Андрология и генитальная хирургия. 2024; 25: 95-104
Новый эффективный способ выделения единичных сперматозоидов из хирургически полученного материала при азооспермии
https://doi.org/10.62968/2070-9781-2024-25-1-95-104Аннотация
Введение. Новый способ обработки хирургически полученного материала из яичка/придатка основан на дифференциальном центрифугировании, которое ранее никогда не применяли для работы со сперматозоидами. Клетки биоптата/аспирата разделяют в соответствии со скоростью седиментации, таким образом сперматозоиды остаются в супернатанте, а осадок с эритроцитами, прочими клетками и дебрисом удаляется. Фракция единичных сперматозоидов из супернатанта может быть получена даже в самых тяжелых случаях.
Материалы и методы. Всего методом дифференциального центрифугирования обработано 436 биологических образцов из яичка/придатка, полученных хирургическим путем у 398 пациентов с азооспермией. Для оценки эффективности нового подхода каждый биологический образец был отнесен к определенной категории качества исходя из содержания сперматозоидов при интраоперационном исследовании. Выделенные сперматозоиды использовали для оплодотворения ооцитов и/или криоконсервировали. Выполнен ретроспективный анализ данных.
Результаты. При микроскопическом исследовании хирургически полученного биологического материала у 237 пациентов (59,5%) был обнаружен по крайней мере один сперматозоид во всем объеме пробы. Эффективность дальнейшей обработки методом дифференциального центрифугирования независимо от качества образца составила 99,6% (сперматозоиды для оплодотворения были получены в 236 случаях, а в 214 из них дополнительно была выполнена криоконсервация – 90,7%). У 199 пациентов при интраоперационном исследовании хирургически полученного биологического материала сперматозоиды не обнаружены. После обработки методом дифференциального центрифугирования единичные сперматозоиды выделены и использованы для оплодотворения ооцитов в 40 случаях (20%).
Заключение. Новый способ позволяет выделить мужские половые клетки из хирургически полученного материала и использовать их для оплодотворения и/или криоконсервировать даже в самых тяжелых случаях, когда стандартный метод, основанный на простой отмывке с последующим отбором сперматозоидов из осадка, малоэффективен.
Список литературы
1. In-Vitro Fertilization. 4th Edition. Elder K., Dale B. Cambridge University Press. 2020. ISBN: 9781108441810
2. Popal W., Nagy Z.P. Laboratory processing and intracytoplasmic sperm injection using epididymal and testicular spermatozoa: what can be done to improve outcomes? Clinics (Sao Paulo). 2013;68; 1:125-30. DOI: 10.6061/clinics/2013(sup01)14
3. Verheyen G., Popovic-Todorovic B., Tournaye H. Processing and selection of surgically-retrieved sperm for ICSI: a review. Basic Clin Androl. 2017;21; 27:6. DOI:10.1186/s12610-017-0050-2
4. WHO laboratory manual for the examination and processing of human semen, sixth edition. World Health Organization; 2021. Licence: CC BY-NC-SA 3.0 IGO. DOI:10.1093/hropen/hoaa020
5. Mangum C.L., Patel D.P., Jafek A.R., Samuel R., Jenkins T.G., Aston K.I., Gale B.K., Hotaling J.M. Towards a better testicular sperm extraction: novel sperm sorting technologies for non-motile sperm extracted by microdissection TESE. Transl Androl Urol. 2020;9: S206-S214. DOI: 10.21037/tau.2019.08.36
6. Berkovitz A, Miller N, Silberman M, Belenky M, Itsykson P. A novel solution for freezing small numbers of spermatozoa using a sperm vitrification device. Hum Reprod. 2018;1;33(11):1975-1983. DOI: 10.1093/humrep/dey304. PMID: 30285105
