Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts

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1 Human Reproduction Vol.16, No.9 pp , 2001 Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts R.R.Yeoman 1,4, B.Gerami-Naini 3, S.Mitalipov 3, K.D.Nusser 3, A.A.Widmann-Browning 3 and D.P.Wolf 1,2,3 1 Andrology/Embryology Laboratory, Departments of Obstetrics/Gynecology and 2 Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201, and 3 Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, Oregon 97006, USA. 4 To whom correspondence should be addressed at: Oregon Primate Research Center, 505 NW 185th Avenue, Beaverton, OR , USA. yeomanr@ohsu.edu BACKGROUND: Vitrification using the cryoloop procedure was evaluated for preservation of non-human primate blastocysts by comparing survival results from two different cryoprotectant mixtures with prior results from controlled rate cooling. METHODS: Rhesus monkey blastocysts were produced by intracytoplasmic sperm injection of mature oocytes from cycling females stimulated with recombinant human hormones. Morphologically wellformed blastocysts were divided between Procedure A (2.8 mol/l dimethylsulphoxide and 3.6 mol/l ethylene glycol with 0.65 mol/l sucrose and 25 µmol/l Ficoll in TALP-HEPES with 20% fetal bovine serum (TH20)) and Procedure B (3.4 mol/l glycerol and 4.5 mol/l ethylene glycol in TH20). After >48 h in liquid nitrogen, the removal of cryoprotectants was accomplished in the presence of a 3-step series of decreasing sucrose concentrations in TH20. Surviving embryos were co-cultured on buffalo rat liver cells. RESULTS: Of 16 blastocysts vitrified via Procedure A, 38% survived with minimal lysis and only one hatched in culture; in contrast, of 33 blastocysts vitrified by Procedure B, 85% survived and 71% hatched. Of 22 blastocysts cryopreserved by conventional slow cooling, 36% survived and 6% hatched. Transfer into three recipients, each with two embryos vitrified with Procedure B, resulted in a successful twin-term pregnancy. CONCLUSION: Modified cryoloop vitrification with a final solution of 3.4 mol/l glycerol and 4.5 mol/l ethylene glycol is a promising procedure for preserving Rhesus monkey blastocysts that is simple, rapid, and inexpensive. Keywords: blastocysts/cryoloop/cryopreservation/monkey/vitrification Introduction Embryo cryopreservation is a vital aspect of assisted reproduction technologies and reliable procedures for zygotes and cleaving embryos have been developed and routinely applied in humans and non-human primates (Kuzan and Quinn, 1988; Wolf et al., 1989). With the ability to culture embryos to the blastocyst stage (Zhang et al., 1994; Gardner and Lane, 1997), the low temperature storage of these advanced embryos becomes a necessity. While in-vitro developed blastocysts may have greater implantation potential, their survival rate after conventional cryostorage in glycerol has been disappointing, to the extent that it diminishes this advantage (Hartshorne et al., 1991; Kaufman et al., 1995). Alternative cryopreservation techniques include vitrification or the rapid cooling of a liquid to achieve a glass-like solid state (Rall and Fahy, 1985). In this approach, damaging ice crystal formation is minimized and water removal is enhanced by short-term exposure to high concentrations of cryoprotectants. Moreover, rapid cooling rates are obtained by minimizing the mass of the embryo suspension and the container. This has been optimized with a small nylon loop adapted from protein crystallography (Teng, 1990). The loop has a volume of 1 µl and no thermoinsulating layer which yields very high cooling rates and the successful vitrification of rodent, bovine and human blastocysts (Lane et al., 1999a,b). Because of the limited success with conventional slow freezing of Rhesus monkey blastocysts, we investigated the use of the cryoloop to cryopreserve Rhesus embryos. Materials and methods All procedures were performed in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Use and Care Committee of the Oregon Regional Primate Research Center, Oregon Health Science University. The procedures for follicular stimulation, fertilization, and embryo culture have been reviewed recently (Ouhibi et al., 2000). Twenty adult Rhesus macaques were entered into a follicular stimulation protocol starting 1 to 4 days after menses (Zelinski-Wooten et al., 1995). European Society of Human Reproduction and Embryology 1965

R.R.Yeoman et al. Figure 1. A pictorial sequence of vitrification with cryoloops is displayed. (1) Embryos were exposed in sequence to the cryoprotectant solutions placed into four wells of the plate.

(3) Embryos are then added to the film within the loop using a pulled glass transfer pipette. (4) The cryoloop is plunged into liquid nitrogen and screwed into place using the magnetic holding rod.

