Article Vitrification versus programmable rate freezing of late stage murine embryos: a randomized comparison prior to application in clinical IVF

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1 RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 16 March 2004 Article Vitrification versus programmable rate freezing of late stage murine embryos: a randomized comparison prior to application in clinical IVF David Walker is a graduate of the Master s programme in Clinical Embryology through the University of Leeds, UK. He has worked in reproductive biology research and development for 22 years, the last 18 of which were spent in the USA working in clinical embryology at the University of Iowa Hospitals and Clinics, Iowa City, Iowa and Mayo Clinic College of Medicine in Scottsdale, Arizona and Rochester, Minnesota. Specific areas of research interest include cryobiology, embryo quality assessment and preimplantation genetic diagnosis. David Walker MSc David L Walker 1, Ian S Tummon 1, Diane G Hammitt 1,2, Donna R Session 1, Daniel A Dumesic 1, Alan R Thornhill 1,3 1 Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Mayo Clinic College of Medicine, Rochester, Minnesota, USA; 2 Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Mayo Clinic College of Medicine, Scottsdale, Arizona, USA 3 Correspondence: Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, MN 55905, USA. Fax ; Thornhill.alan@mayo.edu Abstract A prospective randomized trial was performed to compare post-thaw development of murine blastocysts following programmable rate freezing and two methods of vitrification. Frozen 2-cell murine embryos (n = 429) thawed and cultured for 48 h, were randomly allocated by stage of development into four groups: control (not refrozen), programmable rate freezing (PR) in 0.25 ml straws, vitrification in flexible micropipettes by immersion in super-cooled (VSC) liquid nitrogen (LN 2 ), and vitrification in flexible micropipettes by immersion in LN 2 (VLN). Survival, developmental stage progression, presence or absence of an inner cell mass (ICM), and cell counts were recorded 24 h post-thaw. All measured outcomes were different between embryos from the control group and all freezing methods. Controlled-rate freezing resulted in the lowest total cell counts and fewest embryos with a distinct ICM. A higher percentage of embryos survived 24 h post-thaw, progressed to more advanced developmental stages and had higher total cell counts after VLN compared with PR. Moreover, fewer embryos, frozen by either PR or VSC, contained a detectable ICM compared with VLN. These data demonstrate that vitrification may be a better method for freezing murine blastocysts than PR, and may prove to be a superior method for freezing human blastocysts. Keywords: blastocyst, controlled-rate freezing, cryopreservation, embryo freezing, inner cell mass, murine, programmable rate freezing, vitrification 558 Introduction IVF programmes routinely use embryo cryopreservation to augment cumulative pregnancy rates from a single oocyte retrieval. Since the first successful pregnancy resulting from a frozen human embryo transfer reported in 1983 (Trounson and Mohr, 1983), the significance of cryopreservation in modern IVF has been underscored by a report that in the US, an estimated 350,000 frozen embryos are destined for therapeutic use (Hoffman et al., 2003). Early literature focused on cryopreservation at the pronuclear and cleavage stage. Recent trends in culturing embryos to the blastocyst stage have created the need to refine cryopreservation at this later stage. Advances in embryo culture, particularly sequential media, have resulted in more IVF programmes transferring at the blastocyst stage. Using this strategy, fewer embryos can be selected for transfer, with the aim of increasing implantation rates while simultaneously decreasing multiple gestation rates (Gardner et al., 1998a,b; Behr et al., 1999; Marek et al., 1999; Stalf et al., 2002; Smith, 2003). As implantation rates with fresh blastocyst transfers increase, fewer embryos should be required for transfer, resulting in more embryos available for cryostorage.

