Kumulus Hücresi Ekstraselüler Matriksi ile Desteklenen Sperm Hazırlama Protokollerinin İn Vitro Sperm Kalitesi Üzerindeki Etkisi

Öz

Bu çalışmada, üremeye yardımcı tedavilerde (ÜYTE) uygulanan sperm hazırlama tekniklerinde kültür medyumuna kumulus hücresi ekstraselüler matriksi (CCECM) eklenmesinin; rutin sperm parametreleri, fertilizasyon kapasitesi ve sperm DNA fragmantasyonu üzerindeki etkilerini belirlemek ve sperm ayrıştırma protokollerini karşılaştırmalı olarak analiz etmek amaçlanmıştır. Çalışmada, normozoospermik 30 farklı erkek hastadan alınan ejakülat örnekleri ile bu hastaların eşlerinden toplanan kumulus-oosit kompleksleri kullanıldı. Her hastadan elde edilen semen örneği beş eşit hacme ayrılarak kontrol, dansite gradiyent (DG), dansite gradiyent + kumulus hücresi ekstraselüler matriksi (DG+K), swim-up (SU), swim-up + kumulus hücresi ekstraselüler matriksi (SU+K) olmak üzere beş grup oluşturuldu. Elde edilen sonuçlar, tüm deney gruplarında sperm konsantrasyonunun kontrol grubuna kıyasla belirgin olarak azaldığını; bu azalmanın özellikle swim-up uygulanan grupta anlamlı düzeye ulaştığını gösterdi. Motilite oranı en yüksek SU+K grubunda saptanırken, DG+K ve SU gruplarında da motilitenin anlamlı biçimde arttığı belirlendi. TPMSS değeri ise SU ve SU+K gruplarında anlamlı olarak azaldı. Morfoloji açısından gruplar arasında belirgin bir farklılık izlenmezken, canlılık oranının CCECM ilavesi yapılan gruplarda kontrol grubuna göre dikkat çekici şekilde arttığı görüldü. Akrozomal reaksiyon kapasitesini yansıtan ARIC skorları DG+K ve SU+K gruplarında anlamlı derecede yüksek bulundu. Ayrıca, DNA hasarını gösteren DFI değerleri bu iki grupta anlamlı olarak daha düşük saptandı ve CCECM katkısının sperm DNA bütünlüğünü koruyabileceği ortaya kondu. Sperm yıkama sırasında kullanılan kültür medyumuna CCECM eklenmesi, özellikle swim-up yöntemiyle birlikte uygulandığında sperm kalitesi ve verimliliğini artırdı. Bu yaklaşımın, ÜYTE kapsamında erkek infertilitesi tedavisinde uygulanabilir bir seçenek olabileceğini düşünüyoruz.

Anahtar Kelimeler

• Kumulus hücresi ekstraselüler matriksi
• Sperm kalitesi
• Dansite-gradiyent
• Swim-up
• DNA fragmantasyonu
• Akrozom reaksiyonu

Effects of Cumulus Cell Extracellular Matrix–Assisted Sperm Preparation Protocols on In Vitro Sperm Quality

Öz

This study aimed to determine the effects of adding cumulus cell extracellular matrix (CCECM) to the culture medium used in sperm preparation techniques for assisted reproductive treatments (ART) on routine sperm parameters, fertilization capacity, and sperm DNA fragmentation, and to compare sperm separation protocols. Ejaculate samples from 30 normozoospermic male patients and cumulus–oocyte complexes collected from their spouses were included. Each semen sample was divided into five equal volumes and allocated to five groups: control, density gradient (DG), density gradient plus cumulus cell extracellular matrix (DG+K), swim-up (SU), and swim-up plus cumulus cell extracellular matrix (SU+K). The results showed that sperm concentration was markedly lower in all experimental groups than in the control group, with the decrease reaching statistical significance, particularly in the swim-up group. Motility was highest in the SU+K group and was also significantly increased in the DG+K and SU groups. In contrast, the TPMSS value decreased significantly in the SU and SU+K groups. No marked differences were observed among the groups in terms of morphology. Viability increased notably in the CCECM-supplemented groups, and this increase remained significant across all experimental groups compared with the control group. ARIC scores, reflecting acrosomal reaction capacity, were significantly higher in the DG+K and SU+K groups. Moreover, DFI values, indicating DNA damage, were significantly lower in these two groups, suggesting that CCECM supplementation may help preserve sperm DNA integrity. Adding CCECM to the culture medium used in sperm washing improved sperm quality and efficiency, particularly with the swim-up method. We believe this approach may be a feasible option for treating male infertility within assisted reproductive technologies (ART).

