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 
. In two multi-center placebo controlled phase III trials, 180 and 149 female kidney allograft recipients were given sirolimus for 12 and 6 months respectively and there were no reports of menstrual cycle disturbance or ovarian cysts among these patients 
. This discrepancy is likely explained by the systematic and prospective assessment for menstrual cycle and ovarian cyst formation in our study which was not done in these large multicenter studies.
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 
. Cure and colleagues reported a higher frequency of oligoamenorrhea after transplantation and the occurrence of ovarian cysts in 8 of 13 patients, while serum follicle and luteinizing hormone levels remained in the normal range 
. Alfadhli and colleagues reported on the occurrence of ovarian cysts in 31 of 44 premenopausal patients 8 months after transplantation and then after sirolimus withdrawal, a cyst size reduction in 80% of these 31 patients 
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 clinical practice, sirolimus is often given simultaneously with other immunosuppressants making it difficult to reliably link observed ovarian toxicity with a single immunosuppressive agent.
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 
. There is no evidence that ADPKD affects the menstrual cycle or fertility in woman with normal renal function 
. Cysts can also occur infrequently in organs other than the kidneys;
however, the frequency of ovarian cysts, ovarian size and follicle dimensions are no different in women with ADPKD than in a normal population 
. Our cyst measurements may have included normal pre-ovulatory follicles as false positives. However our analyses show that sirolimus increased the risk of both ovarian cysts and menstrual cycle disturbances in patients receiving sirolimus and these differences between randomized groups suggest that the cysts detected in patients treated with sirolimus were probably not normal.
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 
, the follicle-stimulating hormone signaling pathway 
, granulosa 
and luteal 
cell proliferation and control of primordial oocyte dormancy 
. Shivaswamy and colleagues reported an increased frequency of abnormal cycles and hyperinsulinemia in Sprague-Dawley rats receiving daily 2 milligrams sirolimus subcutaneously for 4 weeks, but a similar frequency of abnormal ovarian follicles in the their sirolimus and control groups 
. We also observed an increased frequency of abnormal cycles in Wistar rats receiving sirolimus for 3 weeks, and a similar number and morphology of ovarian follicles in our sirolimus and control groups.
Polycystic ovary syndrome (PCOS) manifests as menstrual cycle disturbances, ovarian cysts, and insulin resistance 
. Sirolimus causes insulin resistance in rodents 
and in humans 
and we observed cycle disturbance and ovarian cysts in our ADPKD patients when treated with sirolimus, suggesting sirolimus promotes a polycystic ovary like syndrome. In our animal study, sirolimus activated Akt in ovaries. Polymorphisms of the Akt gene were associated with an increased risk for the occurrence of PCOS in epidemiological studies 
and an increase in the insulin-induced IRS/PI 3-kinase/Akt pathway in rat ovaries caused a PCOS like phenotype 
, suggesting this pathway has a role in the development of ovarian cysts.
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.