ALS received consulting fees from Hoffmann–La Roche and Nycomed, and grant support from Wyeth (now Pfizer); DP received travel support from Wyeth (now Pfizer); ADK received lecture fees from Wyeth and Genzyme, and travel support from Amgen; JY is an employee of Biometrical Practice BIOP; PK is an employee of Aardex; RPW received consulting and lecture fees from Genzyme, Novartis and Wyeth, and grant support from Wyeth. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: ALS DP ADK RPW. Performed the experiments: MB CSR FK PK XW YL JL GA AE. Analyzed the data: ALS JY OS. Contributed reagents/materials/analysis tools: JL. Wrote the paper: MB JY ALS RPW.
Sirolimus has been approved for clinical use in non proliferative and proliferative disorders. It inhibits the mammalian target of rapamycin (mTOR) signaling pathway which is also known to regulate ovarian morphology and function. Preliminary observational data suggest the potential for ovarian toxicity but this issue has not been studied in randomized controlled trials. We reviewed the self-reported occurrence of menstrual cycle disturbances and the appearance of ovarian cysts
ClinicalTrials.gov
Sirolimus (Rapamune, Pfizer, New York, NY, USA) is a potent immunosuppressive and anti-proliferative drug which blocks the mammalian target of rapamycin (mTOR). MTOR is a key regulatory kinase which is also known to regulate ovarian function
Most of our knowledge regarding sirolimus toxicity has been derived from kidney transplant efficacy trials. However there were no reports of menstrual cycle disturbances and ovarian cyst formation in three large clinical trials
Aside from organ transplantation, sirolimus is being assessed for clinical effectiveness in several cancers and in other proliferative disorders, including autosomal dominant polycystic kidney disease (ADPKD). ADPKD is characterized by the growth of kidney cysts; the disease itself is not known to affect ovarian morphology and function
Animal and observational data suggest the potential for ovarian toxicity but this issue has not been considered in previous trials
The SUISSE ADPKD study was a randomized controlled phase II trial carried out to determine whether 18 months of treatment with sirolimus slows kidney growth in adults with ADPKD. The occurrence of menstrual cycle disturbances and ovarian cysts were pre-defined secondary endpoints in the statistical analysis plan
After a run-in period of 6 months, patients were randomly assigned to receive either 18 months treatment with sirolimus (target dose 2 mg daily) or standard care. Standard care consisted of blood pressure control (office systolic and diastolic blood pressure targets below 130 and 85 mm Hg respectively), prompt antibiotic treatment of kidney cyst infections, and avoidance of potentially nephrotoxic substances. The sirolimus dose was adjusted to achieve steady-state levels between 4 and 10 µg per liter, determined by liquid chromatography–mass spectrometry from whole blood. Patient adherence to sirolimus was assessed using an electronic system (MEMS™, Aardex Group, Ltd., Sion, Switzerland).
Wyeth (now Pfizer) had no role in the trial design, collection, analysis and interpretation of the data or the writing of the report. Andreas L. Serra and James Young had full access to the study data and take responsibility for the integrity of the data and the accuracy of the data analysis.
At enrollment, randomization, 6, 12 and 18 months patients were asked if they had any menstrual cycle abnormalities in the past 6 months. Oligoamenorrhea was defined as no menstrual period for 3 months or more when one should have occurred, or an interval of more than 35 days between menstrual periods. Our definition of oligoamenorrhea was based on criteria used in epidemiologic studies
Abdominal magnetic resonance imaging (MRI) without contrast material was performed every 6 months using a 1.5T MR scanner (Signa Echospeed Excite HD or HDx, General Electrics (GE) Healthcare, Waukesha, Wisconsin, USA).
