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The effect of maternal serum on phase-change temperature in human embryo cryopreservation
FERTILITY AND STERILITY
Vol. 59, No.2, February 1993
Copyright <> 1993 The American Fertility Society
Printed on acid·free paper in U.S.A.
The effect of maternal serum on phase-change temperature in human embryo cryopreservation
Louise M. Adamson, Ph.D. John P. P. Tyler, Ph.D.* Geoffrey L. Driscoll, M.D. Integrated Fertility Services, Westmead, New South Wales, Australia
Human embryo cryopreservation is now a routine procedure and the practical methods employed are generally similar between infertility units. The source of protein supplementation in the cryomedium varies from bovine serum albumin or human serum albumin to serum obtained from the patient for whom the embryos are being frozen. This latter source is increasingly common because of concerns over cross-contamination (and hence infection) or the increasing difficulty of importing potentially hazardous biological material, at least in Australia. The temperature at which seeding of the cryoprotectant is performed is usually -7°C and most units (in Australia) do this manually. This short study investigates the eutectic point of cryomedium when the concentration of the protein source (from the embryos maternal parent) is varied and when seeding occurs automatically. MATERIALS AND METHODS
The experiments were performed using standard human embryo cryomedium (phosphate-buffered saline containing 1.5 M propandiol and 0.2 M sucrose) and the CryoGenetic CG4400B cryofreezer (CryoGenetic Technology Inc., Yoncalla, OR). This instrument lowers the sample straws through a liquid nitrogen vapor gradient established between room temperature and the liquid state at -196°C. The straws are held in a Cryoprism (CryoGenetic
Received June 2, 1992; revised and accepted October 20, 1992. * Reprint requests: John P. P. Tyler, Ph.D., Integrated Fertility Services, 12 Caroline Street, Westmead, New South Wales 2145, Australia. Vol. 59, No.2, February 1993
Technology Inc.) a copper block that acts to eliminate rapid temperature fluctuations at phase change. Because the ends of each straw protrude from the Cryoprism, medium in this section freezes before that contained inside the Cryoprism where the embryos are situated. When the eutectic point inside the Cryoprism is reached, ice crystals already formed at the ends of the straws induce freezing to continue through the section containing the embryos. A thermocouple positioned in a control straw containing identical cryomedium records changes in temperature. Using this instrument seeding always occurs at the appropriate temperature for the particular serum-supplemented cryomedium. The seeding points are represented by a transient increase in sample temperature that returns to prephase-change temperature within 45 to 60 seconds. Phase-change temperatures were compared for significant variation using the one-factor analysis of variance with repeated measures (Table 1). RESULTS
In the first experiment, the effect of different serum concentrations (10%, 15%, and 20%, vol/vol) from the same patient was investigated in seven different women. Each medium sample was tested in triplicate. No significant difference (P = 0.34) was found in the seeding temperature between replicates for each concentration or between concentrations (P = 0.66) for each patient. Thus, for further experiments patients' serum was added at 15%. In the second experiment, interpatient serum variation was examined. The mean ± SE seeding temperature for cryomedium prepared with 10 difAdamson et al.
Communications-in-brief
451
Table 1 Phase-Change (Eutectic Point) Temperatures for Each Experiment Temperature *
Experiment
Pvalue
·c Serum concentration 10% 15% 20% Variation between patients (15% serum) Collection time in menstrual cycle Days 1 to 5 Days 6 to 10
-6.03 ± 0.13 (7) -6.19 ± 0.15 (7) -6.11 ± 0.16 (7)
0.34
-5.94 ± 0.13 (10)
0.002
-6.00 ± 0.09 (5) -6.04 ± 0.13 (5)
0.83
* Values are means ± SE; values in parentheses are number of patients.
ferent patients' serum was found to be -5.94 ± O.l°C and the temperature range between patients was -5.52 to -6.63°C. A significant difference was found in the seeding temperatures between patients' serum (P = 0.002) at the 15% concentration. Finally, the effect of the day during the ovarian cycle when serum was collected from a patient was tested. Serum from five patients was pooled between days 1 and 5 and between days 6 and 10 of their stimulated cycle but no difference to the phasechange temperature within the subjects could be demonstrated (P = 0.83). DISCUSSION
Sample seeding during embryo cryopreservation does not appear to have been well studied. The majority of Australian units use a Planar Cryosystem (Planer Products, Sunbury, Middlesex, United Kingdom) with manual seeding at -7°C. Theoretically, a system that utilizes self-seeding would allow optimization, particularly if the cryomedium contains a protein source that is nondefined and is as heterogeneous as maternal serum. The results of these simple experiments demonstrate a significant interpatient variation in eutectic point when the protein source is maternal serum. The only other cryomedium constituent that is known to have a concentration-dependent effect on the eutectic point is the cryoprotective agent itself (1). However, these results further demonstrate that serum concentration (10%, 15%, and 20%) and the stage of a woman's ovarian cycle when serum was collected are unimportant considerations in human embryo cryopreservation.
