Abstract
Objectives: The developed sperm cryopreservation technique has opened up a very high chance of having a baby for male infertility cases. Among the cryopreservation methods, vitrification is a widely studied and gradually applied technique. Although sperm vitrification has many advantages, the application of sperm vitrification in IVF is still not a substitute for traditional freezing. Therefore, we performed the study on “Evaluation of blastocyst quality in in vitro fertilization using vitrified-warmed sperm” to assess the effect of vitrification compared with rapid freezing in IVF outcomes.
Materials and Methods: Studying the quality of blastocysts after ICSI on sibling eggs in 18 cycles of IVF treatment at the Center of Reproductive Endocrinology and Infertility, Hue University of Medicine and Pharmacy Hospital from April 2022 to April 2023 with cryopreserved sperm by two methods: conventional rapid freezing (group 1) and vitrification (group 2).
Results: Group 1 had a higher fertilization rate than group 2 (60.67 ± 21.24% versus 56.52 ± 24.27%) with p > 0.05. However, the group using vitrified-warmed sperm had better blastocyst formation rate and good blastocyst rate than the other (78.97 ± 26.13% compared to with 72.27 ± 29.75% and 60.85 ± 34.19% compared to 55.93 ± 37.34, respectively, p > 0.05).
Conclusions: The quality of blastocysts in the group using vitrified-warmed sperm tended to be better than the group using conventional rapid freezing sperm. However, more studies with larger sample sizes are still needed to confirm the advantages and safety of vitrification.
Keywords
References
1. Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008; 136(2):167–73.
2. Sánchez R, Schulz M, Risopatrón J, Isachenko V, Isachenko E. Vitrification sperm: An alternative to intracytoplasmic sperm injection in oligoasthenozoospermic patient. Rev Int Androl. 2013; 11:36–9.
3. Medrano L, Enciso M, Gomez-Torres MJ, Aizpurua J. First birth of a healthy infant following intra-cytoplasmic sperm injection using a new permeable cryoprotectant-free sperm vitrification protocol. Cryobiology. 2019; 87:117–9.
4. Li Y, Zhou L, Lv M, Ge P, Liu Y, Zhou D. Vitrification and conventional freezing methods in sperm cryopreservation: A systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2019; 233:84–92.
5. Hezavehei M, Sharafi M, Kouchesfahani HM, Henkel R, Agarwal A, Esmaeili V, et al. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reprod Biomed Online. 2018; 37(3):327–39.
6. Patel K, Sharma N, Mishra V, Aggarwal R, Suthar A, Sheth H. Comparison between the outcome of sperm vitrification protocol and conventional slow freezing protocol for semen cryopreservation. Hum Reprod. 2021; 36(Supplement_1).
7. Schulz M, Risopatrón J, Uribe P, Isachenko E, Isachenko V, Sánchez R. Human sperm vitrification: A scientific report. Andrology. 2020; 8(6):1642–50.
8. Eastick J, Venetis C, Cooke S, Storr A, Susetio D, Chapman M. Is early embryo development as observed by time-lapse microscopy dependent on whether fresh or frozen sperm was used for ICSI? A cohort study. J Assist Reprod Genet. 2017; 34(6):733–40.
9. Vicdan K, Akarsu C, Sözen E, Buluç B, Vicdan A, Yılmaz Y, et al. Outcome of intracytoplasmic sperm injection using fresh and cryopreserved-thawed testıcular spermatozoa in 83 azoospermic men with Klinefelter syndrome. J Obstet Gynaecol Res. 2016; 42(11):1558–66.
10. Kopeika J, Thornhill A, Khalaf Y. The effect of cryopreservation on the genome of gametes and embryos: Principles of cryobiology and critical appraisal of the evidence. Hum Reprod Update. 2015; 21(2):209–27.
11. Isachenko V, Isachenko E, Petrunkina AM, Sanchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: Birth of two healthy babies. Reprod Fertil Dev. 2012; 24:323–6.
12. Sanchez R, Isachenko V, Petrunkina AM, Risopatron J, Schulz M, Isachenko E. Live birth after intrauterine insemination with spermatozoa from an oligoasthenozoospermic patient vitrified without permeable cryoprotectants. J Androl. 2012; 33(4):559–62.
13. Le MT, Nguyen TTT, Nguyen TT, Nguyen VT, Nguyen TTA, Nguyen VQH, et al. Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects. Eur J Obstet Gynecol Reprod Biol. 2019; 234:14–20.
14. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: Towards a single blastocyst transfer. Fertil Steril. 2000; 73(6):1155-8.
