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Effects of pulsed ultrasound on development and glucose uptake of preimplantation mouse embryos
Ultrasound in Med. & Biol., Vol. 27, No. 7, pp. 999 –1002, 2001 Copyright © 2001 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/01/$–see front matter
PII: S0301-5629(01)00383-0
● Original Contribution EFFECTS OF PULSED ULTRASOUND ON DEVELOPMENT AND GLUCOSE UPTAKE OF PREIMPLANTATION MOUSE EMBRYOS EIJI RYO,* HIDEMI SHIOTSU,* YASUSHI TAKAI,* OSAMU TSUTSUMI,* TAKASHI OKAI,* YUJI TAKETANI,* and YASUHITO TAKEUCHI† *Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and † Department of Information and Computer Science, Faculty of Engineering, Kagoshima University, Kagoshima, Japan (Received 22 May 2000; in final form 6 March 2001)
Abstract—The effects of pulsed ultrasound (US) on early mouse embryos were investigated. Two-cell embryos contained in oviducts were irradiated to US (1.875 MHz with an I SPTA of 2.96 W/cm2) in an experimental unit for either 1 or 5 min (exposure groups). The embryos were cultured to examine the rate of developing to blastocysts, and the uptake of 2-deoxyglucose (2-DG) into blastocysts was measured to evaluate their viability. The rates in the exposure groups were essentially the same, with those of the embryos treated similarly in the unit unless being exposed to US (nonexposure groups). However, they were lower than that of embryos not treated in the experimental unit (a control group). There were no significant differences of 2-DG uptake among the 1-min exposure, 1-min nonexposure, and control groups. The uptake in the 5-min exposure group did not differ from that in the 5-min nonexposure group; however, uptake in both groups was lower than that in the control group. Pulsed US for 1 min did not affect viability of preimplantation mouse embryos. © 2001 World Federation for Ultrasound in Medicine & Biology. Key Words: Bioeffect of ultrasound, Glucose uptake, Mouse embryo, Safety of ultrasound.
ena occur due to the absorption of ultrasonic energy and its conversion to heat. In tissues with a normal blood supply, however, blood flow promotes heat transmission and, thereby, reduces temperature elevation. In contrast, embryos located in the oviduct are floating in the tissue fluid in the absence of blood flow. As for cavitational activities, cavitation-induced free radicals are supposed to be detrimental to tissues. However, in an in vivo setting, they are eliminated by antioxidant enzymes and vitamins. On the other hand, early embryos are considered to be susceptible to them. With this background, along with the dissemination of ultrasonic diagnosis in reproductive medicine, it is of importance to confirm its safety especially for early embryos. In the present study, we intended to investigate the effects of US on morphologic development in early mouse embryos, as well as the amount of glucose uptake as an index of their viability.
INTRODUCTION Ultrasound (US) is a major diagnostic tool in obstetric and gynecological practice. There have been many epidemiologic studies of effects by US exposure. In some studies, an association was identified such as low birth weight (Newnham et al. 1993), delayed speech (Campbell et al. 1993) or increased incidence of left handedness (Salvensen et al. 1993). However, these findings have not been duplicated, and the majority of such studies have been negative. So far, no verified US-induced adverse effects have been reported clinically. However, acoustic output of diagnostic US equipment has increased recently (Duck and Henderson 1998), and US can produce biologic damage if its intensity is high. Therefore, there is increasing concern that deleterious effects may yet occur. The possible mechanisms by which US may produce biologic effects are thermal and cavitational activities. When tissues are exposed to US, thermal phenom-
METHODS Embryos Female mice (6- to 8-week-old Crj; CD-1, ICR) were superovulated with 5 IU pregnant mare serum go-
Address correspondence to: Eiji Ryo, Department of Obstetrics and Gynecology, Japanese Red Cross Medical Center, 4-1-22, Hiroo, Shibuya-ku, Tokyo 150-8935 Japan. 999
1000
Ultrasound in Medicine and Biology
Volume 27, Number 7, 2001
Fig. 2. In situ exposure waveform and exposure parameters measured by needle hydrophone.
