We hypothesize that ovarian follicle depletion by iron and oxygen charged particle radiation is greater than ovarian follicle depletions by gamma-radiation at comparable doses and that silicon, iron, and oxygen charged particle radiation cause ovarian tumors at lower doses than gamma-radiation.

Aim 1: Utilize archived ovaries to compare ovarian tumor induction by irradiation with charged particles or gamma-rays. Our original aim was a comparison of ovarian tumors in 3-4 month old CB6F1 mice irradiated with 0.4, 0.8, 1.2, or 1.6 Gy gamma-rays or 0.04, 0.08, 0.16 and 0.32 Gy 260 MeV/u silicon and concurrent controls at 15-16 months after irradiation. We added analysis of ovarian tumors in mice of the same strain and age irradiated with 0, 0.10, or 0.20 Gy each of silicon, titanium, and iron ions in quick succession (mixed ion beam) and sacrificed 16 months after irradiation. We are conducting detailed histopathology of ovaries and molecular characterization of tumors using immunostaining for tumor markers.

Aim 2: Utilize archived ovaries harvested at various time points after irradiation with low doses of oxygen or iron charged particles to examine the persistence of ovarian oxidative lipid, protein, and DNA damage, archived serum to measure a biomarker of ovarian reserve, and evaluate these as potential early biomarkers of ovarian tumorigenesis. Irradiate mice with low doses of gamma-radiation and harvest ovaries at 1 week after irradiation in order to compare ovarian follicle depletion by charged iron or oxygen particles with gamma-radiation. Our published work (Mishra, Ortiz et al. 2016; Mishra, Ripperdan et al. 2017) demonstrates oxidative damage and dose-dependent apoptotic depletion of ovarian follicles after exposure to 0, 0.05, 0.3, and 0.5 Gy charged iron or oxygen particles. Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) were significantly elevated in 0.5 Gy-irradiated mice 8 wk after irradiation, consistent with loss of negative feedback due to follicle depletion, but serum LH and FSH are not optimal serum markers of ovarian reserve because they vary with estrous cycle stage and are secreted episodically, and we did not perform immunohistochemical analyses of ovaries at 8 wk after irradiation. We will examine oxidative lipid, protein, and DNA damage by immunostaining as potential biomarkers of ovarian tumor risk in archived ovaries from mice sacrificed 8 wk after irradiation with charged iron or oxygen particles. We will measure Anti-Müllerian Hormone (AMH), a serum marker of ovarian reserve that is used clinically, in archived serum from mice sacrificed 1 wk and 15 months after irradiation. 3-month old female C57BL/6J mice will be irradiated with 0, 0.05, 0.15, or 0.5 Gy gamma-rays and sacrificed one week post irradiation for ovarian follicle counts, which will be compared to our published data on charged iron or oxygen particle-irradiated mice.

Materials and Methods

Aim 1: Utilize archived ovaries to compare ovarian tumor induction by irradiation with charged particles or gamma-rays. Fixed ovary samples from female CB6F1 mice irradiated with 0, 0.4, 0.8, 1.2, and 1.6 Gy gamma-rays and 0, 0.04, 0.08, 0.12, and 0.32 Gy silicon charged particles or 0, 0.1, 0.2 Gy each silicon, titanium, and iron (hereafter referred to as mixed beam) and sacrificed 16 months after irradiation will be obtained from the NASA tissue bank. The ovaries will be processed for counting of ovarian follicles, ovarian tumor histopathology, and immunostaining for tumor markers.

Aim 2: Utilize archived ovaries harvested at various time points after irradiation with low doses of oxygen or iron charged particles to examine the persistence of ovarian oxidative lipid, protein, and DNA damage, archived serum to measure a biomarker of ovarian reserve, and evaluate these as potential early biomarkers of ovarian tumorigenesis. Irradiate mice with low doses of gamma-radiation and harvest ovaries at 1 week after irradiation in order to compare ovarian follicle depletion by charged iron or oxygen particles with gamma-radiation.

3-month old female C57BL/6J mice were irradiated with 0.05, 0.15, or 0.5 Gy gamma-rays or transported and restrained in an identical manner and not irradiated (0 Gy). All mice were sacrificed one-week post irradiation. One ovary per mouse was processed for counting ovarian follicles, and the other ovary was processed for immunostaining to measure proliferation, cell death, and oxidative damage. Blood serum was also collected from the gamma-irradiated mice, and together with archived serum from the Principal Investigator’s published studies (Mishra et al. 2016) was analyzed for anti-Müllerian hormone (AMH), as a biomarker of ovarian reserve.

