In vivo delivery of FTY720 prevents radiation-induced ovarian failure and infertility in adult female nonhuman primates

Abstract

Objective: To determine whether sphingosine-1-phosphate (S1P), or the S1P mimetic FTY720 shields ovaries of adult female rhesus monkeys from damage caused by 15 Gy of targeted radiotherapy, allowing for the retention of long-term fertility, and to evaluate whether S1P protects human ovarian tissue (xenografted into mice) from radiation-induced damage.

Design: Research animal study.

Setting: Research laboratory and teaching hospital.

Patient(s): Adult female rhesus macaques (8-14 years of age; n = 21) and two women (24 and 27 years of age) undergoing gynecologic surgery for benign reasons, after informed consent and approval.

Intervention(s): None.

Main outcome measure(s): Ovarian histologic analysis, ovarian reserve measurements, and fertility in mating trials.

Result(s): Rapid ovarian failure was induced in female macaques by ovarian application of 15 Gy of radiation. Females given S1P or FTY720 by direct intraovarian cannulation for 1 week before ovarian irradiation rapidly resumed menstrual cycles because of maintenance of follicles, with greater beneficial effects achieved using FTY720. Monkeys given the S1P mimetic before ovarian irradiation also became pregnant in mating trials. Offspring conceived and delivered by radioprotected females developed normally and showed no evidence of genomic instability, as measured by micronucleus frequency in reticulocytes. Adult human ovarian cortical tissue xenografted into mice also exhibited a reduction in radiation-induced primordial oocyte depletion when preexposed to S1P.

Conclusion(s): S1P and its analogs hold clinical promise as therapeutic agents to preserve ovarian function and fertility in female cancer patients exposed to cytotoxic treatments.

Copyright © 2011 American Society for Reproductive Medicine. All rights reserved.

Figures

FIGURE 1

In-vivo delivery of S1P or FTY720 leads to a rapid return of reproductive cyclicity in adult female primates after OXI. Menstrual cyclicity in unilaterally ovariectomized monkeys that received intraovarian infusion of vehicle (Veh), S1P or FTY720 (FTY) 1 week prior to sham manipulation or OXI (each horizontal line represents one animal). Red bars, ovarian cycles with normal follicular and luteal phases; blue bars, ovarian cycles with a normal follicular phase and no luteal phase (based on circulating estradiol and progesterone levels); M, menses; *, termination of study.

FIGURE 2

In-vivo delivery of S1P or FTY720 attenuates radiation-induced ovarian damage in adult female primates. Representative histological appearance of ovaries from rhesus monkeys 9–10 months after intraovarian infusion of vehicle (A, B), S1P (C) or FTY720 (D) for 1 week prior to sham manipulation (A) or OXI (B–D). Percentage of ovarian follicles remaining in each animal (E), defined as the number of follicles counted in an ovary obtained 9–10 months after sham or OXI divided by the total number of follicles counted in the contralateral ovary from each animal prior to sham or OXI. Bars, represent the mean ± SE (n = 3–5 animals per group) with P-values determined by one-way ANOVA followed by the Neuman-Keuls multiple range test (different superscript letters, P<.05); ND, none detected.

FIGURE 3

Radioprotection of primate ovaries with FTY720 preserves natural fertility. Panels A–F show the first set of offspring conceived and delivered by adult female rhesus monkeys following FTY720-mediated ovarian protection from 15 Gy of radiation exposure (A and B, Hope at 7 and 453 days of age, respectively; C and D, Boneita at 7 and 432 days of age, respectively; E and F, Trinity at 7 and 416 days of age, respectively). Analysis of the frequency of micronucleated reticulocytes in blood of these infants over the first 70 days of age (G) shows no significant differences in offspring conceived by vehicle-infused non-irradiated controls (Veh+Sham) versus radioprotected females (FTY+OXI). Bars are the mean ± SE (n = 3 animals per group sampled at 1, 2, 3, 4 and 10 weeks of age; value range for each group is provided on the x-axis). Birth of the first 3 offspring was followed by a second successful pregnancy in 2 of 3 radioprotected females, with delivery of Victor (H; Hope’s brother) and Victoria (I; Boneita’s sister). As of the 1st of October 2010, the first set of offspring conceived and delivered by the FTY720-radioprotected females were 777 (Hope), 756 (Boneita) and 740 (Trinity) days of age; the second set of offspring were 352 (Victor) and 331 (Victoria) days of age.

FIGURE 4

In-vivo exposure to S1P for 1 hour prevents radiation-induced degeneration of primordial follicles in adult human ovary tissue xenografted into immunodeficient mice. Ovarian cortical biopsies (1–2 mm2) were grafted into NOD-SCID mice. One set of mice were euthanized 1 week after grafting to collect baseline information on oocyte numbers (Control, open bars; n = 6 mice), whereas the remaining mice were subjected to irradiation (2 Gy) 1 hour after injection of the graft sites with either vehicle (VEH+IR, gray bars; n = 6 mice) or S1P (S1P+IR, black bars; n = 6 mice). Grafts were collected from all mice 6 days later and assessed for the number of degenerative oocytes out of the total number of oocytes present by morphological criteria. Bars, represent the mean ± SE of combined data from 6–8 ovarian grafts per group (the total number of follicles scored in each group is provided over the respective bar).