Fig. 1. Schematic sketch (exploded cross-section view) of the exposure system. An oviduct containing two-cell embryos was placed in (4) with medium. During operation (3) and (5) were pressed to the bottom of water chamber with thin layer of coupling water (vertical arrows).
nadotropin, followed 48 h later by 5 IU human chorionic gonadotropin (hCG). Mating with males of the same strain was confirmed by the presence of a vaginal plug. The pregnant mice were killed 48 h after hCG injection and their oviducts containing two-cell embryos were removed. The oviducts were divided into five groups (i.e., 1- and 5-min US exposure groups, 1- and 5-min nonexposure groups, and a control group). Animal care and treatment were conducted in conformity with the institutional guideline, which is in compliance with the NIH Guide for care and use of laboratory animals. Ultrasound exposure unit Figure 1 sketches the laboratory-built US exposure unit used in this study, where a 2-MHz 30-mm dia. 95-mmR concave PZT piczoelectric transducer (1) is located at the top of water chamber (2) filled with degassed water. At the focal zone located at the outside proximity of bottom wall, a sterile detachable exposure chamber unit (3) is placed. It has a 2.5-mm dia. 6-mm depth cylindrical exposure chamber (4) at its center to keep the subject for exposure. The chamber is exactly
placed coaxially at the focal zone. Underneath the exposure chamber unit, is a relatively long terminator rod (5) for anechoic termination of the US irradiation pulse travelling through the exposure chamber. The sonic path component including the exposure chamber unit (3) and terminal rod (5) are all made of polymethylpentene, a well known polyorephin plastic material having the best available acoustic impedance match to the water to have maximum transparency and minimal reflection. The unit at work is enclosed in an external housing (6) to regulate the environmental temperature during the experiment, typically around 37°C. Ultrasound exposures The oviduct containing two-cell embryos in the exposure groups was placed one by one in the exposure chamber containing modified Biggers–Whitten–Wittingham (mBWW) medium kept in a humidified atmosphere of 95% air and 5% CO2 at 37°C. After the sample was closed up without air, 1.875 MHz, four to five cycles burst, IM ⫽ 621 w/cm2, ISPTA ⫽ 2.96 W/cm2 pulsed US in pulse repetition frequency ⫽ 3.3 kHz was irradiated for either 1 min or 5 min (1-min exposure and 5-min exposure groups). The exposure unit at working setup was calibrated using a standard polyvinylidene fluoride hydrophone of needle type (Medicoteknisk) placed at the position of exposure chamber. The measured US waveform and beam profile are shown in Figs. 2 and 3. ISPTA was calculated from measured ISPPI data by hydrophone
Ultrasound effects on mouse embryos ● E. RYO et al.
1001
Table 1. Development rates of blastocysts from 2-cell embryos Group
Development rate (%)
Significance
Control
48.8 (80 of 164)
1-min 1-min 5-min 5-min
22.8 (120 of 526) 25.8 (122 of 472) 27.7 (224 of 809) 27.4 (192 of 702)
p ⬍ 0.0001 vs. 1-min p ⬍ 0.0001 vs. 5-min N.S. vs. non-exposure
exposure nonexposure exposure nonexposure
N.S. vs. nonexposure
Numbers of blastocysts obtained from two-cell embryos appear in parentheses. N.S. ⫽ not significant.
Fig. 3. Lateral beam profile measured by hydrophone.
