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Cryosystem assessment by glucose uptake of murine blastocysts
RBMOnline - Vol 11. No 5. 2005 601–607 Reproductive BioMedicine Online; www.rbmonline.com/Article/1747 on web 31 August 2005
Article Cryosystem assessment by glucose uptake of murine blastocysts 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.
Mr David Walker David L Walker1,6, David K Gardner2, Michelle Lane3, Ian S Tummon1, Donna R Session4, Alan R Thornhill5 1 Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Section of Reproductive Endocrinology and Infertility, Charlton Building Desk 3-A, 200 First Street SW, Mayo Clinic College of Medicine, Rochester, MN 55905; 2Colorado Centre for Reproductive Medicine, Englewood, CO, USA; 3Research Centre for Reproductive Health, Department of Obstetrics and Gynecology, University of Adelaide, Adelaide, Australia and Repromed, Adelaide, Australia; 4Emory Reproductive Centre, 550 Peachtree Street, Suite 1800, Atlanta, GA 30308, USA; 5London Gynaecology and Fertility Centre Limited, Cozens House, 112A Harley Street, London W1G 7JH, UK 6 Correspondence: Tel: +1 507 2663995; Fax: +1 507 2841774; email: [email protected]
Abstract Glucose uptake was used as a measure of metabolic activity and implantation potential to compare vitrification and slow freezing in a prospective randomized trial using murine blastocysts. Frozen 2-cell embryos (n = 132) thawed and cultured for 48 h to the blastocyst stage were randomly divided into four groups: (i) control – not refrozen; (ii) slow freezing using a programmed rate (PR); (iii) vitrification by super-cooled (VSC) liquid nitrogen; and (iv) vitrification in liquid nitrogen (VLN). Upon re-thawing, embryos were cultured individually for 24 h to determine glucose uptake non-invasively. Morphological assessments included total cell counts and inner cell mass (ICM) detection following immunosurgery. Mean glucose uptake was lower for each treatment (PR and VSC, 4.3 pmol/embryo per h; VLN, 4.9 pmol/embryo per h) versus controls (6.8 pmol/embryo per h). PR and VSC embryos had fewer cells (57.4 ± 24.2 and 64.1 ± 31.5) versus controls (85.7 ± 26.2), and fewer embryos containing a detectable ICM (42.9 and 61.8%) compared with controls (88.2%). The only difference between control and VLN embryos was absolute glucose uptake, although in both treatments glucose uptake was increased from embryos with an ICM compared with those without. Glucose uptake appears to be a sensitive, non-invasive method to validate cryopreservation protocols. Keywords: blastocyst, cryopreservation, embryo freezing, inner cell mass, murine, vitrification
Introduction Recent trends in culturing embryos to the blastocyst stage have created the need to refine blastocyst cryopreservation methodologies for improved post-thaw survival and implantation. The introduction of sequential media has resulted in more IVF programmes transferring at the blastocyst stage with fewer embryos being selected for transfer, thus increasing implantation rates while decreasing multiple gestation rates (Gardner et al., 1998a,b; Behr et al., 1999; Marek et al., 1999; Smith et al., 2003). With higher implantation rates using fresh blastocysts, fewer embryos are required for transfer, resulting in more high quality supernumerary blastocysts being available for cryostorage.
At present, programmable rate (PR) freezing is the most widely utilized method of cryopreservation for human blastocysts. This time-consuming method is accomplished using sophisticated, usually expensive, equipment that requires ongoing maintenance and involves prolonged exposure of embryos to cryoprotectants to prevent potentially damaging intercellular ice crystal formation. In contrast, vitrification is rapid, can be accomplished by plunging directly into liquid nitrogen and requires extremely short exposure times to high concentrations of cryoprotectants with no ice crystal formation. Because of the comparative simplicity and brevity of vitrification procedures, a laboratory can perform multiple cryopreservation procedures over time within the same cohort of embryos to optimize survival and implantation potential per embryo.
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Article - Glucose uptake to compare cryopreservation methods - DL Walker et al. To date, no studies have been reported to prospectively compare PR freezing and vitrification of human blastocysts. Indeed, the only prospective comparison between these methods in human embryos involved pronuclear zygotes, with no clear advantage cited for either method. Although pregnancy rates were not different, vitrified embryos had higher post-thaw survival, whereas PR frozen embryos had a higher cleavage rate (Van den Abbeel et al., 1997). Human IVF laboratories rely primarily on development rate and morphology to determine which embryos possess the greatest implantation potential. However, non-invasive, quantitative analysis of metabolites such as glucose and pyruvate has been reported in both human and murine embryos to provide additional selection criteria for identifying embryos with the greatest implantation potential (Gardner and Leese, 1987; Conaghan et al., 1993; Gardner et al., 1996, 2001; Lane and Gardner, 1996) and in predicting blastocyst survival following slow freezing (Gardner et al., 1996). The preimplantation blastocyst relies on glucose to provide energy for biosynthesis activities and in preparation for implantation. Glucose stores within the embryo are utilized immediately prior to implantation, due to a somewhat decreased availability of oxygen in the uterus at the site of implantation; thus, the embryo is able to maintain high glycolytic activity during this critical time period. In-vitrocultured murine blastocysts with a glucose metabolism similar to in-vivo-derived blastocysts had a 4-fold increase in implantation compared with embryos with perturbed glycolytic activity (Lane and Gardner, 1996). Thus increased depletion of glucose from the media serves as a marker for detection of embryos that have increased metabolic activity and higher implantation potential. In a previous study from this laboratory, group cultured murine late stage embryos were used to compare two methods of vitrification by plunging into super-cooled liquid nitrogen (VSC) or standard liquid nitrogen (VLN) and PR freezing (PR). Compared with VSC and VLN, PR resulted in the lowest total cell counts and fewest embryos with a distinct ICM. With VLN, a higher percentage of embryos survived 24 h post-thaw, progressed to more advanced developmental stages and had higher total cell counts compared with PR. Moreover, fewer embryos frozen by both PR and VSC contained a detectable ICM compared with VLN (Walker et al., 2004). Since these embryos were cultured in groups, glucose uptake of individual embryos could not be accurately assessed; hence the current study design. In the present study, identical cryopreservation methods and conditions were used as described previously (Walker et al., 2004), except that embryos were randomized post-thaw for individual culture with the primary purpose of glucose uptake analysis.
