Human oocyte cryopreservation and the fate of cortical granules

Human oocyte cryopreservation and the fate of cortical granules Yehudith Ghetler, Ph.D., a,b,c Ehud Skutelsky, Ph.D., b Isaac Ben Nun, M.D., c Liah Ben Dor, M.Sc., a,b Dina Amihai, M.Sc., b and Ruth Shalgi, Ph.D. a a Department of Cell and Developmental Biology and b Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; and c IVF Unit, Meir Hospital (affiliated with Tel Aviv University), Kfar Saba, Israel Objective: To examine the effect of the commonly used oocyte cryopreservation protocol on the cortical granules (CGs) of human immature germinal vesicle (GV) and mature metaphase II (MII) oocytes. Design: Laboratory study. Setting: IVF unit. Intervention(s): Unfertilized, intracytoplasmic sperm injected (ICSI) oocytes, and immature oocytes were cryopreserved using a slow freezing rapid thawing program with 1,2-propanediol (PROH) as a cryoprotectant. Main Outcome Measure(s): Cortical granule exocytosis (CGE) was assessed by either confocal microscopy or transmission electron microscopy (TEM). Result(s): The survival rates of frozen-thawed oocytes (mature and immature) were significantly lower compared with zygotes. Both mature and immature oocytes exhibited increased fluorescence after cryopreservation, indicating the occurrence of CGE. Mere exposure of oocytes to cryoprotectants induced CGE of 70% the value of control zygotes. The TEM revealed a drastic reduction in the amount of CGs at the cortex of frozen-thawed GV and MII oocytes, as well as appearance of vesicles in the ooplasm. Conclusion(s): The commonly used PROH freezing protocol for human oocytes resulted in extensive CGE. This finding explains why ICSI is needed to achieve fertilization of frozen-thawed human oocytes. (Fertil Steril 2006; 86:210 6. 2006 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, cortical granules, exocytosis, mature human oocyte, immature human oocyte Cryopreservation of human embryos at different developmental stages is successfully applied in IVF treatment programs. However, the cryopreservation of human oocytes still generally yields unsatisfactory results and is therefore considered experimental. The ability to cryopreserve human oocytes is of great importance because it avoids serious ethical and legal issues arising from embryo freezing, such as disposal, donation, or dispute over ownership. Furthermore, cryopreservation of human oocytes could lay the ground for egg donation programs and for establishing oocyte banks, offering the opportunity to preserve the fertility potential of women who face chemotherapy or radiation therapy. Successful cryopreservation of mammalian oocytes had already been reported by several investigators (1, 2). The technologies for cryopreservation of human oocytes used in IVF clinics are derived from traditional protocols of embryo cryopreservation and, as such, render results that are inconsistent and suboptimal. Cryopreservation of oocytes results in low survival rate, hardening of the zona pellucida (ZP), and various types of injuries, including damage to the meiotic spindle and microfilaments (3 7). Attempts have been Received June 8, 2005; revised and accepted December 5, 2005. Reprint requests: Ruth Shalgi, Ph.D., Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel (FAX: 972-3-6406149; E-mail: shalgir@ post.tau.ac.il). made to freeze immature oocytes at the germinal vesicle (GV) stage, where the DNA is still enclosed within the nucleus and protected by a membrane and may be less sensitive to stressful environmental conditions (8). Immature oocytes, excised from unstimulated follicles or from ovarian tissue, could, in part, survive and mature, but their developmental capability is still very low (9, 10). The common protocol used for oocyte cryopreservation is slow freezing rapid thawing in 1,2-propanediol (PROH) (11 13). Other protocols such as slow freezing rapid thawing in dimethyl sulphoxyde (DMSO) (14, 15) and an ultrarapid method (16, 17) have also been introduced. Some research groups have used variations of vitrification (18, 19), including vitrification at specially enhanced cooling rates using open pooled straws (20), electron microscope grids (21), and minimum drop size in supercooled liquid nitrogen (22). Human oocytes that survived cryopreservation had, reportedly, a low fertilization rate (23), indicating that the normal process of the sperm penetration is impaired. Therefore, cryopreserved human oocytes are fertilized by intracytoplasmic sperm injection (ICSI) in an effort to improve their fertilization rate (5, 13). The cortical granules (CGs) are Golgi-derived membranebound spherical or slightly ovoid organelles formed during the early stages of oocyte growth and maturation and are 210 Fertility and Sterility Vol. 86, No. 1, July 2006 0015-0282/06/$32.00 Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2005.12.061