and pulse-repetition frequency. From the beam profile irradiation is sufficiently uniform across the exposure chamber within about ⫺3 dB distribution. The US ⫺6 dB beamwidth at the target was around 3.5 mm. The size of the oviduct was about 1.5 mm and that of two-cell embryos were much less than 1 mm. The oviduct in the nonexposure groups was placed in the same way in the exposure chamber either for 1 min or 5 min without US exposure (1-min nonexposure and 5-min nonexposure groups). The procedures were conducted alternately between the exposure and nonexposure groups. The oviducts in the control group were not placed in the exposure chamber. The temperature in the unit was maintained between 36°C and 38°C during the procedures. Culture and glucose uptake After the abovementioned procedures, the two-cell embryos were collected by flushing the oviducts in mBWW medium. About 5 two-cell embryos were contained in one oviduct. Then, they were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 48 h to obtain blastocysts. The methods of measuring glucose uptake into the blastocysts have been reported elsewhere (Hosoya et al. 1991; Morita et al. 1992). The amount of glucose uptake for 60 min into one blastocyst could be measured by the method. The blastocysts obtained were washed 5 times by transferring each time to 100 L of mBWW medium free of glucose.They were then incubated for 60 min in 4 L of mBWW medium containing 25 M [3H]-2-deoxy-D-glucose (2-DG, Amersham, Littel Chalfont, Buckinghamshire, UK; 17 Ci/ mmol) instead of glucose. After that, they were washed again in the same way, and the amount of 2-DG incorporated into each blastocyst was counted with 1 mL of Aquasol solution in a Beckman scintillation counter. The data were expressed as mean ⫾ SEM. Com-
parison of the values were performed by one-way ANOVA and Scheffe’s method as post hoc test. Categoric data were analyzed by means of 2 test. Statistical significance was considered at p ⬍ 0.05. RESULTS Table 1 shows the blastocyst formation rates from two-cell embryos in each group. In the control group, 80 blastocysts (48.8%) from 164 two-cell embryos were obtained. And 120 blastocysts (22.8%) from 526 twocell embryos and 122 blastocysts (25.8%) from 472 two-cell embryos developed in the 1-min exposure and nonexposure groups, respectively. The blastocyst formation rates in the 1-min treatment groups were significantly lower than that in the control group (p ⬍ 0.0001). However, there was no significant difference (p ⫽ 0.2972) between the 1-min exposure group and nonexposure group. Looking at the 5-min treatment groups, the blastocyst formation rate was 27.7% (224 blastocysts from 809 two-cell embryos) in the exposure group and 27.4% (192 blastocysts from 702 two-cell embryos) in the nonexposure group. Although the formation rates in the 5-min treatment groups were lower than that in the control group (p ⬍ 0.0001), no significant difference was detected between the 5-min exposure and nonexposure groups (p ⫽ 0.9291). The amounts of 2-DG uptake in the control group, 1-min exposure group, and 1-min nonexposure groups are shown in Fig. 4. They were 82.9 ⫾ 5.95 fmol/ embryo/h, 86.3 ⫾ 6.94 fmol/embryo/h, and 99.5 ⫾ 5.75 fmol/embryo/h, respectively. There were no significant differences among these three groups (p ⫽ 0.1508). In the 5-min treatment groups, the amounts of 2-DG uptake were 60.6 ⫾ 3.99 fmol/embryo/h in the exposure group and 53.3 ⫾ 3.90 fmol/embryo/h in the nonexposure group. Although those in the both groups were lower than that in the control group (p ⫽ 0.0053, 0.0002, respectively), there was no significant difference (p ⫽ 0.4522) between the exposure and nonexposure groups (Fig. 4).
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Ultrasound in Medicine and Biology
Volume 27, Number 7, 2001
influence. Glucose is the predominant energy source at blastocyst stage, and the viability of blastocysts depends upon its uptake (Lees and Barton 1984; Brinster 1971). 2-DG is an appropriate tracer for glucose uptake because it is an analog of glucose and not metabolized in cytoplasm (Sokoloff et al. 1977). In this study, there were no significant diffrences as to 2-DG uptake of blastocysts among the control group, 1-min exposure group, and 1-min nonexposure group. On the other hand, 2-DG uptake in the 5-min exposure and nonexposure groups was significantly lower than that in the control group. It was probable that incubation without air for 5 min might diminish the viability of embryos. Therefore, we also could not draw a firm conclusion from 5-min treatment groups. It is difficult to study the effects of US on early embryos because they are very susceptible in vitro. However, it was certain that US exposure for 1 min had no effects on glucose uptake of early mouse embryos. In conclusion, 1-min exposure of pulsed US (intensity 2.96 W/cm2 of SPTA) has no effects on viability of early mouse embryos; thus, reinforcing the perceived safety of US irradiation in routine clinical practice. Acknowledgements—The authors thank Dr. Keiichi Murakami (Fujitsu Laboratory) for production of the transducer driver. We also thank Dr. Miwa Naito (Aloka Industry) for ultrasound calibration and dosimetry.
REFERENCES Fig. 4. 2-Deoxy-D-glucose uptake into blastocysts in the control group, 1-min treatment groups and 5-min treatment groups. The bar and T on the bar indicate mean and SEM, respectively. N.S. ⫽ not significant.
