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. Author manuscript; available in PMC: 2012 Mar 15.
Published in final edited form as: Fertil Steril. 2011 Mar 15;95(4):1440–1445.e7. doi: 10.1016/j.fertnstert.2011.01.012

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

Mary B Zelinski a, Mark K Murphy b, Maralee S Lawson a, Andrea Jurisicova c, K Y Francis Pau a, Natalia P Toscano a, Darla S Jacob d, John K Fanton d, Robert F Casper c, Stephen D Dertinger e, Jonathan L Tilly f
PMCID: PMC3063448  NIHMSID: NIHMS264137  PMID: 21316047

Abstract

Objective

To determine if 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 retention of long-term fertility; and, to evaluate if S1P protects human ovarian tissue xenografted into mice from radiation-induced damage.

Design

Research animal study.

Setting

Research laboratory and teaching hospital.

Animal(s)

Adult female rhesus macaques (8–14 years of age).

Patient(s)

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 histology, 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 prior to ovarian irradiation rapidly resumed menstrual cycles due to maintenance of follicles, with greater beneficial effects achieved using FTY720. Monkeys given the S1P mimetic prior to 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 pre-exposed to S1P.

Conclusion(s)

S1P and its analogs hold clinical promise as therapeutic agents to preserve both ovarian function and fertility in female cancer patients exposed to cytotoxic treatments.

Keywords: Fertility preservation, cancer, ovary, oocyte, monkey, human, phingosine-1-phosphate, FTY720

INTRODUCTION

Premature ovarian failure and infertility are frequently observed in young girls and reproductive age women treated for cancer (1). Attempts to preserve ovarian function in these patients have met with little success, leaving assisted reproduction or cryopreservation of oocytes and ovarian tissue as the only currently-available clinical options other than egg donation (2, 3). However, many patients cannot undergo hormonal stimulation protocols needed to retrieve oocytes due to time constraints or their type of cancer, and pregnancy success rates using frozen-thawed human eggs remain low. Although some of these issues can be circumvented by ovarian tissue banking, this procedure is experimental and presents the risk of re-introducing cancer cells back into the patient following transplantation (2–5).

Studies in mice have shown that anti-cancer treatments activate apoptosis in oocytes (6). Further, targeted deletion of genes encoding pro-apoptotic signaling factors, including acid sphingomyelinase (which generates ceramide), Bax (which disrupts mitochondrial membrane potential) or the protease caspase-2, prevents such oocyte death (7–10). The anti-apoptotic factor sphingosine-1-phosphate (SIP), which is produced through ceramide metabolism (11), recapitulates ovarian protective effects of acid sphingomyelinase deficiency (8). These data, and findings that ceramide is required for radiation-induced germ cell death in Caenorhabditis elegans (12), indicate that ceramide is a conserved mediator of germ cell death triggered by these insults. In fact, massive oocyte loss caused by irradiation of adult female mice can be prevented by prior treatment with S1P delivered into the bursal sac surrounding each ovary (7). Although the ovarian protective actions of S1P have been confirmed in several subsequent studies, all are confined to rodent models (13–15).

Importantly, S1P-protected oocytes do not show DNA damage and remain fully competent for maturation, fertilization and preimplantation embryonic development in vitro and in vivo (7, 8). Follow-up studies showed that F1 offspring conceived by female mice protected from radiation-induced ovarian damage with S1P, as well as F2 offspring conceived by the F1 animals, show no anatomical, histological, behavioral or cytogenetic evidence of transgenerational genomic damage or other abnormalities (8). Here we conducted a preclinical trial to evaluate the effects of S1P and its long-acting mimetic, FTY720 (16), on ovarian function and fertility in adult female rhesus macaques following irradiation.

MATERIALS AND METHODS

Animals

Adult female rhesus monkeys during prime reproductive life (8–14 years of age) and of proven fertility were used under research protocols approved by Oregon National Primate Research Center (ONPRC) institutional animal care and use committee. Each animal was implanted subcutaneously with osmotic minipumps attached to intraovarian catheters [17], which were designed to continuously deliver vehicle (60% polyethylene glycol, 30% ethanol, 10% Tween-20; PET), S1P (0.2 mM in PET, 10 µl per hour; Biomol Research Laboratories, Plymouth Meeting, PA) or FTY720 (0.2 mM in PET, 10 µl per hour; Cayman Chemical Company, Ann Arbor, MI) for 1 week prior to irradiation.