Materials and methods Murine embryos
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One hundred and thirty-two frozen 2-cell murine embryos (B6C3-F1 females × B6D2-F1 males; Embryotech Laboratories, Inc., Wilmington, MA, USA) were used for this study. Upon thawing, embryos were rinsed through five pre-equilibrated
microdrops of 100 μl each, G1.3 (Vitrolife, Inc., Englewood, CO, USA) before being placed into 50 μl droplets of G1.3 in 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’). Compacting morula through early hatching blastocyst stage embryos were placed into groups by stage. Embryos from each group were sequentially divided into four treatment groups: controls – not refrozen; PR freezing (PR) in 0.25 ml straws; vitrification in flexible micropipettes by immersion in super-cooled (VSC) liquid nitrogen (LN2) and vitrification in flexible micropipettes by immersion in LN2 (VLN). Assessments performed 24 h post-thaw or postwarming included developmental stage progression, total cell counts with detection of a morphologically distinct ICM following immunosurgery, and glucose uptake. All chemicals used in this study were purchased from Sigma-Aldrich Chemical Company, Saint Louis, MO, USA, unless otherwise indicated.
Pre-freeze embryo assessment Embryos were assigned to one of five developmental stages prior to cryopreservation, morula, early-blastocyst, midblastocyst, late-blastocyst, and early-hatching blastocyst.
Post-thaw embryo assessment At 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: degenerating, midhatching blastocyst, late-hatching blastocyst and completely hatched blastocyst as described earlier (Walker et al., 2004). 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.
Microsurgical separation of ICM/TE by immunosurgery Disaggregation of trophectoderm (TE) and ICM cells was performed 24 h post-thaw on all but severely degenerating embryos. Briefly, embryos not already completely hatched from the zona pellucida were exposed to acidified Tyrode’s solution for removal of the zona pellucida. Embryos were rinsed in PBS supplemented with 0.1% polyvinyl alcohol (PVA), exposed to a 10 mmol/l solution of trinitrobenzenesulphonic acid (TNBS) in PBS-0.1% PVA followed by incubation in 0.1 mg/ml anti-dinitrophenylated bovine serum albumin (rabbit antibody; Valeant Pharmaceuticals Intl., Costa Mesa, CA, USA, formerly ICN Pharmaceuticals Co.). Embryos were finally incubated in 10% guinea pig complement serum in PBS–0.1% PVA to initiate lysing of the TE cells coated with the antibody. Embryos were then rinsed in PBS–0.1% PVA to stop the lysing process before complete disruption of TE membranes. Using a small-bore pipette (120 μm), the embryonic mass was pipetted, releasing the ICM from the lysed TE cells. The two cell types were easily distinguishable; TE cells appeared pale and were easily disaggregated, while ICM cells remained in a compact formation with maintenance
Article - Glucose uptake to compare cryopreservation methods - DL Walker et al. of membrane integrity and a luminescent appearance. All TE and ICM cell types were separated and mounted on silanated glass microscope slides (Fisher Scientific, Inc., Pittsburgh, PA, USA) for cell count estimates by placing the cells into a small droplet of 0.1% polyoxyethylenesorbitan monolaurate (Tween-20) in 0.01 normal (N) hydrochloric acid (Coonen et al., 1994). 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.
Non-invasive measurement of glucose uptake Immediately after thaw, embryos were incubated individually in 500 nl of fresh G2.3 (containing reduced glucose at 0.5 mmol/l versus standard concentration of 3.15 mmol/l) for overnight culture. All incubations took place in 6% CO2 in air at 37ºC. Following the 24-h culture period, the 500nl drop was aspirated into a washed and sterilized, 1 μl Microcap glass capillary tube (Drummond; Fisher Scientific Inc., Broomall, PA, USA). The microcaps were placed inside cryostraws and heat sealed for storage at –70°C prior to analysis for glucose. Glucose concentration was determined by an ultramicrofluorometric technique, in which biochemical reactions were performed on a siliconized glass slide under oil. The fluorescent reaction was quantified using a photomultiplier and photometer, and was expressed as pmol glucose utilized per embryo per hour. Control media from non-spent microdrops was analysed with each representative treatment run. Samples were read in a blinded fashion to eliminate bias. The analysis procedure is described in detail elsewhere (Leese and Barton, 1984; Gardner and Lane, 2004).
Controls Following the 48-h post-thaw development assessment, control embryos were rinsed through five 100-μl droplets of G2.3 (with reduced glucose) before placement into individual culture microdrops (500 nl), as for treatment embryos.
Programmable rate freezing and thawing (PR) Standard PR freezing and stepwise thawing was performed as previously described (Walker et al., 2004), using a Cryomed freezing system (Forma, Inc., Marietta, OH, USA) and commercially available freezing and thawing solutions used according to the manufacturer’s recommendations (Sage BioPharma, Bedminster, NJ, USA, catalogue nos 8015 and 8016). The total time for this freezing technique, from placement into the first cryopreservation solution to submerging the straws in LN2, was approximately 2 h and 45 min. After thawing, embryos were rinsed through five 100-μl droplets of G2.3 (with reduced glucose) before being randomized and placed into final culture microdrops of the reduced glucose medium.
Vitrification in super-cooled nitrogen and warming (VSC) Embryos were vitrified in LN2 slush, super-cooled to –208°C to –210°C utilizing the Vit-Master (MiniTub, Tiefenbach, Germany) and warmed as described previously (Walker et al., 2004). Following exposure to the final solution, embryos were rinsed through five droplets of G2.3 before being randomized and placed in final culture microdrops of reduced glucose G2.3 medium. The total time for this technique, from placement into the first cryopreservation solution to submerging the straws in LN2, was approximately 5 min.
Vitrification in liquid nitrogen and warming (VLN) Embryos allocated to this treatment were treated identically to those in VSC with the exception of using LN2 (–196°C) 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 24 h post-thaw. 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. The nonparametric Kruskal–Wallis test was 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 statistical software (JMP, Version 4.0.4; SAS Institute Inc., Cary, NC, USA, 1989–2002). Statistical significance was set at the 0.05 level.