DISCUSSION In the present study, the rates of blastocyst formation in the 1-min treatment groups were lower than that in the control group, although there was no significant difference between the 1-min exposure and 1-min nonexposure groups. The same results were obtained in the 5-min treatment groups. We assumed that the procedures, which consisted of placing the oviduct into the exposure chamber, closing it up, and removing it from there, might be fatal, not to all embryos but to some of the embryos. Therefore, we could not draw a firm conclusion, although US exposure had no apparent effects on morphologic development. We further investigated the effect of US exposure to glucose uptake of blastocysts, because the morphologic criteria is not quantitative enough to detect any US
Brinster RL. Oxydation of pyruvate and glucose by oocytes of the mouse and Rhesus monkey. J Reprod Fertil 1971;24:187–191. Campbell JD, Elford RW, Brant RF. Case-control study of prenatal ultrasonography exposure in children with delayed speech. Can Med Assoc J 1993;149:1435–1440. Duck FA, Henderson J. Acoustic output of modern ultrasound equipment: Is it increasing? In: Barnet SB, Kossoff G, eds. Safety of diagnostic ultrasound. Progress in sonography series. Cardiff: Parthenon Publishing, 1998:15–25. Hosoya I, Tsutsumi O, Mizuno M, Kato T. Microdetermination of developmental changes in the uptake of glucose by preimplantation mouse embryo (in Japanese). Acta Obst Gynaecol Jpn 1991;43: 1579 –1580. Lees HJ, Barton AM. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J Reprod Fertil 1984;72:9 –13. Morita Y, Tsutsumi O, Hosoya I, et al. Expression and possible function of glucose transporter protein glut 1 during preimplantation mouse development from oocytes to blastocystes. Biochem Bioph Res Co 1992;188:8 –15. Newnham JP, Evans SF, Michael CA, Stanly FJ, Landau LI. Effects of frequent ultrasound during pregnancy: A randomized control study. Lancet 1993;342:887– 891. Salvensen KA, Vatten LJ, Eik-Ness SH, Hugdahl K, Bakketeig LS. Routine ultrasonography in utero and subsequent handedness and neurological development. Br Med J 1993;307:159 –164. Sokoloff L, Reivich M, Kennedy C, et al. The [14C] deoxyglucose method for the measurement of local cerebral glucose utilization. J Neurochem 1977;28:897.
PII: S0301-5629(01)00383-0
● Original Contribution EFFECTS OF PULSED ULTRASOUND ON DEVELOPMENT AND GLUCOSE UPTAKE OF PREIMPLANTATION MOUSE EMBRYOS EIJI RYO,* HIDEMI SHIOTSU,* YASUSHI TAKAI,* OSAMU TSUTSUMI,* TAKASHI OKAI,* YUJI TAKETANI,* and YASUHITO TAKEUCHI† *Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and † Department of Information and Computer Science, Faculty of Engineering, Kagoshima University, Kagoshima, Japan (Received 22 May 2000; in final form 6 March 2001)
Abstract—The effects of pulsed ultrasound (US) on early mouse embryos were investigated. Two-cell embryos contained in oviducts were irradiated to US (1.875 MHz with an I SPTA of 2.96 W/cm2) in an experimental unit for either 1 or 5 min (exposure groups). The embryos were cultured to examine the rate of developing to blastocysts, and the uptake of 2-deoxyglucose (2-DG) into blastocysts was measured to evaluate their viability. The rates in the exposure groups were essentially the same, with those of the embryos treated similarly in the unit unless being exposed to US (nonexposure groups). However, they were lower than that of embryos not treated in the experimental unit (a control group). There were no significant differences of 2-DG uptake among the 1-min exposure, 1-min nonexposure, and control groups. The uptake in the 5-min exposure group did not differ from that in the 5-min nonexposure group; however, uptake in both groups was lower than that in the control group. Pulsed US for 1 min did not affect viability of preimplantation mouse embryos. © 2001 World Federation for Ultrasound in Medicine & Biology. Key Words: Bioeffect of ultrasound, Glucose uptake, Mouse embryo, Safety of ultrasound.
ena occur due to the absorption of ultrasonic energy and its conversion to heat. In tissues with a normal blood supply, however, blood flow promotes heat transmission and, thereby, reduces temperature elevation. In contrast, embryos located in the oviduct are floating in the tissue fluid in the absence of blood flow. As for cavitational activities, cavitation-induced free radicals are supposed to be detrimental to tissues. However, in an in vivo setting, they are eliminated by antioxidant enzymes and vitamins. On the other hand, early embryos are considered to be susceptible to them. With this background, along with the dissemination of ultrasonic diagnosis in reproductive medicine, it is of importance to confirm its safety especially for early embryos. In the present study, we intended to investigate the effects of US on morphologic development in early mouse embryos, as well as the amount of glucose uptake as an index of their viability.
