Results From a Prospective, Randomized, Placebo-Controlled Clinical Trial
Objective To evaluate effects of simvastatin on selected biochemical parameters and reproductive outcome among patients with polycystic ovary syndrome (PCOS) who undergo in vitro fertilization (IVF).
Methods Patients with PCOS were randomized to receive either oral simvastatin, 20 mg/d (n = 32), or placebo (n = 32) in a prospective, double-blind, randomized clinical trial (NCT 005-75601) in parallel with controlled ovarian hyperstimulation for IVF. All patients were determined to be at average risk for cardiovascular disease, based on high-sensitivity C-reactive protein (hsCRP) measurement at entry. After an 8-week treatment interval concluding at periovulatory human chorionic gonadotropin administration, selected clinical and laboratory parameters were measured.
Results Mean serum total testosterone level decreased by 25% in the simvastatin group, compared to a 10% reduction in the placebo group (P < 0.001). A trend of lower serum luteinizing hormone levels was noted in experimental and control groups (29% vs 22%, respectively), although this difference was not significant (P > 0.05). Neither fasting insulin nor quantitative insulin sensitivity check index were significantly impacted by simvastatin (P > 0.05). As expected, total cholesterol was not modified among placebo patients but was significantly reduced after simvastatin (P = 0.001). In addition, hsCRP and vascular cell adhesion protein-1 were both significantly lower after simvastatin therapy compared to controls (P ≤ 0.005 for both). At study completion, no important change in body mass index was observed in either group (P ≥ 0.60). Although oocyte maturation, fertilization, and clinical pregnancy rates were all higher after simvastatin, none of these improvements were statistically significant.
Conclusions This report presents data from the first prospective, randomized, placebo-controlled clinical investigation of simvastatin in the setting of PCOS and IVF. Simvastatin seems to be compatible with gonadotropin therapy for IVF and can offer beneficial endocrine and cardiovascular effects for patients with PCOS who undergo embryo transfer. Although the observed improvements in reproductive function were mild, the reductions in hsCRP and vascular cell adhesion protein-1 after simvastatin treatment were significant, suggesting the need for further clinical trials to clarify simvastatin's impact on reproductive physiology.
- polycystic ovary syndrome
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Polycystic ovary syndrome (PCOS) is a common multisystem disorder associated with abnormal (or absent) ovulation, affecting up to 10% of reproductive age females.1-4Although the specific cause of PCOS remains poorly understood, a broad range of endocrine, inflammatory, and metabolic derangements have been recognized. Specifically, PCOS is frequently associated with insulin resistance, systemic inflammation, and oxidative stress, which result in endothelial dysfunction. For many women with PCOS, these consequences are multiplied by chronically elevated serum androgens leading to dyslipidemia and cardiovascular sequelae.5For in vitro fertilization (IVF) patients with PCOS, it is not unusual to observe a high cycle cancellation rate due to ovarian hyper-response, as well as large numbers of oocytes retrieved with relatively poor fertilization.6
Dyslipidemia is one of the most common metabolic abnormalities among women with PCOS, with a prevalence approaching 70%.7-9Statins are a pharmacologic class of agents that selectively inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway.5Because reduced serum testosterone and improvement of hirsutism have been reported after simvastatin treatment of patients with PCOS,10-12this has evoked some speculation as to how this medication might affect ovarian function. To date, statins have not been systematically studied in an assisted reproductive context, however. By directly inhibiting cholesterol production, simvastatin alters the downstream availability of testosterone, resulting in markedly reduced ovarian androgens. In addition, simvastatin may attenuate intracellular insulin and insulin-like growth factor-I signaling in the ovary13as well as reverse theca hyperplasia and diminished steroidogenic enzyme production. From these earlier investigations, we hypothesized that incorporation of simvastatin into an IVF regime for patients with PCOS would yield desirable metabolic effects, translating to improved oocyte quality, and potentially improve reproductive outcome after embryo transfer.14,15
Against this background, the dyslipidemia observed in patients with PCOS suggested a worthwhile role for statin therapy.16Unfortunately, the use of this class of medication in females of reproductive age, and particularly, the inclusion of statins into an assisted reproduction sequence, presents a special challenge, owing to its pregnancy contraindication. Unlike oral contraceptive pills, which render the risk of unplanned pregnancy very low, patients with PCOS treated with statins might still establish a pregnancy, and any possible teratogenic consequences secondary to statin use would be unacceptable. Accordingly, a novel study design was explored where statin administration was incorporated with IVF but limited to an 8-week interval terminating with periovulatory human chorionic gonadotropin (hCG) administration. This effort represents the first known randomized, double-blind, placebo-controlled clinical trial to evaluate simvastatin during IVF.