15. Lê Minh Tâm, Nguyễn Văn Trung, Nguyễn Thị Thái Thanh, Đặng Thị Hồng Nhạn. Chương 4. Kỹ thuật nuôi cấy, đánh giá và các can thiệp trên phôi. Các vấn đề trọng yếu trong Hỗ trợ Sinh sản. Tập 2: Dành cho Phôi học lâm sàng. Nhà xuất bản Y học; 2022; 274–355.
16. Luyet BJ, Hodapp EL. Revival of Frog’s Spermatozoa Vitrified in Liquid Air. Exp Biol Med. 1938; 39(3):433–4.
17. Bunge RG, Sherman JK. Fertilizing capacity of frozen human spermatozoa. Nature. 1953; 172(4382):767–8.
18. Mohamed MSA. Slow cryopreservation is not superior to vitrification in human spermatozoa; An experimental controlled study. Iran J Reprod Med. 2015; 13(10):633-44.
19. Pabón D, Meseguer M, Sevillano G, Cobo A, Romero JL, Remohí J, et al. A new system of sperm cryopreservation: evaluation of survival, motility, DNA oxidation, and mitochondrial activity. Andrology. 2019; 7(3):293–301.
20. Spis E, Bushkovskaia A, Isachenko E, Todorov P, Sanchez R, Skopets V, et al. Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births. Cryobiology. 2019; 90:100–2.
21. Endo Y, Fujii Y, Shintani K, Seo M, Motoyama H, Funahashi H. Simple vitrification for small numbers of human spermatozoa. Reprod Biomed Online. 2012; 24(3):301–7.
22. Ohno M, Tanaka A, Nagayoshi M, Yamaguchi T, Takemoto Y, Tanaka I, et al. Modified permeable cryoprotectant-free vitrification method for three or fewer ejaculated spermatozoa from cryptozoospermic men and 7-year follow-up study of 14 children born from this method. Hum Reprod. 2020; 35(5):1019-28.
23. Lê Minh Tâm, Nguyễn Văn Trung, Nguyễn Thị Thái Thanh, Đặng Thị Hồng Nhạn. Chương 5. Kỹ thuật trữ lạnh trong hỗ trợ sinh sản. Các vấn đề trọng yếu trong Hỗ trợ Sinh sản. Tập 2: Dành cho Phôi học lâm sàng. 2022; 335–95.
1. Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008; 136(2):167–73.
2. Sánchez R, Schulz M, Risopatrón J, Isachenko V, Isachenko E. Vitrification sperm: An alternative to intracytoplasmic sperm injection in oligoasthenozoospermic patient. Rev Int Androl. 2013; 11:36–9.
3. Medrano L, Enciso M, Gomez-Torres MJ, Aizpurua J. First birth of a healthy infant following intra-cytoplasmic sperm injection using a new permeable cryoprotectant-free sperm vitrification protocol. Cryobiology. 2019; 87:117–9.
4. Li Y, Zhou L, Lv M, Ge P, Liu Y, Zhou D. Vitrification and conventional freezing methods in sperm cryopreservation: A systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2019; 233:84–92.
5. Hezavehei M, Sharafi M, Kouchesfahani HM, Henkel R, Agarwal A, Esmaeili V, et al. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reprod Biomed Online. 2018; 37(3):327–39.
6. Patel K, Sharma N, Mishra V, Aggarwal R, Suthar A, Sheth H. Comparison between the outcome of sperm vitrification protocol and conventional slow freezing protocol for semen cryopreservation. Hum Reprod. 2021; 36(Supplement_1).
7. Schulz M, Risopatrón J, Uribe P, Isachenko E, Isachenko V, Sánchez R. Human sperm vitrification: A scientific report. Andrology. 2020; 8(6):1642–50.
8. Eastick J, Venetis C, Cooke S, Storr A, Susetio D, Chapman M. Is early embryo development as observed by time-lapse microscopy dependent on whether fresh or frozen sperm was used for ICSI? A cohort study. J Assist Reprod Genet. 2017; 34(6):733–40.
9. Vicdan K, Akarsu C, Sözen E, Buluç B, Vicdan A, Yılmaz Y, et al. Outcome of intracytoplasmic sperm injection using fresh and cryopreserved-thawed testıcular spermatozoa in 83 azoospermic men with Klinefelter syndrome. J Obstet Gynaecol Res. 2016; 42(11):1558–66.
10. Kopeika J, Thornhill A, Khalaf Y. The effect of cryopreservation on the genome of gametes and embryos: Principles of cryobiology and critical appraisal of the evidence. Hum Reprod Update. 2015; 21(2):209–27.