INTRODUCTION Ultrasound (US) is a major diagnostic tool in obstetric and gynecological practice. There have been many epidemiologic studies of effects by US exposure. In some studies, an association was identified such as low birth weight (Newnham et al. 1993), delayed speech (Campbell et al. 1993) or increased incidence of left handedness (Salvensen et al. 1993). However, these findings have not been duplicated, and the majority of such studies have been negative. So far, no verified US-induced adverse effects have been reported clinically. However, acoustic output of diagnostic US equipment has increased recently (Duck and Henderson 1998), and US can produce biologic damage if its intensity is high. Therefore, there is increasing concern that deleterious effects may yet occur. The possible mechanisms by which US may produce biologic effects are thermal and cavitational activities. When tissues are exposed to US, thermal phenom-
METHODS Embryos Female mice (6- to 8-week-old Crj; CD-1, ICR) were superovulated with 5 IU pregnant mare serum go-
Address correspondence to: Eiji Ryo, Department of Obstetrics and Gynecology, Japanese Red Cross Medical Center, 4-1-22, Hiroo, Shibuya-ku, Tokyo 150-8935 Japan. 999
1000
Ultrasound in Medicine and Biology
Volume 27, Number 7, 2001
Fig. 2. In situ exposure waveform and exposure parameters measured by needle hydrophone.
Fig. 1. Schematic sketch (exploded cross-section view) of the exposure system. An oviduct containing two-cell embryos was placed in (4) with medium. During operation (3) and (5) were pressed to the bottom of water chamber with thin layer of coupling water (vertical arrows).
nadotropin, followed 48 h later by 5 IU human chorionic gonadotropin (hCG). Mating with males of the same strain was confirmed by the presence of a vaginal plug. The pregnant mice were killed 48 h after hCG injection and their oviducts containing two-cell embryos were removed. The oviducts were divided into five groups (i.e., 1- and 5-min US exposure groups, 1- and 5-min nonexposure groups, and a control group). Animal care and treatment were conducted in conformity with the institutional guideline, which is in compliance with the NIH Guide for care and use of laboratory animals. Ultrasound exposure unit Figure 1 sketches the laboratory-built US exposure unit used in this study, where a 2-MHz 30-mm dia. 95-mmR concave PZT piczoelectric transducer (1) is located at the top of water chamber (2) filled with degassed water. At the focal zone located at the outside proximity of bottom wall, a sterile detachable exposure chamber unit (3) is placed. It has a 2.5-mm dia. 6-mm depth cylindrical exposure chamber (4) at its center to keep the subject for exposure. The chamber is exactly
placed coaxially at the focal zone. Underneath the exposure chamber unit, is a relatively long terminator rod (5) for anechoic termination of the US irradiation pulse travelling through the exposure chamber. The sonic path component including the exposure chamber unit (3) and terminal rod (5) are all made of polymethylpentene, a well known polyorephin plastic material having the best available acoustic impedance match to the water to have maximum transparency and minimal reflection. The unit at work is enclosed in an external housing (6) to regulate the environmental temperature during the experiment, typically around 37°C. Ultrasound exposures The oviduct containing two-cell embryos in the exposure groups was placed one by one in the exposure chamber containing modified Biggers–Whitten–Wittingham (mBWW) medium kept in a humidified atmosphere of 95% air and 5% CO2 at 37°C. After the sample was closed up without air, 1.875 MHz, four to five cycles burst, IM ⫽ 621 w/cm2, ISPTA ⫽ 2.96 W/cm2 pulsed US in pulse repetition frequency ⫽ 3.3 kHz was irradiated for either 1 min or 5 min (1-min exposure and 5-min exposure groups). The exposure unit at working setup was calibrated using a standard polyvinylidene fluoride hydrophone of needle type (Medicoteknisk) placed at the position of exposure chamber. The measured US waveform and beam profile are shown in Figs. 2 and 3. ISPTA was calculated from measured ISPPI data by hydrophone
Ultrasound effects on mouse embryos ● E. RYO et al.