2 There are several methods of cryopreservation for human blastocysts, with programmable rate freezing being the most widely utilized. This method requires a substantial amount of time for dehydration of the embryo to prevent ice crystal formation. Moreover, slow rate cooling is accomplished using expensive equipment requiring ongoing maintenance. In contrast, vitrification utilizes an extremely rapid cooling rate accomplished by plunging directly into liquid nitrogen. Vitrification may allow a laboratory to perform multiple cryopreservations rapidly within the same cohort of embryos to optimize survival and implantation potential. Several vitrification methods used to increase the rate of cooling, thereby reducing exposure time and potential toxicity of cryoprotectants to improve post-thaw outcomes have been described. A reduction in cryoprotectant volume (1 2 µl) has been achieved using the open pulled straw (Vajta et al., 1998; Kuleshova et al., 1999; Kong et al., 2000; Selman and El- Danasouri, 2002). This system was also successfully used to vitrify human embryonic stem cells (Reubinoff et al., 2001), which may be indirect evidence to support the safety of vitrification for ICM cells of blastocyst stage embryos. Other vitrification vessels used successfully include electron microscope (EM) grids (Cho et al., 2002), and nylon cryoloops (Lane et al., 1999a,b; Mukaida et al., 2003). There are several reports of successful pregnancies following vitrification of human oocytes, day 1, day 3, morula and blastocyst stage embryos (Kuleshova et al., 1999; El- Danasouri and Selman, 2001; Mukaida et al., 2001; Jelinkova et al., 2002; Isachenko, 2003a,b), as well as with programmable rate freezing (Fehilly et al., 1985; Menezo et al., 1992; Kaufmann et al., 1995; Nakayama et al., 1995; Quinn, 1990, 1995; Menezo and Veiga, 1997; Behr et al., 1999; Chi et al., 2002; Veeck, 2003). Although no studies prospectively comparing vitrification and programmable rate freezing of human blastocyst could be found, a comparison between these methods has been made for human pronucleate stage embryos with no clear advantage cited for either method. Although pregnancy rates were not significantly different between the two methods, vitrified embryos had significantly higher post-thaw survival, whereas programmable rate frozen embryos had a higher cleavage rate (Van den Abbeel et al., 1997). The goal of the present study was to determine optimal blastocyst cryopreservation techniques by evaluating postthaw development of murine blastocysts following programmable rate freezing or vitrification by two different methods. If vitrification were shown to be at least equivalent to programmable rate freezing in terms of post-thaw outcomes, vitrification may be a more practical alternative for freezing blastocysts due to the significant saving in time and the decreased financial investment for cryopreservation equipment. Materials and methods Murine embryos thawing, embryos were rinsed through 5 pre-equilibrated microdrops of 100 µl each, G1.3 (Vitrolife, Inc., Englewood, CO, USA) before being placed into 50 µl droplets of G1.3 by groups of approximately 30 embryos each. After 24 h of culture post-thaw (48-h embryo age ), embryos were rinsed and placed into pre-equilibrated G2.3 (Vitrolife). The stage of development of surviving embryos was assessed at 48 h postthaw (72 h embryo age ). Embryos reaching the morula through early hatching blastocyst stages (n = 429, 97.5%) were divided by stage and randomly placed into four treatment groups: controls (not refrozen); programmable rate freezing (PR) in 0.25 ml straws; vitrification in flexible micropipettes by immersion in super-cooled (VSC) liquid nitrogen (LN 2 ) and vitrification in flexible micropipettes by immersion in LN 2 (VLN). Assessments performed 24 h post-thaw included developmental stage progression and total cell counts with detection of a morphologically distinct inner cell mass (ICM) following immunosurgery. All chemicals used in this study were purchased from Sigma-Aldrich Chemical Company, St Louis, MO, USA, unless otherwise indicated. Pre-freeze embryo assessment Embryos were assigned to one of five developmental stages prior to cryopreservation. Embryos classified as morula had all cells appearing flattened with cell boundaries becoming vague or complete disappearance of intercellular spaces, with or without small vacuoles. Early blastocysts had a blastocoele occupying one-third of the embryo area or less. Midblastocysts had a blastocoele occupying one-third to twothirds of the embryo area. Late blastocysts had a blastocoele occupying greater than two-thirds