Results Overall survival and continued development 24 h post-thaw was not different between the non-refrozen controls and the treatments, with the exception of VSC showing slightly lower development (82.4%) compared with control embryos (97.1%, Table 1). Mean glucose uptake was lower for each treatment (PR and VSC, 4.3 pmol/embryo per h; VLN, 4.9 pmol/embryo per h) compared with controls (6.8 pmol/embryo per h, Table 2). PR and VSC embryos had fewer cells (57.4 ± 24.2 and 64.1 ± 31.5) than controls (85.7 ± 26.2, Table 3), and fewer embryos containing a detectable ICM (42.9 and 61.8%) compared with controls (88.2%, Figure 1). However, survival rates, presence of an ICM, and cell counts were similar between control and VLN embryos. VLN embryos had higher cell counts (70.9 ± 30.4) and more embryos with a detectable ICM (79.3%) than PR embryos (57.4 ± 24.2 and 42.9% respectively). When comparing glucose uptake between all embryos with and without an ICM, embryos with an ICM had a higher glucose uptake (5.7 pmol/embryo per h) than those without (3.8 pmol/embryo per h). Although the absolute glucose uptake between control and VLN embryos was different, both control and VLN embryos showed higher glucose uptake
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Table 1. Embryo survival and development 24 h post-thaw. Treatment
Degenerating embryos
Early to late blastocysts
Early to complete hatching blastocysts
Percentage survival and re-expansion (n)
Control PR VSC VLN
1 6 6 3
4 9 8 5
29 20 20 21
97.1 (33/34) 82.9 (29/35)b 82.4 (28/34)a 89.7 (26/29)
P < 0.05 versus control. P < 0.0508 versus control. Embryos frozen at morula to early hatching stage: PR = programmable rate freezing, VSC = vitrification with super-cooled nitrogen, VLN = vitrification with liquid nitrogen.
a
b
Table 2. Twenty-four hour post-thaw glucose uptake in embryos cultured individually (pmol glucose utilized by treatment). Treatment
No. embryos
Mean pmol/embryo per h (±SD)
95% CI
Median pmol/embryo per h (range)
Control PR VSC VLN
34 35 34 29
6.8 (2.5) 4.3 (2.3)a 4.3 (1.9)a 4.9 (2.3)a
5.9–7.6 3.5–5.1 3.7–5.0 4.1–5.8
7.2 (0.8–10.6) 4.6 (0.0–9.0) 4.2 (0.2–7.9) 5.2 (0.7–9.1)
a P < 0.02 versus control. PR = programmable rate freezing, VSC = vitrification with super-cooled nitrogen, VLN = vitrification with liquid nitrogen.
Table 3. Total cell counts, all embryos. Treatment
No. of embryos
Mean no. cells/embryo (±SD)
95% mean CI
Median no. cells/embryo (range)
Control PR VSC VLN
34 35 34 29
85.7 (±26.2) 57.4 (±24.2)a,b 64.1 (±31.5)a 70.9 (±30.4)
76.6–94.8 49.1–65.7 53.1–75.1 59.2–82.4
90 (29–138) 56 (19–114) 65 (11–132) 80 (9–111)
Control versus PR (P < 0.0001) and VSC (P = 0.0032). Versus VLN (P = 0.0417). PR = programmable rate freezing, VSC = vitrification with super-cooled nitrogen, VLN = vitrification with liquid nitrogen.
a
b
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Figure 1. Embryos with a countable inner cell mass. aP < 0.02 versus control; bP < 0.01 versus VLN. PR = programmable rate freezing; VSC = vitrification with super-cooled nitrogen; VLN = vitrification with liquid nitrogen.
Table 4. Twenty-four hour post-thaw glucose uptake in embryos cultured individually (pmol glucose utilized compared in embryos with and without an ICM). Treatment
No. embryos
Mean pmol/embryo per h (±SD)
95% CI
Median pmol/embryo per h (range)
With ICM Without ICM
89 43
5.7 (2.4) 3.8 (2.0)a
5.2–8.0 3.2–3.4
5.9 (0.0–10.6) 4.1 (0.0–7.1)
a
P < 0.0001 versus ‘With ICM’.
Table 5. Glucose uptake for embryos with (+) and without (–) ICM. Treatment
ICM
No. embryos
Mean of pmol/embryo per h (±SD)
95% CI
Median pmol/embryo per h (range)
Control
+ – + – + – + –
30 4 15 20 21 13 23 6
7.1 (2.3) 4.4 (2.6)a 4.5 (2.8) 4.1 (2.0)c 4.8 (1.7) 3.5 (1.9)d 5.5 (2.1) 2.9 (1.8)b
6.3–8.0 0.2–8.5 3.0–6.1 3.2–5.1 4.0–5.6 2.4–4.7 4.6–6.4 1.0–4.7
7.4 (2.3–10.6) 4.8 (0.80–7.1) 5.0 (0.0–9.0) 4.5 (0.0–6.7) 4.3 (1.9–7.9) 3.6 (0.2–6.2) 5.6 (1.4–9.1) 2.7 (0.7–5.2)
PR VSC VLN
P < 0.05 versus With ICM. P < 0.01 versus With ICM. P = 0.5154 versus With ICM (NS). d P = 0.0556 versus With ICM (NS). Within each treatment, control and VLN embryos maintained a difference in glucose uptake between embryos with and without an ICM (P < 0.05 and 0.01 respectively). However, glucose uptake was not different between embryos with and without an ICM from the PR and VSC treatments (P = 0.5154 and 0.0556 respectively). a
b c
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Article - Glucose uptake to compare cryopreservation methods - DL Walker et al. for embryos with an ICM compared with those that did not. In contrast, the presence or absence of an ICM had no effect on glucose uptake in embryos from either PR or VSC treatments (Tables 4 and 5).