11. Isachenko V, Isachenko E, Petrunkina AM, Sanchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: Birth of two healthy babies. Reprod Fertil Dev. 2012; 24:323–6.
12. Sanchez R, Isachenko V, Petrunkina AM, Risopatron J, Schulz M, Isachenko E. Live birth after intrauterine insemination with spermatozoa from an oligoasthenozoospermic patient vitrified without permeable cryoprotectants. J Androl. 2012; 33(4):559–62.
13. Le MT, Nguyen TTT, Nguyen TT, Nguyen VT, Nguyen TTA, Nguyen VQH, et al. Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects. Eur J Obstet Gynecol Reprod Biol. 2019; 234:14–20.
14. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: Towards a single blastocyst transfer. Fertil Steril. 2000; 73(6):1155-8.
15. Lê Minh Tâm, Nguyễn Văn Trung, Nguyễn Thị Thái Thanh, Đặng Thị Hồng Nhạn. Chương 4. Kỹ thuật nuôi cấy, đánh giá và các can thiệp trên phôi. Các vấn đề trọng yếu trong Hỗ trợ Sinh sản. Tập 2: Dành cho Phôi học lâm sàng. Nhà xuất bản Y học; 2022; 274–355.
16. Luyet BJ, Hodapp EL. Revival of Frog’s Spermatozoa Vitrified in Liquid Air. Exp Biol Med. 1938; 39(3):433–4.
17. Bunge RG, Sherman JK. Fertilizing capacity of frozen human spermatozoa. Nature. 1953; 172(4382):767–8.
18. Mohamed MSA. Slow cryopreservation is not superior to vitrification in human spermatozoa; An experimental controlled study. Iran J Reprod Med. 2015; 13(10):633-44.
19. Pabón D, Meseguer M, Sevillano G, Cobo A, Romero JL, Remohí J, et al. A new system of sperm cryopreservation: evaluation of survival, motility, DNA oxidation, and mitochondrial activity. Andrology. 2019; 7(3):293–301.
20. Spis E, Bushkovskaia A, Isachenko E, Todorov P, Sanchez R, Skopets V, et al. Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births. Cryobiology. 2019; 90:100–2.
21. Endo Y, Fujii Y, Shintani K, Seo M, Motoyama H, Funahashi H. Simple vitrification for small numbers of human spermatozoa. Reprod Biomed Online. 2012; 24(3):301–7.
22. Ohno M, Tanaka A, Nagayoshi M, Yamaguchi T, Takemoto Y, Tanaka I, et al. Modified permeable cryoprotectant-free vitrification method for three or fewer ejaculated spermatozoa from cryptozoospermic men and 7-year follow-up study of 14 children born from this method. Hum Reprod. 2020; 35(5):1019-28.
23. Lê Minh Tâm, Nguyễn Văn Trung, Nguyễn Thị Thái Thanh, Đặng Thị Hồng Nhạn. Chương 5. Kỹ thuật trữ lạnh trong hỗ trợ sinh sản. Các vấn đề trọng yếu trong Hỗ trợ Sinh sản. Tập 2: Dành cho Phôi học lâm sàng. 2022; 335–95.
2. Sánchez R, Schulz M, Risopatrón J, Isachenko V, Isachenko E. Vitrification sperm: An alternative to intracytoplasmic sperm injection in oligoasthenozoospermic patient. Rev Int Androl. 2013; 11:36–9.
3. Medrano L, Enciso M, Gomez-Torres MJ, Aizpurua J. First birth of a healthy infant following intra-cytoplasmic sperm injection using a new permeable cryoprotectant-free sperm vitrification protocol. Cryobiology. 2019; 87:117–9.
4. Li Y, Zhou L, Lv M, Ge P, Liu Y, Zhou D. Vitrification and conventional freezing methods in sperm cryopreservation: A systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2019; 233:84–92.
5. Hezavehei M, Sharafi M, Kouchesfahani HM, Henkel R, Agarwal A, Esmaeili V, et al. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reprod Biomed Online. 2018; 37(3):327–39.
6. Patel K, Sharma N, Mishra V, Aggarwal R, Suthar A, Sheth H. Comparison between the outcome of sperm vitrification protocol and conventional slow freezing protocol for semen cryopreservation. Hum Reprod. 2021; 36(Supplement_1).
7. Schulz M, Risopatrón J, Uribe P, Isachenko E, Isachenko V, Sánchez R. Human sperm vitrification: A scientific report. Andrology. 2020; 8(6):1642–50.
8. Eastick J, Venetis C, Cooke S, Storr A, Susetio D, Chapman M. Is early embryo development as observed by time-lapse microscopy dependent on whether fresh or frozen sperm was used for ICSI? A cohort study. J Assist Reprod Genet. 2017; 34(6):733–40.