present in the cortex of mature unfertilized oocytes of most animal species (24). Both density and distribution of CGs change when they translocate to the cortical region of the oocyte during the process of oocyte maturation (25, 26). The CG s content is released into the perivitelline space (PVS) immediately after oocyte fertilization or activation and is believed to alter the ZP glycoproteins, establishing the block to polyspermy, and preventing the penetration of additional spermatozoa (27). Due to the major role of CGs in inducing the block to polyspermy, their fate in frozen-thawed oocytes is of great importance, but research results have been inconsistent, ranging from a reduction in the number of CGs following cryopreservation (3, 28) to an apparently normal density and distribution of CGs in frozen-thawed oocytes (29 31). In view of this inconsistency, we aimed to examine the affect of the commonly used oocyte PROH cryopreservation protocol on the CGs of human immature GV and mature metaphase II (MII) oocytes by using confocal microscopy as well as transmission electron microscopy (TEM). MATERIALS AND METHODS Oocytes included in this study were donated by consenting patients undergoing IVF-ICSI procedures and represent material that is otherwise discarded. The study was approved by the local review board. The ovulation induction (by a long protocol of GnRH analog and hmg) and the ICSI procedure used were as described previously (32, 33). Oocytes that failed to be fertilized by ICSI (unfertilized [UF]) and immature oocytes not suitable for ICSI were used. The UF oocytes were reassessed 24 h after ICSI to rule out late fertilization occurrence and only then were they frozen. The immature oocytes were handled according to their developmental state: GV oocytes were frozen on the retrieval day, whereas oocytes at the first metaphase (MI) stage were further cultured for 24 h in the routinely used culture media (Cook Culture System; Cook, Brisbane, Australia). Only oocytes that were able to mature in vitro to the MII stage were cryopreserved. The UF as well as in vitro matured MII oocytes served as a model of mature oocytes throughout this research. Abnormally fertilized (one pronucleus [1PN] or three pronuclei [3PN]) oocytes served as positive control for occurrence of cortical granule exocytosis (CGE). Study Groups Several experimental groups were included in the research. Group 1: Untreated mature oocytes do not undergo CGE (34) served as a negative control for assessing CG status (n 13). Group 2: Mature oocytes that were subjected to the freezing-thawing solutions, according to the protocol, but not to the actual freezing procedure, served as a control for the effect of the solutions on the CGs fate, (n 16). Group 3 consisted of mature oocytes that were frozen 1 week before thawing (n 23). Group 4 consisted of GV oocytes that were frozen 1 week before thawing (n 10). Group 5: Abnormally fertilized oocytes (1PN or 3PN) after ICSI served as a positive control for CGE occurrence (n 11); in a previous study we demonstrated that these abnormally fertilized ICSI oocytes exhibit an intense CGE (34). All oocytes were assessed for CGE by confocal microscopy or TEM. Cryopreservation Protocol The slow freezing rapid thawing method was used throughout the study. The freezing/thawing solutions (Cook) and protocols were the routine protocols used in our IVF program for zygote and early embryo cryopreservation. In brief, a three-step cryopreservation system was used with HEPESbuffered salt solution (HBSS) containing PROH and sucrose as cryoprotectants: step 1: incubation in prewarmed (37 C) HBSS for 10 minutes at room temperature (RT); step 2: incubation in HBSS containing 1.5 mol/l PROH for 10 minutes at RT; step 3: transfer into HBSS containing 1.5 mol/l PROH and 0.1 mol/l sucrose, loading the samples into straws at RT, and transfering them into an automated Kryo 10 biologic vertical freezer (Kryo 10 series