7. Aydos K., Aydos O.S. Sperm selection procedures for optimizing the outcome of ICSI in patients with NOA. J Clin Med. 2021;18;10(12):2687. DOI: 10.3390/jcm10122687
8. Esteves SC, Varghese AC. Laboratory handling of epididymal and testicular spermatozoa: What can be done to improve sperm injections outcome. J Hum Reprod Sci. 2012;5(3):233-43. DOI: 10.4103/0974-1208.106333
9. Nagy Z.P., Verheyen G., Tournaye H., Devroey P., Van Steirteghem A.C. An improved treatment procedure for testicular biopsy specimens offers more efficient sperm recovery: case series. Fertil Steril. 1997;68(2):376-9. DOI: 10.1016/s0015-0282(97)81534-8
10. Corona G., Minhas S., Giwercman A., Bettocchi C, Dinkelman-Smit M, Dohle G, Fusco F, Kadioglou A, Kliesch S, Kopa Z, Krausz C, Pelliccione F, Pizzocaro A, Rassweiler J, Verze P, Vignozzi L, Weidner W, Maggi M, Sofikitis N. Sperm recovery and ICSI outcomes in men with non-obstructive azoospermia: a systematic review and metaanalysis. Hum Reprod Update. 2019;5;25(6):733-757. DOI: 10.1093/humupd/dmz028
11. Aydos K., Demirel L.C., Baltaci V., Unlü C. Enzymatic digestion plus mechanical searching improves testicular sperm retrieval in nonobstructive azoospermia cases. Eur J Obstet Gynecol Reprod Biol. 2005;1;120(1):80-6. DOI: 10.1016/j.ejogrb.2004.10.009. PMID: 15866091
12. Achermann A.P.P., Pereira T.A., Esteves S.C. Microdissection testicular sperm extraction (micro-TESE) in men with infertility due to nonobstructive azoospermia: summary of current literature. Int Urol Nephrol. 2021;53(11):2193-2210. DOI: 10.1007/s11255-021-02979-4
13. Ramasamy R., Reifsnyder J.E., Bryson C., Zaninovic N., Liotta D., Cook C.A., Hariprashad J., Weiss D., Neri Q., Palermo G.D., Schlegel P.N. Role of tissue digestion and extensive sperm search after microdissection testicular sperm extraction. Fertil Steril. 2011;96(2):299-302. DOI: 10.1016/j.fertnstert.2011.05.033
14. Verheyen G., De Croo I., Tournaye H., Pletincx I., Devroey P., van Steirteghem A.C. Comparison of four mechanical methods to retrieve spermatozoa from testicular tissue. Hum Reprod. 1995;10(11):2956-9. DOI: 10.1093/oxfordjournals.humrep.a135828
15. Arshad M.A., Majzoub A., Esteves S.C. Predictors of surgical sperm retrieval in non-obstructive azoospermia: summary of current literature. Int Urol Nephrol. 2020;52(11):2015-2038. DOI: 10.1007/s11255-020-02529-4.
16. Патент РФ на изобретение № 2762489/21.12.21. Бюл. № 36. Захарова Е.Е. Способ выделения сперматозоидов из материала аспирации и/или биопсии из придатка и/или яичка для использования в программах экстракорпорального оплодотворения и/или криоконсервации.
17. Zakharova E. New effective method of spermatozoa recovery from surgically-retrieved testicular and epididymal specimens by differential centrifugation. MedRxiv. 2022;11.03.22281832. DOI: 10.1101/2022.11.03.22281832
18. Zakharova E. Separating spermatozoa from debris, non-germ and red blood cells during differential centrifugation as a new effective approach of sperm recovery from surgically-retrieved specimens. Human Reproduction. 2022;37;1; 105:128. DOI: 10.1093/humrep/deac105.128
19. Simopoulou M., Gkoles L., Bakas P., Giannelou P., Kalampokas T., Pantos K., Koutsilieris M. Improving ICSI: A review from the spermatozoon perspective. Syst Biol Reprod Med. 2016;62(6):359-371. DOI: 10.1080/19396368.2016.1229365
20. Huang C., Gan R.X., Hu J.L., Liu F., Hong Y., Zhu W.B., Li Z. Clinical benefit for cryopreservation of single human spermatozoa for ICSI: A systematic review and meta-analysis. Andrology. 2022;10(1):82-91. DOI: 10.1111/andr.13091
21. Письмо МЗ РФ №15-4/И/2-1908 от 5 марта 2019 г. «Вспомогательные репродуктивные технологии и искусственная инсеминация».
