1 Department of Gynaecological Endocrinology and Reproductive Medicine, University of Bonn, Bonn, Germany and 2 Gynaecological Clinic Ma-Ma, Moscow, Russia
3 To whom correspondence should be addressed at: Department of Gynaecological Endocrinology and Reproductive Medicine, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Email: vovaisachenko{at}yahoo.com
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Abstract |
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Key words: aseptic application/microbial contamination/oocytes/open-pulled straw/vitrification
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Introduction |
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For the cryopreservation of embryos or oocytes, standard 0.25 ml insemination straws were used almost exclusively (Kuleshova and Shaw, 2000). The achievable cooling rate by direct plunging into liquid nitrogen (Palasz and Mapletoft, 1996
) was
2500°C/min. However, new methods based on the immersion of very small amounts of solutions in liquid nitrogen were developed and most authors have attributed the improved developmental rate of cells after cryopreservation to an increased (>10 000°C/min) speed of freezing and warming (Martino et al., 1996
; Vajta et al., 1998
; Lane et al., 1999
; Lane and Gardner, 2001
; Dinnyes et al., 2000
; Kuwayama and Kato, 2000
).
All methods mentioned above have a common parameter: exposure of the vitrification solution holding the biological object directly to liquid nitrogen during freezing. Isolation of vitrifying samples from liquid nitrogen in order to avoid direct contact results in a decreased speed of cooling which, in the opinion of the majority of authors, can decrease the efficacy of the vitrification protocol.
Bielanski et al., (2000) reported the contamination of bovine embryos by viral pathogens during storage of these embryos in liquid nitrogen. Even the vapour of liquid nitrogen can be contaminated by microorganisms (Fountain et al., 1997
). Consequently, long-term banking of spermatozoa and embryos in liquid nitrogen can have a potential risk of cross-contamination when specimens are not sealed properly (Bielanski et al., 2003
).
The aim of our investigations was to assess the viability of vitrified human pronuclear oocytes after slow cooling of samples which were protected from direct contact with liquid nitrogen followed by rapid warming.
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Materials and methods |
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Patients with unexplained infertility were stimulated for IVF-ICSI with triptorelin (Decapeptyl®; Ferring, Kiel, Germany) and recombinant FSH (Puregon®; Organon, Oss, The Netherlands) according to the long protocol. Ovulation was induced by the administration of 10 000 IU of HCG (Pregnil®; Organon). Oocytes were retrieved 3436 h later and inseminated with the husband's sperm through conventional IVF or ICSI techniques. Fertilization was assessed 1418 h after insemination. Abnormally fertilized oocytes [at the three-pronuclei (PN) stage, n=102] or 2PN stage (from patients who did not want cryopreservation of oocytes, n=12) were used for vitrification, warming and subsequent culture.
In total, 62 patients ranging in age from 24 to 39 years (median age 32±3.9) volunteered to have their oocytes cryopreserved by vitrification at the 2PN or 3PN stage.
Twelve oocytes with 2PN and 102 oocytes with 3PN were assigned randomly to a control (n=28) and two experimental groups. As we found no difference in the developmental rate of embryos derived from these two stages, all data were combined and presented together. The oocytes in the experimental groups were non-equilibrium cryopreserved (vitrified) by plunging into liquid nitrogen. All the cryopreservation solutions were prepared in TCM-199 supplemented with 20% serum supplement (Irvine Scientific, Santa Aria, CA). Oocyte cryopreservation was performed in open-pulled-straws (OPS) (Medical Technology GmbH, Altdorf, Germany) (Vajta et al., 1998) with a four-step exposure in 20, 33, 50 and 100% vitrification solution [20% ethylene glycol +20% dimethylsulphoxide (DMSO) +0.75 mol/l sucrose) for 2, 1 and 1 min, and 3050 s, respectively at 37°C and cooled. The volume of final vitrification solution in the OPS was 22.5 µl. For group 1, rapid cooling of oocytes (n=42) was realized by direct plunging of OPS into liquid nitrogen. For group 2, OPS with oocytes (n=44) were first placed inside a sterile 0.5 ml insemination straw (Medical Technology GmbH), which was hermetically closed by metal balls at both sides (Figure 1), and then plunged into liquid nitrogen. It should be noted that there was no contact between the metal ball and the vitrification solution holding the oocytes due to the presence of a meniscus of the vitrification solution (Figure 1). The potential risk of contact of vitrification solution with the metal ball is not a big problem because the metal balls and 0.5 ml straws are sterile; oocytes locate in the upper part of a column of vitrification solution (Figure 1). The speed of cooling in straw in straw vitrification was determined using a Testo 950 electrical thermometer (Testo AG, Lenzkirch, Germany). For rapid warming, the OPS of group 1 were removed from liquid nitrogen and immediately plunged into a 1.5 ml microcentrifuge tube (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) containing 1 ml of 1 mol/l sucrose. Before plunging into sucrose solution, the OPS of group 2 were first removed from the 0.5 ml straws in the following way. On removal of the ball using scissors, the part of the 0.5 ml straw which was still half submerged in liquid nitrogen is exposed and then, using fine forceps, OPS were taken out of the 0.5 ml straw and plunged into sucrose solution as described above. This mode of thawing allows for the simultaneous removal of cryoprotectant and rapid warming. After warming, oocytes of both groups were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) for gradual removal of the cryoprotectant in five 2.5 min steps. All the oocytes were then cultured for an additional 6 days in 5% CO2 at 37°C in Blast-Assist Medium (I and II) (Medicult, Denmark) according to a standard protocol.