Analyses of prevalence and incidence were carried out using logistic and Cox proportional hazards regression in SAS version 9.2 (Cary, NC, USA). Outcomes for logistic regression were any patient report of oligoamenorrhea and any detected ovarian cyst greater than 2 cm in diameter during the 18 months treatment period. Outcomes for Cox proportional hazards regression were time to a first report of oligoamenorrhea in those without oligoamenorrhea at randomization, and time to a first ovarian cyst greater than 2 cm in diameter in those without such cysts at randomization. Since these outcomes can only be assessed at each visit we used a discrete time version of the Cox proportional hazards model.
The sirolimus-associated effects on the mTOR signaling pathway in ovaries were examined by Western blotting and immunohistochemistry in rats given oral sirolimus. The animal study was approved by the animal health regulatory agency of the Canton Zürich, Switzerland.
Female 4 week old Wistar rats were given daily 3.0 milligram per kilogram body weight sirolimus (N = 4) or vehicle (N = 4) by gavage feeding. The rats were sacrificed during pro-estrus three weeks after initiation of treatment at six PM and serum levels of follicle and luteal stimulating hormones were measured by enzyme linked immunoassay according to the manufactures' protocol.
Ovaries were homogenized with ice-cold lysis buffer containing 40 mM Hepes, 120 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM potassium pyrophosphate, 10 mM glycerol phosphate, 50 mM NaF, 0.5 mM NaVO3, 1% Triton, and protease inhibitor (pH 7.6). Tissue lysates were cleared by centrifugation. Equal amounts of lysates were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, and probed with antibodies. Cell Signaling Technology supplied antibodies against Akt Ser473, p70 S6K Thr421/Ser424 and p70 S6K; Abcam supplied an anti-Akt antibody; Millipore supplied an anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody.
Paraffin-embedded sections were cut and stained by the immunoperoxidase technique, following standard methods of deparaffinization, antigen retrieval, primary antibody incubation with either anti-Akt Ser473 (Cell Signaling) or anti-p70 S6K Thr421 (GenWay), secondary antibody incubation, 3,3-diaminobenzidine (DAB) incubation, and counterstaining with methyl green.
Of the 39 females enrolled, 21 were randomized to receive sirolimus and 18 to receive standard care. Follow-up was complete in all but one patient receiving sirolimus who withdrew at 6 months (
Characteristic – values are means (standard deviation) or numbers (percent) | Sirolimus | Control |
(N = 21) | (N = 18) | |
31 (8) | 32 (7) | |
22 (3) | 22 (3) | |
Systolic | 132 (12) | 120 (11) |
Diastolic | 83 (11) | 78 (10) |
88 (20) | 96 (16) | |
1 | 10 (48) | 11 (61) |
2 | 10 (48) | 7 (39) |
3 | 1 (5) | 0 |
13 (2) | 13 (2) | |
Nulliparous | 13 (62) | 14 (78) |
1–2 | 7 (33) | 4 (22) |
>2 | 1 (5) | 0 |
Hormonal | 10 (48) | 9 (50) |
Barrier method | 11 (52) | 9 (50) |
From New England Journal of Medicine, Ovarian toxicity from sirolimus, Braun M, Young J, Reiner CS, Poster D, Wüthrich RP, Serra AL. 366(11):1062-4. Copyright © (2012) Massachusetts Medical Society. Reprinted with permission.
The body-mass index (BMI) is the weight in kilograms divided by the square of the height in meters.
The glomerular filtration rate (GFR) was estimated by using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation
Chronic kidney disease was classified according to the Kidney Disease Outcomes Quality Initiative of the National Kidney Foundation. Stage 1 denotes a glomerular filtration rate (GFR) of 90 ml or more per minute per 1.73 m2 of body-surface area; stage 2, a GFR of 60 to 89 ml per minute; and stage 3, a GFR of 30 to 59 ml per minute.
Patients' self-reported medical histories at randomization.