of the embryo area with signs of zona thinning. Early hatching blastocysts had an expanded blastocoele with moderate to significant zona thinning and protrusion of cells less than one-quarter the size of the zona-encased embryo (see Figure 1b f) Post-thaw embryo assessment Since embryonic cells respond osmotically to cryopreservation solutions and may appear shrunken immediately post-thaw, survival and developmental progression was recorded 24 h post-thaw. One of nine development stages was assigned to each embryo. Stages included the five stages described above with the following four additions. Embryos classified as degenerating had all or most cells appearing dark, shrunken and necrotic. Mid-hatching blastocysts had an expanded blastocoele with significant zona thinning and protrusion of cells between one-quarter and one-half the size of the zonaencased embryo. Late-hatching blastocysts had an expanded blastocoele with significant zona thinning and protrusion of cells greater than one-half the size of the zona-encased embryo and may appear as a figure-of-eight shape. Completely hatched blastocysts had an expanding embryonic mass completely free from the zona encasement (see Figure 1a, g i). Embryos were categorized according to these criteria 24 h post-thaw and the percentage of embryos continuing to develop is reported as a survival rate (see Table 1 on p. 564). Four hundred and forty frozen 2-cell murine embryos (B6C3- F1 females B6D2-F1 males; Embryotech Laboratories, Inc., Wilmington, MA, USA) were used for this study. Upon 559

3 A B C D E F G H I Figure 1. (a) Degenerating murine embryo. (b) Morula stage murine embryo. (c) Early blastocyst stage murine embryo. (d) Mid-blastocyst stage murine embryo. (e) Late blastocyst stage murine embryo. (f) Early hatching blastocyst stage murine embryo. (g) Mid-hatching blastocyst stage murine embryo. (h) Late hatching blastocyst stage murine embryo. (i) Complete hatched blastocyst stage murine embryo. 560 Microsurgical separation of ICM/TE by immunosurgery Disaggregation of trophectoderm (TE) and ICM cells was performed 24 h post-thaw on all but severely degenerative embryos by a modified technique as described previously (Hardy et al., 1989). Embryos not already completely hatched from the zona pellucida were exposed for approximately 30 s to acidified Tyrode s solution for removal of the zona pellucida. Embryos were rinsed in phosphate-buffered saline (PBS; Sage BioPharma, Bedminster, NJ, USA) supplemented with 0.1% PVA and exposed to a 10 mmol/l solution of trinitrobenzenesulphonic acid (TNBS) in PBS 0.1% PVA on ice for 15 min (see Figure 2a), followed by rinsing with PBS 0.1% PVA and incubated in 0.1 mg/ml anti-dnp BSA (rabbit antibody: ICN Flow Co., Costa Mesa, CA, USA) for 15 min at 37ºC. Embryos were then rinsed with PBS 0.1% PVA and finally incubated in 10% guinea pig complement in PBS 0.1% PVA for 15 min at 37ºC (see Figure 2b). At this point, the embryos were carefully rinsed in PBS-0.1% PVA to stop the lysing process before complete disruption of the cell membranes. Using a small-bore pipette, the embryonic mass was pipetted, releasing the ICM cells from the lysed TE cells (see Figure 2c). The two cell types were readily distinguishable; TE cells appeared pale and were easily disaggregated, while ICM cells remained in a compact formation with maintenance of membrane integrity. All TE and ICM cell types were separated and mounted for cell count estimates by placing the cells into a small droplet of 0.1% polyoxyethylenesorbitan monolaurate (Tween-20) in 0.01 N hydrochloric acid (HCL) on a silanated glass microscope slide (Fisher Scientific Inc., Pittsburgh, PA, USA). After air-drying, the area was flooded with a 0.08% Giemsa stain in PBS, a glass coverslip added and the stained nuclei were counted using bright field 320 magnification (see Figure 2d,e). Controls Following the 48-h post-thaw development assessment, control embryos were rinsed through five 100-µl droplets of G2.3 before being placed into final culture microdrops of the same medium. Programmable rate freezing and thawing (PR) Standard programmable rate freezing was performed using a Cryomed freezing system (Forma Inc., Marietta, OH, USA). Commercially available freezing and thawing solutions were used according to the manufacturer s recommendations (Sage BioPharma; catalogue nos and 8016). Solutions were warmed to 37ºC and embryos were placed into human tubal fluid (HTF) HEPES supplemented with 12 mg/ml human serum albumin (HSA) for 5 min, followed by a 10-min incubation of HTF HEPES HSA with 5% glycerol and 10 min in HTF HEPES HSA with 9% glycerol and 0.2 mol/l sucrose.