Ovarian X-Irradiation (OXI)

Ovaries were externalized from anesthetized animals and exposed to X-ray-based radiation for 13 min to generate 1,500 rads of absorbed dose (15 Gy), and then returned to the peritoneal cavity.

Steroid Hormone Measurements

Serum steroid levels were assayed in the ONPRC Endocrine Services Laboratory to monitor menstrual cyclicity.

Follicle Counts

Ovarian sections were analyzed using a Marianas™ digital imaging workstation and the Stereology module of Slidebook 4.2 (Intelligent Imaging Innovations).

Mating Trials

Females were paired with a single male. Pregnancies and vaginal delivery of offspring continued without intervention in all animals except for one FTY720-infused OXI-treated female who underwent Cesarean section 1 week after her expected due date.

Micronucleus Assay

Micronuclei frequency in blood reticulocytes was assessed by flow cytometry (18).

Human ovarian xenografting

Cortical biopsies obtained from ovaries of two patients (24 and 27 years of age) after ovariectomy under an IRB-approved protocol were grafted into NOD-SCID mice (19), treated as indicated, and analyzed for the incidence of oocyte degeneration (20).

Data Analysis

Compiled data from the replicate experiments were analyzed using ANOVA followed by the Neuman-Keuls multiple range test, unpaired t-tests or Fisher's exact test, with differences in mean values considered significant at P<.05.

RESULTS

Menstrual Cyclicity and Ovarian Reserve

One ovary was removed from each animal before manipulation to provide baseline information on ovarian architecture and follicular dynamics in each female. The remaining ovary was subjected to treatment and then removed 316 ± 7 days later for analysis. Sham-manipulated control females infused with vehicle (n = 3) resumed normal menstrual cyclicity within 92 ± 21 days post-sham manipulation (Fig. 1). Acyclicity was observed throughout the post-irradiation interval in 2 of 3 vehicle-infused females subjected to OXI. The remaining female had a single menstrual cycle starting on day 139 post-OXI (Fig. 1), which was likely due to inadvertent slippage of her ovary out of the radiation field during treatment. No further cycles were observed in this animal. Five of 6 S1P-infused females resumed menstrual cyclicity within 163 ± 30 days post-OXI, whereas 1 animal remained acyclic (Fig. 1).

FIGURE 1.

FIGURE 1

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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.

Ovaries from vehicle-infused control females appeared normal and resembled ovaries collected prior to manipulation (Fig. 2). Ovaries obtained post-OXI from vehicle-infused females were small and completely devoid of oocytes and follicles (Fig. 2). In contrast, ovaries collected from S1P-infused females that resumed menstrual cyclicity following OXI retained a small number of immature follicles with normal morphology (Fig. 2). Corpora lutea from the cycle prior to ovariectomy were noted in 3 of these females, indicating the occurrence of endogenous ovulatory cycles (data not shown).

FIGURE 2.

FIGURE 2

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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.

In parallel we tested the long-acting S1P mimetic, FTY720, for its ability to protect ovaries from radiation-induced damage. In females whose ovaries were infused with FTY720 for 1 week prior to OXI (n = 3), all animals resumed menstrual cyclicity within 78 ± 5 days post-OXI (Fig. 1). Ovaries of females infused with FTY720 and collected 317 ± 10 days post-OXI contained significantly more resting (primordial) and growing (primary, secondary) follicles than ovaries of SIP-infused OXI females (Fig. 2), indicating that more pronounced protective effects are observed with FTY720 compared with S1P.