1001
Table 1. Development rates of blastocysts from 2-cell embryos Group
Development rate (%)
Significance
Control
48.8 (80 of 164)
1-min 1-min 5-min 5-min
22.8 (120 of 526) 25.8 (122 of 472) 27.7 (224 of 809) 27.4 (192 of 702)
p ⬍ 0.0001 vs. 1-min p ⬍ 0.0001 vs. 5-min N.S. vs. non-exposure
exposure nonexposure exposure nonexposure
N.S. vs. nonexposure
Numbers of blastocysts obtained from two-cell embryos appear in parentheses. N.S. ⫽ not significant.
Fig. 3. Lateral beam profile measured by hydrophone.
and pulse-repetition frequency. From the beam profile irradiation is sufficiently uniform across the exposure chamber within about ⫺3 dB distribution. The US ⫺6 dB beamwidth at the target was around 3.5 mm. The size of the oviduct was about 1.5 mm and that of two-cell embryos were much less than 1 mm. The oviduct in the nonexposure groups was placed in the same way in the exposure chamber either for 1 min or 5 min without US exposure (1-min nonexposure and 5-min nonexposure groups). The procedures were conducted alternately between the exposure and nonexposure groups. The oviducts in the control group were not placed in the exposure chamber. The temperature in the unit was maintained between 36°C and 38°C during the procedures. Culture and glucose uptake After the abovementioned procedures, the two-cell embryos were collected by flushing the oviducts in mBWW medium. About 5 two-cell embryos were contained in one oviduct. Then, they were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 48 h to obtain blastocysts. The methods of measuring glucose uptake into the blastocysts have been reported elsewhere (Hosoya et al. 1991; Morita et al. 1992). The amount of glucose uptake for 60 min into one blastocyst could be measured by the method. The blastocysts obtained were washed 5 times by transferring each time to 100 L of mBWW medium free of glucose.They were then incubated for 60 min in 4 L of mBWW medium containing 25 M [3H]-2-deoxy-D-glucose (2-DG, Amersham, Littel Chalfont, Buckinghamshire, UK; 17 Ci/ mmol) instead of glucose. After that, they were washed again in the same way, and the amount of 2-DG incorporated into each blastocyst was counted with 1 mL of Aquasol solution in a Beckman scintillation counter. The data were expressed as mean ⫾ SEM. Com-
parison of the values were performed by one-way ANOVA and Scheffe’s method as post hoc test. Categoric data were analyzed by means of 2 test. Statistical significance was considered at p ⬍ 0.05. RESULTS Table 1 shows the blastocyst formation rates from two-cell embryos in each group. In the control group, 80 blastocysts (48.8%) from 164 two-cell embryos were obtained. And 120 blastocysts (22.8%) from 526 twocell embryos and 122 blastocysts (25.8%) from 472 two-cell embryos developed in the 1-min exposure and nonexposure groups, respectively. The blastocyst formation rates in the 1-min treatment groups were significantly lower than that in the control group (p ⬍ 0.0001). However, there was no significant difference (p ⫽ 0.2972) between the 1-min exposure group and nonexposure group. Looking at the 5-min treatment groups, the blastocyst formation rate was 27.7% (224 blastocysts from 809 two-cell embryos) in the exposure group and 27.4% (192 blastocysts from 702 two-cell embryos) in the nonexposure group. Although the formation rates in the 5-min treatment groups were lower than that in the control group (p ⬍ 0.0001), no significant difference was detected between the 5-min exposure and nonexposure groups (p ⫽ 0.9291). The amounts of 2-DG uptake in the control group, 1-min exposure group, and 1-min nonexposure groups are shown in Fig. 4. They were 82.9 ⫾ 5.95 fmol/ embryo/h, 86.3 ⫾ 6.94 fmol/embryo/h, and 99.5 ⫾ 5.75 fmol/embryo/h, respectively. There were no significant differences among these three groups (p ⫽ 0.1508). In the 5-min treatment groups, the amounts of 2-DG uptake were 60.6 ⫾ 3.99 fmol/embryo/h in the exposure group and 53.3 ⫾ 3.90 fmol/embryo/h in the nonexposure group. Although those in the both groups were lower than that in the control group (p ⫽ 0.0053, 0.0002, respectively), there was no significant difference (p ⫽ 0.4522) between the exposure and nonexposure groups (Fig. 4).