III, Planer, Middlesex, UK). Cooling steps: starting at 20 C, cooling at 2 C/min down to 7 C, soaking for 10 minutes, manual seeding, holding 10 minutes, cooling at 0.3 C/min down to 30 C, cooling at 50 C/min down to 150 C and plunging into liquid nitrogen ( 196 C). Samples were stored in liquid nitrogen for 1 week. For thawing, straws were removed from liquid nitrogen and kept 30 seconds at RT before being immersed in a 30 C water bath until all the ice melted. The cryoprotectant was removed with four consecutive steps of 5 minutes each incubation at RT: step 1: HBSS containing 1.0 mol/l PROH and 0.2 mol/l sucrose; step 2: HBSS containing 0.5 mol/l PROH and 0.2 mol/l sucrose; step 3: HBSS containing 0.2 mol/l sucrose; step 4: only HBSS followed by additional 5 minutes at 37 C. Finally, the oocytes were incubated in culture medium (Cook) at 37 C with 5% CO 2 in humidified air for 1 hour and then assessed morphologically. Only oocytes that survived the whole process of freezing-thawing were further examined. Assessment of CGE The assessment of cortical reaction was performed by labeling the oocytes with the lectin Lens culinaris agglutinin (LCA), which binds specifically to CG content and exudate (35). The studied oocytes were briefly exposed to 0.25% pronase (Sigma, St. Louis, MO) for ZP removal, followed by three rinses in HTF-HEPES medium (Irvine, Santa Ana, CA) supplemented with 10% synthetic serum substitute (Irvine). The ZP-free oocytes were fixed with 3% paraformaldehyde in DPBS for 30 minutes at RT and washed three times in DPBS supplemented with 1% bovine serum albumin (Fraction V; Sigma). The fixated oocytes were stained for 30 minutes with Fertility and Sterility 211

TABLE 1 Survival rate of frozen thawed oocytes and zygotes. Study group Number of oocytes/ zygotes Survival rate* (mean % SEM) Zygotes 173 74.7 4.2 a Mature ocytes 109 40.8 4.9 b GV oocytes 92 31.4 7.1 b Note: Survival of thawed oocytes and zygotes was morphologically determined after 1 h of culture. a,b Different superscripts within column denote significant differences (P.001). Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006. 5 g/ml LCA-biotin (Vector, Burlingame, CA), washed, and stained with 2 g/ml Texas red streptavidin (Vector) for 30 minutes, thus labeling the membrane of exocytosed granules and the exudate. For high-quality assessment of the CGE, a confocal laser scanning microscope (CLSM) (LSM 410; Ziess, Oberkochen, Germany) equipped with a 25 mw krypton-argon laser was used. The oocytes were scanned every 5 m and the composite image of micrographs taken at all focal planes of each oocyte was displayed. The fluorescence intensity of each individual oocyte was quantified, with the aid of the LSM software, and presented as the ratio between the fluorescent area and the total area of the oocyte, which was then related to the fluorescence intensity of the positive control (group 5) which was set arbitrarily as 1. Assessment of Meiotic Status Oocytes were incubated for 10 minutes in the presence of 2 g/ml DNA-specific fluorochrome (Hoechst 33342, Sigma) and then washed three times in DPBS. Analysis of each oocyte was performed at the time of CGE assessment, using the CLSM. Electron Microscopy (EM) The ultrastructure of CGs of oocytes from the various experimental groups was examined using TEM. At least three oocytes from each group were sectioned and analyzed. The oocytes were fixed for 1 hour in Karnovsky fixative (36), washed twice in 0.2 mol/l cacodylate buffer, ph 7.4, postfixed in 1% osmium tetroxide, washed, and gradually dehydrated through ascending grades of ethanol and propylene oxide. Each oocyte was individually embedded in Araldite (Polysciences, Warrington, PA) and sectioned (0.075 0.015 nm) on an LKB III Ultratome. The ultrathin sections were mounted on grids and stained with uranyl acetate followed by lead citrate. The sections were examined using a JOEL-100CX TEM at 80 kv (37). Statistical Analysis The significance of differences between experimental groups was determined by one-way analysis of variance after arcsin square root of proportion transformation (for survival study) and ln transformation (for the fluorescence study) to achieve normal distribution, combined with Tukey s method for multiple comparisons. RESULTS Survival rate of the frozen-thawed oocytes was compared to the survival rate of zygotes (2 PN) cryopreserved in our IVF program (Table 1). The