2 R.R.Yeoman et al. Figure 1. A pictorial sequence of vitrification with cryoloops is displayed. (1) Embryos were exposed in sequence to the cryoprotectant solutions placed into four wells of the plate. Blastocysts are transferred through these sequential solutions using a glass pipette. (2) A film on the loop is made by dipping into the last cryoprotectant solution. (3) Embryos are then added to the film within the loop using a pulled glass transfer pipette. (4) The cryoloop is plunged into liquid nitrogen and screwed into place using the magnetic holding rod. The cryoloop consisted of a 20 µm nylon filament in a mm diameter loop mounted on a stainless steel tube fixed to the inside of a 1.8 ml cryovial cap (Hampton Research, Laguna Niguel, CA, USA). Figure 2. The percentages of warmed Rhesus monkey blastocysts that survived, expanded, hatched and attached are displayed after low temperature storage in liquid nitrogen. Slow is the group that was frozen slowly by programmed freezing. Vit.1 is the group vitrified using the cryoloop with solutions and sequence of Procedure A.. Vit.2 is the group vitrified using the cryoloop with the solutions and sequence of Procedure B. The results with Vit.2 were significantly improved versus Slow and Vit.1 (P 0.05). The numbers in parentheses denote the number of blastocysts preserved. Cycling females received twice daily injections of recombinant human FSH (rfsh; 30 IU i.m., Ares Advanced Technology, Norwell, MA, USA) and once daily injections of Antide (a gonadotrophin-releasing hormone antagonist; 0.5 mg/kg s.c., Ares Advanced Technology) for 8 consecutive days. On the last two days of rfsh/antide stimulation, animals also received twice daily injections of recombinant human LH, 30 IU i.m. (Ares Advanced Technology). On the last day of hormonal stimulation, ovarian morphology was evaluated by ultrasonography (Ultrasound Unit, ATL, Bothell, WA, USA) and animals with follicles 3 mm indiameter received an injection of recombinant human chorionic gonadotrophin, 1000 IU i.m. (Ares Advanced Technology). Cumulus oocyte complexes were collected laparoscopically h later and held in HEPES-buffered TALP (modified Tyrode s solution with albumin, lactate and pyruvate) medium (Bavister et al., 1983) containing 3 mg fatty acid free, 1966 fraction V bovine serum albumin per ml (TH3) (Sigma, St Louis, MO, USA). After 1 min exposure to hyaluronidase (1 mg/ml) (Sigma), intracytoplasmic sperm injection was performed under embryo-tested mineral oil (Sigma) in a chamber (Falcon 1009, Becton-Dickinson, Franklin Lakes, NJ, USA) containing two drops: a sperm drop consisting of 4 µl of 10% polyvinylpyrrolidone (Irvine Scientific, Santa Ana, CA, USA) in TH3 and 1 µl of sperm suspension ( /ml) and an oocyte drop, 20 µl of TH3, into which mature oocytes were placed. Individual spermatozoa were injected using a 7 µm outer diameter micropipette (Humagen, Charlettesville, VA, USA) using micromanipulators (Narishige, Tokyo, Japan) on an inverted microscope equipped with Hoffman optics (IX70, Olympus, Melville, NY, USA). Injected oocytes were placed in co-culture with buffalo rat liver (BRL) cells (American Type Culture Collection, Manassas, VA, USA) in CMRL (Connaught Medical Research Laboratories) medium (Life Technologies, Rockville, MD, USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA), 10 mmol/l L-glutamine, 5 mmol/l sodium pyruvate, 1 mmol/l sodium lactate, 100 units/ml of penicillin, and 100 µg/ml streptomycin (Sigma) at 37 C in5%co 2 (Zhang et al., 1994). Embryos were transferred to fresh plates of BRL cells every other day to reduce nitrogen accumulation. Blastocysts, defined as the expansion of the embryo after compaction and cavitation to include both a discernable trophectoderm and inner cell mass, were distributed evenly among the low temperature storage groups. Blastocyst preservation Conventional slow cooling involved exposure of embryos at room temperature for 10 minutes to 0.68 mol/l (5%, volume/volume) glycerol (Sigma) in TH3 followed by 10 min in 1.22 mol/l (9% volume/volume) glycerol (Ménézo et al., 1992). Samples were loaded into cryovials (Nalgene, Rochester, NY, USA) with 1 ml of the final solution, cooled at 3 C/min to 8 C for seeding, and further cooled at 0.3 C/min to 30 C before plunging into liquid nitrogen for storage. For recovery, vials were warmed until liquefied in a 37 C water bath and recovered embryos were then incubated sequentially in 0.5 mol/l and 0.2 mol/l sucrose (Sigma) at room temperature for 10 min each before being placed in co-culture with BRL cells in CMRL media.