Discussion The primary aim of this study was to compare glucose uptake from individually cultured blastocyst stage murine embryos frozen by conventional PR freezing and two methods of vitrification. Other outcome measures included survival, developmental stage progression, total cell counts and presence or absence of an inner cell mass. In accordance with previous results comparing these methods (Walker et al., 2004), overall survival and development was nearly identical for all treatments using a larger number of embryos. Non-refrozen control embryos showed better results than treatment embryos for all 24-h post-thaw parameters evaluated, with the exception of VLN embryos, in which the only significant difference between control and VLN embryos was glucose uptake. It is speculated that outcomes across all treatments may have improved if fresh, rather than re-frozen, embryos had been utilized. However, non-cryopreserved mouse embryos were not available for this research. In agreement with previous results (Walker et al., 2004), a lower number of embryos frozen by the PR contained a detectable ICM than controls. However, among embryos with a detectable ICM, no difference was seen in the ICM to total cell ratio between treatments (data not shown), indicating that those embryos containing an ICM were developing both cell types at a similar rate regardless of the cryopreservation method used. Programmable rate freezing resulted in embryos with the lowest total cell counts and fewest embryos progressing to more advanced stages of development. Trophectoderm cells may have sustained more damage from this cryopreservation method and would thus account for the lower number of embryos with an ICM. Alternatively, the low number of ICM cells, which primarily consume glucose (Hewitson and Leese, 1993) could reflect changes in total glucose utilization post-cryopreservation. Although it was not possible to assess pregnancy outcomes for frozen–thawed embryos in this study, it can be speculated that embryos frozen by the PR method would have lower birth rates based on fewer embryos containing an ICM and lower overall glucose uptake. Previous studies (Nowshari and Brem, 2001; Walker et al., 2004), showed that post-thaw outcomes were significantly improved after cryostorage by VLN versus VSC. However, with the exception of glucose uptake between embryos with and without an ICM, these differences were not seen in the present study.
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Embryos vitrified by VSC did fare slightly better post-warming than those frozen by the PR method, with total cell counts and the number of embryos containing a detectable inner cell mass being higher in the VSC group. However, these differences did not achieve statistical significance, perhaps as a result of the small sample size.
To date, there have been no published comparisons of PR freezing and vitrification in human blastocysts. In a prospective randomized comparison of PR freezing and vitrification using bovine blastocysts (Kaidi et al., 2001), there was no difference between cryopreservation methods for post-thaw re-expansion and hatching. Embryos from PR freezing had lower glucose and pyruvate uptake, higher lactate release and lower glycolytic activity than controls. There were fewer total cells and trophectoderm cells in PR frozen embryos than in vitrified embryos, but no difference in the ICM cell numbers. Another study using bovine embryos showed higher (nearly double) post-thaw hatching rates in PR freezing versus vitrification, but cell counts and number of live cells were not different (Sommerfeld and Niemann, 1999). A striking feature of all the reports utilizing bovine embryos was the generally longer exposure time for vitrified embryos at higher vitrification solution concentrations compared with the methods used for this study. Together, these factors could have led to increased toxicity stress on the vitrified embryos, contributing to the poor outcomes. Many previous studies have shown the utility of glucose uptake as a marker of embryo viability (Gardner and Leese, 1987; Gardner et al., 1996; Lane and Gardner, 1996; Uechi et al., 1999). The use of glucose uptake as a measure of post-thaw embryo viability was validated in the present study. When considering all embryos (regardless of treatment arm), hatching blastocysts showed a higher glucose uptake versus non-hatching (earlier developmental stage) blastocysts in accordance with previous findings. Both embryos at an advanced stage of development and higher quality embryos have increased glucose uptake compared with earlier stage embryos or poor quality blastocyst stage embryos. Similarly, when considering all results (regardless of treatment arm), embryos with a detectable ICM (presumably having greater viability) also showed higher glucose uptake. When considering comparisons between treatments, not only were the proportions of ICM detectable from control and VLN embryos higher than those in the VSC and PR groups but both control and VLN embryos showed higher glucose uptake for embryos with an ICM compared with those that did not. In contrast, the presence or absence of an ICM had no effect on glucose uptake in embryos from either PR or VSC treatments. A previous larger data set of ‘group-cultured’ embryos demonstrated that VLN resulted in more embryos with an ICM than PR or VSC (Walker et al., 2004). Since glucose uptake in this study was higher in embryos with an ICM, it is possible that additional glucose uptake data might further demonstrate VLN’s superiority over PR and VSC. Furthermore, vitrification offers considerable time savings, approximately 2 h and 40 min, compared with PR freezing. In conclusion, the data demonstrate vitrification to be a superior method for cryopreserving murine blastocysts compared with the current method of PR freezing. Glucose uptake analysis was confirmed to be a useful marker of embryo viability. It can be used to assess the efficacy of cryopreservation protocols, since it compares favourably with other post-thaw viability markers such as total cell count and the presence or absence of an inner cell mass, with the added advantage that it is non-invasive.
Article - Glucose uptake to compare cryopreservation methods - DL Walker et al.
