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  • Print publication year: 2010
  • Online publication date: July 2010

Chapter 15 - Cryopreservation of oocytes by slow cooling

from Section 4 - Cryopreservation of oocytes


Sperm cryopreservation plays an important role in the field of male infertility and reproductive medicine. A donor sperm cryopreservation program has been developed and improved in mainland China. The conventional approach to sperm cryopreservation is to simply dilute semen with cryoprotectant and cryopreserve in liquid nitrogen until the sperm samples are thawed for use. Patients with spinal cord injuries often have a problem with poor sperm production as well as ejaculation after the damage. Electroejaculation is usually performed under a general anesthesia while the patient is placed in lateral decubitus. With the advance of new approaches for sperm vitrification, treatment of male infertility will become more effective without using sperm donors. Using vitrification for cryopreservation of sperm obtained from testicular biopsy, epididymal fluid, or a semen sample after electroejaculation could create new hope for infertile men.


1. ChenC. Pregnancy after human oocyte cryopreservation. Lancet 1986; i: 884–886.
2. Al-HasaniS, DiedrichK, van der VenH, KrebsD. [Initial results of the cryopreservation of human oocytes]. Geburtshilfe Frauenheilkd 1986; 46: 643–644.
3. WhittinghamDG. Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at −196 degrees C. J Reprod Fertil 1977; 49: 89–94.
4. PorcuE, FabbriR, SeracchioliR, et al. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997; 68: 724–726.
5. BoriniA, BonuMA, CoticchioG, et al. Pregnancies and births after oocyte cryopreservation. Fertil Steril 2004; 82: 601–605.
6. PaynterSJ, O ’ NeilL, FullerBJ, ShawRW. Membrane permeability of human oocytes in the presence of the cryoprotectant propane-1,2-diol. Fertil Steril 2001; 75: 532–538.
7. FabbriR, PorcuE, MarsellaT, et al. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001; 16: 411–416.
8. BianchiV, CoticchioG, DistratisV, et al. Differential sucrose concentration during dehydration (0.2 mol/l) and rehydration (0.3 mol/l) increases the implantation rate of frozen human oocytes. Reprod Biomed Online 2007; 14: 64–71.
9. BoriniA, SciajnoR, BianchiV, et al. Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration. Hum Reprod 2006; 21: 512–517.
10. De SantisL, CinoI, RabellottiE, et al. Oocyte cryopreservation: clinical outcome of slow-cooling protocols differing in sucrose concentration. Reprod Biomed Online 2007; 14: 57–63.
11. Levi SettiPE, AlbaniE, NovaraPV, CesanaA, MorrealeG. Cryopreservation of supernumerary oocytes in IVF/ICSI cycles. Hum Reprod 2006; 21: 370–375.
12. PaynterSJ, BoriniA, BianchiV, et al. Volume changes of mature human oocytes on exposure to cryoprotectant solutions used in slow cooling procedures. Hum Reprod 2005; 20: 1194–1199.
13. BoriniA, BianchiV, BonuMA, et al. Evidence-based clinical outcome of oocyte slow cooling. Reprod Biomed Online 2007; 15: 175–181.
14. CoboA, KuwayamaM, PerezS, et al. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 2008; 89: 1657–1664.
15. CoboA, BellverJ, DomingoJ, et al. New options in assisted reproduction technology: the Cryotop method of oocyte vitrification. Reprod Biomed Online 2008; 17: 68–72.
16. De SantisL, CoticchioG, PaynterS, et al. Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation. Hum Reprod 2007; 22: 2776–2783.
17. KasaiM, MukaidaT. Cryopreservation of animal and human embryos by vitrification. Reprod Biomed Online 2004; 9: 164–170.
18. NottolaSA, CoticchioG, de SantisL, et al. Ultrastructure of human mature oocytes after slow cooling cryopreservation with ethylene glycol. Reprod Biomed Online 2008; 17: 368–377.