Mating Trials

Natural mating trials were then conducted with female monkeys given bilateral intraovarian vehicle infusion followed by sham manipulation (n = 3) or bilateral intraovarian FTY720 infusion followed by OXI (n = 3). Females receiving vehicle infusion followed by OXI were not included since female macaques lacking ovarian function exhibit decreased proceptivity, attractivity and receptivity to males (21). Although mounting can be observed in the absence of estrogen, bona fide mating with intromission and ejaculation is absent and pregnancy does not occur (22; M.B. Zelinski, unpublished observations). A resumption of menstrual cyclicity was observed within 65 ± 9 days in the vehicle-infused sham control group and 69 ± 5 days in the FTY720-infused OXI group (Supplementary Table 1). Each female was individually mated with a fertile male during the second ovarian cycle post-treatment for up to 5 consecutive cycles. Successful pregnancies, as confirmed by evidence of fetal cardiac activity during ultrasound at 33 days post-mating, were established in all vehicle-infused sham females within 104 ± 11 days post-treatment and in all FTY720-infused OXI-treated females within 123 ± 17 days post-treatment (Supplementary Table 1). With the exception of femur length at day 145 of gestation, fetal growth parameters and final birth weights were similar for both groups (Supplementary Tables 1 and 2).

Offspring Analysis

All offspring were delivered within 1 week of their expected due date, and developed past the first year of life without complications, abnormalities or behavioral differences (Fig. 3A–F). Although femur growth between gestational days 101–145 in offspring of radioprotected females was approximately 10 mm less than that of controls (Supplementary Table 2), femur length is now similar in the two groups (Supplementary Table 3). Cytogenetic instability measured by frequency of micronucleated reticulocytes revealed no differences in offspring conceived by radioprotected mothers compared with those delivered by non-irradiated sham controls (Fig. 3G). The 3 females whose ovaries were protected from radiotherapy-induced damage with FTY720 were mated again after weaning their first set of infants. Two of the 3 females became pregnant and delivered a second set of offspring (Fig. 3H, I). Like the first set of offspring, the second set of offspring exhibited normal gestational parameters, birth weights and neonatal development, and no evidence of elevated micronuclei frequency (data not shown). The third radioprotected female has failed to conceive again after repeated mating attempts, despite continuation of menstrual cyclicity.

FIGURE 3.

FIGURE 3

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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.

Human Ovarian Radioprotection by S1P

In a final set of experiments, we assessed if S1P exerts similar protective effects against radiation-induced destruction of human ovarian follicles using a mouse xenograft model. An approximate 4-fold increase in the incidence of degenerative (atretic) primordial follicles was detected in human ovarian grafts 6 days after exposure to 2 Gy of radiation; however, this response was completely absent in human ovaries pre-exposed to S1P for 1 hour prior to irradiation (Fig. 4). Radiation-induced damage to the early growing (primary) follicles in human ovaries was not inhibited by S1P pre-exposure (Fig. 4).

FIGURE 4.

FIGURE 4

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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).

DISCUSSION

Approximately 1 of 49 human females in the United States between birth and age 40, which spans the pre-reproductive and reproductive period, will be diagnosed with cancer (23). Many of these patients will be treated with cytotoxic therapies known to cause significant side-effect damage to the ovaries. Despite the importance of this problem, little is available to female cancer patients seeking fertility preservation other than assisted reproduction or cryopreservation of embryos, eggs or ovary tissue. Even if these approaches prove successful for birth of a child, these patients are left with the very real possibility of developing significant health problems associated with premature ovarian failure. The only method proposed thus far for protecting the ovaries in situ involves gonadotropin-releasing hormone analog therapy during anti-cancer treatment; however, clinical data supporting the usefulness of this approach for fertility preservation in female cancer patients have been a subject of considerable debate (24–27).

More than a decade of work with rodent models has consistently demonstrated a remarkable efficacy of S1P in preserving ovarian function and fertility in vivo in females exposed to anti-cancer treatments 97–9, 13–15). Despite these encouraging results, possible human application has remained uncertain. Aside from traditional problems often associated with extrapolating drug efficacy studies in rodents to primates, the fact that the primate ovary is not enclosed within a bursal sac (as is the case with rodents) presents an anatomical hurdle to confining S1P to the ovaries. Through the use of an intraovarian catheter-osmotic mini-pump system, we showed that direct and controlled long-term delivery of S1P or FTY720 to the ovaries of adult female primates can be successfully achieved. In turn, this approach has provided important translational proof-of-concept that S1P, and in particular its mimetics, protect the primate ovary against radiation-induced damage.