References Behr B, Pool TB, Milki AA et al. 1999 Preliminary clinical experience with human blastocyst development in vitro without co-culture. Human Reproduction 14, 454–457. Conaghan J, Hardy K, Handyside AH et al. 1993 Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology. Journal of Assisted Reproduction and Genetics 10, 21–30. Coonen E, Dumoulin JC, Ramaekers FC, Hopman AH 1994 Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescence in-situ hybridization. Human Reproduction 9, 533–537. Gardner DK, Lane M 2004 Nutrient uptake and metabolite production and enzyme activity and regulation. In: Gardner DK, Lane M, Watson AJ (eds) A Laboratory Guide to the Mammalian Embryo. Oxford University Press, Oxford, pp. 139–153. Gardner DK, Leese HJ 1987 Assessment of embryo viability prior to transfer by the noninvasive measurement of glucose uptake. Journal of Experimental Zoology 242, 103–105. Gardner DK, Lane M, Stevens J, Schoolcraft WB 2001 Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential. Fertility and Sterility 76, 1175–1180. Gardner DK, Schoolcraft WB, Wagley L et al. 1998a A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Human Reproduction 13, 3434–3440. Gardner DK, Vella P, Lane M et al. 1998b Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertility and Sterility 69, 84–88. Gardner DK, Pawelczynski M, Trounson AO 1996 Nutrient uptake and utilization can be used to select viable day 7 bovine blastocysts after cryopreservation. Molecular Reproduction and Development, 44, 472–475. Hewitson LC, Leese HJ 1993 Energy metabolism of the trophectoderm and inner cell mass of the mouse blastocyst. Journal of Experimental Zoology 267, 337–343. Kaidi S, Bernard S, Lambert P et al. 2001 Effect of conventional
controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro. Biology of Reproduction 65, 1127–1134. Lane M, Gardner DK 1996 Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Human Reproduction 11,1975–1978. Leese HJ, Barton AM 1984 Pyruvate and glucose uptake by mouse ova and preimplantation embryos. Journal of Reproduction and Fertility 72, 9–13. Marek D, Langley M, Gardner DK et al. 1999 Introduction of blastocyst culture and transfer for all patients in an in vitro fertilization program. Fertility and Sterility 72, 1035–1040. Nowshari MA, Brem G 2001 Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos. Human Reproduction 16, 2368–2373. Smith K, Roots E, Dorsett JO et al. 2003 Day 6 blastocyst transfers can increase implantation, pregnancy and live birth rates compared to day 5 blastocyst transfers. The Embryologists’ Newsletter 6, 4–9. Sommerfeld V, Niemann H 1999 Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification. Cryobiology 38, 95–105. Uechi H, Tsutsumi O, Morita Y et al. 1999 Comparison of the effects of controlled-rate cryopreservation and vitrification on 2-cell mouse embryos and their subsequent development. Human Reproduction 14, 2827–2832. Van den Abbeel E, Camus M, Van Waesberghe L et al. 1997 A randomized comparison of the cryopreservation of one-cell human embryos with a slow controlled-rate cooling procedure or a rapid cooling procedure by direct plunging into liquid nitrogen. Human Reproduction 12, 1554–1560. Walker DL, Tummon IS, Hammitt DG et al. 2004 Vitrification versus programmable rate freezing of late stage murine embryos: a randomized comparison prior to application in clinical IVF. Reproductive BioMedicine Online 8, 558–568. Received 15 February 2005; refereed 24 March 2005; revised and resubmitted 12 July 2005; accepted 29 July 2005.
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Article Cryosystem assessment by glucose uptake of murine blastocysts 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.
Mr David Walker David L Walker1,6, David K Gardner2, Michelle Lane3, Ian S Tummon1, Donna R Session4, Alan R Thornhill5 1 Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Section of Reproductive Endocrinology and Infertility, Charlton Building Desk 3-A, 200 First Street SW, Mayo Clinic College of Medicine, Rochester, MN 55905; 2Colorado Centre for Reproductive Medicine, Englewood, CO, USA; 3Research Centre for Reproductive Health, Department of Obstetrics and Gynecology, University of Adelaide, Adelaide, Australia and Repromed, Adelaide, Australia; 4Emory Reproductive Centre, 550 Peachtree Street, Suite 1800, Atlanta, GA 30308, USA; 5London Gynaecology and Fertility Centre Limited, Cozens House, 112A Harley Street, London W1G 7JH, UK 6 Correspondence: Tel: +1 507 2663995; Fax: +1 507 2841774; email: [email protected]
Abstract Glucose uptake was used as a measure of metabolic activity and implantation potential to compare vitrification and slow freezing in a prospective randomized trial using murine blastocysts. Frozen 2-cell embryos (n = 132) thawed and cultured for 48 h to the blastocyst stage were randomly divided into four groups: (i) control – not refrozen; (ii) slow freezing using a programmed rate (PR); (iii) vitrification by super-cooled (VSC) liquid nitrogen; and (iv) vitrification in liquid nitrogen (VLN). Upon re-thawing, embryos were cultured individually for 24 h to determine glucose uptake non-invasively. Morphological assessments included total cell counts and inner cell mass (ICM) detection following immunosurgery. Mean glucose uptake was lower for each treatment (PR and VSC, 4.3 pmol/embryo per h; VLN, 4.9 pmol/embryo per h) versus controls (6.8 pmol/embryo per h). PR and VSC embryos had fewer cells (57.4 ± 24.2 and 64.1 ± 31.5) versus controls (85.7 ± 26.2), and fewer embryos containing a detectable ICM (42.9 and 61.8%) compared with controls (88.2%). The only difference between control and VLN embryos was absolute glucose uptake, although in both treatments glucose uptake was increased from embryos with an ICM compared with those without. Glucose uptake appears to be a sensitive, non-invasive method to validate cryopreservation protocols. Keywords: blastocyst, cryopreservation, embryo freezing, inner cell mass, murine, vitrification
Introduction Recent trends in culturing embryos to the blastocyst stage have created the need to refine blastocyst cryopreservation methodologies for improved post-thaw survival and implantation. The introduction of sequential media has resulted in more IVF programmes transferring at the blastocyst stage with fewer embryos being selected for transfer, thus increasing implantation rates while decreasing multiple gestation rates (Gardner et al., 1998a,b; Behr et al., 1999; Marek et al., 1999; Smith et al., 2003). With higher implantation rates using fresh blastocysts, fewer embryos are required for transfer, resulting in more high quality supernumerary blastocysts being available for cryostorage.
At present, programmable rate (PR) freezing is the most widely utilized method of cryopreservation for human blastocysts. This time-consuming method is accomplished using sophisticated, usually expensive, equipment that requires ongoing maintenance and involves prolonged exposure of embryos to cryoprotectants to prevent potentially damaging intercellular ice crystal formation. In contrast, vitrification is rapid, can be accomplished by plunging directly into liquid nitrogen and requires extremely short exposure times to high concentrations of cryoprotectants with no ice crystal formation. Because of the comparative simplicity and brevity of vitrification procedures, a laboratory can perform multiple cryopreservation procedures over time within the same cohort of embryos to optimize survival and implantation potential per embryo.