452
Adamson et al.
Communications-in-brief
The negligible effect of serum concentration is encouraging because in vitro fertilization units have published methods with differing serum concentrations but similar success rates (2, 3). The mean phase-change temperature in our studies was considerably lower than that used by most programs (-5.9°C rather than -7°C) and it is interesting to speculate whether survival rates of embryos might be improved if attention was given to this finding. Similarly, it would have been interesting to compare embryo survival and pregnancy rates using the selfseeding method described here with a manual method at -7°C in the same vapor gradient system. However, the design and operation of the CryoGenetic CG4400B Cryofreezer does not allow this. To conclude, two points derive from this simple study. First, these results suggest that the practice of manually seeding embryos at -7°C, when the mean optimal eutectic point is closer to -6°C, should be reconsidered. Second, because of significant interpatient variation in phase-change temperature when maternal serum is used as the protein source, instruments utilizing self-seeding devices may confer a significant advantage to survival and pregnancy rates using cryopreserved human embryos. SUMMARY
The effect of addition of maternal serum to cryomedium on the phase-change temperature for embryo cryopreservation has been investigated. No effect on the eutectic point (i.e., earliest seeding temperature) was found within patients because of serum concentration or the time in the ovarian cycle when the/serum was collected. However, the optimum seeding temperature did vary between patients, which suggests that self-seeding devices for embryo cryopreservation may be practically beneficial. Key Words: Embryo cryopreservation, human, protein source. REFERENCES 1. Whittingham DG. Some factors affecting embryo storage in
laboratory animals. Ciba Found Symp 1977;52:97-127. 2. Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhom JD, et al. High pregnancy rate after early human embryo freezing. Fertil Steril 1986;46:268-72. 3. Marrs RP, Brown J, Sato F, Ogawa T, Vee B, Paulson R, et al. Successful pregnancies from cryopreserved human embryos produced by in vitro fertilization. Am J Obstet Gynecol 1987;156:1503-8.
Fertility and Sterility
Vol. 59, No.2, February 1993
Copyright <> 1993 The American Fertility Society
Printed on acid·free paper in U.S.A.
The effect of maternal serum on phase-change temperature in human embryo cryopreservation
Louise M. Adamson, Ph.D. John P. P. Tyler, Ph.D.* Geoffrey L. Driscoll, M.D. Integrated Fertility Services, Westmead, New South Wales, Australia
Human embryo cryopreservation is now a routine procedure and the practical methods employed are generally similar between infertility units. The source of protein supplementation in the cryomedium varies from bovine serum albumin or human serum albumin to serum obtained from the patient for whom the embryos are being frozen. This latter source is increasingly common because of concerns over cross-contamination (and hence infection) or the increasing difficulty of importing potentially hazardous biological material, at least in Australia. The temperature at which seeding of the cryoprotectant is performed is usually -7°C and most units (in Australia) do this manually. This short study investigates the eutectic point of cryomedium when the concentration of the protein source (from the embryos maternal parent) is varied and when seeding occurs automatically. MATERIALS AND METHODS
The experiments were performed using standard human embryo cryomedium (phosphate-buffered saline containing 1.5 M propandiol and 0.2 M sucrose) and the CryoGenetic CG4400B cryofreezer (CryoGenetic Technology Inc., Yoncalla, OR). This instrument lowers the sample straws through a liquid nitrogen vapor gradient established between room temperature and the liquid state at -196°C. The straws are held in a Cryoprism (CryoGenetic
Received June 2, 1992; revised and accepted October 20, 1992. * Reprint requests: John P. P. Tyler, Ph.D., Integrated Fertility Services, 12 Caroline Street, Westmead, New South Wales 2145, Australia. Vol. 59, No.2, February 1993
Technology Inc.) a copper block that acts to eliminate rapid temperature fluctuations at phase change. Because the ends of each straw protrude from the Cryoprism, medium in this section freezes before that contained inside the Cryoprism where the embryos are situated. When the eutectic point inside the Cryoprism is reached, ice crystals already formed at the ends of the straws induce freezing to continue through the section containing the embryos. A thermocouple positioned in a control straw containing identical cryomedium records changes in temperature. Using this instrument seeding always occurs at the appropriate temperature for the particular serum-supplemented cryomedium. The seeding points are represented by a transient increase in sample temperature that returns to prephase-change temperature within 45 to 60 seconds. Phase-change temperatures were compared for significant variation using the one-factor analysis of variance with repeated measures (Table 1). RESULTS
In the first experiment, the effect of different serum concentrations (10%, 15%, and 20%, vol/vol) from the same patient was investigated in seven different women. Each medium sample was tested in triplicate. No significant difference (P = 0.34) was found in the seeding temperature between replicates for each concentration or between concentrations (P = 0.66) for each patient. Thus, for further experiments patients' serum was added at 15%. In the second experiment, interpatient serum variation was examined. The mean ± SE seeding temperature for cryomedium prepared with 10 difAdamson et al.