3 Cryoloop vitrification of monkey blastocysts Figure 3. Micrographs of Rhesus monkey blastocyst after storage in liquid nitrogen. (A) A thawed blastocyst frozen in glycerol-based medium via a slow controlled rate cooling protocol. Note the presence of degenerating cells and cracked zona. (B) A surviving blastocyst vitrified by cryoloop Procedure A with notable cellular granularity. (C) Two blastocysts from cryoloop Procedure B which expanded and hatched. Note the presence of an empty zona pellucida. (D) A hatched blastocyst vitrified by the modified cryoloop protocol and maintained in culture for 3 days. Bars designate scale. Vitrification with the cryoloop involved exposure to a solution of 1.4 mol/l (10%, volume/volume) dimethylsulphoxide (DMSO) (Sigma) and 1.8 mol/l (10%, volume/volume) ethylene glycol (Sigma) in HEPES-buffered TALP medium containing 20% fetal bovine serum (TH20) for 2 min before transfer to a solution of 2.8 mol/l (20%, volume/volume) DMSO, 3.6 mol/l (20%, volume/volume) ethylene glycol, 25 µmol/l (10 mg/ml) Ficoll ( molecular wt., Sigma), and 0.65 mol/l sucrose in TH20 for ~25 s during which time the blastocysts were placed onto a film of this solution within the loop. The cryoloop consisted of a 20 µm nylon filament in a mm diameter loop mounted on a stainless steel tube fixed to the inside of a 1.8 ml cryovial cap (Hampton Research, Laguna Niguel, CA, USA) (Figure 1). The loop was plunged immediately into liquid nitrogen within the cryovial which was then fixed onto a cane in liquid nitrogen for storage (Procedure A; Lane et al., 1999a). Warming was performed by placing the loop in a solution of 0.25 mol/l sucrose in TH20 and allowing the blastocysts to fall to the bottom of the dish for a 2 min exposure. Further cryoprotectant removal occurred during 3 min in mol/l sucrose in TH20 prior to rinsing twice in media for 5 min before co-culture on BRL cells in CMRL media as above. Alternatively, Procedure B (Agca et al., 1998) involved incubation for 3 min in 1.36 mol/l (10%) glycerol in TH20 and then 3 min in combined 1.36 mol/l (10%) glycerol and 2.7 mol/l (20%) ethylene glycol in TH20. During the last 20 s of this last step, the loop was dipped into a cryoprotectant solution of 3.4 mol/l (25%) glycerol plus 4.5 mol/l (25%) ethylene glycol in TH20. At the completion of the last step, the blastocysts were rinsed quickly in the cryoprotectant solution, placed into the loop and plunged into liquid nitrogen within 25 s. Warming and recovery of the blastocysts was performed as with Procedure A except that the initial incubation occurred in a 0.5 mol/l sucrose solution for 3 min followed by 0.25 mol/l sucrose and mol/l sucrose for 3 min each before the blastocysts were rinsed and then cultured. Whenever sufficient blastocysts were available, vitrifications involving the two alternative solutions were carried out in parallel with blastocysts of equal quality and maturity evenly distributed. After 4 h in culture, survival was based on clarity of blastomeres and blastocoel re-expansion. Further development was recorded when applicable with attachment assessed at 24 h after hatching and outgrowth assessed after 48 h. Embryo transfers were conducted on multiparous female recipients 4 5 days after the midcycle oestradiol peak. Pregnancies were detected by measuring hormone concentrations and subsequent fetal development was monitored periodically by ultrasonography. Data analysis Statistical significance of survival and further growth after warming among treatment groups was compared using a χ 2 analysis (SigmaStat, Jandel Scientific, San Rafael, CA, USA). Results The developmental stages of the 71 blastocysts available for cryopreservation varied: 31% were early blastocysts, 15% blastocysts, 42% expanded blastocysts, and 10% were hatched. The distribution of developmental stages within the three treatments did not differ significantly from this overall distribution (P NS, χ 2 -test). Cryoloop vitrification of 16 blastocysts with Procedure A resulted in 38% of the embryos surviving after warming (Figure 2). Only one blastocyst developed further with co-culture on BRL cells and hatched. With our conventional slow freezing method, 36% of 22 blastocysts 1967