4 A B C D E Figure 2. (a) Zona-free murine blastocyst incubating in TNBS. (b) Lysing of TE cells after anti-dnp-bsa exposure and incubation in complement. Note ICM is unaffected. (c) Careful pipetting can separate ICM from lysed TE cells. (d) TE cells are fixed with Tween 20/HCL and stained with Giemsa for counting. (e) ICM cells fixed and stained for counting. Four or five embryos were then loaded into a 150 µl continuous column of HTF HEPES HSA with 9% glycerol/0.2 mol/l sucrose within standard 0.25 ml IVM straws (Instruments-Medicine-Veterinarian IMV International, Minneapolis, MN, USA) using a flexible micropipette attached to a Stripper syringe (Mid-Atlantic Diagnostics, Marlton, NJ, USA). Both ends of the straw were heat-sealed using a Nyclave Impulse Heat Sealer (Lorvic Corp. St Louis, MO, USA) and the straws placed horizontally into a toploading Cryomed freezing chamber at room temperature. Straws were cooled from room temperature to 7ºC at a programmed rate of 2.0ºC per minute. Straws were seeded using a super-cooled metal rod following a stable temperature of 7ºC for 5 min. Following the heat of fusion ( 4 to 5ºC) and stabilization of the sample temperature back to 7ºC for 5 min, the programme was advanced to cool at a rate of 0.3ºC/min to 37ºC. Straws were then cooled at a maximum rate of 99.9ºC/min to 140ºC and plunged into liquid nitrogen (LN 2 ), in which they remained for a minimum of 30 min before thawing. The total time required for this freezing technique, from placement into the first cryopreservation solution to submerging the straws in LN 2, was approximately 2 h and 45 min. Three solutions for step-down dilution at 37ºC were used for thawing. Straws were removed from LN 2 and placed at room temperature for 30 s before submerging in a 30ºC bath until all ice crystals disappeared. Straw contents were emptied into a 35 mm Petri dish, embryos located and placed into HTF- HEPES HSA with 0.5 mol/l sucrose for 10 min, then into 0.2 mol/l sucrose for 10 min and finally into HTF-HEPES HSA without sucrose for 1 min. Embryos were then rinsed through five 100-µl droplets of G2.3 before being placed into final culture microdrops of the same medium. Vitrification in super-cooled nitrogen and thawing (VSC) Embryos were vitrified in liquid nitrogen slush, super-cooled to 208ºC to 210ºC utilizing the Vit-Master (MiniTub, Tiefenbach, Germany). Vitrification solutions were modified from a published version from Interface Multigrad Technology, Ltd (IMT, Ness Ziona, Israel) consisting of 38% ethylene glycol and 0.5 mol/l trehalose in PBS supplemented with 20% serum substitute supplement (SSS; Irvine Scientific), prepared in the laboratory with 10, 50 and 100% dilutions. The freezing vessel was a flexible Stripper-Tip pipette (MidAtlantic Diagnostics Inc., Marlton, NJ, USA), that had been shortened from 9 cm to 6 cm by the manufacturer. The Stripper-Tip pipette was used because it is commercially available, gamma-sterilized, and FDA approved for use in human IVF. In addition, the small size of the pipette is appropriate for the embryo size and the minute volumes of vitrification solutions used; it also allows storage within a separate vessel that can more easily be hermetically sealed for biosecurity during storage in liquid nitrogen. Embryos were first placed into 10% vitrification solution (VS) for 2 4 min and then rinsed briefly in 50% VS followed by brief rinsing in 100% VS (see Figure 3a d). Within 30 s of exposure to 100% VS, embryos were drawn into a modified Stripper-Tip pipette attached to a Stripper syringe (MidAtlantic Diagnostics Inc.) with approximately 2 µl 100% VS (see Figure 4a). The pipette containing the embryos was removed from the syringe and directly plunged into the supercooled LN 2 ( 208ºC to 210ºC). For secure storage, the Stripper-Tip pipets containing the vitrified embryos were inserted and sealed into CBS High Security straws (Cryo Bio System; IMV Technologies Group, L Aigle, France, distributed by Conception Technologies, Inc., San Diego, CA, USA). With one end of the CBS straw containing an identification rod and pre-sealed, the straw was wrapped in an aluminium foil sleeve and placed in LN 2 to ensure that the Stripper-Tip pipette containing embryos would not warm to damaging temperatures when placed into the final storage straw (see Figure 4b). The foil-wrapped straw was removed from the LN 2, quickly sealed and placed back into LN 2 for storage (see Figure 4c,d). This process was pre-tested revealing that, while the CBS straw was submerged in the foil and LN 2, the internal temperature measured 192 C. Once the 561

