This is me Şule Çetinel This is me Şule Çetinel This is me Elif Kervancıoğlu Demirci This is me Elif Kervancıoğlu Demirci This is me Gülçin Ekter Kanter This is me Gülçin Ekter Kanter This is me Ertan Kervancıoğlu This is me M. Ertan Kervancıoğlu This is me
Publication Date	July 2, 2015
Published in Issue	Year 2015 Volume: 28 Issue: 1

Cite

APA	Kervancıoğlu, G., Kervancıoğlu, G., Çetinel, Ş., Çetinel, Ş., et al. (2015). Prepubertal testis hücre süspansiyonun dondurulmasında seeding etkisi. Marmara Medical Journal, 28(1), 13-20. https://doi.org/10.5472/MMJ.2015.03894.1
AMA	Kervancıoğlu G, Kervancıoğlu G, Çetinel Ş, Çetinel Ş, Kervancıoğlu Demirci E, Kervancıoğlu Demirci E, Ekter Kanter G, Ekter Kanter G, Kervancıoğlu E, Kervancıoğlu ME. Prepubertal testis hücre süspansiyonun dondurulmasında seeding etkisi. Marmara Med J. August 2015;28(1):13-20. doi:10.5472/MMJ.2015.03894.1
Chicago	Kervancıoğlu Gülnaz, Kervancıoğlu Gülnaz, Şule Çetinel, Şule Çetinel, Elif Kervancıoğlu Demirci, Elif Kervancıoğlu Demirci, Ekter Kanter Gülçin, Ekter Kanter Gülçin, Ertan Kervancıoğlu, and M. Ertan Kervancıoğlu. “Prepubertal Testis hücre süspansiyonun dondurulmasında Seeding Etkisi”. Marmara Medical Journal 28, no. 1 (August 2015): 13-20. https://doi.org/10.5472/MMJ.2015.03894.1.
EndNote	Kervancıoğlu G, Kervancıoğlu G, Çetinel Ş, Çetinel Ş, Kervancıoğlu Demirci E, Kervancıoğlu Demirci E, Ekter Kanter G, Ekter Kanter G, Kervancıoğlu E, Kervancıoğlu ME (August 1, 2015) Prepubertal testis hücre süspansiyonun dondurulmasında seeding etkisi. Marmara Medical Journal 28 1 13–20.
IEEE	G. Kervancıoğlu, “Prepubertal testis hücre süspansiyonun dondurulmasında seeding etkisi”, Marmara Med J, vol. 28, no. 1, pp. 13–20, 2015, doi: 10.5472/MMJ.2015.03894.1.
ISNAD	Kervancıoğlu Gülnaz et al. “Prepubertal Testis hücre süspansiyonun dondurulmasında Seeding Etkisi”. Marmara Medical Journal 28/1 (August 2015), 13-20. https://doi.org/10.5472/MMJ.2015.03894.1.
JAMA	Kervancıoğlu G, Kervancıoğlu G, Çetinel Ş, Çetinel Ş, Kervancıoğlu Demirci E, Kervancıoğlu Demirci E, Ekter Kanter G, Ekter Kanter G, Kervancıoğlu E, Kervancıoğlu ME. Prepubertal testis hücre süspansiyonun dondurulmasında seeding etkisi. Marmara Med J. 2015;28:13–20.
MLA	Kervancıoğlu Gülnaz et al. “Prepubertal Testis hücre süspansiyonun dondurulmasında Seeding Etkisi”. Marmara Medical Journal, vol. 28, no. 1, 2015, pp. 13-20, doi:10.5472/MMJ.2015.03894.1.
Vancouver	Kervancıoğlu G, Kervancıoğlu G, Çetinel Ş, Çetinel Ş, Kervancıoğlu Demirci E, Kervancıoğlu Demirci E, Ekter Kanter G, Ekter Kanter G, Kervancıoğlu E, Kervancıoğlu ME. Prepubertal testis hücre süspansiyonun dondurulmasında seeding etkisi. Marmara Med J. 2015;28(1):13-20.

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