19. StacheckiJJ, CohenJ, WilladsenS. Detrimental effects of sodium during mouse oocyte cryopreservation. Biol Reprod 1998; 59: 395–400.
20. StacheckiJJ, CohenJ, WilladsenSM. Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology 1998; 37: 346–354.
21. StacheckiJJ, WilladsenSM. Cryopreservation of mouse oocytes using a medium with low sodium content: effect of plunge temperature. Cryobiology 2000; 40: 4–12.
22. StacheckiJJ, CohenJ, GarrisiJ, et al. Cryopreservation of unfertilized human oocytes. Reprod Biomed Online 2006; 13: 222–227.
23. BoldtJ, TidswellN, SayersA, KilaniR, ClineD. Human oocyte cryopreservation: 5-year experience with a sodium-depleted slow freezing method. Reprod Biomed Online 2006; 13: 96–100.
24. QuintansCJ, DonaldsonMJ, BertolinoMV, PasqualiniRS. Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium. Hum Reprod 2002; 17: 3149–3152.
25. PetraccoA, AzambujaR, OkadaL, et al. Comparison of embryo quality between sibling embryos originating from frozen or fresh oocytes. Reprod Biomed Online 2006; 13: 497–503.
26. ZiebeS, PetersenK, LindenbergS, et al. Embryo morphology or cleavage stage: how to select the best embryos for transfer after in-vitro fertilization. Hum Reprod 1997; 12: 1545–1549.
27. EdgarDH, ArcherJ, McBainJ, BourneH. Embryonic factors affecting outcome from single cryopreserved embryo transfer. Reprod Biomed Online 2007; 14: 718–723.
28. CoticchioG, SereniE, SerraoL, et al. What criteria for the definition of oocyte quality?Ann New York Acad Sci 2004; 1034: 132–144.
29. NagyZP, ChangCC, ShapiroDB, et al. Clinical evaluation of the efficiency of an oocyte donation program using egg cryo-banking. Fertil Steril 2009; 92: 520–526.
30. NottolaSA, MacchiarelliG, CoticchioG, et al. Ultrastructure of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations. Hum Reprod 2007; 22: 1123–1133.
31. GookDA, OsbornSM, JohnstonWI. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993; 8: 1101–1109.
32. GhetlerY, SkutelskyE, Ben NunI, et al. Human oocyte cryopreservation and the fate of cortical granules. Fertil Steril 2006; 86: 210–216.
33. GualtieriR, IaccarinoM, MolloV, et al. Slow cooling of human oocytes: ultrastructural injuries and apoptotic status. Fertil Steril 2009; 91: 1023–1034.
34. FukuE, XiaL, DowneyBR. Ultrastructural changes in bovine oocytes cryopreserved by vitrification. Cryobiology 1995; 32: 139–156.
35. HochiS, KozawaM, FujimotoT, et al. In vitro maturation and transmission electron microscopic observation of horse oocytes after vitrification. Cryobiology 1996; 33: 300–310.
36. CoticchioG, DistratisV, BoriniA, et al. Reduced ultrastructural damage in human oocytes cryopreserved after dehydration involving single-step propanediol exposure. Hum Reprod 2008; 23(Suppl 1): i150.
37. NottolaSA, CoticchioG, SciajnoR, et al. Ultrastructural changes in human metaphase II oocytes cryopreserved by vitrification. Hum Reprod 2008; 23(Suppl 1): i34.
38. PickeringSJ, BraudePR, JohnsonMH, CantA, CurrieJ. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54: 102–108.
39. CoticchioG, de SantisL, RossiG, et al. Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configurations after slow-cooling cryopreservation. Hum Reprod 2006; 21: 1771–1776.
40. CoboA, PerezS, de los SantosMJ, ZulateguiJ, DomingoJ, RemohiJ. Effect of different cryopreservation protocols on the metaphase II spindle in human oocytes. Reprod Biomed Online 2008; 17: 350–359.
41. WangWH, MengL, HackettRJ, KeefeDL. Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination. Hum Reprod 2001; 16: 1464–1468.
