Oocyte freezing: basics, current status and potential applications in reproductive biology

International Journal of Animal Biotechnology, Vol.1, No.1 (Dec. 2011) ISSN 2277-4122 General article Oocyte freezing: basics, current status and potential applications in reproductive biology S. K. Gautam a*, B. Singh a, V. Verma a, R.S. Manik a, M. S. Chauhan a a Animal Biotechnology Centre, National Dairy Research Institute, Karnal-136119 Accepted 15 Oct.2011, Available online 1 Dec. 2011 Abstract Over the past several decades, considerable effort has been expended toward the successful cryopreservation of various cells. While attempts at cryopreservation have been directed at different tissue types, one of the most vigorously pursued targets has been reproductive tissue. Historically, cryopreservation of sperm has existed for several decades. The earliest reports of pregnancies (Trounson et al., 1983) and births (Zeilmaker et al., 1984) from the cryopreservation of embryos occurred in the early 1980s. Presently, the freezing and storage of embryos following in vitro fertilization (IVF) is standard practice at most fertility clinics. In 2003, the CDC Assisted Reproductive Technology success rates report stated that 4,246 live births occurred out of 17,517 non-donor frozen embryo cycles.. Because the egg is a relatively voluminous cell with abundant cytoplasm, crystallization at the time of freezing may result in damage to the organelles. Secondly, a mature metaphase II oocyte contains a fragile spindle apparatus involved in cleavage. The purpose of this research study is to evaluate a method of freezing and thawing oocytes. This evaluation will be made by comparing the survival rates and rates of fertilization, cleavage and embryo quality of fresh oocytes and frozen-thawed oocytes which will be inseminated during the IVF (in vitro fertilization) treatment cycle. In addition, the same comparisons will be made between frozen oocytes from infertile women and those of egg donors. You are being asked to be in this study because you are currently undergoing in vitro fertilization Keywords: Oocyte, Freezing, Croprotectant 1. Introduction Cryobiology is the study of the effects of low temperatures on living organisms. People long believed that very low temperatures would only exert negative effects on cells and tissues. They could not possibly imagine the advancements in cryobiology to be achieved and possibilities of the future in this area. In the future, it may be possible to cryopreserve human cells, whole human organs, such as kidneys, hearts and livers for subsequent transplantation, preserve corneas and other delicate tissues with minimal damage long enough to allow them to be shared all over the world and protect fragile and rare plants from extinction through ice-free preservation. Before going further let us try to define the term freezing that is also known as cryopreservation. Cryopreservation may be defined as arresting all biological processes and placing biological material such as cells, eggs also known as oocytes, embryos, spermatozoa or tissues etc. into a suspended state of animation at ultra low temperatures. 1.1 The concept of oocyte Freezing Oocyte freezing has the potential to be an important adjunct to assisted reproductive technologies (ART) in humans and domestic animals. However, the ease and success of cryopreservation programmes for sperm and embryos contrast markedly with the problems associated with freezing mammalian oocytes. The results of numerous studies suggest that the survival of oocytes after cryopreservation can be affected by their stage of maturation, quality or by biophysical factors resulting from the cryopreservation procedure used. For example, the maturity, quality and size of the oocyte are particularly important characteristics affecting the outcome of cryopreservation. However, oocyte freezing has been slow to be adopted clinically as the number of oocytes which survive the freeze-thaw process is extremely variable and less than 1% of fertilized cryopreserved oocytes have developed to term. 1.2Basics of oocyte Freezing Developing protocols that optimize the survival, fertilization and developmental rates of oocytes following exposure to the extreme chemical and physical stresses associated with cryopreservation has proved to be a major challenge. Cryopreservation of biological specimens

causes complex changes in structure and cellular composition, and no single approach has yet proved to be universally effective. In addition, there are significant stage and species-specific differences between freezing oocytes. A number of parameters of normal oocyte physiology have been highlighted as potential targets for injury that may result from various cryopreservation methods. Changes in any of these parameters will contribute to loss of developmental competence of the stored oocytes through cellular injury or even to irreversible loss of viability during the cryopreservation process. 