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Results |
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Figure 3 shows the appearance of the vitrified pronuclear oocytes subjected to vitrification using OPS located inside the closed 0.5 ml straw. Just after warming, a dispersion of nucleoli was occasionally detected; later nucleoli were re-organized to a more polarized pattern and the initial dispersion had no influence on the further development of embryos.
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Discussion |
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All combinations of slow or quick cooling with slow warming were the main cause for a decreasing viability of vitrified mouse embryos (Rall and Fahy, 1985; Rall, 1987
; Rall and Wood, 1994
).
Elevated warming rates and the absence of visible crystal formation during thawing of oocytes was shown to be most effective when the warming/cooling proportion was >1.3. For example, cooling at 200°C/min followed by warming at 360°C/min (warming/cooling proportion 1.3) is a more effective regime than cooling at 2000°C/min followed by warming at 2400°C/min (warming/cooling proportion 1.2) (Isachenko et al., 1998, 1999
).
In the opinion of the majority of authors, the high effectiveness of different protocols for the vitrification of oocytes and embryos with decreased volumes of cooled medium can be explained by the combination of high speeds of cooling and warming (Martino et al., 1996; Vajta et al., 1998
; Lane et al., 1999
; Lane and Gardner, 2001
; Mukaida et al., 2003; Son et al., 2003
; Vanderzwalmen et al., 2003
). The results of our investigations with human oocytes do not support this point of view. For human oocytes, the relatively slow cooling rate in combination with rapid warming is as efficient as conventional vitrification with regard to survival rates and embryo development.
Cremades et al., (2004) reported the successful vitrification of human embryos using fine diameter plastic micropipettes, which were cooled in the vapour of liquid nitrogen and subsequently placed inside a pre-cooled cryotube. Similarly to our findings, this methodology also includes the parameter slow cooling and rapid warming; however, this methodology does not prevent the contact with nitrogen of an open system holding the biological objects.
Slow cooling of biological objects can also be used for cooling of a relatively large volume. To prevent the 0.25 ml straw from coming into direct contact with liquid nitrogen and eliminating the potential contamination risk associated with storage in liquid nitrogen, Kuleshova and Shaw (2000) have reported straw in straw vitrification of mouse embryos. In their protocol, a standard 0.25 ml straw containing vitrification medium was located inside another 0.5 ml straw which was hermetically closed before plunging into liquid nitrogen. However, this method, in contrast to the one described herein, does not allow for rapid warming of a small volume and simultaneous removal of the cryoprotectant during the thawing process. Other data rgarding straw in straw cryopreservation of ovine germinal vesicle stage oocytes (Isachenko et al., 2001
) and mouse embryos (Walker et al., 2004
) have also been published.
Our finding regarding the minor importance of the speed of cooling for the vitrification of oocytes and embryos can probably be applied to all reproductive human cells including spermatozoa. We recently reported that vitrification of human spermatozoa by fast (20 000°C/min) or relatively slow (200°C/min) cooling resulted in similar post-thaw characteristics (Isachenko et al., 2004b).
The major point of the work presented here is that the described technique completely avoids direct contact between liquid nitrogen and the oocytes. In fact, any technology in reproductive biology and especially in a therapeutic medical approach must ensure and guarantee the full protection of biological objects from microorganisms (Gardner, 1998; Bielanski et al., 2000
). Liquid nitrogen, which is used for storage of frozen material, can be a source of contamination by these microorganisms (Tedder et al., 1995; Bielanski et al., 2000
). Filtration or ultraviolet treatment of liquid nitrogen cannot guarantee the absence of contamination of biological material by viruses including human immunodeficiency virus (HIV). For example, Tedder et al. reported the contamination of blood probes by hepatitis virus during the time of storage of probes in liquid nitrogen (Tedder et al., 1995). Different types of viruses, which are simple and very cryo-stable structures, may increase their virulence after direct plunging and storage in liquid nitrogen, like hepatitis virus (Hawkins et al., 1996
), papova virus (Charles and Sire, 1971
), vesicular stomatitis virus (Schaffer et al., 1976
) and herpes virus (Jones and Darville, 1989
).
