Evaluation Of Fecundity And Safety Of Offspring After Orthotopic Transplantation Of Frozen Ovaries In Mice

The ovary contains a large number of egg cells and it is a good repository of female reproductive cells.If a healthy ovarian tissue is preserved by vitrification,then a rapid recovery is performed when needed,and then be transplanted back into the pelvic cavity in situ,the mother’s natural fertility is expected to recover.There have been sporadic reports of human ovarian cryopreservation and the successful production of newborn babies,and there have been many attempts in animals.However,there is no clear report on whether there is a certain adverse effect on the recipient and it’s offspring from the frozen ovary in situ transplantation.In this paper,the establishment of mice frozen ovary in situ transplantation model was used to explore the reproductive ability of female B6 mice and to detect the safety of their offspring about genetic、growth、development and reproduction.This provides an experimental basis for exploring the application of this technique to human reproductive safety.The major findings are as follows:(1)In experimental group,put the black B6 mice ovary orthotopic transplantation into the hybrid F1 mice B6FVBF1 pelvic whose colour is ash black,then let them mate with the male B6 mice again,gave birth to the offspring of the pure black hair color.In the control group,female B6FVBF1 mice mated with male B6 mice and gave birth to the offspring of hair color separated.In the other control group,female B6 mice were transplanted with ovary from B6 mice,then were mated with male B6 mice,and the offspring were all black hair mice.(2)The B6 mice of 2 months old were divided into three groups:experimental group、operation control group and blank group.After cryopreservation of the donor ovary,the operation group recovered quickly and underwent the orthotopic transplantation.In the control group,fresh ovary was transplanted in situ.The blank control group did not do any treatment.Observe the ovarian tissue structure after the freezing and recovery,and to check estrus restore after operation,to detect the fertility of postoperative mice.Growth and development and fertility and ethology and genetic traits were observed of the offspring.From HE stained paraffin tissue section,it can be observed that the appearance of the frozen ovary was intact,and the follicles at all levels of the cortex are clearly visible,and the granule cells are arranged in an orderly manner.Through transmission electron microscopy,it can be observed that the cytoplasm in frozen ovarian cytoplasm is normal,the endoplasmic reticulum and mitochondrial structure are intact,and the matrix electron density is even.There wasn’t any obvious histological damage caused by the freezing recovery process.The mice in the experimental group were able to recover the estrus cycle after three weeks of operation,and there was no significant difference in the stillbirths and infantophagia between the groups.Comparison between groups of per litter size、four months litter size、six months litter size found that per litter size of the experimental group decreased than the control group and blank control group,four months litter size of the experimental group decreased than in blank control group.In the experimental group and the control group,there was a decrease in the number of offspring in the six months compared with the blank control group.The offspring of the frozen ovary transplantation mice still has the normal fertility.(3)The following six behavioral tests were performed between frozen ovary in situ transplanted mice and normal adult B6 mice:rotarod test、open field test、novel object recognition test、elevated plus-maze test、social behavior test、water maze test.There was no significant ethological difference between the offspring mice and normal B6 mice.It is indicated that the offspring of the frozen ovary in situ transplantation is equivalent to normal mice in the level of psychological anxiety、study ability、social skills、sport skills、physical endurance、memory capacity.So the reproductive safety of vitrfication in these aspects was verified.(4)No allograft rejection was found in offspring through skin transplantation.The whole genome resequencing of frozen ovary offspring was found to be low in variation and the risk of protein function change was low.(5)The ovary of young mice was transplanted into aged mice,which could improve the reproductive hormone level.But after mating,the offspring were not observed to produce successfully.This may be related to the excessive aging of the reproductive system in aged mice.Ovarian vitrification of cryopreservation and in-situ transfer techniques have restored natural fertility in mice.Although to some extent,vitrification makes the per litter size decreased,but their offspring has no significant harmful effects in terms of ethology and genetics.Although the technique can only increased the level of reproductive hormone secretion in the 1.5 year old mice,it failed to restore the ability to give birth.Overall,cryopreservation of ovarian looks like a convenient and practical method of fertility maintain.