By John G. Wilcox, MD, FACOG Board Certified, Reproductive Endocrinology and Infertility
Human oocyte cryopreservation, or “egg freezing,” has long been an elusive goal of cryobiologists. Although sperm and embryos (fertilized eggs) have been successfully frozen and subsequently thawed to create healthy children for decades, egg freezing has only recently become a successful reality. The cryopreservation of human oocytes is highly beneficial for several reasons, most importantly to preserve a woman’s fertility. Oocyte storage allows: (i) women at risk of becoming sterile due to cancer to preserve their oocytes prior to radio- or chemotherapy or ovariectomy; (ii) the salvage of an in-vitro fertilization (IVF) cycle when no sperm is available; (iii) the alleviation of religious and ethical concerns of embryo storage; (iv) the elimination of donor-recipient synchronization problems; (v) a “quarantine period” on donated oocytes similar to that of donated semen; and (vi) women to delay reproduction until later in life, providing them with more reproductive choices.
The theoretical basis for cryopreservation of cells was first proposed in 1965 (Mazur, 1965). These theories were not tested, however, until 1972 resulting in the first births of mice from cryopreserved embryos (Whittingham et al., 1972). In 1976, the first successful birth of mice from frozen-thawed oocytes was reported (Parkening et al., 1976). More recently, a review of the literature reported embryos from at least 22 other mammalian species have now been successfully cryopreserved (Rall, 2001; Leibo et al., 2002). In the early eighties, the first human pregnancy (Trounson et al., 1983) and births (Zeilmaker et al., 1984) were reported from cryopreserved human embryos. The freezing and storage of human embryos is now standard practice in clinical invitro fertilization (IVF) laboratories around the world (Shaw et al., 1993). According to the CDC website, 5,047 children were born in the year 2001 alone from the transfer of frozen-thawed human embryos (Wright et al., 2004).
The first report of a pregnancy from a frozen-thawed human oocyte was in 1986 (Chen, 1986). Until recently, progress with frozen oocytes was slow with few births reported (Al-Hasani et al., 1987; Van Uem et al., 1987; Chen, 1988). This was due mainly to two factors: concerns of a higher incidence of chromosomal abnormalities in the cryopreserved oocyte as compared to fresh oocytes (Johnson et al., 1987; Pickering et al., 1987; Sathananthan et al., 1988), and poor fertilization due to “zona hardening” (Vincent et al., 1990; Wood et al., 1992; George et al., 1993). Following the development of intracytoplasmic sperm injection (ICSI) to assist fertilization following cryopreservation of oocytes in the 1990’s, significant progress has been reported in cryopreservation of oocytes (Gook et al., 1995; Kazem et al.,1995). Subsequent research demonstrated cryopreservation of oocytes was not as detrimental as previously believed (Gook et al., 1993,1994). By 2000, there were over 20 normal children born
from frozen-thawed oocytes (Tucker et al.,1996, 1998; Porcu et al.,1997, 1998, 1999a,b; Antinori et al.,1998; Borini et al., 1998; Nawroth et al.,1998; Polk de Fried et al.,1998; Vidali et al.,1998;Yang et al.,1998,1999;Young et al.,1998; Porcu,1999). Currently, there are approximately 100 children reported following oocyte freezing (Leibo, 2003; Stachecki et al., 2004).
Slow freezing is the most reported method for cryopreservation of oocytes, resulting in the vast majority of babies born worldwide. Slow freezing methods, with only slight modifications, are based on the procedure first described by Willadsen (1977) for the freezing of ovine and bovine embryos. The equilibration time and amount of permeating cryoprotective additive (CPA) as well as the concentration of sucrose are among most common changes between various protocols (Fabbri et al., 2001). Recent alterations include the use of low or sodium free freezing solutions (Azambuja et al., 2002; Quintans et al., 2002; Boldt et al., 2003). However, the basic principles remain the same. Oocytes are suspended in a solution containing CPAs, cooled and then seeded to induce ice formation promoting dehydration of the oocyte as the solution is cooled slowly at 0.3° C/min to -30° C or below. Finally, the oocyte is plunged into liquid nitrogen solution for storage
Ultra Rapid Freezing (Vitrification)
Vitrification, or achieving a glass-like state, represents a potential alternative to slow freezing (first described in 1985 (Rall et al., 1985)). Since chilling injury to oocytes is time dependent, the rationale is to prevent ice formation and injury by freezing at a rate fast enough to solidify the intracellular water before it can crystallize (Martino et al., 1996). This is accomplished by exposing the cell to high concentrations of CPAs for a very short equilibration followed by very rapid cooling by plunging into liquid nitrogen. The high osmolarity of the vitrification solution rapidly dehydrates the cell and the submersion into liquid nitrogen quickly solidifies the cell before the remaining intracellular water has time to form damaging ice crystals. Two important concerns with this technique are the increased toxicity of high levels of CPAs at room temperature (Shaw et al., 1992) and the ability to freeze and thaw fast enough to avoid crystal formation and
devitrification (Vajta et al., 1998). Several recent reports describe their ability to overcome these issues resulting in healthy children from vitrified oocytes (Kuleshova et al., 1999; Kuwayama et al., 2000; Yoon et al., 2000, 2003; Katayama et al., 2003).