1.3 Cryopreservation Protocols Cryopreservation protocols for oocytes can be broadly classified as equilibrium (slow freezing) or nonequilibrium (vitrification and ultra rapid freezing) according to the cooling rates and cryoprotective agents (CPAs) used. Slow freezing is a method that requires a long slow controlled cooling before the sample is stored in liquid nitrogen (LN 2 ), a refrigerant commonly used for low temperature storage (-196 C). Vitrification, by contrast, enables rapid cooling of samples by direct plunging into LN 2 by use of a highly concentrated solution of cryoprotectants. However, the basic concepts of these two protocols are the same, as they both aim to protect the cells from the effects of intracellular ice crystal formation, cellular dehydration and drastic changes in solute concentrations at both high and low temperatures. 1.4 Slow freezing The slow freezing method (also known as programmable freezing) involves cooling the oocytes at a slow controlled rate before these are plunged and stored in LN 2. Slow freezing is a conventional equilibrium method using permeating cryoprotectants. In this method, the samples are suspended in 1-2 mol/l cryoprotectant dissolved in a physiological solution, ice seeded, and cooled very slowly so that the cellular contents become concentrated by gradual dehydration in response to the concentration of the extracellular unfrozen fraction during the growth of extracellular ice. The slow freezing method has the following distinctive steps: i.)the oocytes are exposed at room temperature to an appropriate concentration of a cryoprotectant until equilibrium is reached between the cryoprotectant solution and the oocytes. ii) The oocytes are loaded in 0.25 ml French plastic straw. iii) The straws are kept in the programmable freezing machine where the temperature is lowered @ 0.3 to 0.5 C/min to around - 5 C at a controlled fashion. iv) Seeding (induction of ice crystals) is carried out at 5 C to 7 C. v)the temperature is reduced to around 30 to -70 C at rate of around 0.2 to 2 C/min. vi) Finally the sample is plunged into LN 2 for storage. Fig. 1 Protocol of Cryopreservation 2. Conventional Vitrification Vitrification is a physical process by which a solution is transformed into a stable amorphous glass by rapid cooling bypassing ice crystal formation while maintaining the properties of a liquid in a solidified form. In most of the studies on vitrification of oocytes or embryos, a 0.25 ml straw is used. The straw is cooled by direct plunging in LN 2. In this case the cooling rate is as high as >2500 C/min. Vitrification method has the following distinctive steps: The oocytes are exposed at room temperature to a high concentration of a cryoprotectant until equilibrium is reached between the cryoprotectant solution and the oocytes. The oocytes are exposed to a high concentration of the cryoprotectant for a very brief period. Oocytes are loaded in French straws. In last, sample is plunged into LN 2 for storage. Though vitrification gives better result than slow freezing still it is associated with poor survivability rates when compared with the latest ultrarapid vitrification protocols. 2.1Ultrarapid Vitrification As a container for conventional vitrification, a plastic insemination straw (0.25 ml) is widely used. As the straw is relatively small (2.0 mm in outer diameter,13 cm long) when it is cooled by direct plunging in LN 2 and warmed by immersion in water (Thawing), the cooling and warming rate is as high as ~2500 C/min (between 25 and 175 C). Nevertheless, cells that are sensitive to chilling still suffer from chilling injury. An effective way of obtaining a much higher cooling rate and warming rate would be to minimize the volume of the solution and the container. In order to increase the survivability rate and handling oocytes with a minimal volume of solution, various methods have been devised. These include vitrification by open pulled straw (OPS), Cryoloop, microdropes, electron microscope grids etc. These are now a day followed widely in a lot of labs in the world. 108

1. Open pulled straw (OPS) Vitrification The insemination straws (0.25 ml) are heat softened and pulled manually until the inner diameter and the wall thickness of the central part is decreased from 1.7 to ~0.8 mm and from 0.15 to ~0.07 mm, respectively. Then the straws are cut at the narrowest point with a razor blade. Oocytes are loaded by means of the capillary effect by placing the narrow end in a drop of vitrification solution containing oocytes, and aspirating the solution in a 2 3 cm long column (1.0 1.5 L). Although it would be difficult to measure the precise rates of cooling and warming, it is estimated that the liquid column in OPS is cooled at 22, 500 0 C/min (between -25 and -175 0 C), whereas the rate in the conventional straw is 2500 0 C/min. 2. Cryoloop Vitrification Another strategy to increase the cooling rate is to use a refined system called the cryoloop, which consists of a minute nylon loop (20 m wide, 0.5 0.7 mm in diameter) mounted on a stainless steel pipe inserted into the lid of a cryovial. Oocytes are placed on loops that have been loaded with a thin film of vitrification solution, the amount of which is< 1 L. The cryovial is submerged in LN 2, the loop containing the oocytes is immersed in LN 2 in the cryovial, and then the cryovial is screw sealed. As the sample is enclosed in a cryovial, it can be labeled and stored easily. 