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Article - Glucose uptake to compare cryopreservation methods - DL Walker et al. To date, no studies have been reported to prospectively compare PR freezing and vitrification of human blastocysts. Indeed, the only prospective comparison between these methods in human embryos involved pronuclear zygotes, with no clear advantage cited for either method. Although pregnancy rates were not different, vitrified embryos had higher post-thaw survival, whereas PR frozen embryos had a higher cleavage rate (Van den Abbeel et al., 1997). Human IVF laboratories rely primarily on development rate and morphology to determine which embryos possess the greatest implantation potential. However, non-invasive, quantitative analysis of metabolites such as glucose and pyruvate has been reported in both human and murine embryos to provide additional selection criteria for identifying embryos with the greatest implantation potential (Gardner and Leese, 1987; Conaghan et al., 1993; Gardner et al., 1996, 2001; Lane and Gardner, 1996) and in predicting blastocyst survival following slow freezing (Gardner et al., 1996). The preimplantation blastocyst relies on glucose to provide energy for biosynthesis activities and in preparation for implantation. Glucose stores within the embryo are utilized immediately prior to implantation, due to a somewhat decreased availability of oxygen in the uterus at the site of implantation; thus, the embryo is able to maintain high glycolytic activity during this critical time period. In-vitrocultured murine blastocysts with a glucose metabolism similar to in-vivo-derived blastocysts had a 4-fold increase in implantation compared with embryos with perturbed glycolytic activity (Lane and Gardner, 1996). Thus increased depletion of glucose from the media serves as a marker for detection of embryos that have increased metabolic activity and higher implantation potential. In a previous study from this laboratory, group cultured murine late stage embryos were used to compare two methods of vitrification by plunging into super-cooled liquid nitrogen (VSC) or standard liquid nitrogen (VLN) and PR freezing (PR). Compared with VSC and VLN, PR resulted in the lowest total cell counts and fewest embryos with a distinct ICM. With VLN, a higher percentage of embryos survived 24 h post-thaw, progressed to more advanced developmental stages and had higher total cell counts compared with PR. Moreover, fewer embryos frozen by both PR and VSC contained a detectable ICM compared with VLN (Walker et al., 2004). Since these embryos were cultured in groups, glucose uptake of individual embryos could not be accurately assessed; hence the current study design. In the present study, identical cryopreservation methods and conditions were used as described previously (Walker et al., 2004), except that embryos were randomized post-thaw for individual culture with the primary purpose of glucose uptake analysis.
Materials and methods Murine embryos
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One hundred and thirty-two frozen 2-cell murine embryos (B6C3-F1 females × B6D2-F1 males; Embryotech Laboratories, Inc., Wilmington, MA, USA) were used for this study. Upon thawing, embryos were rinsed through five pre-equilibrated
microdrops of 100 μl each, G1.3 (Vitrolife, Inc., Englewood, CO, USA) before being placed into 50 μl droplets of G1.3 in 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’). Compacting morula through early hatching blastocyst stage embryos were placed into groups by stage. Embryos from each group were sequentially divided into four treatment groups: controls – not refrozen; PR freezing (PR) in 0.25 ml straws; vitrification in flexible micropipettes by immersion in super-cooled (VSC) liquid nitrogen (LN2) and vitrification in flexible micropipettes by immersion in LN2 (VLN). Assessments performed 24 h post-thaw or postwarming included developmental stage progression, total cell counts with detection of a morphologically distinct ICM following immunosurgery, and glucose uptake. All chemicals used in this study were purchased from Sigma-Aldrich Chemical Company, Saint Louis, MO, USA, unless otherwise indicated.
Pre-freeze embryo assessment Embryos were assigned to one of five developmental stages prior to cryopreservation, morula, early-blastocyst, midblastocyst, late-blastocyst, and early-hatching blastocyst.
Post-thaw embryo assessment At 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: degenerating, midhatching blastocyst, late-hatching blastocyst and completely hatched blastocyst as described earlier (Walker et al., 2004). 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.
Microsurgical separation of ICM/TE by immunosurgery Disaggregation of trophectoderm (TE) and ICM cells was performed 24 h post-thaw on all but severely degenerating embryos. Briefly, embryos not already completely hatched from the zona pellucida were exposed to acidified Tyrode’s solution for removal of the zona pellucida. Embryos were rinsed in PBS supplemented with 0.1% polyvinyl alcohol (PVA), exposed to a 10 mmol/l solution of trinitrobenzenesulphonic acid (TNBS) in PBS-0.1% PVA followed by incubation in 0.1 mg/ml anti-dinitrophenylated bovine serum albumin (rabbit antibody; Valeant Pharmaceuticals Intl., Costa Mesa, CA, USA, formerly ICN Pharmaceuticals Co.). Embryos were finally incubated in 10% guinea pig complement serum in PBS–0.1% PVA to initiate lysing of the TE cells coated with the antibody. Embryos were then rinsed in PBS–0.1% PVA to stop the lysing process before complete disruption of TE membranes. Using a small-bore pipette (120 μm), the embryonic mass was pipetted, releasing the ICM from the lysed TE cells. The two cell types were easily distinguishable; TE cells appeared pale and were easily disaggregated, while ICM cells remained in a compact formation with maintenance
Article - Glucose uptake to compare cryopreservation methods - DL Walker et al. of membrane integrity and a luminescent appearance. All TE and ICM cell types were separated and mounted on silanated glass microscope slides (Fisher Scientific, Inc., Pittsburgh, PA, USA) for cell count estimates by placing the cells into a small droplet of 0.1% polyoxyethylenesorbitan monolaurate (Tween-20) in 0.01 normal (N) hydrochloric acid (Coonen et al., 1994). 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.
Non-invasive measurement of glucose uptake Immediately after thaw, embryos were incubated individually in 500 nl of fresh G2.3 (containing reduced glucose at 0.5 mmol/l versus standard concentration of 3.15 mmol/l) for overnight culture. All incubations took place in 6% CO2 in air at 37ºC. Following the 24-h culture period, the 500nl drop was aspirated into a washed and sterilized, 1 μl Microcap glass capillary tube (Drummond; Fisher Scientific Inc., Broomall, PA, USA). The microcaps were placed inside cryostraws and heat sealed for storage at –70°C prior to analysis for glucose. Glucose concentration was determined by an ultramicrofluorometric technique, in which biochemical reactions were performed on a siliconized glass slide under oil. The fluorescent reaction was quantified using a photomultiplier and photometer, and was expressed as pmol glucose utilized per embryo per hour. Control media from non-spent microdrops was analysed with each representative treatment run. Samples were read in a blinded fashion to eliminate bias. The analysis procedure is described in detail elsewhere (Leese and Barton, 1984; Gardner and Lane, 2004).
Controls Following the 48-h post-thaw development assessment, control embryos were rinsed through five 100-μl droplets of G2.3 (with reduced glucose) before placement into individual culture microdrops (500 nl), as for treatment embryos.