22. Holstein A.F., Schulze W., Davidoff M. Understanding spermatogenesis is a prerequisite for treatment. Reprod Biol Endocrinol. 2003; 1:107. DOI: 10.1186/1477-7827-1-107
23. Захарова Е.Е., Сухачева Т.С., Мельник Я.И., Дорощук Н.А., Капралова И.В., Биканов Р.А., Фердман Д.К., Залетова В.В. Рождение ребенка у пациента с необструктивной азооспермией и носительством реципрокной транслокации (описание случая). Проблемы репродукции. 2023;29(2):110 115. DOI: 10.17116/repro202329021110
24. Salama N., Sirelkhatim Hassan O. Staged Laboratory Processing of Testicular Tissue in Non-Obstructive Azoospermia May Rescue Retrieving an Existing Sperm: A Case Report and Literature Review. Clin Med Insights Case Rep. 2023; 16:11795476231178353. DOI: 10.1177/11795476231178353
25. Amer M., Fakhry E. Fresh vs frozen testicular sperm for assisted reproductive technology in patients with non-obstructive azoospermia: A systematic review. Arab J Urol. 2021;19(3):247-254. DOI: 10.1080/2090598X.2021.1932303
26. O’Neill H.C., Nikoloska M., Ho H., Doshi A., Maalouf W. Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage. J Assist Reprod Genet. 2019;36(8):1713-1720. DOI: 10.1007/s10815-019-01505-x
27. Ramasamy R., Fisher E.S., Ricci J.A., Leung R.A., Schlegel P.N. Duration of microdissection testicular sperm extraction procedures: relationship to sperm retrieval success. J Urol. 2011;185(4):1394-7. DOI: 10.1016/j.juro.2010.11.074
Andrology and Genital Surgery. 2024; 25: 95-104
A new effective method for isolating single spermatozoa from surgically obtained material in azoospermia
https://doi.org/10.62968/2070-9781-2024-25-1-95-104Abstract
Introduction. Conventional methods of processing testicular and epididymal specimens are based on simple washing, so that spermatozoa are contaminated with other cells and difficult to recover from the sediment. The newly method is based on differential centrifugation, which has never been used for male germ cells before. Cell particles are separated according to their sedimentation rate so that spermatozoa remain in the supernatant, whereas the sediment with extra cells and debris is removed. A pure fraction of spermatozoa from supernatant is retrieved even in severe cases.
Methods. A total of 436 specimens surgically obtained from epididymis/testis in 398 patients were treated using method of differential centrifugation. To evaluate the effectiveness of the new approach, each biological sample was assigned to a specific quality category based on sperm content during intraoperative examination. The recovered spermatozoa were used to fertilize the fresh spouse’s oocytes and/or cryopreserved. A retrospective data analysis was performed.
Results. The microscopic examination surgically obtained specimens revealed at least one spermatozoon in all fields of view in 237 patients (59,5%). The further processing by differential centrifugation was 99,6% efficient (spermatozoa for fertilization were recovered in 236 cases, including cryopreservation in 214 cases). In 199 patients no spermatozoa were found during intraoperative examination. After treatment by differential centrifugation, single spermatozoa were isolated and used for oocyte fertilization in 40 cases (20%).
Conclusion. The newly proposed method of processing cell suspensions for spermatozoa recovery allows obtaining male germ cells from biopsy samples and using them for fertilization, especially if they are critically low in number and conventional methods for sperm recovery do no work or inefficient.