5 A C B D Figure 3. (a) Murine blastocyst prior to exposure of three step-wise vitrification solutions. (b) Murine blastocyst following 3-min exposure to 10% vitrification solution (VS), 3.8% EG mol/l trehalose. (c) Murine blastocyst following s exposure to 50% VS, 19% EG M trehalose. (d) Murine blastocyst following s exposure to 100% VS, 38% EG mol/l trehalose. Figure 4. (a) Loading embryos into the Stripper Tip pipette immediately prior to plunging into LN 2 slush. (b) After vitrifying in LN 2 slush the Stripper Tip pipette is placed into a pre-cooled CBS straw. (c) The pre-cooled CBS straw is hermetically sealed for biosecure storage in LN 2. (d) The Stripper Tip pipette containing the vitrified embryos is securely sealed in a CBS straw for storage in LN 2. A C B D 562 foil and straw were removed from the LN 2 for sealing, the temperature rose to 190 C after 1 min and to 186 C after 2 min. Since the sealing process takes approximately 7 s, this method ensures appropriate temperatures within the straw during this procedure. At thawing, while remaining partially submerged in LN 2, the sealed end containing the identification rod was cut, the CBS straw removed from LN 2 and the end containing the Stripper- Tip pipette was cut and the straw gently tapped to expose the pipette containing the embryos. The pipette was quickly placed onto a Stripper syringe, the tip submerged into the first of four warming solutions (0.6 mol/l trehalose in PBS 20% SSS) and the plunger depressed, expelling the embryos. Embryos were mixed in the solution and held at 37ºC for 2.5 min before being sequentially transferred to each of three more thawing solutions (0.5, 0.3 and 0.1 mol/l trehalose) with 2.5-min incubation intervals. Following exposure to the final solution, embryos were rinsed through 5 droplets of G2.3 before being placed in final culture microdrops of G2.3. Vitrification in liquid nitrogen and thawing (VLN) Embryos allocated to this treatment were treated identically to those in VSC, with the exception of using liquid nitrogen ( 196ºC) rather than super-cooled nitrogen for the vitrification process. Statistical methods Wilcoxon rank-sum tests were used for pairwise comparisons of embryo-stage distribution between treatment groups. This analysis was performed separately for pre-freeze and 24 h post-thaw stages. The proportion of embryos with a countable ICM 24 h post-thaw was compared among all treatment groups and between each pair of treatments using chi-squared or Fisher s exact tests, as appropriate. Side-by-side box plots and the non-parametric Kruskal Wallis tests were used to compare total cell counts among the four treatments. Pairwise Wilcoxon rank-sum tests were then used to assess differences between groups. Statistical analyses were performed using JMP 4

6 statistical software (JMP, Version 4.0.4; SAS Institute Inc., Cary, NC, USA, ). P-values <0.05 were considered statistically significant. Results The developmental stage distribution of embryos pre-freeze was not different (P = 0.9) among all treatment groups (Figure 5a). However, 24 h after thawing more advanced stages of development (P < , Figure 5b), increased survival and continued development (P < , Table 1), presence of an ICM (P < , Figure 6) and higher total cell counts (P < , Figure 7) were observed in the control embryos compared with all re-freezing treatments. Among the freezing treatments, differences in embryo stage distribution were observed between VLN and PR methods (P = ) with more embryos developed to advanced stages in the VLN group. Similarly, the per cent survival and continued development post-thaw was higher in VLN than PR (P = , Table 1). The proportion of embryos containing a distinct ICM after immunosurgery was higher in the VLN group (65.7%) compared with the PR group (39.2%, P < 0.001) and the VSC group (51.9%, P = , Figure 6). In addition, higher total cell counts were measured for the VLN treatment group embryos compared with PR embryos (P < 0.001, Figure 7, Table 2). The only parameter that was not different among any of the treatments was the ratio of ICM cells to total cells for embryos possessing a detectable ICM (P = , Table 3). Discussion Use of murine embryos as a model for human embryo cryopreservation The primary aim of this controlled, randomized study was to compare survival, developmental stage progression, total cell counts and detection of an inner cell mass, from late stage murine embryos frozen by conventional programmable rate freezing and two methods of vitrification. The limited numbers of normal human embryos available for research coupled with unreliable and variable development of abnormally fertilized