42. WangWH, MengL, HackettRJ, OdenbourgR, KeefeDL. The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril 2001; 75: 348–353.
43. CookeS, TylerJP, DriscollGL. Meiotic spindle location and identification and its effect on embryonic cleavage plane and early development. Hum Reprod 2003; 18: 2397–2405.
44. MoonJH, HyunCS, LeeSW, et al. Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI. Hum Reprod 2003; 18: 817–820.
45. RienziL, UbaldiF, MartinezF, et al. Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI. Hum Reprod 2003; 18: 1289–1293.
46. de SantisL, CinoI, RabellottiE, et al. Polar body morphology and spindle imaging as predictors of oocyte quality. Reprod Biomed Online 2005; 11: 36–42.
47. Rama RajuGA, PrakashGJ, KrishnaKM, MadanK. Meiotic spindle and zona pellucida characteristics as predictors of embryonic development: a preliminary study using Polscope imaging. Reprod Biomed Online 2007; 14: 166–174.
48. ShenY, StalfT, MehnertC, et al. Light retardance by human oocyte spindle is positively related to pronuclear score after ICSI. Reprod Biomed Online 2006; 12: 737–751.
49. MadaschiC, de SouzaBonetti TC, de Almeida Ferreira Braga DP, et al. Spindle imaging: a marker for embryo development and implantation. Fertil Steril 2008; 90: 194–198.
50. BianchiV, CoticchioG, FavaL, FlamigniC, BoriniA. Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration. Hum Reprod 2005; 20: 1078–1083.
51. RienziL, MartinezF, UbaldiF, et al. Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures. Hum Reprod 2004; 19: 655–659.
52. CoticchioG, SciajnoR, HuttKJ, et al. Comparative analysis of the metaphase II spindle of human oocytes through polarized light and high performance confocal microscopy. Fertil Steril 2009; epub ahead pf print.
53. BarrittJ, LunaM, DukeM, et al. Report of four donor-recipient oocyte cryopreservation cycles resulting in high pregnancy and implantation rates. Fertil Steril 2007; 87: e13–17.
54. La SalaGB, NicoliA, VillaniMT, et al. Outcome of 518 salvage oocyte-cryopreservation cycles performed as a routine procedure in an in vitro fertilization program. Fertil Steril 2006; 86: 1423–1427.
55. ChenSU, LienYR, ChenHF, et al. Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI. Hum Reprod 2005; 20: 1975–1980.
56. LucenaE, BernalDP, LucenaC, et al. Successful ongoing pregnancies after vitrification of oocytes. Fertil Steril 2006; 85: 108–111.
57. GookDA, EdgarDH. Cryopreservation of the human female gamete: current and future issues. Hum Reprod 1999; 14: 2938–2940.
58. LassalleB, TestartJ, RenardJP. Human embryo features that influence the success of cryopreservation with the use of 1,2-propanediol. Fertil Steril 1985; 44: 645–651.
59. ParmegianiL, CognigniGE, BernardiS, et al. Freezing within 2 h from oocyte retrieval increases the efficiency of human oocyte cryopreservation when using a slow freezing/rapid thawing protocol with high sucrose concentration. Hum Reprod 2008; 23: 1771–1778.
60. DucibellaT. Biochemical and cellular insights into the temporal window of normal fertilization. Theriogenology 1998; 49: 53–65.
61. BoriniA, CattoliM, BullettiC, CoticchioG. Clinical efficiency of oocyte and embryo cryopreservation. Ann New York Acad Sci 2008; 1127: 49–58.
62. PorcuE, FabbriR, DamianoG, et al. Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 2000; 169: 33–37.
63. BoriniA, CattoliM, MazzoneS, et al. Survey of 105 babies born after slow-cooling oocyte cryopreservation. Fertil Steril 2007; 88: S13–S14.
64. Tur-KaspaI, GalM, HorwitzA. Genetics and health of children born from cryopreserved oocytes. Fertil Steril 2007; 87: S14.
65. MaherB. Little consensus on egg freezing. Nature 2007; 449: 958.