9. Vicdan K, Akarsu C, Sözen E, Buluç B, Vicdan A, Yılmaz Y, et al. Outcome of intracytoplasmic sperm injection using fresh and cryopreserved-thawed testıcular spermatozoa in 83 azoospermic men with Klinefelter syndrome. J Obstet Gynaecol Res. 2016; 42(11):1558–66.
10. Kopeika J, Thornhill A, Khalaf Y. The effect of cryopreservation on the genome of gametes and embryos: Principles of cryobiology and critical appraisal of the evidence. Hum Reprod Update. 2015; 21(2):209–27.
11. Isachenko V, Isachenko E, Petrunkina AM, Sanchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: Birth of two healthy babies. Reprod Fertil Dev. 2012; 24:323–6.
12. Sanchez R, Isachenko V, Petrunkina AM, Risopatron J, Schulz M, Isachenko E. Live birth after intrauterine insemination with spermatozoa from an oligoasthenozoospermic patient vitrified without permeable cryoprotectants. J Androl. 2012; 33(4):559–62.
13. Le MT, Nguyen TTT, Nguyen TT, Nguyen VT, Nguyen TTA, Nguyen VQH, et al. Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects. Eur J Obstet Gynecol Reprod Biol. 2019; 234:14–20.
14. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: Towards a single blastocyst transfer. Fertil Steril. 2000; 73(6):1155-8.
15. Lê Minh Tâm, Nguyễn Văn Trung, Nguyễn Thị Thái Thanh, Đặng Thị Hồng Nhạn. Chương 4. Kỹ thuật nuôi cấy, đánh giá và các can thiệp trên phôi. Các vấn đề trọng yếu trong Hỗ trợ Sinh sản. Tập 2: Dành cho Phôi học lâm sàng. Nhà xuất bản Y học; 2022; 274–355.
16. Luyet BJ, Hodapp EL. Revival of Frog’s Spermatozoa Vitrified in Liquid Air. Exp Biol Med. 1938; 39(3):433–4.
17. Bunge RG, Sherman JK. Fertilizing capacity of frozen human spermatozoa. Nature. 1953; 172(4382):767–8.
18. Mohamed MSA. Slow cryopreservation is not superior to vitrification in human spermatozoa; An experimental controlled study. Iran J Reprod Med. 2015; 13(10):633-44.
19. Pabón D, Meseguer M, Sevillano G, Cobo A, Romero JL, Remohí J, et al. A new system of sperm cryopreservation: evaluation of survival, motility, DNA oxidation, and mitochondrial activity. Andrology. 2019; 7(3):293–301.
20. Spis E, Bushkovskaia A, Isachenko E, Todorov P, Sanchez R, Skopets V, et al. Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births. Cryobiology. 2019; 90:100–2.
21. Endo Y, Fujii Y, Shintani K, Seo M, Motoyama H, Funahashi H. Simple vitrification for small numbers of human spermatozoa. Reprod Biomed Online. 2012; 24(3):301–7.
22. Ohno M, Tanaka A, Nagayoshi M, Yamaguchi T, Takemoto Y, Tanaka I, et al. Modified permeable cryoprotectant-free vitrification method for three or fewer ejaculated spermatozoa from cryptozoospermic men and 7-year follow-up study of 14 children born from this method. Hum Reprod. 2020; 35(5):1019-28.
23. Lê Minh Tâm, Nguyễn Văn Trung, Nguyễn Thị Thái Thanh, Đặng Thị Hồng Nhạn. Chương 5. Kỹ thuật trữ lạnh trong hỗ trợ sinh sản. Các vấn đề trọng yếu trong Hỗ trợ Sinh sản. Tập 2: Dành cho Phôi học lâm sàng. 2022; 335–95.
1. Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008; 136(2):167–73.
2. Sánchez R, Schulz M, Risopatrón J, Isachenko V, Isachenko E. Vitrification sperm: An alternative to intracytoplasmic sperm injection in oligoasthenozoospermic patient. Rev Int Androl. 2013; 11:36–9.
3. Medrano L, Enciso M, Gomez-Torres MJ, Aizpurua J. First birth of a healthy infant following intra-cytoplasmic sperm injection using a new permeable cryoprotectant-free sperm vitrification protocol. Cryobiology. 2019; 87:117–9.
4. Li Y, Zhou L, Lv M, Ge P, Liu Y, Zhou D. Vitrification and conventional freezing methods in sperm cryopreservation: A systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2019; 233:84–92.
