| OBJECTIVE To investigate the availability of early pregnancy termination with low frequency ultrasound . The early embryos were irradiated directly with low frequency focused ultrasound (353 kHz ) and embryonic mortality and morphology changes of placenta and uterus were observed . At the same time , the apoptosis and expression of regulatory genes of apoptosis of placenta were tested with methods of TUNEL , immunohistochemistry and in situ hybridization respectively at 24, 48, 72, 96, 120,168, 192 hours after irradiating . The studies mightprovide important experimental foundation for clinical studies .METHODS1. The implantation sites of left uterus of 10th day rabbit embryos were exposured with an intensity of 30 W/cm~2, 35 W/cm~2, 40 W/cm~2, 45 W/cm~2, 50 W/cm~2 and 55 W/cm~2 for 60 seconds . At same time , the embryos of right uterus belonged to the control group . At 20 th day of gestation , the animals were sacrificed . Embryonic mortality was observed .2. Choosing three rabbits on 10 th day and three on 20 th day of pregnancy of control group , three at once and three 10 days after irradiating of different experimental groups , opening the abdominal cavity of pregnant rabbits , choosing part of the placenta and putting them into 4 % paraformaldehyde for 24 hours and 2.5 % glutaraldehyde for more than 2 hours respectively . Paraffin embedding , slices and dye with HE as routine procedure . Morphology changes of placenta and uterus were observed under optical microscope . Embedding using for electron microscope as routine procedure and ultrathin slices . The ultrastructure changes of placenta and uterus were observed under electron microscope .3. The embryos of right uterus , as control group , were not irradiated . The embryos of left uterus of 10th day rabbit were irradiated with 40 W/cm~2 for 60 seconds which had been proved asan totally pregnancy terminating intensity . The apoptosis of placenta cells were tested with TUNEL at 24, 48, 72, 96, 120, 168,192 hours after irradiating . Choosing part of the placenta of 10 th day of control group and 24,48,72, 96, 120, 168, 192 hours after irradiating , dipping them in 4 % paraformaldehyde for 24 hours , paraffin embedding , slices and dye with HE as routine procedure . The apoptosis of placenta cells was detected with TUNEL reaction .4. The expression of Fas, FasL, Bax and Bcl-2 proteins of placenta cells was detected with immunohistochemistry at 24, 48, 72, 96,120, 168, 192 hours after irradiating with 40 W/cm~2 for 60 seconds .5. The expression of p53 mRNA, Bax mRNA, Bcl-2 mRNA of placenta cells was detected with In situ hybridization at 24, 48, 72,96, 120, 168, 192 hours after irradiating with 40 W/cm~2 for 60 seconds.RESULTS1. It was found that total embryonic mortality of control group was 12.65 % ( 32/253 ) and those of experimental group were 25.00 % (12/48 ), 72.88 % (43/59 ) and 100 % ( 64/64, 43/43 , 40/40 , 35/35 ) respectively with an intensity of 30 W/cm~2, 35W/cm~2 and equal or more than 40 W/cm~2. There was a rough increase of embryonic mortality from the group of 35 W/cm~2 and ther after . Ther was a very significant difference compared with control group .2. Under optical microscope , necrosis and hemorrhage were observed in placenta (villosity ang decidua) at once after irradiating . Under electron microscope , the cells of placenta were found to have lost normal structures and there was mitochondrial swelling in endometrial and myometrial cells at once after irradiating . On 10 th day after irradiating , degeneration and necrosis were also observed in villosity , decidua cells and there was no obvious change in endometrial, myometrial and vascula endothelial cells under optical microscope.3. There was a significant apoptosis increase at 24,48,72, 96, 120, 168, 192 hours after irradiating compared with control group . The incidence of placenta cell apoptosis was increased at 24 hours after irradiating and peaked at 96 hours , but then decreased gradually , and almost recovered to the level of controls by 192 hours after irradiating .4. There was a significant increase of expression of Fas protein, FasL protein,p53 mRNA,Bax protein and mRNA at 24,48,72,96,120,168,192 hours after irradiating comparingto control group . The rate of these genes expression of placenta cells was increased at 24 hours after irradiating and peaked at 96 hours, but then decreased gradually , and almost recovered to control levels by 192 hours . The incidence of Bcl-2 protein and mRNA was decreased at 24,48,72,96,120,168 hours after irradiating compared with those of control group and least at 96 hours after irradiating. But then recovered gradually, and to almost same levels as control by 192 hours .CONCLUSIONS1. It is feasible for early pregnancy termination of rabbit with low frequency ultrasound . But the further studies were needed in the future.2. The placenta was more sensitive and injured to low frequency ultrasonic irradiation than uterus . There was a slight and reparable changes in uterus after irradiating .3. The low frequency ultrasound may result in more placenta cells apoptosis in rabbits and the number of apoptosis of placenta cell can be recovered gradually.4. The low frequency ultrasoundmay result in more Fas, FasL, p53 expressions of placenta cell and less Bcl-2 expression . That might result in more apoptosis in placenta cells, but the changes were reparable.In summary , we concluded the points of creation as follows :1. For the first time , we applied low frequency ultrasound for terminating animal pregnancy.2. For the first time , we detected the apoptosis changes in placenta cells after irradiating rabbit embryos with low frequency ultrasoundmay.3. For the first time , we detected the expression changes of regulatory genes of apoptosis in placenta cells after irradiating rabbit embryos with low frequency ultrasound .
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