In conclusion, vitrification of human pronuclear oocytes in OPS which are placed inside a hermetically closed container before plunging into liquid nitrogen allows a reliable isolation of oocytes from liquid nitrogen and avoids contamination by pathogenic microorganisms. Although this technique is associated with a relatively slow cooling rate, the developmental potential of these pronuclear oocytes is not compromised if the thawing process involves rapid warming and simultaneous removal of cryoprotectants.
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Acknowledgements |
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References |
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---|
Bielanski A, Bergeron H, Lau PCK and Devenish J (2003) Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 46, 146152.[CrossRef][ISI][Medline]
Charles GN and Sire DJ (1971) Transmission of papova virus by cryotherapy applicator. J Am Med Assoc 218, 1435.[CrossRef]
Cremades N, Sousa M, Silva J, Viana P, Sousa S, Olivera C, Teixeira da Silva J and Barros A (2004) Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes. Hum Reprod 19, 300305.
Darvelid U, Gustafsson H, Shamsuddin M, Larsson B and Rodriquez-Martinez H (1994) Survival rate and ultrastructure of vitrified bovine in vitro and in vivo developed embryos. Acta Vet Scand 35, 417426.[ISI][Medline]
Dinnyes A, Wallace GA and Rall WF (1995) Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing. Mol Reprod Dev 40, 429435.[ISI][Medline]
Dinnyes A, Dai Y, Jiang S and Yang X (2000) High development rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Biol Reprod 63, 513518.
Donnay I, Auquier P, Kaidi S, Carolan C, Lonergan P, Mermillod P and Massip A (1998) Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity. Anim Reprod Sci 52, 93104.[CrossRef][ISI][Medline]
Fountain D, Ralston M, Higgins N, Gorlin JB, Uhl L, Wheeler C, Antin JH, Churchill WH and Benjamin RJ (1997) Liquid nitrogen freezers: a potential sourse of microbial contamination of hematopoietic stem cell components. Transfusion 37, 587591.
Gardner DK (1998) Development of serum-free media for the culture and transfer of human blastocysts. Hum Reprod 13, 218285.[Medline]
Hawkins AE, Zuckerman MA, Briggs M, Gilson RJ, Goldstone AH, Brink NS and Tedder RS (1996) Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank. J Virol Methods 60, 8188.[CrossRef][ISI][Medline]
Isachenko V, Isachenko E, Ostashko F and Grishchenko V (1997) Ultra-rapid freezing of rat embryos with rapid dilution of permeable cryoprotectants. Cryobiology 34, 157164.[CrossRef][ISI][Medline]
Isachenko V, Perez-Sanchez F, Isachenko V, Grishchenko V and Soler C (1998) Vitrification of GV-porcine oocytes with intact intracellular lipids: effect of the cryoprotectant saturation/dilution stepping, elevated temperature and cytoskeletal inhibitor. Cryobiology 36, 250253.[CrossRef][ISI][Medline]
Isachenko V, Gorbunov L, Isachenko E, Ostashko F and Bezugly N (1999) Some physical and technological aspects of GV porcine oocyte vitrification. Cryobiology 39, 35 abstract.[CrossRef]
Isachenko V, Alabart JL, Isachenko E, Michelmann HW and Bezugly N (2000) Ultra-rapid freezing and storage of rat embryos in an electric refrigerator at 130°C without liquid cryo-agents with ultra-short exposure in the freezing medium and direct rehydration after thawing. CryoLetters 21, 1318.[ISI][Medline]
Isachenko V, Alabart JL, Nawroth F, Isachenko E, Vajta G and Folch J (2001) The open pulled straw vitrification of ovine GV-oocytes: positive effect of rapid cooling or rapid thawing or both? CryoLetters 22, 157162.[ISI][Medline]
Isachenko V, Folch J, Nawroth F, Krivokharchenko A, Vajta G, Dattena M and Alabart JL (2003a) Double vitrification of rat embryos at different developmental stages using an identification protocol. Theriogenology 60, 445452.[CrossRef][ISI][Medline]
Isachenko V, Selman H, Isachenko E, Montag M, El-Danasouri I and Nawroth F (2003b) Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure. RBM Online 7, 211216.[Medline]
Isachenko V, Montag M, Isachenko E, Nawroth, Dessole S and Van der Ven H (2004a) Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration. Hum Reprod 19, 660665.
Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F and Van der Ven H (2004b) Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod 71, 11671173.
Jelinkova L, Selman HA, Arav A, Strehler E, Reeka N and Sterzik K (2002) Twin pregnancy after vitrification of 2-pronuclei human embryos. Fertil Steril 77, 412414.[CrossRef][ISI][Medline]
Jones SK and Darville JM (1989) Transmission of virus-particles by cryo-therapy and multi-use caustic pencils: a problem to a dermatologist? Br J Dermatol 121, 481486.[ISI][Medline]
Kuleshova LL and Shaw JM (2000) A strategy for rapid cooling of mouse embryos within a double straw to eliminate the risk of contamination during storage in liquid nitrogen. Hum Reprod 15, 26042609.