3. Microdrops This approach is a completely containerless method called microdrops (or microdroplets). Originally, this method was reported by Landa and Tepla in 1990, without intending to increase the cooling/ warming rate. It reported that this method is more effective than conventional straw vitrification for bovine embryos at early cleavage stages, which are sensitive to chilling. With this method, oocytes suspended in vitrification solution are aspirated in a pipette and expelled onto the surface of LN 2 as a microdrop. Then, the 4 8 L microdrop vitrifies without any thermo insulating layers Advantage of ultrarapid vitrification One advantage of ultrarapid vitrification is that the formation of intracellular ice (will be discussed later on) can be prevented with a smaller amount of intracellular cryoprotectant. Therefore, the use of a lower concentration of the permeating cryoprotectant, thus a less toxic solution, is possible. In actual fact, the concentration of the permeating cryoprotectant in ultrarapid vitrification is lower than that adopted for conventional vitrification. In conventional vitrification, solutions with 40% (or more) permeating cryoprotectant(s) are widely used, whereas in ultrarapid vitrification, those with ~30% cryoprotectant are more frequently used. Use of Cryoprotectants Cryoprotectants are the compounds that are necessary in freezing solutions to prevent cell damage during freezing and thawing. For the cryopreservation of oocytes or embryos, the inclusion of a cryoprotectant that can permeate into the cell is essential. The mechanism of the protective action of permeating agents is considered the same, but their toxicities are different. For slow freezing, the concentration is limited to 1 2 mol/l, and the toxicity is relatively low. In vitrification, however, the concentration can be as high as 8 mol/l, and the selection of a low toxicity agent is more important. In addition to permeating agents, small saccharides and macromolecules are frequently added to the solution. However, the toxicity of these non-permeating agents is quite low. Types of Cryoprotectants Cryoprotectants can be broadly classified into the following three categories 1. Low molecular weight (MW) permeating cryoprotectants that include ethylene glycol, propylene glycol, dimethyl sulphoxide and glycerol. 2. Low molecular weight non-permeating cryoprotectants including cryoprotectants such as galactose, glucose, sucrose and trehalose. 3. High molecular weight non-permeating cryoprotectants including polyvinylpyrrolidone and polyvinylalcohol etc. Mode of Action of Cryoprotectants Although cryoprotective actions of these different compounds are not entirely understood, they play different roles during cooling and thawing processes. The presence of low MW cryoprotectants is absolutely necessary. They osmotically replace intracellular water in cells before cooling and reduce cell volume, changes and minimize formation of ice crystals within the cells. The cryoprotective actions of non permeating cryoprotectants are based on the dehydration of cells prior to cooling which results in reduced ice crystal formation. However, low MW nonpermeating cryoprotectants must be combined with low MW permeating cryoprotectants to effectively protect the cells during freezing. Cryoprotectants of high MW group protect the cells during freezing and warming by altering ice crystal formation to an innocuous size and shape. Moreover they are also thought to stabilize the intracellular proteins Applications of Cryopreservation Because of an increased incidence of childhood malignancy and improved survival rate, there is an increasing number of individuals who are not able to have their own children. If oocyte freezing is feasible, their oocytes could be extracted before chemotherapy, which may induce menopause if commenced, for later use when the patient would like to have a child. Secondly, egg 109

banks, similar to sperm banks could be established to help infertile women who require oocyte donation. These are primarily the one percent of women who experience premature menopause before 40 years of age. Moreover, oocyte freezing will provide young women the option of freezing their oocytes to be used when they are ready to start a family. The optimal age physiologically to have a child is also the time when human beings in general are building their careers. Therefore, some women wait until they are in their late thirties or early forties to begin to have a family. Unfortunately, as women age, the number and quality of their oocytes get diminished; they, therefore, have an increased incidence of infertility, miscarriage and abnormal babies. Besides this, cryopreservation of oocytes offers many other advantages like treatment of congenital infertility disorders; prevent fertility loss through surgery, treatment of premature ovarian follicle and oocyte donation programmes. Oocyte cryopreservation has a lot of applications in domestic animals as well. Like oocytes can be retrieved from the live animals and subjected to freezing, or for embryo cloning and production of transgenic animals. Stored oocytes could potentially be used for several purposes (see table).routine oocyte freezing would greatly benefit ART as it would provide an ethically acceptable alternative to embryo freezing Table1 Applications