Communications-in-brief
451
Table 1 Phase-Change (Eutectic Point) Temperatures for Each Experiment Temperature *
Experiment
Pvalue
·c Serum concentration 10% 15% 20% Variation between patients (15% serum) Collection time in menstrual cycle Days 1 to 5 Days 6 to 10
-6.03 ± 0.13 (7) -6.19 ± 0.15 (7) -6.11 ± 0.16 (7)
0.34
-5.94 ± 0.13 (10)
0.002
-6.00 ± 0.09 (5) -6.04 ± 0.13 (5)
0.83
* Values are means ± SE; values in parentheses are number of patients.
ferent patients' serum was found to be -5.94 ± O.l°C and the temperature range between patients was -5.52 to -6.63°C. A significant difference was found in the seeding temperatures between patients' serum (P = 0.002) at the 15% concentration. Finally, the effect of the day during the ovarian cycle when serum was collected from a patient was tested. Serum from five patients was pooled between days 1 and 5 and between days 6 and 10 of their stimulated cycle but no difference to the phasechange temperature within the subjects could be demonstrated (P = 0.83). DISCUSSION
Sample seeding during embryo cryopreservation does not appear to have been well studied. The majority of Australian units use a Planar Cryosystem (Planer Products, Sunbury, Middlesex, United Kingdom) with manual seeding at -7°C. Theoretically, a system that utilizes self-seeding would allow optimization, particularly if the cryomedium contains a protein source that is nondefined and is as heterogeneous as maternal serum. The results of these simple experiments demonstrate a significant interpatient variation in eutectic point when the protein source is maternal serum. The only other cryomedium constituent that is known to have a concentration-dependent effect on the eutectic point is the cryoprotective agent itself (1). However, these results further demonstrate that serum concentration (10%, 15%, and 20%) and the stage of a woman's ovarian cycle when serum was collected are unimportant considerations in human embryo cryopreservation.
452
Adamson et al.
Communications-in-brief
The negligible effect of serum concentration is encouraging because in vitro fertilization units have published methods with differing serum concentrations but similar success rates (2, 3). The mean phase-change temperature in our studies was considerably lower than that used by most programs (-5.9°C rather than -7°C) and it is interesting to speculate whether survival rates of embryos might be improved if attention was given to this finding. Similarly, it would have been interesting to compare embryo survival and pregnancy rates using the selfseeding method described here with a manual method at -7°C in the same vapor gradient system. However, the design and operation of the CryoGenetic CG4400B Cryofreezer does not allow this. To conclude, two points derive from this simple study. First, these results suggest that the practice of manually seeding embryos at -7°C, when the mean optimal eutectic point is closer to -6°C, should be reconsidered. Second, because of significant interpatient variation in phase-change temperature when maternal serum is used as the protein source, instruments utilizing self-seeding devices may confer a significant advantage to survival and pregnancy rates using cryopreserved human embryos. SUMMARY
The effect of addition of maternal serum to cryomedium on the phase-change temperature for embryo cryopreservation has been investigated. No effect on the eutectic point (i.e., earliest seeding temperature) was found within patients because of serum concentration or the time in the ovarian cycle when the/serum was collected. However, the optimum seeding temperature did vary between patients, which suggests that self-seeding devices for embryo cryopreservation may be practically beneficial. Key Words: Embryo cryopreservation, human, protein source. REFERENCES 1. Whittingham DG. Some factors affecting embryo storage in
laboratory animals. Ciba Found Symp 1977;52:97-127. 2. Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhom JD, et al. High pregnancy rate after early human embryo freezing. Fertil Steril 1986;46:268-72. 3. Marrs RP, Brown J, Sato F, Ogawa T, Vee B, Paulson R, et al. Successful pregnancies from cryopreserved human embryos produced by in vitro fertilization. Am J Obstet Gynecol 1987;156:1503-8.
Fertility and Sterility