MATERIALS AND METHODS
Patient Selection and Study Design
After approval by the institutional review board at the Tehran University Faculty of Medicine, subjects (n = 64) were enrolled in a prospective, double-blind, randomized, placebo-controlled clinical trial [NCT 005-75601]. Written informed consent was obtained from all study patients. Criteria to enter the study included female patient aged between 18 and 40 years, PCOS diagnosis confirmed by meeting 2004 Rotterdam criteria,17and currently undergoing IVF + ICSI for a male factor indication. Exclusion criteria were cycle day 3 serum follicle stimulating hormone (FSH) greater than 10 mIU/L, stage III or IV endometriosis, congenital adrenal hyperplasia, thyroid disease, Cushing syndrome, or hyperprolactinemia. Commercially available immunoassay kits were used to measure serum luteinizing hormone and FSH. Serum dehydroepiandrosterone sulfate (DHEAS), testosterone, and prolactin levels were determined via chemiluminescence assay (DiaSorin; Turin, Italy). Sex hormone-binding globulin was measured by enzyme-linked immunosorbent assay (ELISA; DRG, Marburg, Germany). In addition, fasting plasma glucose and fasting insulin were recorded, and a standard 75-g oral glucose load was used to perform a glucose tolerance test with calculation of area under the curve for insulin and glucose. Insulin sensitivity was estimated via quantitative insulin-sensitivity check index.18A photometry assay (Pars Azmon, Tehran, Iran) was used to measure serum high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, and a high-sensitivity C-reactive protein ELISA kit (Diazyme Laboratories, Poway, CA) was used to record high-sensitivity C-reactive protein (hsCRP). Serum vascular cell adhesion protein-1 (VCAM-1) was measured via ELISA kit (Abcam, Cambridge, MA). Intra-assay and interassay coefficients of variation were less than 10% for all evaluations performed. Study patients did not receive oral contraceptives, steroids, or other medications affecting ovarian function, insulin sensitivity, or lipid metabolism for a minimum of 3 months before the investigation. Blood samples were uniformly obtained between 7:00 and 8:00 am after a 12-hour fast. Specimens were taken in the follicular phase of spontaneous menses or after oral medroxyprogesterone acetate. Randomization was by sequentially numbered opaque, sealed envelope methodology; envelopes were opened sequentially after participant (assignment) details were written on the envelope. Aluminum foil was used inside envelopes to shield contents from intense light. In this noncrossover study, allocation was blinded both for patients and clinic staff. Group A received 20 mg oral simvastatin (n = 32), whereas group B received placebo (n = 29). Both groups received study medication for 8 weeks before initiating their IVF treatment sequence.
Controlled Ovarian Hyperstimulation Sequence
Both patient groups underwent pituitary down-regulation followed by ovulation induction via daily injection of 150 IU recombinant FSH (Gonal-F, Serono, Bari, Italy), which was initiated on cycle day 3. Periovulatory hCG was administered when serum estradiol levels exceeded 500 pg/mL and if there were at least 2 follicles with mean diameter 18 mm or greater. Both simvastatin and placebo were discontinued on the day of hCG injection. Transvaginal ultrasound-guided oocyte retrieval was performed 36 hours after hCG administration, and transfer of 1 to 2 embryos was carried out 2 days after oocyte retrieval. Cyclogest, 400 mg (Actavis Pharmaceuticals, Barnstaple, UK) twice daily, was used for postembryo transfer luteal support, beginning the day after oocyte retrieval and continuing until the day of clinical pregnancy assessment. Clinical pregnancy was defined as at least one intrauterine gestational sac with fetal cardiac activity documented by transvaginal ultrasound approximately 5 weeks after embryo transfer. Supplementary progesterone was discontinued when a negative hCG test was registered. Repeat endocrine and metabolic evaluations were obtained 8 weeks after embryo transfer.
Assuming simvastatin results in a 10% improvement in the number of metaphase II (MII, "mature") oocytes, a sample of 30 in each arm was calculated to yield a significance of 0.05 with power of 0.8 (Sigmastat/Jandel Scientific; San Rafael, CA). The primary outcome was improvement in metaphase II oocytes, and the secondary outcome was decrease in testosterone and inflammatory markers. Comparisons were carried out according to intention to treat by Mann-Whitney, χ2, or Fisher exact test, as appropriate. Statistical analysis was performed using SPSS Version 12.0 (IBM; Chicago, IL). A P <0.05 was considered statistically significant.