Over the past nine years, the cryopreservation of more than 5,000 human oocytes have been described in over 20 articles and abstracts resulting in over 100 births (summarized in reviews: Leibo, 2004; Stachecki et al., 2004). However, these numbers are conservative and anecdotal evidence points to over 200 healthy live births to date. Extrapolating from reports, estimations of the average oocyte survival rates, fertilization rates, and clinical pregnancy rates are 54%, 61%, and 35% respectively (Kazem et al., 1995; Gook et al., 1995; Tucker et al., 1996, 1998; Porcu et al., 1997, 1999, 2000; Polak de Fried et al., 1998; Young et al., 1998; Kulehova et al., 1999; Hong et al., 1999; Yoon et al., 2000, 2003; Fabbri et al., 2001; Winslow et al., 2001; Yang et al., 2002; Boldt et al., 2003; Katayama et al., 2003; Fosas et al., 2003; Borini et al., 2004). In May 2005, an abstract presented by HRC at the annual Pacific Coast Reproductive Society demonstrated human oocyte
survival and fertilization rates as high as 79% and 81% respectively. Also, in June 2005, Porcu presented his experience with over 500 cycles at the European Society for Human Reproduction and Embryology (ESHRE). His results supported pregnancy success rates with cryopreserved oocytes that were comparable to cryopreserved embryos.
HRC Fertility Oocyte Cryopreservation Program
HRC Fertility is a medical practice committed to furthering the advancement of oocyte cryopreservation research and providing services to women in need of preserving their fertility due to cancer, age, or other medical conditions.
Experience & Innovation
HRC has partnered with five premier medical centers across the U.S., including Stanford University’s Reproductive Endocrinology and Infertility program (Northern CA), Reproductive Medicine Associates of New York (NY), IVF New Jersey (NJ), Reproductive Science Center (MA) and Texas Fertility Center (TX), to further research of oocyte cryopreservation through multi-center clinical trials. HRC provides state of the art technology for women interested in cryopreservation of oocytes. An IRB protocol has been established to continue researching new techniques for cryopreserving oocytes. Any woman under the age of 38 can be screened to determine whether she is a candidate to cryopreserve oocytes for future reproduction.
- A aAl-Hasini S, Diedrich K, van der Ven H et al. 1987 Cryopreservation of human oocytes. Human Reproduction 2, 695-700
- Antinori S, Dani G, Selman H et al. 1998 Pregnancy after sperm injection into cryopreserved human oocytes. Human Reproduction 13, 157-158.
- Azambuja R, Stachecki J, Badalotti M et al. 2002 Nascimento de feto a termo apos de oocitos. Journal Brasileiro De Reproducao Assistida 6, 199-121.
- Behr B, Le A, Khoury C, Boostanfar B, Feinman M, Frederick J. 2005 Comparison between two different human oocyte cryopreservation protocols on survival, fertilization and embryo development. Abstract, Presented at Pacific Coast Reproductive Society annual meeting, May 2005.
- Boldt J, Cline D, McLaughlin D 2003 Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles. Human Reproduction 18, 1250-1255.
- Borini A, Baffaro M, Bonu M et al. 1998 Pregnancies after freezing and thawing human oocytes. Preliminary data. Human Reproduction 13, 124-125.
- Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M, Flamigni C. 2004 Pregnancies and births after oocyte cryopreservation. Fertility and Sterility 82(3):
- Chen C 1986 Pregnancy after human oocyte cryopreservation. Lancet 1, 884-886.
- Fabbri R, Porcu E, Marsella T et al. 2001 Human oocyte cryopreservation: new perspectives regarding oocyte survival. Human Reproduction 16, 411-416.
- George MA, Johnson MH 1993 Use of fetal bovine serum substitutes for the protection of the mouse zona pellucida against hardening during cryoprotectant
addition. Human Reproduction 8, 1898-1900.
- Gook DA, Osborn SM, Johnson WI 1993 Cryopreservation of mouse and human oocytes using 1,2 propanediol and the configuration of the meiotic spindle. Human Reproduction 8, 1101-1109.
- Gook DA, Osborn SM, Bourne H et al. 1994 Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Human Reproduction 9, 684-691.
- Katayama KP, Stehlik J, Kuwayama M, et al. 2003 High survival rate of vitrified human oocytes results in clinical pregnancy. Fertility and Sterility 80, 223-224.