References
1. In-Vitro Fertilization. 4th Edition. Elder K., Dale B. Cambridge University Press. 2020. ISBN: 9781108441810
2. Popal W., Nagy Z.P. Laboratory processing and intracytoplasmic sperm injection using epididymal and testicular spermatozoa: what can be done to improve outcomes? Clinics (Sao Paulo). 2013;68; 1:125-30. DOI: 10.6061/clinics/2013(sup01)14
3. Verheyen G., Popovic-Todorovic B., Tournaye H. Processing and selection of surgically-retrieved sperm for ICSI: a review. Basic Clin Androl. 2017;21; 27:6. DOI:10.1186/s12610-017-0050-2
4. WHO laboratory manual for the examination and processing of human semen, sixth edition. World Health Organization; 2021. Licence: CC BY-NC-SA 3.0 IGO. DOI:10.1093/hropen/hoaa020
5. Mangum C.L., Patel D.P., Jafek A.R., Samuel R., Jenkins T.G., Aston K.I., Gale B.K., Hotaling J.M. Towards a better testicular sperm extraction: novel sperm sorting technologies for non-motile sperm extracted by microdissection TESE. Transl Androl Urol. 2020;9: S206-S214. DOI: 10.21037/tau.2019.08.36
6. Berkovitz A, Miller N, Silberman M, Belenky M, Itsykson P. A novel solution for freezing small numbers of spermatozoa using a sperm vitrification device. Hum Reprod. 2018;1;33(11):1975-1983. DOI: 10.1093/humrep/dey304. PMID: 30285105
7. Aydos K., Aydos O.S. Sperm selection procedures for optimizing the outcome of ICSI in patients with NOA. J Clin Med. 2021;18;10(12):2687. DOI: 10.3390/jcm10122687
8. Esteves SC, Varghese AC. Laboratory handling of epididymal and testicular spermatozoa: What can be done to improve sperm injections outcome. J Hum Reprod Sci. 2012;5(3):233-43. DOI: 10.4103/0974-1208.106333
9. Nagy Z.P., Verheyen G., Tournaye H., Devroey P., Van Steirteghem A.C. An improved treatment procedure for testicular biopsy specimens offers more efficient sperm recovery: case series. Fertil Steril. 1997;68(2):376-9. DOI: 10.1016/s0015-0282(97)81534-8
10. Corona G., Minhas S., Giwercman A., Bettocchi C, Dinkelman-Smit M, Dohle G, Fusco F, Kadioglou A, Kliesch S, Kopa Z, Krausz C, Pelliccione F, Pizzocaro A, Rassweiler J, Verze P, Vignozzi L, Weidner W, Maggi M, Sofikitis N. Sperm recovery and ICSI outcomes in men with non-obstructive azoospermia: a systematic review and metaanalysis. Hum Reprod Update. 2019;5;25(6):733-757. DOI: 10.1093/humupd/dmz028
11. Aydos K., Demirel L.C., Baltaci V., Unlü C. Enzymatic digestion plus mechanical searching improves testicular sperm retrieval in nonobstructive azoospermia cases. Eur J Obstet Gynecol Reprod Biol. 2005;1;120(1):80-6. DOI: 10.1016/j.ejogrb.2004.10.009. PMID: 15866091
12. Achermann A.P.P., Pereira T.A., Esteves S.C. Microdissection testicular sperm extraction (micro-TESE) in men with infertility due to nonobstructive azoospermia: summary of current literature. Int Urol Nephrol. 2021;53(11):2193-2210. DOI: 10.1007/s11255-021-02979-4
13. Ramasamy R., Reifsnyder J.E., Bryson C., Zaninovic N., Liotta D., Cook C.A., Hariprashad J., Weiss D., Neri Q., Palermo G.D., Schlegel P.N. Role of tissue digestion and extensive sperm search after microdissection testicular sperm extraction. Fertil Steril. 2011;96(2):299-302. DOI: 10.1016/j.fertnstert.2011.05.033
14. Verheyen G., De Croo I., Tournaye H., Pletincx I., Devroey P., van Steirteghem A.C. Comparison of four mechanical methods to retrieve spermatozoa from testicular tissue. Hum Reprod. 1995;10(11):2956-9. DOI: 10.1093/oxfordjournals.humrep.a135828
15. Arshad M.A., Majzoub A., Esteves S.C. Predictors of surgical sperm retrieval in non-obstructive azoospermia: summary of current literature. Int Urol Nephrol. 2020;52(11):2015-2038. DOI: 10.1007/s11255-020-02529-4.