It is important to mention that while radiotherapy was selected as the gonadotoxic agent for this study, many chemotherapeutic drugs cause equally concerning irreversible damage to the ovaries (1). However, the costs of running preclinical trials with monkeys did not permit us to conduct a parallel analysis of both treatments, and thus we focused on radiotherapy-induced ovarian failure for the following reasons. First, the extent of damage to the gonads caused by 15 Gy of direct radiation treatment is at least equivalent to, if not considerably worse than, that resulting from treatment with most chemotherapeutic regimens 1, 28; see also http://www.fertilehope.org). In fact, the LD50 for radiation-induced oocyte destruction in humans is less than 2 Gy (29). Second, radiation can be directed specifically to the gonads, thus minimizing the potential for widespread systemic toxicity – and in particular, immunologic failure – resulting from intravenous chemotherapy. Third, irradiation provides a means to achieve ovarian failure in a simple and consistent manner, whereas chemotherapy protocols vary widely, not only in terms of which particular drug to focus on but also of whether single agent or combination chemotherapy should be used. Finally, work with mice has shown that S1P appears equally effective at sparing the ovaries from damage caused by chemotherapy or radiotherapy (7, 8, 13, 14). Still, it remains to be determined if S1P or FTY720 can protect primate ovaries from cytotoxic drug-induced damage in vivo.

Fertility preservation in adult female primates exposed to sterilizing doses of radiation through the use of the potent S1P mimetic, FTY720, offers many advantages over other experimental fertility preservation strategies being evaluated that rely on ovarian tissue cryopreservation or in-vitro follicle maturation (5, 30–32). These latter methods are designed to provide a possible chance at conception through assisted reproduction, but do not alleviate the onset of premature ovarian failure and its associated health complications in cancer patients post-treatment. Further, for those patients with blood cell-borne cancers or metastatic disease, the risk of introducing cancer cells back into the patient post-therapy by ovarian tissue auto-transplantation is of concern (4, 5). The present findings offer compelling evidence that natural conception and delivery of healthy offspring by adult female primates whose ovaries were exposed to very high levels of radiation can be achieved with a small molecule delivered directly to the gonads prior to treatment. Using a xenograft model, we further observed that radiation-induced degeneration of primordial follicles in adult human ovary tissue transplanted into immunodeficient mice is prevented by injection of S1P at the graft site 1 hour prior to irradiation. These findings collectively support the feasibility of ovarian protection in situ through the use of S1P or its mimetics as a promising option for improving the quality of life in female cancer patients in the future.

Supplementary Material

1
NIHMS264137-supplement-1.eps (1,006.1KB, eps)
2

Acknowledgments

We thank D. Miller (in memorium), V. Warren, J. Dewane, D. Allen and T. Hobbs, as well as the animal technicians and surgical staff, of the ONPRC Division of Animal Resources for assistance; A. Cornea, B. Mason and E. Ritchie (ONPRC Morphology and Imaging Core) for assistance with ovarian histology and follicle counting; D. Hess and associates (ONPRC Endocrine Services Laboratory) for performing the steroid assays; J. Penner (Battelle Laboratories) and R.K. Piper (Pacific Northwest National Laboratories) for veterinary care and dosimetry; T. Hannam for assistance with the xenograft surgeries; and W. Sutton for animal photography. We also thank P. Slavin, J.S. Loeffler, I. Schiff and F. Frigoletto Jr. for critical reading of the manuscript before submission.

This work was supported by the United States National Institutes of Health (R01-HD45787, U54-HD18185, NCRR-RR00163), Canada Research Chair Program, Canadian Institutes of Health Research (Operating Grants MOP 14058 and MOP 84328), and Vincent Memorial Research Funds.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

M.B.Z. has nothing to disclose. M.K.M. has nothing to disclose. M.S.L. has nothing to disclose. A.J. has nothing to disclose. K.Y.F.P. has nothing to disclose. N.P.T. has nothing to disclose. D.S.J. has nothing to disclose. J.K.F. has nothing to disclose. R.F.C. has nothing to disclose. S.D.D. has nothing to disclose. J.L.T. declares interest in the intellectual property associated with U.S. Patent Number 7,195,775 and its associated Continuation-in-Part (Supplemental Notice of Allowability) describing the use of S1P and its analogs as fertility preservation agents.

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Associated Data

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Supplementary Materials

1
NIHMS264137-supplement-1.eps (1,006.1KB, eps)
2
NIHMS264137-supplement-2.doc (87.5KB, doc)

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