Programmable rate freezing and thawing (PR) Standard PR freezing and stepwise thawing was performed as previously described (Walker et al., 2004), using a Cryomed freezing system (Forma, Inc., Marietta, OH, USA) and commercially available freezing and thawing solutions used according to the manufacturer’s recommendations (Sage BioPharma, Bedminster, NJ, USA, catalogue nos 8015 and 8016). The total time for this freezing technique, from placement into the first cryopreservation solution to submerging the straws in LN2, was approximately 2 h and 45 min. After thawing, embryos were rinsed through five 100-μl droplets of G2.3 (with reduced glucose) before being randomized and placed into final culture microdrops of the reduced glucose medium.
Vitrification in super-cooled nitrogen and warming (VSC) Embryos were vitrified in LN2 slush, super-cooled to –208°C to –210°C utilizing the Vit-Master (MiniTub, Tiefenbach, Germany) and warmed as described previously (Walker et al., 2004). Following exposure to the final solution, embryos were rinsed through five droplets of G2.3 before being randomized and placed in final culture microdrops of reduced glucose G2.3 medium. The total time for this technique, from placement into the first cryopreservation solution to submerging the straws in LN2, was approximately 5 min.
Vitrification in liquid nitrogen and warming (VLN) Embryos allocated to this treatment were treated identically to those in VSC with the exception of using LN2 (–196°C) 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 24 h post-thaw. 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. The nonparametric Kruskal–Wallis test was 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 statistical software (JMP, Version 4.0.4; SAS Institute Inc., Cary, NC, USA, 1989–2002). Statistical significance was set at the 0.05 level.
Results Overall survival and continued development 24 h post-thaw was not different between the non-refrozen controls and the treatments, with the exception of VSC showing slightly lower development (82.4%) compared with control embryos (97.1%, Table 1). Mean glucose uptake was lower for each treatment (PR and VSC, 4.3 pmol/embryo per h; VLN, 4.9 pmol/embryo per h) compared with controls (6.8 pmol/embryo per h, Table 2). PR and VSC embryos had fewer cells (57.4 ± 24.2 and 64.1 ± 31.5) than controls (85.7 ± 26.2, Table 3), and fewer embryos containing a detectable ICM (42.9 and 61.8%) compared with controls (88.2%, Figure 1). However, survival rates, presence of an ICM, and cell counts were similar between control and VLN embryos. VLN embryos had higher cell counts (70.9 ± 30.4) and more embryos with a detectable ICM (79.3%) than PR embryos (57.4 ± 24.2 and 42.9% respectively). When comparing glucose uptake between all embryos with and without an ICM, embryos with an ICM had a higher glucose uptake (5.7 pmol/embryo per h) than those without (3.8 pmol/embryo per h). Although the absolute glucose uptake between control and VLN embryos was different, both control and VLN embryos showed higher glucose uptake
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Table 1. Embryo survival and development 24 h post-thaw. Treatment
Degenerating embryos
Early to late blastocysts
Early to complete hatching blastocysts
Percentage survival and re-expansion (n)
Control PR VSC VLN
1 6 6 3
4 9 8 5
29 20 20 21
97.1 (33/34) 82.9 (29/35)b 82.4 (28/34)a 89.7 (26/29)
P < 0.05 versus control. P < 0.0508 versus control. Embryos frozen at morula to early hatching stage: PR = programmable rate freezing, VSC = vitrification with super-cooled nitrogen, VLN = vitrification with liquid nitrogen.
a
b
Table 2. Twenty-four hour post-thaw glucose uptake in embryos cultured individually (pmol glucose utilized by treatment). Treatment
No. embryos
Mean pmol/embryo per h (±SD)
95% CI
Median pmol/embryo per h (range)
Control PR VSC VLN
34 35 34 29
6.8 (2.5) 4.3 (2.3)a 4.3 (1.9)a 4.9 (2.3)a
5.9–7.6 3.5–5.1 3.7–5.0 4.1–5.8
7.2 (0.8–10.6) 4.6 (0.0–9.0) 4.2 (0.2–7.9) 5.2 (0.7–9.1)
a P < 0.02 versus control. PR = programmable rate freezing, VSC = vitrification with super-cooled nitrogen, VLN = vitrification with liquid nitrogen.
Table 3. Total cell counts, all embryos. Treatment
No. of embryos
Mean no. cells/embryo (±SD)
95% mean CI
Median no. cells/embryo (range)
Control PR VSC VLN
34 35 34 29
85.7 (±26.2) 57.4 (±24.2)a,b 64.1 (±31.5)a 70.9 (±30.4)
76.6–94.8 49.1–65.7 53.1–75.1 59.2–82.4
90 (29–138) 56 (19–114) 65 (11–132) 80 (9–111)
Control versus PR (P < 0.0001) and VSC (P = 0.0032). Versus VLN (P = 0.0417). PR = programmable rate freezing, VSC = vitrification with super-cooled nitrogen, VLN = vitrification with liquid nitrogen.
a
b
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Figure 1. Embryos with a countable inner cell mass. aP < 0.02 versus control; bP < 0.01 versus VLN. PR = programmable rate freezing; VSC = vitrification with super-cooled nitrogen; VLN = vitrification with liquid nitrogen.
Table 4. Twenty-four hour post-thaw glucose uptake in embryos cultured individually (pmol glucose utilized compared in embryos with and without an ICM). Treatment
No. embryos
Mean pmol/embryo per h (±SD)
95% CI
Median pmol/embryo per h (range)
With ICM Without ICM
89 43
5.7 (2.4) 3.8 (2.0)a
5.2–8.0 3.2–3.4
5.9 (0.0–10.6) 4.1 (0.0–7.1)
a
P < 0.0001 versus ‘With ICM’.