4 R.R.Yeoman et al. survived with one hatching under similar culture conditions (Figure 2). In contrast, vitrification of 33 blastocysts after processing with Procedure B resulted in 28 surviving (85%) at 4 h, 25 expanding (77%), and 23 hatching (71%) (Figure 2). Twelve of these blastocysts were cultured further and attached with outgrowth of cells (Figure 3). Cracked zonae were occasionally seen with the slow freezing protocol but were not observed with either vitrification procedure. Three transfers, each composed of two expanded blastocysts from vitrification Procedure B, were made into recipients 4 5 days after the estimated time of ovulation. A twin pregnancy with two live births has resulted. Discussion The current study presents a superior method to preserve primate blastocysts by vitrification with a nylon loop. The cryoprotectant system with a final concentration of 3.4 mol/l glycerol and 4.5 mol/l ethylene glycol in TH20 protected embryos sufficiently such that with warming, blastocysts reexpanded and developed at high rates. Successful vitrification was first achieved using 8-cell stage mouse embryos in 0.25 ml straws (Rall and Fahy, 1985). With sufficiently rapid warming, a high survival rate was obtained, presumably by avoiding damaging ice crystal reformation which occurs with slower warming rates (Rall, 1987). More recently, vitrified mouse blastocysts cryopreserved using cryoloops and open-pulled straws have yielded high pregnancy rates upon transfer (Nowshari and Brem, 1998; Cseh et al., 1999; Lane et al., 1999a; Kong et al., 2000). Similarly, high pregnancy rates (21 39%) following vitrification of domestic livestock blastocysts have been reported (Ishimori et al., 1993; Agca et al., 1998; Vajta et al., 1998). Cryopreservation of human embryos by conventional slow freezing methods has yielded numerous pregnancies and fullterm births (Ménézo et al., 1992). However, embryo survival post-thaw is generally 50%. With a vitrification method using an electron microscope grid as a support (Martino et al., 1996) and ethylene glycol and sucrose as cryoprotectants, 48 of 93 blastocysts survived warming and five of 20 patients became pregnant (Choi et al., 2000). Additionally, a case study has reported a pregnancy from two blastocysts frozen in straws using ethylene glycol and DMSO as cryoprotectants (Yokota et al., 2000). Vitrification using nylon loops allows for a very small volume of liquid to encase the blastocyst and eliminates the thermo-insulating effect of a pipette or an electron microscope grid resulting in extremely high rates of cooling. Additionally, fracture damage observed in the zona pellucida, which is caused by mechanical stresses produced from non-uniform volume changes of the suspending medium (Rall and Meyer, 1989; Kasai et al., 1996; Van den Abbeel and Van Steirteghem, 2000), was not seen in our vitrification groups. Cryoprotectant toxicity and osmotic swelling during water re-entry may have reduced survival of monkey blastocysts vitrified by Procedure A since blastomere darkening was frequently observed immediately after warming. These outcomes may relate to speciesspecific membrane and microtubule fragility. Vitrification 1968 Procedure B utilized cryoprotectant formulations and procedures developed in the bovine (Agca et al., 1998; Donnay et al., 1998), a species known to be sensitive to chilling injury (Martino et al., 1996). The longer exposure and higher cryoprotectant concentrations employed in Procedure B suggest that more extensive permeation is desirable. Of course, superimposed on this conclusion is the knowledge that embryos have different permeabilities to various cryoprotectants (Kaidi et al., 1999; Paynter et al., 1999). Additionally, cryoprotectant removal in a higher sucrose concentration may have slowed water diffusion reducing damage related to osmotic shock. Thus, a promising procedure for vitrifying Rhesus monkey blastocysts has been developed that is simple, rapid, and inexpensive and may also have clinical applications. Acknowledgments The authors gratefully acknowledge the contributions of Dr David Hess for hormone assays and Dr John Fanton and the Division of Animal Resources for surgical procedures and animal care. The Assisted Reproduction core is recognized for assistance in providing semen samples. Information on vitrification with cryoloops from Dr Michelle Lane, Colorado Center for Reproductive Medicine, Englewood, Colorado, USA and Dr David Battaglia, University of Washington, Seattle, Washington, USA was appreciated. This work was supported by NIH RR12804, RR00163, and product donation GF9662 from Ares Advanced Technology, Inc., Norwell, MA, USA. References Agca, Y., Monson, R.L., Northey, D.L. et al. (1998) Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos. Theriogenology, 50, Bavister, B.D., Boatman, D.E., Leifried, L. et al. (1983) Fertilization and cleavage of rhesus monkey oocytes in vitro. Biol. Reprod., 28, Choi, D.H., Chung, H.M., Lim, J.M. et al. (2000) Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVF-ET program. Fert. Steril., 74, Cseh, S., Horlacher, W., Brem, G. et al. (1999) Vitrification of mouse embryos in two cryoprotectant solutions. Theriogenology, 52, Donnay, I., Auquier, P., Kaidi, S. et al. (1998) Vitrification of in vitro produced bovine blasocysts: methodological studies and developmental capacity. Anim. Reprod. Sci., 52, Gardner, D.K. and Lane, M. (1997) Culture and selection of viable blastocysts; a feasible proposition for human IVF? Hum. Reprod. Update, 3, Hartshorne, G.M., Elder, K., Crow, J. et al. (1991) The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts. Hum. Reprod., 6, Ishimori, H., Saeki, K., Inai, M. et al. (1993) Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide. Theriogenology, 40, Kaidi, S., Van Langedonckt, A., Massip, A. et al. (1999) Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitroproduced bovine embryos. Theriogenology, 52, Kasai, M., Zhu, S.E., Pedro, P.B. et al. (1996) Fracture damage of embryos and its prevention during vitrification and warming. Cryobiology, 33, Kaufman, R.A., Menezo, Y., Hazout, A. et al. (1995) Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil. Steril., 64, Kong, I.K., Lee, S.I., Cho, S.G. et al. (2000) Comparison of open pulled straws vs glass micropipette vitrification in mouse blastocysts. Theriogenology, 53, Kuzan, F.B. and Quinn, P. (1988) Cryopreservation of mammalian embryos. In Wolf, D.P., Bavister, B.D., Gerrity, M. and Kopf, G.S. (eds), In Vitro Fertilization and Embryo Transfer: A Manual of Basic Techniques. Plenum Press, New York, pp