Number of patients (percent) [missing if any] | Sirolimus N = 21 | Control N = 18 |
|
||
Enrollment | 3 (14) | 2 (12) |
Randomization | 2 (10) |
1 (7) |
6 months | 9 (43) | 2 (11) |
12 months | 5 (25) |
2 (11) |
18 months | 5 (26) |
1 (6) |
Any visit after randomization | 11 (52) | 3 (17) |
|
||
Enrollment | 4 (19) | 4 (25) |
Randomization | 2 (10) | 4 (24) |
6 months | 6 (29) | 2 (14) |
12 months | 6 (30) |
3 (18) |
18 months | 7 (39) |
2 (12) |
Any visit after randomization | 12 (57) | 5 (28) |
From New England Journal of Medicine, Ovarian toxicity from sirolimus, Braun M, Young J, Reiner CS, Poster D, Wüthrich RP, Serra AL. 366(11):1062-4. Copyright © (2012) Massachusetts Medical Society. Reprinted with permission.
Patients' self-reported events during the 6 months before the visit.
Assessed by two independent observers – there was no disagreement between observers.
Median (interquartile range [IQR]) follow up time after randomization was 19 months (19–20) and 19 months (18–20) in the sirolimus and control groups, respectively. Both the numbers of missing reports of menstrual cycle disturbances and the number of unreadable MRI scans were lower among patients in the sirolimus group (
A total of 11 out of 21 patients in the sirolimus group reported oligoamenorrhea at any visit after randomization, compared to 3 out of 18 patients in the control group (
Ovarian cysts were observed in 12 out of 21 patients in the sirolimus group, compared to 5 out of 18 patients in the control group (
Differences in cycle disturbances were apparent in those not on oral contraceptives – 8 out of 11 and 2 out of 9 patients in the sirolimus and control groups; but were less apparent in those on oral contraceptives – 3 out of 10 and 1 out of 9 patients in the sirolimus and control groups. Differences in ovarian cysts between sirolimus and control did not seem to depend on the contraceptive method (barrier methods: 7 out of 11 and 3 out of 9 patients in the sirolimus and control groups; oral contraceptives: 5 out of 10 and 2 out of 9 patients in the sirolimus and control groups).
Although imprecise, estimates of odds and hazard ratios suggest that the prevalence and incidence of both oligoamenorrhea and ovarian cysts were higher among patients receiving sirolimus (
Oligoamenorrhea | Ovarian cysts | |||
Estimate | 95% CI | Estimate | 95% CI | |
|
||||
Profile likelihood | 5.5 | 1.3 to 29 | 3.5 | 0.94 to 14 |
Exact | 5.3 | 1.0 to 37 | 3.4 | 0.76 to 17 |
|
||||
Profile likelihood | 4.3 | 1.1 to 29 | 4.0 | 1.1 to 26 |
Exact |
4.4 | 0.75 to 48 | 4.3 | 0.82 to 43 |
Exact hazard ratios were estimated using a model with a logit link function and no offset. Profile likelihood estimates for this alternative model were 4.5, (95% confidence interval [CI] 1.1 to 31) and 4.6, (95% CI 0.99 to 34) for oligoamenorrhea and ovarian cysts respectively.
Mean sirolimus doses were between 1.2 and 1.5 milligram per day and the sirolimus steady-state blood levels between 3.5 and 4.7 microgram per liter (
The persistence curve shows a Kaplan Meier estimation of the proportion of patients on treatment. The adherence curve indicates the day-to-day proportion of patients who took sirolimus as prescribed. Data from 21 individual dosing histories were used for this analysis.
Months | Sirolimus dosage |
Sirolimus level |
Continuation |
Adherence |
|
1.1 (0.5) | 3.5 (1.0) | 100 | 97.6 |
|
1.3 (0.4) | 3.5 (1.1) | 100 | 96.9 |
|
1.2 (0.6) | 3.7 (1.1) | 95.2 | 95.1 |
|
1.4 (0.4) | 4.2 (1.9) | 95.2 | 93.9 |
|
1.5 (0.5) | 4.7 (1.6) | 95.2 | 92.2 |
Values are means (standard deviation).
Continuation was defined as the proportion of patients who remained on the sirolimus treatment.
Adherence was defined for each specific time interval as the average daily proportion of patients who took their sirolimus dose as prescribed among those still on the sirolimus treatment.