of oocyte freezing S.No Applications 1 Creation of oocyte banks 2 Alternative to embryo freezing 3 Oocyte preservation for patients with ovarian hyper stimulation syndrome 4 Oocyte donation programme 5 Prevent fertility loss through surgery 6 The treatment of congenital infertility disorders 7 Conservation of endangered species 8 Extension of fertile life of oocyte 9 Conservation of germplasm from superior genetic merit animals 10 Improve the efficiency of IVF Risks and Disadvantages of Cryopreservation For every upside there is a down. The frozen environment of LN 2 can be very harsh on an oocyte. We can expect to see a reduction in viability of the oocyte. This can be from numerous factors. A reduction in temperature can result in membrane lipid phase changes and depolymerisation of the cytoskelton. The increase in solute concentration will cause osmotic shrinking, and an increase in ionic concentration can have a direct effect on the membranes, this includes the solubility of the membrane proteins. Dehydration can cause destabilization of the lipid layers. Gas bubble formation causes mechanical damage. As the solution becomes extremely viscous, diffusion processes, including osmosis can become very limited. The changes in ph can denature the proteins and as cells become closely packed membranes may get damaged. In addition to all the damage or oocyte loss that can take place, you will increase your cost and have to keep more extensive records. References Asada, M. and Fukui, Y. (2000). Effect on fertilization and development by re-culture after freezing and thawing of bovine oocytes matured in vitro. Theriogenology 54: 889-898. Bernard, A. and Fuller, B.J. (1996). Cryopreservation of human oocytes: a review of current problems and perspectives. Human Reprod. Update 2 (3): 193 207. Chauhan, M.S., Nadir, S., Bailey, T.L., Pryor A.W., Butler, S.P., Notter, D.R., Valander, W.H and Gwazdauskas. 1999. Bovine follicular dynamics, oocytes recovery, and development of oocytes microinjected with a green fluorescent protein construct. J. Dairy Sci. 82; 918-926 Das, S.K. (1997).Vitrification procedures for cryopreservation of oocytes. In in vitro fertilization, embryo transfer and associated techniques in farm animals. Manik et al (eds). NDRI press /007/05-97/600.pp68-72. Dhali, A., Manik, R.S., Singla, S.K. and Palta, P. (2000). Vitrification of buffalo (Bubalus bubalis) oocytes. Theriogenology 53(6): 1295-1303. Dhali, A., Manik, R.S., Singla, S.K. and Palta, P. (2000). Post-vitrification survival and in vitro maturation rate of buffalo (Bubalus bubalis) oocytes: effect of ethylene glycol concentration and exposure time. Anim. Reprod. Sci. 63(3-4): 159-165. Fabbri, R.. and Porcu, E. (2001).Human oocyte cryopreservation: New perspectives regarding oocyte survival. Human Reprod.16 (3): 411-416. Fuku, E., Kozima, T., Shioya, Y., Mercus, G.J. and Downey, B.R. (1992). In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology.29: 485-492. Gal, F.L. and Massip, A. (1999). Cryopreservation of cattle oocytes: Effects of meiotic stage; cycloheximide treatment, and vitrification procedure. Cryobiology.38: 290-300. Gordon, I. (1994). Laboratory Production of Cattle Embryos. Cambridge, CAB International, University Press. p 71. Kasai, M. (2002). Advances in the cryopreservation of mammalian oocytes and embryos: Development of ultra rapid vitrification. Reprod. Med. Biol. 1: 1 9. Landa V, Tepla O. Cryopreservation of mouse 8-cell embryos in microdrops. Folia Biol 1990; 36: 153 158. 110

Leibo, S.P., Martino, A., Kobayashi, S and Pollard, J.W. (1996). Stage dependent sensitivity of oocytes and embryos to low temperatures. Anim. Reprod.Sci.: 42: 45 53. Manik, R.S., Chauhan, M.S., Singla, S.K. and Palta, P. (2002). Transvaginal ultrasound-guided aspiration of follicles from Indian buffaloes (Bubalus bubalis) with reproductive problems. Vet. Rec. 150: 22-24. Manik, R.S (1997). Cryopreservation of embryos. In in vitro fertilization, embryo transfer and associated techniques in farm animals. Manik et al (eds). NDRI press /007/05-97/600.pp pp 62-68. Mavrides, A. and Morroll, D. (2002). Cryopreservation of bovine oocytes: is cryoloop vitrification the future to preserving the female gamete? Reprod. Nutr. Dev. 42: 73 80 Otoi, T., Tachikawa, S., Kondo, S. and Suzuki, T. (1993). Developmental capacity of bovine oocytes frozen in different cryoprotectants. Theriogenology. 40: 801-807. Parks, J.E. Ruffing, N.A. (1992).Factors affecting low temperature survival of mammalian oocytes. Theriogenology: 37: 59 73 Picton, M.H., Gosden, R.G. and Leibo,S.P. (2003).Cryopreservation of oocytes and ovarian tissue. Gamete source, manip and disp.30:142-151. Rall,W.F. and Fahy,G.M. (1985).Ice-free cryopreservation of mouse embryos at -196 C by vitrification. Nature. 313: 573 575. Vajta, G., Helm, P. Greve,T. and Callesen, H.(1996) Overall efficiency of in vitro embryo production and vitrification in cattle. Theriogenology.45:683-689. Vajta, G. (2000). Vitrification of the oocytes and embryos of domestic animals. Anim. Reprod. Sci.60 61: 357 364 Luster S. M. (2004). Cryopreservation of bovine and caprine oocytes by vitrification. M Sc.Thesis submitted to Louisiana State University and Agricultural and Mechanical College USA. 111