1002
Ultrasound in Medicine and Biology
Volume 27, Number 7, 2001
influence. Glucose is the predominant energy source at blastocyst stage, and the viability of blastocysts depends upon its uptake (Lees and Barton 1984; Brinster 1971). 2-DG is an appropriate tracer for glucose uptake because it is an analog of glucose and not metabolized in cytoplasm (Sokoloff et al. 1977). In this study, there were no significant diffrences as to 2-DG uptake of blastocysts among the control group, 1-min exposure group, and 1-min nonexposure group. On the other hand, 2-DG uptake in the 5-min exposure and nonexposure groups was significantly lower than that in the control group. It was probable that incubation without air for 5 min might diminish the viability of embryos. Therefore, we also could not draw a firm conclusion from 5-min treatment groups. It is difficult to study the effects of US on early embryos because they are very susceptible in vitro. However, it was certain that US exposure for 1 min had no effects on glucose uptake of early mouse embryos. In conclusion, 1-min exposure of pulsed US (intensity 2.96 W/cm2 of SPTA) has no effects on viability of early mouse embryos; thus, reinforcing the perceived safety of US irradiation in routine clinical practice. Acknowledgements—The authors thank Dr. Keiichi Murakami (Fujitsu Laboratory) for production of the transducer driver. We also thank Dr. Miwa Naito (Aloka Industry) for ultrasound calibration and dosimetry.
REFERENCES Fig. 4. 2-Deoxy-D-glucose uptake into blastocysts in the control group, 1-min treatment groups and 5-min treatment groups. The bar and T on the bar indicate mean and SEM, respectively. N.S. ⫽ not significant.
DISCUSSION In the present study, the rates of blastocyst formation in the 1-min treatment groups were lower than that in the control group, although there was no significant difference between the 1-min exposure and 1-min nonexposure groups. The same results were obtained in the 5-min treatment groups. We assumed that the procedures, which consisted of placing the oviduct into the exposure chamber, closing it up, and removing it from there, might be fatal, not to all embryos but to some of the embryos. Therefore, we could not draw a firm conclusion, although US exposure had no apparent effects on morphologic development. We further investigated the effect of US exposure to glucose uptake of blastocysts, because the morphologic criteria is not quantitative enough to detect any US
Brinster RL. Oxydation of pyruvate and glucose by oocytes of the mouse and Rhesus monkey. J Reprod Fertil 1971;24:187–191. Campbell JD, Elford RW, Brant RF. Case-control study of prenatal ultrasonography exposure in children with delayed speech. Can Med Assoc J 1993;149:1435–1440. Duck FA, Henderson J. Acoustic output of modern ultrasound equipment: Is it increasing? In: Barnet SB, Kossoff G, eds. Safety of diagnostic ultrasound. Progress in sonography series. Cardiff: Parthenon Publishing, 1998:15–25. Hosoya I, Tsutsumi O, Mizuno M, Kato T. Microdetermination of developmental changes in the uptake of glucose by preimplantation mouse embryo (in Japanese). Acta Obst Gynaecol Jpn 1991;43: 1579 –1580. Lees HJ, Barton AM. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J Reprod Fertil 1984;72:9 –13. Morita Y, Tsutsumi O, Hosoya I, et al. Expression and possible function of glucose transporter protein glut 1 during preimplantation mouse development from oocytes to blastocystes. Biochem Bioph Res Co 1992;188:8 –15. Newnham JP, Evans SF, Michael CA, Stanly FJ, Landau LI. Effects of frequent ultrasound during pregnancy: A randomized control study. Lancet 1993;342:887– 891. Salvensen KA, Vatten LJ, Eik-Ness SH, Hugdahl K, Bakketeig LS. Routine ultrasonography in utero and subsequent handedness and neurological development. Br Med J 1993;307:159 –164. Sokoloff L, Reivich M, Kennedy C, et al. The [14C] deoxyglucose method for the measurement of local cerebral glucose utilization. J Neurochem 1977;28:897.