Rats given sirolimus 3.0 milligram per kilogram per day for three weeks, a dose that produces blood concentrations similar to those in patients
Panel A. Gel electrophoresis of proteins carried out to assess sirolimus-associated changes in the activity of key signaling pathways thought to regulate ovarian function and morphology. Sirolimus treatment amplified signaling through phosphatidylinositol 3-kinase (AktSer743), thought to be associated with human polycystic ovarian syndrome, and blocked the mammalian target of rapamycin pathway (p70 S6KThr421). Panel B. The ratios of phosphorylated to non-phosphorylated molecules are expressed in arbitrary densitometric units (Sirolimus minus control – mean (95% confidence interval): p70 S6KThr421/Ser424 to p70 S6K −0.28 (−0.57 to 0.02) and AktSer473 to Akt 0.34 (0.09 to 0.59). Panel C: Immunohistochemical analysis shows positive staining with a nuclear pattern for AktSer743 in granulosa cells of rats receiving sirolimus and for p70 S6KThr421 in granulosa cells of control rats receiving vehicle.
Serum levels of follicle stimulating and luteinizing hormones (FSH, LH) were similar between groups: FSH: 1.8±0.3 and 2.2±0.3 nanograms per milliliter in the sirolimus and control groups respectively; LH: 3.4±0.7 and 3.1±0.4 nanograms per milliliter in the sirolimus and control groups respectively.
When given sirolimus at a relatively low dose for a median of 19 months, 52% of 21 patients with ADPKD reported menstrual cycle abnormalities, compared to 17% of 18 patients in the control group. Sirolimus also increased the risk of ovarian cysts, and one patient was cystectomized after receiving sirolimus for five months. With relatively few female patients, it is not possible to make precise estimates but these increases in relative risk are potentially large – with a fourfold increase or more possible.
Reports from other studies suggest a lower ovarian toxicity or make no mention of it at all. In a multi-center placebo controlled phase II trial, Kahan and colleagues did not report menstrual cycle abnormalities in 47 female kidney allograft recipients receiving sirolimus for 12 months
Our findings of sirolimus associated ovarian toxicity are supported by two reports of a higher frequency of oligoamenorrhea and ovarian cysts in pancreatic islet recipients treated with a dual therapy of sirolimus and tacrolimus
Sirolimus-associated adverse events of this sort are usually not assessed in randomized trials, even though normal menstrual cycle recovery after successful organ transplantation is a major benefit and a biological marker of general health, and menstrual cycle abnormalities are a sign of lower fertility and are associated with cardiovascular disease and bone loss.
In our phase II trial, we enrolled patients with early stage autosomal dominant polycystic kidney disease. The disease is characterized by the cystic enlargement of both kidneys while glomerular filtration rate remains preserved up to age 40 in most patients
The sirolimus target mTOR is a key regulatory kinase of cell growth, proliferation and differentiation and is also expressed in non-T-cells, which can lead to unexpected toxicities including the ovaries. Animal studies indicate that the mTOR signaling pathway regulates luteal hormone receptor action
Polycystic ovary syndrome (PCOS) manifests as menstrual cycle disturbances, ovarian cysts, and insulin resistance
Our results should be interpreted in the context of the trial setting. Even though the achieved sirolimus dose was approximately 35% lower than the intended dose, mainly because of dose-limiting gastrointestinal side effects, our findings show evidence of sirolimus-associated ovarian toxicity. Close monitoring is therefore prudent for patients receiving sirolimus and this might help guide clinical use of mTOR inhibitors.
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We thank Marian Struker, Ruth Russi, Charlotte Burger and Adrian Wüthrich for technical support; Janine Suter for trial monitoring (KMS GmbH, Zug); Dr. Natascia Corti, Dr. Gérald Keusch, Dr. Lukas Zimmerli and Dr. Oliver Senn (chair) were members of the data and safety monitoring board. We thank the referring physicians in Switzerland for their support in making this trial possible.