overall survival rate of oocytes was low and inconsistent (0% 92%). The survival rate of zygotes in our IVF unit during the same period of time was 74.7% 4.2% (mean SEM), similar to other reports (38) and significantly (P.001) higher than the survival rate of either immature (31.4% 7.1%) or mature oocytes (40.8% 4.9%). There was no significant difference between the survival rate of mature and immature oocytes. We used the CLSM for assessing the CGE occurrence as an indicator of the fate of CGs in frozen-thawed oocytes. The treated oocytes (groups 2, 3, 4) and the negative control FIGURE 1 Detection of cortical granule exocytosis (CGE) by labeling with biotinylated lectin Lens culinaris agglutinin and Texas red strepavidin. Composite confocal microscopy images (A D) and light microscopy images (a d) of untreated mature oocyte (A, a), untreated 3PN zygote (B, b), frozenthawed (slow freezing rapid thawing in 1,2 propanediol) mature oocyte (C, c), and frozenthawed (slow freezing rapid thawing in 1,2 propanediol) germinal vesicle stage oocyte (D, d), illustrating the degree of CGE at the various treatments. Bar 35 m. Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006. 212 Ghetler et al. Cortical granules in cryopreserved human oocytes Vol. 86, No. 1, July 2006

TABLE 2 Fluorescence intensity of frozen-thawed oocytes. Study group Number of oocytes Fluorescence intensity (mean SEM) 1 14 0.23 0.09 a 2 16 0.73 0.21 b 3 23 1.51 0.33 b,c 4 10 2.51 1.08 c 5 11 1 Note: Relative fluorescence intensity expressed as ratio of the abnormally ICSI-fertilized oocytes (group 5). Group 1: untreated mature oocytes. Group 2: mature oocytes following exposure to freezingthawing solutions. Group 3: frozen-thawed mature oocytes. Group 4: frozen-thawed germinal vesicle oocytes. Group 5: abnormally fertilized oocytes, fluorescence intensity arbitrarily defined as 1. a,b,c Different superscripts within column denote significant differences P.001). Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006. (group 1) did not show activation, as indicated by Hoechst staining. However, our results demonstrate that both mature and immature frozen-thawed oocytes underwent CGE (Figs. 1C and D, respectively) compared with untreated oocytes (Fig. 1A) or fertilized oocytes (Fig. 1B). The calculated CGE intensity is presented in Table 2. Untreated mature oocytes (group 1, negative control) exhibited very low fluorescence relative to the positive control group of abnormally fertilized oocytes (group 5). The mere exposure to cryopreservation solutions (group 2) resulted in a strong CGE: about 70% of the positive control value and significantly different from the untreated mature oocytes (P.001). The CGE of oocytes, both mature (group 3) and immature (group 4), passing through the complete freezingthawing procedure was significantly stronger than that of untreated oocytes. The GV frozen-thawed oocytes exhibited a higher (but not statistically different) degree of CGE relative to the frozen-thawed mature oocytes. We examined ultrathin sections of mature and immature untreated oocytes by TEM and compared them to sections of oocytes that survived the freezing-thawing procedure. Mature untreated oocytes exhibited an abundant number of CGs consisting of dark (electron-dense) and light subpopulations (Fig. 2, top). The dark granules were restricted only to the cortical region, whereas light granules were scattered all through the cytoplasm as well. The GV untreated oocytes contained few granules at the cortex (mostly dark) and many clusters of light granules throughout the cytoplasm (Fig. 3, top). A change in the CG distribution pattern was observed in both mature and immature oocytes after freezing-thawing procedure (Figs. 2, bottom, and 3, bottom). Only very few dark CGs were observed within frozen-thawed oocytes. Mature frozen-thawed oocytes had only a few scattered CGs deeper in the cytoplasm, whereas GV frozen-thawed oocytes had a few CGs at the cortex and aggregates of light granules throughout the cytoplasm. All the frozen-thawed oocytes formed membrane-coated electron-transparent vesicles, which in some cases were aggregated with CGs (Figs. 2, bottom, and 3, bottom). No such vesicles could be observed within the oocytes before cryopreservation. FIGURE 2 The effect of cryopreservation on mature oocytes. (Top) An untreated oocyte: Abundant dark electron-dense granules (white arrow) are present in the cortex, and light (black arrow) granules are present mainly deeper in the cytoplasm. (Bottom) A frozen-thawed oocyte: Very few granules are present at the cortex. Light (black arrow) and a few dark (white arrow) granules are in the inner region. A large number of membrane-coated transparent vesicles (V) are observed, scattered throughout the ooplasm. ZP zona pellucida. Bar 2 m. Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006. Fertility and Sterility 213