Sixty-four IVF patients were enrolled and randomized in this investigation, but 3 subjects did not complete IVF for personal reasons. Of the remaining 61 women, 32 were assigned to group A (simvastatin), and 29 women entered group B (placebo) as shown in Figure 1. Patients in both IVF groups received the same cumulative gonadotropin dose (1500 IU). Pretreatment and posttreatment comparisons for both groups are summarized in Table 1. The median number of retrieved oocytes during IVF was similar for both groups (12.3 ± 6.96 in group A vs 8.8 ± 4.5 in group B; P = 0.066). As depicted in Table 2, the median number (and proportion) of MII oocytes observed in the 2 groups was not significantly different (8.42 ± 5.1 [76.13%] in group A vs 6.71 ± 3.7 [68.2%] in group B; P = 0.296). The 2 pronuclear zygote fertilization rates for patients in groups A and B were similar (74.1% ± 17.25% for patients receiving statin vs 64.9% ± 24.1% for patients receiving placebo; P = 0.128). The clinical pregnancy rate was somewhat higher for patients in the statin group (28% vs 21% for patients receiving placebo), although this difference was not statistically significant (P = 0.25). The observed incidence of mild or moderate ovarian hyperstimulation syndrome was similar in both groups (0.66% in statin group vs 0.77% in placebo group; P = 0.65). No appreciable change in body mass index was noted in either group.
During this study, significant decreases in serum (total) testosterone were observed in both groups, with an average reduction of 25% among statin patients and an average reduction of 10% in the placebo group (P = 0.001, for both). Of note, the difference in serum testosterone reduction between the 2 groups was highly significant (P = 0.0001). Likewise posttreatment serum DHEAS was lower in both groups, but a mean DHEAS reduction of 30% was noted in statin patients versus 44% in placebo patients; P = 0.03. Assessment of pituitary hormones revealed a significant decline of serum luteinizing hormone (LH) but a limited effect on serum FSH. Specifically, patients in the statin group demonstrated an average serum LH decline of 29% and a 30% reduction of the LH/FSH ratio. As summarized in Table 2, these effects were significantly greater than those observed in the placebo group. Total serum cholesterol was reduced by an average of 24% in group A (P = 0.001), but a mean cholesterol reduction of only 1% was observed among group B (control) patients (P > 0.05). In group A, mean serum LDL levels decreased by 15% (P = 0.001), but among patients in the placebo group (group B), LDL cholesterol increased by an average of 8% (P = 0.91). In this study, serum HDL levels increased by an average of 15% and 14% in the statin and placebo groups, respectively (P = 0.98, for difference between groups). Simvastatin did not significantly impact fasting insulin or quantitative insulin sensitivity check index marker compared to placebo control during this investigation. In contrast, simvastatin was observed to have a significant beneficial effect on systemic inflammatory markers, as statin therapy was noted to result in a 58% decline in hsCRP compared to a mean 28% reduction in hsCRP among patients in the placebo group (P = 0.0001). Similarly, we measured mean decreases in serum VCAM-1 of 18% and 7% in the statin and placebo groups, respectively (P = 0.004). All patients tolerated study medications well; there were no discontinuations due to adverse effects.
These data summarize the first prospective, randomized, placebo-controlled clinical trial of simvastatin in women with PCOS who undergo IVF. From this investigation, simvastatin seems to be compatible with gonadotropin therapy with beneficial endocrine and cardiovascular effects for these patients. Whereas the observed improvements in reproductive function were mild, the reductions in hsCRP and VCAM-1 after simvastatin treatment were nevertheless significant. Whereas strategies to reduce androgens suppress follicular development and/or ovulation, and attenuate insulin insensitivity have influenced PCOS treatments,19the present work opens a new potential therapeutic approach for patients with PCOS who contemplate IVF.