16. Patent RF na izobretenie № 2762489/21.12.21. Byul. № 36. Zakharova E.E. Sposob vydeleniya spermatozoidov iz materiala aspiratsii i/ili biopsii iz pridatka i/ili yaichka dlya ispol'zovaniya v programmakh ekstrakorporal'nogo oplodotvoreniya i/ili kriokonservatsii.
17. Zakharova E. New effective method of spermatozoa recovery from surgically-retrieved testicular and epididymal specimens by differential centrifugation. MedRxiv. 2022;11.03.22281832. DOI: 10.1101/2022.11.03.22281832
18. Zakharova E. Separating spermatozoa from debris, non-germ and red blood cells during differential centrifugation as a new effective approach of sperm recovery from surgically-retrieved specimens. Human Reproduction. 2022;37;1; 105:128. DOI: 10.1093/humrep/deac105.128
19. Simopoulou M., Gkoles L., Bakas P., Giannelou P., Kalampokas T., Pantos K., Koutsilieris M. Improving ICSI: A review from the spermatozoon perspective. Syst Biol Reprod Med. 2016;62(6):359-371. DOI: 10.1080/19396368.2016.1229365
20. Huang C., Gan R.X., Hu J.L., Liu F., Hong Y., Zhu W.B., Li Z. Clinical benefit for cryopreservation of single human spermatozoa for ICSI: A systematic review and meta-analysis. Andrology. 2022;10(1):82-91. DOI: 10.1111/andr.13091
21. Pis'mo MZ RF №15-4/I/2-1908 ot 5 marta 2019 g. «Vspomogatel'nye reproduktivnye tekhnologii i iskusstvennaya inseminatsiya».
22. Holstein A.F., Schulze W., Davidoff M. Understanding spermatogenesis is a prerequisite for treatment. Reprod Biol Endocrinol. 2003; 1:107. DOI: 10.1186/1477-7827-1-107
23. Zakharova E.E., Sukhacheva T.S., Mel'nik Ya.I., Doroshchuk N.A., Kapralova I.V., Bikanov R.A., Ferdman D.K., Zaletova V.V. Rozhdenie rebenka u patsienta s neobstruktivnoi azoospermiei i nositel'stvom retsiproknoi translokatsii (opisanie sluchaya). Problemy reproduktsii. 2023;29(2):110 115. DOI: 10.17116/repro202329021110
24. Salama N., Sirelkhatim Hassan O. Staged Laboratory Processing of Testicular Tissue in Non-Obstructive Azoospermia May Rescue Retrieving an Existing Sperm: A Case Report and Literature Review. Clin Med Insights Case Rep. 2023; 16:11795476231178353. DOI: 10.1177/11795476231178353
25. Amer M., Fakhry E. Fresh vs frozen testicular sperm for assisted reproductive technology in patients with non-obstructive azoospermia: A systematic review. Arab J Urol. 2021;19(3):247-254. DOI: 10.1080/2090598X.2021.1932303
26. O’Neill H.C., Nikoloska M., Ho H., Doshi A., Maalouf W. Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage. J Assist Reprod Genet. 2019;36(8):1713-1720. DOI: 10.1007/s10815-019-01505-x
27. Ramasamy R., Fisher E.S., Ricci J.A., Leung R.A., Schlegel P.N. Duration of microdissection testicular sperm extraction procedures: relationship to sperm retrieval success. J Urol. 2011;185(4):1394-7. DOI: 10.1016/j.juro.2010.11.074