Table 5. Glucose uptake for embryos with (+) and without (–) ICM. Treatment
ICM
No. embryos
Mean of pmol/embryo per h (±SD)
95% CI
Median pmol/embryo per h (range)
Control
+ – + – + – + –
30 4 15 20 21 13 23 6
7.1 (2.3) 4.4 (2.6)a 4.5 (2.8) 4.1 (2.0)c 4.8 (1.7) 3.5 (1.9)d 5.5 (2.1) 2.9 (1.8)b
6.3–8.0 0.2–8.5 3.0–6.1 3.2–5.1 4.0–5.6 2.4–4.7 4.6–6.4 1.0–4.7
7.4 (2.3–10.6) 4.8 (0.80–7.1) 5.0 (0.0–9.0) 4.5 (0.0–6.7) 4.3 (1.9–7.9) 3.6 (0.2–6.2) 5.6 (1.4–9.1) 2.7 (0.7–5.2)
PR VSC VLN
P < 0.05 versus With ICM. P < 0.01 versus With ICM. P = 0.5154 versus With ICM (NS). d P = 0.0556 versus With ICM (NS). Within each treatment, control and VLN embryos maintained a difference in glucose uptake between embryos with and without an ICM (P < 0.05 and 0.01 respectively). However, glucose uptake was not different between embryos with and without an ICM from the PR and VSC treatments (P = 0.5154 and 0.0556 respectively). a
b c
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Article - Glucose uptake to compare cryopreservation methods - DL Walker et al. for embryos with an ICM compared with those that did not. In contrast, the presence or absence of an ICM had no effect on glucose uptake in embryos from either PR or VSC treatments (Tables 4 and 5).
Discussion The primary aim of this study was to compare glucose uptake from individually cultured blastocyst stage murine embryos frozen by conventional PR freezing and two methods of vitrification. Other outcome measures included survival, developmental stage progression, total cell counts and presence or absence of an inner cell mass. In accordance with previous results comparing these methods (Walker et al., 2004), overall survival and development was nearly identical for all treatments using a larger number of embryos. Non-refrozen control embryos showed better results than treatment embryos for all 24-h post-thaw parameters evaluated, with the exception of VLN embryos, in which the only significant difference between control and VLN embryos was glucose uptake. It is speculated that outcomes across all treatments may have improved if fresh, rather than re-frozen, embryos had been utilized. However, non-cryopreserved mouse embryos were not available for this research. In agreement with previous results (Walker et al., 2004), a lower number of embryos frozen by the PR contained a detectable ICM than controls. However, among embryos with a detectable ICM, no difference was seen in the ICM to total cell ratio between treatments (data not shown), indicating that those embryos containing an ICM were developing both cell types at a similar rate regardless of the cryopreservation method used. Programmable rate freezing resulted in embryos with the lowest total cell counts and fewest embryos progressing to more advanced stages of development. Trophectoderm cells may have sustained more damage from this cryopreservation method and would thus account for the lower number of embryos with an ICM. Alternatively, the low number of ICM cells, which primarily consume glucose (Hewitson and Leese, 1993) could reflect changes in total glucose utilization post-cryopreservation. Although it was not possible to assess pregnancy outcomes for frozen–thawed embryos in this study, it can be speculated that embryos frozen by the PR method would have lower birth rates based on fewer embryos containing an ICM and lower overall glucose uptake. Previous studies (Nowshari and Brem, 2001; Walker et al., 2004), showed that post-thaw outcomes were significantly improved after cryostorage by VLN versus VSC. However, with the exception of glucose uptake between embryos with and without an ICM, these differences were not seen in the present study.
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Embryos vitrified by VSC did fare slightly better post-warming than those frozen by the PR method, with total cell counts and the number of embryos containing a detectable inner cell mass being higher in the VSC group. However, these differences did not achieve statistical significance, perhaps as a result of the small sample size.
To date, there have been no published comparisons of PR freezing and vitrification in human blastocysts. In a prospective randomized comparison of PR freezing and vitrification using bovine blastocysts (Kaidi et al., 2001), there was no difference between cryopreservation methods for post-thaw re-expansion and hatching. Embryos from PR freezing had lower glucose and pyruvate uptake, higher lactate release and lower glycolytic activity than controls. There were fewer total cells and trophectoderm cells in PR frozen embryos than in vitrified embryos, but no difference in the ICM cell numbers. Another study using bovine embryos showed higher (nearly double) post-thaw hatching rates in PR freezing versus vitrification, but cell counts and number of live cells were not different (Sommerfeld and Niemann, 1999). A striking feature of all the reports utilizing bovine embryos was the generally longer exposure time for vitrified embryos at higher vitrification solution concentrations compared with the methods used for this study. Together, these factors could have led to increased toxicity stress on the vitrified embryos, contributing to the poor outcomes. Many previous studies have shown the utility of glucose uptake as a marker of embryo viability (Gardner and Leese, 1987; Gardner et al., 1996; Lane and Gardner, 1996; Uechi et al., 1999). The use of glucose uptake as a measure of post-thaw embryo viability was validated in the present study. When considering all embryos (regardless of treatment arm), hatching blastocysts showed a higher glucose uptake versus non-hatching (earlier developmental stage) blastocysts in accordance with previous findings. Both embryos at an advanced stage of development and higher quality embryos have increased glucose uptake compared with earlier stage embryos or poor quality blastocyst stage embryos. Similarly, when considering all results (regardless of treatment arm), embryos with a detectable ICM (presumably having greater viability) also showed higher glucose uptake. When considering comparisons between treatments, not only were the proportions of ICM detectable from control and VLN embryos higher than those in the VSC and PR groups but both control and VLN embryos showed higher glucose uptake for embryos with an ICM compared with those that did not. In contrast, the presence or absence of an ICM had no effect on glucose uptake in embryos from either PR or VSC treatments. A previous larger data set of ‘group-cultured’ embryos demonstrated that VLN resulted in more embryos with an ICM than PR or VSC (Walker et al., 2004). Since glucose uptake in this study was higher in embryos with an ICM, it is possible that additional glucose uptake data might further demonstrate VLN’s superiority over PR and VSC. Furthermore, vitrification offers considerable time savings, approximately 2 h and 40 min, compared with PR freezing. In conclusion, the data demonstrate vitrification to be a superior method for cryopreserving murine blastocysts compared with the current method of PR freezing. Glucose uptake analysis was confirmed to be a useful marker of embryo viability. It can be used to assess the efficacy of cryopreservation protocols, since it compares favourably with other post-thaw viability markers such as total cell count and the presence or absence of an inner cell mass, with the added advantage that it is non-invasive.
Article - Glucose uptake to compare cryopreservation methods - DL Walker et al.
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