Kuwayama M and Kato O (2000) Successful vitrification of human oocytes. Fertil Steril 74, 549.
Lane M, Bavister BD, Lyons EA and Forest KT (1999) Containerless vitrification of mammalian oocytes. Nat Biotechnol 17, 12341236.[CrossRef][ISI][Medline]
Lane M and Gardner DK (2001) Vitrification of mouse oocytes using a nylon loop. Mol Reprod Dev 58, 342347.[CrossRef][ISI][Medline]
Liebermann J and Tucker MJ (2002) Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification. Reproduction 124, 483489.
Liebermann J, Tucker MJ, Graham JR, Han T, Davis A and Levy MJ (2002a) Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. RBM Online 4, 148152.
Liebermann J, Nawroth F, Isachenko V, Isachenko E, Rahimi G and Tucker M (2002b) Potential importance of vitrification in reproductive medicine. Biol Reprod 67, 16711680.
Martino A, Songansen N and Leibo SP (1996) Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 54, 10591069.[Abstract]
Mukaida T, Wada M, Takahashi K, Pedro PB, An TZ and Kasai M (1998) Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod 13, 28742879.
Mukaida T, Nakamura S, Tomiyama T, Wada S, Oka C, Kasai M and Takahashi K (2003) Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 18, 384391.
Palasz AT and Mapletoft RJ (1996) Cryopreservation of mammalian embryos and oocytes: recent advances. Biotechnol Adv 14, 127149.[CrossRef][ISI]
Park SP, Kim EY, Oh JH, Nam HK, Lee KS, Park SY, Park EM, Yoon SH, Chung KS and Lim JH (2000) Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids. Hum Reprod 15, 17871790.
Rall WF (1987) Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24, 387402.[ISI][Medline]
Rall WF (1993) Advances in the cryopreservation of embryos and prospects for alication to the conservation of salmonid fishes. In Cloud JG and Thorgaard GH (eds), Genetic conservation of salmonid fishes. Plenum Press, New York, pp. 137158.
Rall WF and Fahy GM (1985) Ice-free cryopreservation of mouse embryos at 196°C by vitrification. Nature 313, 573575.[ISI][Medline]
Rall WF and Wood MJ (1994) High in vitro and in vivo survival of day 3 mouse embryos vitrified in a non-toxic solution of glycerol and albumin. J Reprod Fertil 101, 681688.[ISI][Medline]
SAS Stat Software (1996) Changes and enhancement through release 6.11. SAS Institute, Cary, NC.
Schaffer TW, Everen J, Silver GH and Came PE (1976) Biohazard: virus-contaminated liquid nitrogen. Science 192, 2526.
Selman HA and El-Danasouri I (2002) Pregnancies derived from vitrified human zygotes. Fertil Steril 77, 422423.[CrossRef][ISI][Medline]
Son WY, Yoon SH, Yoon HJ, Lee SM and Lim JH (2003) Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod 18, 137139.
Szell A, Zhang J and Hudson R (1990) Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at 180°C. Reprod Fertil Dev 2, 613618.[ISI][Medline]
Tedder RS, Zuckerman MA, Goldstone AH, Hawkins AE, Fielding A, Briggs EM, Irwin D, Blair S, Gorman AM and Patterson KG et al. (1995) Hepatitis-B transmission from contaminated cryopreservation tank. Lancet 346, 137140.[ISI][Medline]
Vajta G, Holm P, Greve T and Callesen H (1996) Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration. Anim Reprod Sci 45, 191200.[CrossRef][ISI][Medline]
Vajta G, Kuwayama M, Holm P, Booth PJ, Jacobsen H, Greve T and Callesen H (1998) Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 51, 5358.[CrossRef][ISI][Medline]
Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, Bollen N, Rosendaal van E, Vandervorst M, Schoysman R and Zech N (2003) Vitrification of human blastocysts with hemi-straw carrier: application of assisted hatching after thawing. Hum Reprod 18, 15041511.
Walker D, Tummon S, Hammitt DG, Session DR, Dumesic P and Thornhill A (2004) Vitrification versus programmable rate freezing of late stage murine embryos: a randomized comparison prior to application in clinical IVF. RBM Online 8, 558568.[Medline]
Yokota Y, Sato S, Yokota M and Araki Y (2001) Birth of a healthy baby following vitrification of human blastocysts. Fertil Steril 75, 10271029.[CrossRef][ISI][Medline]
Submitted on July 12, 2004; resubmitted on August 13, 2004; accepted on October 15, 2004.
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