FIGURE 3 The effect of cryopreservation on immature (geminal vesicle stage) oocytes. (Top) An untreated oocyte: Dark electron-dense granules (white arrow) are present in the cortex. Numerous aggregated light (black arrow) granules appear throughout the cytoplasm. (Bottom) A frozenthawed oocyte: Very few dark (white arrow) granules are present at the cortex. Light (black arrow) aggregated granules are in the inner cytoplasm. A large number of membrane-coated transparent vesicles (V) were observed scattered throughout the ooplasm. ZP zona pellucida. Bar 2 m. Ghetler. Cortical granules in cryopreserved human oocytes. Fertil Steril 2006. DISCUSSION Oocyte cryopreservation is a feasible procedure yielding birth of healthy babies, although clinical efficiency is still low (39). In the present research we tried to elucidate one of the pitfalls of fertilizing frozen-thawed oocytes, i.e., the premature occurrence of CGE. We demonstrated that the most commonly used procedure of cryopreserving human oocytes, the PROH slow freezing-rapid thawing procedure, has a detrimental effect on the CGs. Our findings explain the low fertilization rate of human frozen-thawed oocytes and justify the need for ICSI for this population of oocytes. The initial step of the cryopreservation procedure, the dehydration step, which included exposure to cryoprotectants at RT, had already caused the release of content from a significant number of CGs. Similar effect on CGs of mouse and human oocytes was demonstrated following exposure to PROH or DMSO (28). Further exocytosis was demonstrated after the freezing-thawing process, as indicated by the fluorescence of CG exudate in mature as well as immature cryopreserved oocytes. Although not statistically significant, GV oocytes appear more susceptible than MII oocytes to the harch procedure of freezing-thawing regarding their survival rate and degree of CGE. The difference might be attributed to possible differences in the composition of membranes of GV and MII oocytes. Cortical granule exocytosis might occur because of changes in the properties and permeability of the oocyte s membrane after chilling injury (40), toxicity of the cryoprotectant solutions (41), or osmotic shock induced by osmotic pressure changes that involve an Na /H exchange mediated signal transduction pathway (42). Mattioli et al. (43) demonstrated a clear biphasic Ca 2 rise in immature pig oocytes during cooling from 30 to 14 C which was related to DNA fragmentation. Ben-Yosef et al. (44) demonstrated cooling-induced rat egg activation, as manifested by intracellular calcium concentration (Ca 2 i) transients and second polar body extrusion. Such a rise in Ca 2 i may trigger CGE as well. The overall fluorescence of frozen-thawed oocytes (mature and immature) was higher than that of ICSI-fertilized (1PN and 3PN) positive control oocytes. It is possible that owing to changes in membrane properties and/or permeability that occur during cryopreservation the content of granules located deeper within the oocyte is also released into the PVS, whereas only cortically located granules are released during fertilization, or that there is leakage of lectin (LCA) binding molecules, which contributes to the massive fluorescent labeling. We demonstrated, by TEM analysis, changes in the distribution pattern of the CGs as well as cytoplasmic structural changes following cryopreservation. The cryopreservation procedure resulted in the loss of CGs from the cortical area and in the appearance of vesicles within the cytoplasm of both immature and mature cryopreserved oocytes, which might indicate structural damage occurring from the freezing-thawing process. Similar findings were reported after exposure of bovine oocytes to vitrification solutions (45). Further research is needed for determining the origin and meaning of the newly formed vesicles. Standard embryo cryopreservation technologies appear to have limitations when applied to oocytes. Cryosurvival is low and inconsistent, and oocytes that manage to survive this procedure exhibit structural and functional damage. An improved survival rate for human oocytes was obtained by raising the seeding temperature to 4.5 C, as close as possible to the melting point of the solution (46), indicat- 214 Ghetler et al. Cortical granules in cryopreserved human oocytes Vol. 86, No. 1, July 2006