5 Cryoloop vitrification of monkey blastocysts Lane, M., Schoolcraft, W.B. and Gardner, D.K. (1999a) Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil. Steril., 72, Lane, M., Bavister, B.D., Lyons, E.A. et al. (1999b) Containerless vitrification of mammalian oocytes and embryos. Nat. Biotech., 17, Martino, A., Songasen, N. and Leibo, S.P. (1996) Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol. Reprod., 54, Ménézo, Y., Nicollet, B., Herbaut, N. et al. (1992) Freezing cocultured human blasocysts. Fertil. Steril., 58, Nowshari, M.A. and Brem, G. (1998) Effect of cryoprotectants and their concentration on post-thaw survival and development of expanded mouse blastocysts frozen by a simple rapid-freezing procedure. Theriogenology, 50, Ouhibi, N., Zelinski-Wooten, M.B., Thomson, J.A. et al. (2000) Assisted fertilization and nuclear transfer in nonhuman primates. In Wolf, D.P. and Zelinski-Wooten, M. (eds), Assisted Fertilization and Nuclear Transfer in Mammals. Contemporary Endocrinology, Humana Press, 2001, pp Paynter, S.J., Cooper, A. and Gregory, L. (1999) Permeability characteristics of human oocytes in the presence of the cryoprotectant dimethylsulphoxide. Hum. Reprod., 14, Rall, W.F. (1987) Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology, 24, Rall, W.F. and Fahy, G.M. (1985) Ice-free cryopreservation of mouse embryos at 196C by vitrification. Nature, 313, Rall, W.F. and Meyer, T.K. (1989) Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos. Theriogenology, 31, Teng, T.-Y. (1990) Mounting of crystals for macromolecular cryocrystallography in a free-standing thin film. J. Appl. Cryst., 23, Vajta, G., Holm, P., Kuwayama, M. et al. (1998) Open pulled straw vitrification: A new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev., 51, Van den Abbeel, E. and Van Steirteghem, A. (2000) Zona pellucida damage to human embryos after cryopreservation and the consequences for their blastomere survival and in-vitro viability. Hum. Reprod., 15, Wolf, D.P., Vandevoort, C.A., Meyer-Haas, G.R. et al. (1989) In vitro fertilization and embryo transfer in the rhesus monkey. Biol. Reprod., 41, Yokota, Y., Sato, S., Yokota, M. et al. (2000) Successful pregnancy following blastocyst vitrification. Hum. Reprod., 15, Zhang, L., Weston, A.M., Denniston, R.S. et al. (1994) Developmental potential of rhesus monkey embryos produced by in vitro fertilization. Biol. Reprod., 51, Received on January 11, 2001; accepted on June 8,

Colorado Center for Reproductive Medicine, Englewood, Colorado

Colorado Center for Reproductive Medicine, Englewood, Colorado FERTILITY AND STERILITY VOL. 72, NO. 6, DECEMBER 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. REPRODUCTIVE BIOLOGY