Anahtar Kelimeler

• Cumulus cell extracellular matrix
• Sperm quality
• Density gradient
• Swim-up
• DNA fragmentation
• Acrosome reaction

Kaynakça

1. 1. European Association of Urology. Male infertility. In: EAU Guidelines on Sexual and Reproductive Health. Arnhem (The Netherlands): EAU Guidelines Office; 2022. Accessed 2026 Feb 22.
2. 2. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 6th ed. Geneva: World Health Organization; 2021.
3. 3. Henkel RR, Schill WB. Sperm preparation for ART. Reprod Biol Endocrinol. 2003;1:108.
4. 4. Naknam W, Salang L, Sothornwit J, Amnatbuddee S, Seejorn K, Pongsritasana T, Sukkasame S. Effect of sperm selection method by cumulus oophorus complexes and conventional sperm preparation method on sperm quality and DNA fragmentation for assisted reproduction technology. Eur J Obstet Gynecol Reprod Biol. 2019;243:46-50.
5. 5. Zhuo L, Kimata K. Cumulus oophorus extracellular matrix: its construction and regulation. Cell Struct Funct. 2001;26(4):189-196.
6. 6. Huszar G, Ozenci CC, Cayli S, Zavaczki Z, Hansch E, Vigue L. Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status. Fertil Steril. 2003;79(Suppl 3):1616-1624.
7. 7. Parmegiani L, Cognigni GE, Bernardi S, Troilo E, Ciampaglia W, Filicori M. "Physiologic ICSI": hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality. Fertil Steril. 2010;93(2):598-604.
8. 8. Majzoub A, Agarwal A, Cho CL, Esteves SC. Sperm DNA fragmentation testing: a cross-sectional survey on current practices of fertility specialists. Transl Androl Urol. 2017;6(Suppl 4):S710-S719.

9. 9. Ozaki T, Takahashi K, Kanasaki H, Miyazaki K. Evaluation of acrosome reaction and viability of human sperm with two fluorescent dyes. Arch Gynecol Obstet. 2002;266(2):114-117.
10. 10. Aizpurua J, Medrano L, Enciso M, Sarasa J, Romero A, Fernández MA, Gómez-Torres MJ. New permeable cryoprotectant-free vitrification method for native human sperm. Hum Reprod. 2017;32(10):2007-2015.
11. 11. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril. 1988;49(1):112-117.
12. 12. Ünal MS, Kabukçu C. Isolation of human cumulus granulosa cells. Van Tıp Dergisi. 2022;29(1):84-89.
13. 13. Agarwal A, Gupta S, Sharma R. Acrosome Reaction Measurement. In: Agarwal A, Gupta S, Sharma R, editors. Andrological Evaluation of Male Infertility. Cham: Springer; 2016. p. 143-146.
14. 14. Abou-Haila A, Tulsiani DR. Mammalian sperm acrosome: Formation, contents, and function. Arch Biochem Biophys. 2000;379(2):173-182.
15. 15. Soto-Heras S, Sakkas D, Miller DJ. Sperm selection by the oviduct: Perspectives for male fertility and assisted reproductive technologies. Biol Reprod. 2023;108(3):538-552.
16. 16. Talevi R, Gualtieri R. Biologia e Tecnologie della Riproduzione Umana. Vol 1. Padova: Piccin; ISBN 978-88-299-2965-8, 2019.
17. 17. Eberhardt M, Prochowska S, Duszewska AM, Van Soom A, Olech W, Nizański W. The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa. BMC Vet Res. 2022;18:305.
18. 18. Tanghe S, Van Soom A, Nauwynck H, Coryn M, de Kruif A. Minireview: Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol Reprod Dev. 2002;61(3):414-424.
19. 19. Turathum B, Gao EM, Chian RC. The function of cumulus cells in oocyte growth and maturation and in subsequent ovulation and fertilization. Cells. 2021;10(9):2292.
20. 20. Jayaraman V, Upadhya D, Narayan PK, Adiga SK. Sperm processing by swim-up and density gradient is effective in elimination of sperm with DNA damage. J Assist Reprod Genet. 2012;29(6):557-563.
21. 21. Raad G, Serhal R, Prost E, et al. Differential impact of four sperm preparation techniques on sperm motility, morphology, DNA fragmentation, acrosome status, oxidative stress and mitochondrial activity. Andrology. 2021.
22. 22. Volpes A, Sammartano F, Rizzari S, Gullo S, Marino A, Allegra A. The pellet swim-up is the best technique for sperm preparation during in vitro fertilization procedures. J Assist Reprod Genet. 2016;33(6):765-770.
23. 23. Menkveld R, Swanson RJ, Kotze TJ, Kruger TF. Comparison of a discontinuous Percoll gradient method versus a swim-up method: Effects on sperm morphology and other semen parameters. Andrologia. 1990;22(2):152-158.
24. 24. Bibi R, Jahan S, Afsar T, et al. Analyzing the differential impact of semen preparation methods on the outcomes of assisted reproductive techniques. Biomedicines. 2023;11(2):467.
25. 25. Lishko PV, Botchkina IL, Kirichok Y. Progesterone activates the principal Ca2+ channel of human sperm. Nature. 2011;471:387-391.
26. 26. Henkel R, Müller C, Miska W, Gips H, Schill WB. Determination of the acrosome reaction in human spermatozoa is predictive of fertilization in vitro. Hum Reprod. 1993;8(12):2128-2132.
27. 27. Cummins JM, Pember SM, Jequier AM, Yovich JL, Hartmann PE. A test of the human sperm acrosome reaction following ionophore challenge: Relationship to fertility and other seminal parameters. J Androl. 1991;12(2):98-103.