ing that the commonly practiced seeding temperature of 7 C is not optimal. Because the initial step of cryopreservation (i.e., dehydration by exposure to cryoprotectants) had already caused an extensive release of CGs, effort should be directed toward modifying that step to minimize the damage and optimize the results. A new freezing protocol should be tailored for the cryopreservation of human oocytes to avoid oocyte damage and improve oocyte post-thaw survival rate. Acknowledgements: The authors thank Ruth Kaplan-Kraicer, M.Sc., Sackler Faculty of Medicine, Tel Aviv University, and Tal Rom, M.Sc., and Ayelet Itzkovitz, M.Sc., IVF Unit, Sapir Medical Center, Kfar Saba, for their technical assistance and Leonid Mittelman, M.D., Sackler Faculty of Medicine, for his assistance in confocal microscopy. This work is in partial fulfillment of the requirements for the Ph.D. degree of Y. Ghetler, Sackler Faculty of Medicine, Tel Aviv University. REFERENCES 1. Parkering TA, Tsunoda Y, Chang MC. Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse eggs. J Exp Zool 1976;197:369 74. 2. Al-Hasani S, Diedrich K, van der Ven H, Reinecke A, Hartje M, Krebs D. Cryopreservation of human oocytes. Hum Reprod 1987;2:695 700. 3. Vincent C, Pickering SJ, Johnson MH. The hardening effect of dimethylsulphoxyde on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present. J Reprod Fertil 1990;89:253 9. 4. Baka SG, Toth TH, Veek LL, Jones HW, Muasher SJ, Lanzendorf SE. Evaluation of the spindle apparatus of in vitro matured human oocytes following cryopreservation. Hum Reprod 1995;10:1816 20. 5. Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M, Flamigni C. Pregnancies and births after oocyte cryopreservation. Fertil Steril 2004; 82:601 5. 6. Rienzi L, Martinez F, Ubaldi F, Minasi MG, Iacobelli M, Tesarik J, Greco E. Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures. Hum Reprod 2004;19:655 9. 7. Bianchi V, Coticchio G, Fava L, Flamigni C, Borini A. Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration. Hum Reprod 2005;20:1078 83. 8. Tucker MJ, Morton PC, Wright G, Massey JB. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil Steril 1998;70:578 9. 9. Kim SS, Soules MR, Bataglia DE. Follicular development, ovulation, and corpus luteum formation in cryopreserved ovarian tissue after xenotransplantation. Fertil Steril 2002;78:77 82. 10. Oktay K, Economos K, Kan M, Rucinski J, Veek L, Rosenwaks Z. Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strip to the forearm. JAMA 2001;286:1490 3. 11. Porcu E, Fabrri R, Seracchioli R, Chiotti P, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997;68:724 30. 12. Tucker MJ, Morton PC, Wright G, Sweitzer CL, Massey JB. Clinical application of human egg cryopreservation. Hum Reprod 1998;13: 3156 9. 13. Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S, Flamigni C. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001;16:411 6. 14. Hunter JE, Bernard A, Fuller B, Amso N, Shaw RW. Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperatures and cryopreservation: a comparison of two techniques. Hum Reprod 1991;6:1460 5. 15. Paynter SJ, Cooper A, Gregory L, Fuller BJ, Shaw RW. Permeability characteristics of human oocytes in the presence of dimethylsulphoxide. Hum Reprod 1999;14:2338 42. 16. Trounson A, Sjoblom P. Cleavage and development of human embryos in vitro after ultrarapid freezing and thawing. Fertil Steril 1988;50: 373 6. 17. Hsieh YY, Tsai HD, Chang CC, Chang CC, LO HU, Lai ACH. Ultrarapid cryopreservation of human embryos: experience with 1582 embryos. Fertil Steril 1999;72:253 6. 18. Hunter JE, Fuller BJ, Bernard A, Jackson A, Shaw RW. Vitrification of human oocytes following minimal exposure to cryoprotectants; initial studies on fertilization and embryonic development. Hum Reprod 1995; 10:1184 8. 19. Jelinkova L, Selman HA, Arav A, Strehler E, Reeka N, Sterzik K. Twin pregnancy after vitrification of 2-pronuclei human embryos. Fertil Steril 2002;77:412 4. 20. Vajta G. Vitrification of the oocytes and embryos of domestic animals. Anim Reprod Sci 2000;60 61:357 64. 