oocytes prompted the selection of murine embryos for this study. Commercially available murine embryos provide high quality test material, allowing for thaw synchronization and good experimental design with remarkably consistent and reliable development under conditions optimized for human embryo culture (Walker et al., 2003). As expected, non-refrozen control embryos showed better results than treatment embryos for all 24-h post-thaw parameters evaluated. It can be speculated that outcomes across all treatments may have improved if fresh, rather than re-frozen, embryos had been utilized. While freezing and, to a greater extent re-freezing, may compromise embryo development, it is also possible that added stress on embryos previously frozen at the 2-cell stage may improve the sensitivity to detect differences between cryopreservation methods or culture conditions. Use of refrozen murine embryos has previously been validated where both once- and twice-frozen embryos showed no difference regarding implantation rate and number of live fetuses compared with non-frozen controls (Nowshari and Brem, 2000). Biosecurity of cryopreserved embryos While post-thaw viability and concomitant implantation potential are of primary importance in a successful embryofreezing programme, biosecurity of cryopreserved embryos during long-term storage should also be considered. With the possibility of viral contamination during storage in liquid nitrogen reported previously (Tedder et al., 1995; Bielanski et al., 2000), there are concerns regarding the use of open systems such as grids (Park et al., 1999; Son et al., 2003) or cryoloops (Lane, 1999b) for storage of human embryos. While obtaining preliminary data using the modified Stripper-Tip pipette, there was an encouraging report in which flexible denuding pipettes, sealed within a 0.25 ml straw, were successfully used with human embryo cryopreservation and storage (Liebermann et al., 2002). This straw-in-straw method was also successful when vitrifying human blastocysts within a Hemi-straw and being stored within a CBS high security straw (Vanderzwalmen et al., 2003). Use of the CBS high security straw for storage of the Stripper-Tip pipets containing vitrified embryos is an additional measure which would likely reduce the chance of viral transmission during storage in liquid nitrogen. Programmable rate freezing may not be optimal for blastocyst cryopreservation Programmable rate freezing of blastocysts has been the standard in many established IVF programmes. Success using this method may be the result of freezing only high quality embryos that developed into blastocysts on days 5 and 6 prior to freezing (Veeck, 2003) or slightly modifying the start temperature and rate of cooling during the freeze (Gardner et al., 2003). The most alarming outcome observed in this study is that only 39% of embryos frozen using a programmable rate method contained a detectable inner cell mass. The ICM is responsible for the formation of cellular layers that eventually develop into a fetus, while TE cells are largely responsible for formation of placental tissue. Cell lineage tracing experiments have shown that ICM formation is governed by cell polarity during the early and late morula stages. As differentiating cells are forming the ICM, they are capable of forming trophectoderm cells if, for some reason, TE cells become damaged. In contrast, TE cells are not capable of replenishing ICM cells if the latter become damaged. This exchange of repair has only been observed up to the late blastocyst stage (Balakier and Pedersen, 1982; Cruz and Pedersen, 1985; Winkel and Pedersen, 1988). A range of embryo stages was used, from early morula to early hatching embryos, for the present study. Following programmable rate freezing, there may have been a higher incidence of irreversible effects on cell polarity within the morula and early blastocyst stage embryos. This may have caused a disruption in the allocation of cells to the ICM. It can be speculated that later stage embryos containing an established ICM would likely be unaffected, but it was not possible to assess this in the present study, due to group culturing. Among embryos with a countable ICM there was no difference seen in the ICM to total cell ratio between treatments indicating that those embryos containing an ICM were developing both cell types at a similar rate regardless of the freezing method used. 563