Results

Aim 1: Utilize archived ovaries to compare ovarian tumor induction by irradiation with mixed heavy ion beam of silicon, titanium, and iron ions, silicon charged particles only, or gamma-rays. Fixed ovaries were received from 55 mice sacrificed at 16 months after irradiation with 0.3 or 0.6 Gy mixed heavy ion beam and compared to control, unirradiated mice. The ovaries were embedded in paraffin, serially sectioned, and every 10th section was stained with hematoxylin and eosin. Thus far, slides with ovaries from 39 mice have been reviewed by a board-certified veterinary pathologist. There was a dose-dependent, highly statistically significant increase in ovarian tubular adenomas, with 91% of mice in the 0.6 Gy mixed beam group having unilateral or bilateral tumors, 8% having a unilateral tumor in the 0.3 Gy mixed beam group, and no ovarian tumors found in the control mice. Tubular adenomas are epithelial ovarian tumors, and positive immunostaining for epithelial markers using a pancytokeratin antibody confirmed that the tumors are epithelial.

Aim 2: Utilize archived ovaries harvested at various time points after irradiation with low doses of oxygen or iron charged particles to examine the persistence of ovarian oxidative lipid, protein, and DNA damage, archived serum to measure a biomarker of ovarian reserve, and evaluate these as potential early biomarkers of ovarian tumorigenesis. Irradiate mice with low doses of gamma-radiation and harvest ovaries at 1 week after irradiation in order to compare ovarian follicle depletion by charged iron or oxygen particles with gamma-radiation.

Ovarian follicle counts in the gamma-irradiated mice have been completed on 4-5 mice per group. Thus far, there are statistically significant effects of dose on primordial, primary, and secondary follicle numbers, with fewer primordial, primary, and secondary follicles in the 0.50 Gy compared to the 0 Gy group.

Serum AMH concentrations at one week after irradiation did not vary significantly with dose of gamma- or 56Fe-radiation. There was a statistically significant effect of 16O irradiation dose (P=0.040), with dose-dependent decrease in serum AMH concentrations at 0.30 Gy compared to 0 Gy, but no difference in concentrations of AMH between the 0 compared to 0.05 and 0.50 Gy groups. The lack of dose-dependent decrease in serum AMH contrasts with the pronounced dose-dependent decrease in ovarian follicle numbers at one week after irradiation in the same mice exposed to 56Fe or 16O in our prior studies or the mice exposed to 0.5 Gy gamma-radiation described above.

Conclusions

We conclude that mixed heavy ion irradiation at 0.6 Gy total dose potently induces ovarian tumors, with 91% of the mice having ovarian tumors at 16 months after irradiation, while the 0.3 Gy total dose was much less effective at inducing ovarian tumors. We also conclude that serum AMH concentration at one week after irradiation does not correlate with primordial follicle numbers and therefore is not a useful biomarker of ovarian reserve at this time point after irradiation.

References

Mishra, B., L. Ortiz and U. Luderer (2016). "Charged Iron Particles, Typical of Space Radiation, Destroy Ovarian Follicles." Human Reproduction 31(8): 1816-1826.

Mishra, B., R. Ripperdan, L. Ortiz and U. Luderer (2017). "Very Low Doses of Heavy Oxygen Ion Radiation Induce Premature Ovarian Failure." Reproduction 154(2): 123-133.

Women made up 50% and 45%, respectively, of the 2013 and 2017 NASA astronaut classes. Astronauts are exposed to galactic cosmic rays during travel in deep space. Galactic cosmic rays consist of protons, helium ions, and charged particles heavier than helium, such as silicon, iron,,, and oxygen. Our published work demonstrates profound sensitivity of the ovary to charged particle radiation, with destruction of the irreplaceable ovarian follicle pool and 4-fold as many ovarian tumors as in control non-irradiated mice. Comparison of our data with published studies of ovarian follicle depletion and ovarian tumorigenesis by exposure to gamma radiation suggest that charged particle radiation may be a more potent inducer of both premature ovarian follicle depletion and ovarian tumors, but this has not been directly tested.

We hypothesize that ovarian follicle depletion by iron and oxygen charged particle radiation is greater than ovarian follicle depletions by gamma-radiation at comparable doses and that silicon, iron, and oxygen charged particle radiation cause ovarian tumors at lower doses than gamma-radiation.

Aim 1: Utilize archived ovaries to compare ovarian tumor induction by irradiation with silicon charged particles or gamma-rays. Comparison of ovarian tumors in 3-4 month old CB6F1 mice irradiated with 0.4, 0.8, 1.2, or 1.6 Gy gamma-rays or 0.04, 0.08, 0.16, and 0.32 Gy 260 MeV/u silicon and concurrent controls at 15-16 months after irradiation. We will conduct detailed histopathology of ovaries and molecular characterization of tumors using immunostaining for tumor markers.