21. Martino A, Songsagen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54:1059 69. 22. Arav A, Yavin S, Zeron J, Natan D, Dekel I, Gacitua H. New trends in gamete s cryopreservation. Mol Cell Endocrinol 2002;187:77 81. 23. Kazem R, Thompson LA, Srikantharajah A, Laing MA, Hamilton MPR, Templeton A. Cryopreservation of human oocytes and fertilization by two techniques: in vitro fertilization and intracytoplasmic sperm injection. Hum Reprod 1995;10:2650 4. 24. Cran DG, Esper CR. Cortical granules and the cortical reaction in mammals. J Reprod Fertil Suppl 1990;42:177 88. 25. Ducibella T, Duffy P, Buetow J. Quantification and localization of cortical granules during oogenesis in the mouse. Biol Reprod 1994;50: 467 73. 26. Connors SA, Kanatsu-Shinohara M, Schultz RM, Kopf G. Involvement of the cytoskeleton in the movement of cortical granules during oocyte maturation, and cortical granules anchoring in mouse eggs. Dev Biol 1998;200:103 15. 27. Wessel GM, Brooks JM, Green E, Haley S, Voronina E, Wong J, et al. The biology of cortical granules. Int Rev Cytol 2001;209:117 206. 28. Schalkoff ME, Oskowitz SP, Powers RD. Ultrastructural observations of human and mouse oocytes treated with cryopreservatives. Biol Reprod 1989;40:379 93. 29. Van Blerkom J, Davis PW. Cytogenetic, cellular and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Techn 1994;27:165 93. 30. Jones A, Van Blerkom J, Davis P, Toledo AA. Cryopreservation of metaphase II human oocytes effects mitochondrial membrane potential: implications for developmental competence. Hum Reprod 2004;19: 1861 6. 31. Wood MJ, Whittingham DG, Lee SH. Fertilization failure of frozen mouse oocytes is not due to premature cortical granule release. Biol Reprod 1992;46:1187 95. 32. Shulman A, Ghetler Y, Beyth Y, Ben Nun I. The significance of early (premature) rise of plasma progesterone in in-vitro fertilization cycles induced by a long protocol of gonadotropin releasing hormone analogue and human menopausal gonadotropins. J Assist Reprod Genet 1996;13:207 11. 33. Van Steirteghem A, Nagy ZP, Joris H, Liu J, Staessen C, Smitz J, Wisato A, Devroy P. High fertilization and implantation rate after intracytoplasmic sperm injection. Hum Reprod 1993;8:1061 6. 34. Ghetler Y, Raz T, Ben Nun I, Shalgi R. Cortical granules reaction after intracytoplasmic sperm injection. Mol Hum Reprod 1998;4:289 94. 35. Cherr GN, Drobnis EZ, Katz DF. Localization of cortical granule constituents before and after exocytosis in the hamster egg. J Exp Zool 1988;246:81 93. 36. Karnovsky MJ. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 1965;27:137a. Fertility and Sterility 215

37. Raz T, Skutelsky E, Amihai D, Hammel I, Shalgi R. Mechanisms leading to cortical reaction in the mammalian egg. Mol Reprod Dev 1998;51:295 303. 38. Al-Hasani S, Ludwig M, Gagsteiger F, Kupfer W, Sturm R, Yilmaz A, et al. Comparison of cryopreservation of supernumerary pronuclear human oocytes obtained after intracytoplasmic sperm injection and after conventional in-vitro fertilization. Hum Reprod 1998;11:604 7. 39. Van der Elst J. Oocyte freezing: here to stay? Hum Reprod Update 2003;5:463 70. 40. Zeron Y, Pearl M, Borochov A, Arav A. Kinetic and temporal factors influence chilling injury to germinal vesicle and mature bovine oocytes. Cryobiology 1999;38:35 42. 41. Wusteman MC, Pegg DE, Robinson MP, Wang LH, Fitch P. Vitrification media: toxicity, permeability, and dielectric properties. Cryobiology 2002;44:24 37. 42. Inagaki N, Suzuki S, Kuji N, Kitai H, Nagatogawa N, Nozawa S. Egg activation induced by osmotic pressure change and the effect of amiloride on the cryopreservation of mouse oocytes. Mol. Hum Reprod 1996;2:835 43. 43. Mattioli M, Barboni B, Luisa G, Loi P. Cold-induced calcium elevation triggers DNA fragmentation in immature pig oocytes. Mol Reprod Dev 2003;65:289 97. 44. Ben-Yosef D, Oron Y, Shalgi R. Low temperature and fertilizationinduced Ca 2 changes in rat eggs. Mol Reprod Dev 1995;42:122 9. 45. Hyttel P, Vajta G, Callesen H. Vitrification of bovine oocytes with the open pulled straw method: ultrastructural consequences. Mol Reprod Dev 2000;56:80 8. 46. Trad FS, Toner M, Biggers JD. Effects of cryoprotectants and iceseeding temperature on intracellular freezing and survival of human oocytes. Hum Reprod 1999;14:1569 77. 216 Ghetler et al. Cortical granules in cryopreserved human oocytes Vol. 86, No. 1, July 2006