7 Figure 5. a. Pre-freeze embryo stage distribution. No difference in embryo stage distribution among treatment groups (P = ). b. Twenty-four hours post-thaw embryo stage distribution. Development greater for control versus all other groups, P < Development greater for VLN versus PR, P = PR = programmable rate freezing; VSC = vitrification in supercooled LN 2 ; VLN = vitrification in liquid nitrogen; Deg = degenerating; Mor = morula; EB = early blastocyst; MB = midblastocyst; LB = late blastocyst; EH = early hatching blastocyst; MH = mid-hatching blastocyst; LH = late-hatching blastocyst; CH = completely hatched blastocyst. Table 1. Embryo survival and development 24 h post-thaw. Non-viable embryos 24 h post-thaw were designated as degenerating. PR = programmable rate freezing; VSC = vitrification in super-cooled LN 2 ; VLN = vitrification in liquid nitrogen. Treatment Number Number early Number early Survival and degenerating to late to complete continued blastocyst hatching development blastocyst Control /104 (99.0) PR /108 (80.6) a,b VSC /110 (81.8) a VLN /107 (90.7) a Values in parentheses are percentages. a P < versus control. b P = versus VLN. 564 Figure 6. Embryos with detectable inner cell mass (ICM). a P < versus control; b P < versus VLN. c P = versus VLN. PR = programmable rate freezing; VSC = vitrification in super-cooled LN 2 ; VLN = vitrification in liquid nitrogen.

8 Figure 7. Total cell counts per embryo. a P < versus control. b P = versus VLN. All viable embryos included regardless of the presence or absence of ICM. PR = programmable rate freezing; VSC = vitrification in super-cooled LN 2 ; VLN = vitrification in liquid nitrogen. Note: the side-byside box plots display particular features of the distribution within each treatment group and allow a visual comparison of total cell count among the groups. The line across the middle of the box identifies the median within each group. The ends of the box are the 25th and 75th percentiles, also called the quartiles. The difference between the quartiles is the interquartile range. Lines, often called whiskers, extend from the ends of the box to the outermost data point that falls within the distances computed as [upper quartile (interquartile range)] and [lower quartile 1.5 (interquartile range)]. (SAS Institute Inc JMP Statistics and Graphics Guide, Cary, NC, USA.) Table 2. Total cell counts per embryo. All viable embryos included, regardless of the presence or absence of ICM. Treatment Number Mean number Median number 95% mean of embryos of cells per of cells per confidence embryo (±SD) embryo (range) interval Control (±27.8) 94.0 (24 149) PR (±20.9) a,b 52.5 (10 114) VSC (±20.9) a 60.0 (4 132) VLN (±20.9) a 67.0 (9 130) a P < versus control. b P = versus VLN. Table 3. Ratio of inner cell mass and total cell counts. Only embryos containing an ICM included. PR = programmable rate freezing; VSC = vitrification in super-cooled LN 2 ; VLN = vitrification in liquid nitrogen. Treatment Number Mean ratio of ICM and Median ratio of ICM and 95% mean of embryos total cell count (±SD) total cell count (range) confidence interval Control (±0.078) ( ) PR (±0.071) ( ) VSC (±0.063) ( ) VLN (±0.051) ( ) No significant difference between all treatments; P =

9 566 Programmable rate freezing also resulted in embryos with the lowest total cell counts and fewer embryos progressing to more advanced stages of development. Since the laboratory used for this work is not equipped to perform murine embryo transfers, pregnancy and implantation outcomes could not be determined for embryos in this study. It is possible that embryos frozen by the programmable rate method would have lower birth rates based on fewer embryos containing an ICM. No advantage using super-cooled liquid nitrogen slush In the present study, more total cells were found in VLN embryos than in VSC embryos. Other outcomes were more favourable with VLN than with VSC, but the differences were not statistically significant. A previous study using murine zygote stage embryos reported the VLN method having higher post-thaw survival (91%) than when super-cooled liquid nitrogen was used (70%), but no difference in cleavage and development between the two methods (Nowshari and Brem, 2001) is in agreement with the present study. Embryos in the present study frozen by VSC, however, did fare better post-thaw than those frozen by the programmable rate freezing method (PR) with total cell counts being higher in the VSC group. Several recent publications have advocated the use of super-cooled liquid nitrogen slush for vitrifying a variety of biological tissues: in-vitro matured bovine oocytes (Arav et al., 2002), ovine cumulus oocyte complexes (Isachenko et al., 2001), human spermatozoa (Nawroth et al., 2002), and murine blastocysts (Nowshari and Brem, 2001). However, the present data do not support