- Kazem R, Thompson LA, Srikantharajah A et al. 1995 Cryopreservation of human oocytes and fertilization by two techniques: in-vitro fertilization and intracyoplasmic sperm injection. Human Reproduction 10, 2650-2654.
- Kuleshova L, Gianaroli L, Magli C et al. 1999a Birth following vitrification of a small number of human oocytes: case report. Human Reproduction 14, 3077-3079.
- Leibo SP, Songsasen N. Cryopreservation of gametes and embryos of non-domestic species. Theriogenology 2002;57:303-326.
- Leibo SP. Cryobiology: preservation of mammalian embryos. In: Genetic Engineering of Animals. JW Evans, A.
- Mazur P 1965 Causes of injury in frozen and thawed cells. Federation Proceedings 24, 175-182.
- Parkening TA, Tsunoda Y, Chang MC. 1976 Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs. J Exp Zool 197:369-374.
- Pickering SJ, Brude PR, Johnson MH et al. 1990 Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocytes. Fertility and Sterility 54, 102-108.
- Polak de Fried E, Notrica J, Robinstein M et al. Pregnancy after human donor oocyte cryopreservation and thawing in association with Intracytoplasmic sperm injection into a patient with ovarian failure. Fertility and Sterility 69, 555-557.
- Porcu E, Fabbri R, Seracchioli R et al. 1997 Birth of a healthy female after intracyoplasmic sperm injection of cryopreserved human oocytes. Fertility and Sterility 68, 724-726.
- Porcu E, Fabbri R, Ciotti PM et al. 1999b Ongoing pregnancy after intracytoplasmic sperm injection of epididymal spermatozoa into cryopreserved human oocytes. Journal of Assisted Reproduction and Genetics 16, 283-285.
- Porcu E 2001 Oocyte freezing. Seminars in Reproductive Medicine 19, 221-230.
- Quintans CJ, Donaldson MJ, Bertolino MV et al. 2002 Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium. Human Reproduction 17, 3149-3152
- Rall WF, Fahy GM 1985 Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature 313, 573-575.
- Rall WF. Cryopreservation of mammalian embryos, gametes, and ovarian tissues. In Wolf DP, Zelinski-Wooten M, eds. Contemporary Endocrinology: Assisted Fertilization and Nuclear Transfer in Mammals. Towata, NJ: Human Press, 2001:173-87
- Shaw JM, Oranratnachai A, Trounson A. Cryopreservation of oocytes and embryos. In Trounson A, Gardner DK, eds. In Vitro Fertilization. Boca Raton, FL: CRC Press, 1993:213-236.
- Stachecki J, Cohen J 2004 An overview of oocyte cryopreservation. Reproductive BioMedicine Online 9, 152-163.
- Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryos. Fertility and Sterility 1984;42: 293-296.
- Tucker M, Wright G, Morton P et al. 1996 Preliminary experience with human oocyte cryopreservation using 1,2-propandiol and sucrose. Human Reproduction 11, 1513-1515.
- Tucker MJ, Wright G, Morton PC et al. 1998 Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertility and Sterility 70, 578-579.
- Vajta G, Holm P, Kuwayama M et al 1998 Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Molecular Reproduction and Development 51, 53-58.
- Van Uem JF, Siebzehnrubl ER, Schuh B et al. 1987 Birth after cryopreservation of unfertilized oocytes [letter] Lancet 1, 752-753.
- Vincent C, Pickering SJ, Johnson MH 1990 The hardening effect of dimethylsulphoxide on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present. Journal of Reproduction and Fertility 89, 253-259.
- Whittingham DG, Leibo SP, Mazur P. 1972 Survival of mouse embryos frozen to -196 and -269 C. Science 178: 411-414.
- Willadsen SM. Factors affecting the survival of sheep embryos during deep-freezing and thawing. In The Freezing of Mammalian Embryos. Ciba
Foundation, Symposium 52 (New Series). Amsterdam: Elsevier Excerpta Media/North Holland, Amsterdam, 1977:175-201.
- Wright VC, Schiere LA, Reynolds MA, Jeng G, Kissin D, Assisted Reproductive Technology Surveillance- United
- Yang D, Blohm P, Winslow L et al. 1998 A twin pregnancy after microinjection of human cryopreserved oocyte with a specially developed oocyte cryopreservation regime. Fertility and Sterility 70, S239.
- Yoon TK, Kim TJ, Park SE et al. 2003 Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program: freezing immature oocytes. Fertility and Sterility 79 1323-1326.
- Young E, Kenny A, Puigdomenech E et al. 1998 Triplet pregnancy after Intracytoplasmic sperm injection of cryopreserved oocytes: case report. Fertility and Sterility 70, 360-361.