5. Hezavehei M, Sharafi M, Kouchesfahani HM, Henkel R, Agarwal A, Esmaeili V, et al. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reprod Biomed Online. 2018; 37(3):327–39.
6. Patel K, Sharma N, Mishra V, Aggarwal R, Suthar A, Sheth H. Comparison between the outcome of sperm vitrification protocol and conventional slow freezing protocol for semen cryopreservation. Hum Reprod. 2021; 36(Supplement_1).
7. Schulz M, Risopatrón J, Uribe P, Isachenko E, Isachenko V, Sánchez R. Human sperm vitrification: A scientific report. Andrology. 2020; 8(6):1642–50.
8. Eastick J, Venetis C, Cooke S, Storr A, Susetio D, Chapman M. Is early embryo development as observed by time-lapse microscopy dependent on whether fresh or frozen sperm was used for ICSI? A cohort study. J Assist Reprod Genet. 2017; 34(6):733–40.
9. Vicdan K, Akarsu C, Sözen E, Buluç B, Vicdan A, Yılmaz Y, et al. Outcome of intracytoplasmic sperm injection using fresh and cryopreserved-thawed testıcular spermatozoa in 83 azoospermic men with Klinefelter syndrome. J Obstet Gynaecol Res. 2016; 42(11):1558–66.
10. Kopeika J, Thornhill A, Khalaf Y. The effect of cryopreservation on the genome of gametes and embryos: Principles of cryobiology and critical appraisal of the evidence. Hum Reprod Update. 2015; 21(2):209–27.
11. Isachenko V, Isachenko E, Petrunkina AM, Sanchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: Birth of two healthy babies. Reprod Fertil Dev. 2012; 24:323–6.
12. Sanchez R, Isachenko V, Petrunkina AM, Risopatron J, Schulz M, Isachenko E. Live birth after intrauterine insemination with spermatozoa from an oligoasthenozoospermic patient vitrified without permeable cryoprotectants. J Androl. 2012; 33(4):559–62.
13. Le MT, Nguyen TTT, Nguyen TT, Nguyen VT, Nguyen TTA, Nguyen VQH, et al. Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects. Eur J Obstet Gynecol Reprod Biol. 2019; 234:14–20.
14. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: Towards a single blastocyst transfer. Fertil Steril. 2000; 73(6):1155-8.
15. Lê Minh Tâm, Nguyễn Văn Trung, Nguyễn Thị Thái Thanh, Đặng Thị Hồng Nhạn. Chương 4. Kỹ thuật nuôi cấy, đánh giá và các can thiệp trên phôi. Các vấn đề trọng yếu trong Hỗ trợ Sinh sản. Tập 2: Dành cho Phôi học lâm sàng. Nhà xuất bản Y học; 2022; 274–355.
16. Luyet BJ, Hodapp EL. Revival of Frog’s Spermatozoa Vitrified in Liquid Air. Exp Biol Med. 1938; 39(3):433–4.
17. Bunge RG, Sherman JK. Fertilizing capacity of frozen human spermatozoa. Nature. 1953; 172(4382):767–8.
18. Mohamed MSA. Slow cryopreservation is not superior to vitrification in human spermatozoa; An experimental controlled study. Iran J Reprod Med. 2015; 13(10):633-44.
19. Pabón D, Meseguer M, Sevillano G, Cobo A, Romero JL, Remohí J, et al. A new system of sperm cryopreservation: evaluation of survival, motility, DNA oxidation, and mitochondrial activity. Andrology. 2019; 7(3):293–301.
20. Spis E, Bushkovskaia A, Isachenko E, Todorov P, Sanchez R, Skopets V, et al. Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births. Cryobiology. 2019; 90:100–2.
21. Endo Y, Fujii Y, Shintani K, Seo M, Motoyama H, Funahashi H. Simple vitrification for small numbers of human spermatozoa. Reprod Biomed Online. 2012; 24(3):301–7.
22. Ohno M, Tanaka A, Nagayoshi M, Yamaguchi T, Takemoto Y, Tanaka I, et al. Modified permeable cryoprotectant-free vitrification method for three or fewer ejaculated spermatozoa from cryptozoospermic men and 7-year follow-up study of 14 children born from this method. Hum Reprod. 2020; 35(5):1019-28.
23. Lê Minh Tâm, Nguyễn Văn Trung, Nguyễn Thị Thái Thanh, Đặng Thị Hồng Nhạn. Chương 5. Kỹ thuật trữ lạnh trong hỗ trợ sinh sản. Các vấn đề trọng yếu trong Hỗ trợ Sinh sản. Tập 2: Dành cho Phôi học lâm sàng. 2022; 335–95.
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