Aim 2: Utilize archived ovaries harvested at various time points after irradiation with low doses of oxygen or iron charged particles to examine the persistence of ovarian oxidative lipid, protein, and DNA damage, archived serum to measure a biomarker of ovarian reserve, and evaluate these as potential early biomarkers of ovarian tumorigenesis. Irradiate mice with low doses of gamma-radiation and harvest ovaries at 1 week after irradiation in order to compare ovarian follicle depletion by charged iron or oxygen particles with gamma-radiation. Our published work demonstrates oxidative damage and dose-dependent apoptotic depletion of ovarian follicles after exposure to 0, 0.05, 0.3, and 0.5 Gy charged iron or oxygen particles. Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) were significantly elevated in 0.5 Gy-irradiated mice 8 wk after irradiation, consistent with loss of negative feedback due to follicle depletion, but serum LH and FSH are not optimal serum markers of ovarian reserve because they vary with estrous cycle stage and are secreted episodically, and we did not perform immunohistochemical analyses of ovaries at 8 wk after irradiation. We will examine oxidative lipid, protein, and DNA damage by immunostaining as potential biomarkers of ovarian tumor risk in archived ovaries from mice sacrificed 8 wk after irradiation with charged iron or oxygen particles. We will measure Anti-Müllerian Hormone (AMH), a serum marker of ovarian reserve that is used clinically, in archived serum from mice sacrificed 1 wk and 15 months after irradiation. 3-month old female C57BL/6J mice will be irradiated with 0, 0.05, 0.15, or 0.5 Gy gamma-rays and sacrificed one week post irradiation for ovarian follicle counts, which will be compared to our published data on charged iron or oxygen particle-irradiated mice.

Materials and Methods

Aim 1: Utilize archived ovaries to compare ovarian tumor induction by irradiation with silicon charged particles or gamma-rays.

Fixed ovary samples from female CB6F1 mice irradiated with 0, 0.4, 0.8, 1.2 ,and 1.6 Gy gamma-rays and 0, 0.04, 0.08, 0.12, and 0.32 Gy silicon charged particles and sacrificed 16 months after irradiation will be obtained from the NASA tissue bank. The ovaries will be processed for histomorphometric analyses of ovarian follicle numbers, ovarian tumor histopathology, and immunostaining for tumor markers.

Aim 2: Utilize archived ovaries harvested at various time points after irradiation with low doses of oxygen or iron charged particles to examine the persistence of ovarian oxidative lipid, protein, and DNA damage, archived serum to measure a biomarker of ovarian reserve, and evaluate these as potential early biomarkers of ovarian tumorigenesis. Irradiate mice with low doses of gamma-radiation and harvest ovaries at 1 week after irradiation in order to compare ovarian follicle depletion by charged iron or oxygen particles with gamma-radiation.

3-month old female C57BL/6J mice were irradiated with 0.05, 0.15, or 0.5 Gy gamma-rays or transported and restrained in an identical manner and not irradiated (0 Gy). All mice were sacrificed one-week post irradiation (N=8 per dose). One ovary per mouse was processed for counting ovarian follicles, and the other ovary was processed for immunostaining to measure proliferation, cell death, and oxidative damage. Blood serum was also collected from the gamma-irradiated mice, and together with archived serum from the Principal Investigator’s published studies will be analyzed for follicle stimulating hormone (FSH), luteinizing hormone (LH), and anti-Müllerian hormone (AMH), as biomarkers of ovarian dysfunction.

Results

Aim 1: Utilize archived ovaries to compare ovarian tumor induction by irradiation with silicon charged particles or gamma-rays. We have not been able to begin work on this aim due to the COVID-19 pandemic.

Aim 2: Utilize archived ovaries harvested at various time points after irradiation with low doses of oxygen or iron charged particles to examine the persistence of ovarian oxidative lipid, protein, and DNA damage, archived serum to measure a biomarker of ovarian reserve, and evaluate these as potential early biomarkers of ovarian tumorigenesis. Irradiate mice with low doses of gamma-radiation and harvest ovaries at 1 week after irradiation in order to compare ovarian follicle depletion by charged iron or oxygen particles with gamma-radiation.

Body weights, ovarian weights, and uterine weights were measured at the time of sacrifice. The uterus was evaluated for presence of ballooning, which is an increase in uterine fluid and tissue mass characteristic of the proestrous stage of the estrous cycle. There was a statistically significant effect of gamma-radiation dose on body weight, with 0.5 to 1 g decreased body weight in all three gamma-irradiated groups compared to unirradiated controls. The mean paired ovary/oviduct weight was 1 to 1.5 mg lower in irradiated groups than in the control group. However, the effect of irradiation on paired ovary/oviduct weight was not statistically significant.

Because the mice were sacrificed at one week after irradiation without regard to estrous cycle stage, uterine weights were analyzed with adjustment for ballooning. The effect of radiation dose was statistically significant for the non-ballooned uteri, with the irradiated groups having 13 to 16 mg lower uterine weights on average than the sham-irradiated group.

Ovarian follicle counts on one ovary from each of these mice are in progress. Counts on 5 of 32 ovaries were completed before the COVID-19 pandemic research shut-down.

Conclusion

The decreased uterine weights observed one week after gamma-radiation in the present study are consistent with prior studies that showed that uterine irradiation decreases uterine size and thickness in premenopausal women and rats. Our finding of non-significantly decreased ovarian weights is consistent with our published observations that antral follicle numbers are decreased after charged iron particle-irradiation. Antral follicles are large structures, and a decrease in their number could reasonably be expected to reduce ovarian weight.