the use of supercooled liquid nitrogen (slush) for vitrification of late stage murine embryos compared with vitrifying in liquid nitrogen. Programmable rate versus vitrification in liquid nitrogen In an extensive literature search, no studies could be found using human blastocyst stage embryos to directly compare programmable rate freezing and vitrification. However, one study using human pronuclear stage embryos reported vitrification having higher post-thaw survival (83.3%) than programmable rate freezing (66.4%). Although the cleavage rate was lower for vitrified embryos (50.9%) compared with programmable rate freezing (65.6%), the pregnancy rate from vitrified embryos (7.4%) was not different from programmable rate frozen embryos (16%) (Van den Abbeel et al., 1997). In comparison, the present vitrification methods utilized higher cryoprotectant concentrations (by 3.3%), a substantially shorter exposure time (30 s versus 4 min) and a lower solution volume (2 µl versus 20 µl). The increased exposure time and solution volume used in the previous study may have affected the thermodynamics of the vitrification process, allowing increased exposure of toxic concentrations of the cryoprotectants with a subsequent lower cleavage rate. Several studies utilizing animal models have been reported comparing programmable rate and vitrification methods. Using 2-cell murine embryos, fewer vitrified embryos developed to the blastocyst stage and had lower glucose uptake. Although blastocysts from both methods were morphologically indistinguishable, vitrified embryos also had a lower implantation rate, suggesting possible detrimental effects of vitrification on early stage embryos (Uechi et al., 1999). Again, increased exposure time (5 min) and higher volumes (250 µl), as well as a slow rate of thawing may have contributed to the poor results from the vitrified embryos. Late stage sheep and bovine embryos have also shown detrimental effects of extended cryoprotectant exposure during vitrification when compared with programmable rate freezing (Sommerfeld and Niemann, 1999; Isachenko et al., 2003a). These reports showed longer exposure at higher VS concentrations than in the present study, which may have led to toxic stress on the vitrified embryos. A study utilizing bovine blastocysts provides data from a prospective randomized comparison of programmable rate freezing and vitrification similar to the present study (Kaidi et al., 2001). There was no difference between the two freezing methods for post-thaw re-expansion and hatching. Embryos from programmable rate freezing, however, had lower glucose and pyruvate uptake, higher lactate release and lower glycolytic activity than controls, while embryos from vitrification were not different from either controls or programmable rate frozen embryos. There were fewer total cells and trophectoderm cells noted in programmable rate frozen embryos than in vitrified embryos, but no difference in the ICM cell numbers. They postulated that this decrease seen in TE cell numbers and not in ICM could be due to possible damage of the cell membrane transport mechanisms caused by the slow rate of freezing and prolonged exposure to cryoprotectants. Short exposure and decreased vitrification solution volumes are critical In contrast to a number of studies in several different species that show poorer outcomes for vitrification versus programmable rate freezing, the present study (using murine blastocysts) shows that liquid nitrogen vitrification yields superior post-thaw outcomes when compared with both programmable rate freezing and super-cooled liquid nitrogen slush vitrification. A brief exposure to high concentrations of cryoprotectants and a minimal volume of VS were probably responsible for the substantial improvement in post-thaw outcomes. Confirmation of these results with human blastocysts, as well as pronuclear and cleavage stage embryos, could lead to higher frozen embryo delivery rates and a significant decrease in the time required to perform embryo cryopreservation. In conclusion, these data demonstrate vitrification to be a superior method for freezing murine blastocysts than PR, and may prove to be as advantageous for freezing human blastocysts. Acknowledgements The authors wish to acknowledge gratefully Ms Tanya Hoskin from the Division of Biostatistics for assistance with statistical analyses. Thanks also to Drs Helen Picton and Lucy Jenner (University of Leeds, UK, Master s in Clinical Embryology programme) for support and guidance, as this work was submitted as part of the first author s thesis.

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