Although patients with PCOS undergoing IVF typically produce increased numbers of oocytes for retrieval, these often are of poor quality and associated with impaired fertilization and implantation. Several authors have speculated on potential metabolic mechanisms leading to increased miscarriages among IVF patients with PCOS.20-22It has been postulated that LH hypersecretion, elevated androgens, and/or relative insulin excess may negatively affect the granulosa cell-oocyte interaction, oocyte maturation, and potential embryonic developmental competence, thus contributing to poor outcomes for patients with PCOS undergoing assisted reproduction.23Prior research in a non-IVF setting has demonstrated that simvastatin can decrease serum testosterone, serum LH, and the LH/FSH ratio.24-26These corrections would be beneficial for women with PCOS who require IVF because high testosterone and the "inverted" LH/FSH ratio are considered hallmarks of hypothalamic-pituitary-ovary dysfunction often seen in PCOS. The capacity of simvastatin to attenuate serum testosterone derives from its mevalonate pathway inhibition, which reduces testosterone from decreased upstream availability of cholesterol (a necessary substrate for androgen production) as well as suppression of the theca compartment in the ovary.27,28Statins may also modulate serum LH by hypothalamic and/or pituitary effects. For example, studies of rat pituitary tumor have shown that statins can influence plasma membrane Gs and Gi proteins as well as adenyl cyclase activity.29With respect to overall reproductive outcome, an improved clinical pregnancy rate among patients who received simvastatin was noted in this study, but this increase was too limited to reach statistical significance. We also observed a small but insignificant improvement in MII oocyte yield after simvastatin treatment compared to placebo (76.1% vs 68.2%; P > 0.05). Additional research is needed to validate the impact of statin-mediated serum lipid improvements on the reproductive outcome of patients with PCOS in IVF.
Not surprisingly, simvastatin in tandem with gonadotropin treatment for IVF produced more pronounced effects on serum lipids in our study, as significant reductions in total serum cholesterol and LDL cholesterol were noted among patients with PCOS receiving simvastatin. In particular, we recorded a significant increase in HDL cholesterol after statin therapy, although no important change was noted in serum triglyceride levels. It should be noted that improvements in HDL (and DHEAS) were also seen among the control patients, and this finding requires further clarification. Improvement in serum HDL is especially beneficial for patients with PCOS in whom dyslipidemia and other cardiovascular risk factors have special relevance. High-density lipoprotein is believed to play a key role in human oocyte health, influencing embryo fragmentation and embryo cell number.30It has been hypothesized that oocytes from women with PCOS could have lower numbers of meiotic spindles compared to controls because of impairments in antioxidant defense in follicular fluid.23This would seem to agree with earlier observations that have placed HDL as the sole lipoprotein present in follicular fluid, where the membrane is permeable to serum proteins up to 300 kd but excludes LDL, very low-density lipoprotein, and larger HDL2s.31,32Serum HDL correlates well with HDL present in follicular fluid, and HDL particle concentration has been shown to be negatively associated with embryo fragmentation score on postfertilization day 3.30
Evidence is also accumulating that endothelial dysfunction and low-grade chronic inflammation are important components of PCOS.33Recent work has suggested that hsCRP is a useful marker for adverse cardiovascular events in women,29,34and in our investigation, all study patients had baseline hsCRP levels consistent with "average" cardiovascular disease risk (where <1 mg/L = low risk, 1-3 mg = average risk, and >3 mg/L = high risk).35The endothelial dysfunction seen in PCOS coexists with (and seems to be influenced by) VCAM-1.36Vascular cell adhesion protein-1 is expressed by vascular endothelium under proinflammatory conditions and plays a key role in the pathogenesis of atherosclerosis by mediating adhesion of activated leukocytes to the vessel wall.37Our data confirmed a significant decrease in mean hsCRP levels among the patients with PCOS who received simvastatin during IVF while also producing pronounced decreases in soluble VCAM-1 levels. These observed lower VCAM-1 levels may be more germane to circulatory and/or perfusion parameters rather than blastocyst-endometrial interactions, because VCAM-1 is not absolutely required for embryo implantation.38Instead, the VCAM-1 attenuation noted in our study patients would lead to reduced migration and adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium.
The physiologic impact of PCOS on reproductive outcome after IVF remains the target of active investigation. One meta-analysis of clinical IVF studies reported that, although a significantly higher number of oocytes were recovered from the patients with PCOS, the number of good-quality embryos available for transfer was not significantly different between controls and the subjects with PCOS.6Other investigators have found a significantly lower number of mature oocytes and reduced fertilization rate in patients with PCOS compared to controls.39Yet, others have reported comparable numbers of MII oocytes harvested at retrieval, similar fertilization rates, and comparable embryo development when women with PCOS were compared against controls undergoing intracytoplasmic sperm injection.40
Because studies of statins and reproductive physiology are rare, data from the current investigation are particularly useful. Although the current classification of statins as contraindicated in pregnancy41can raise substantial barriers to research in this arena, there are precedents in routine clinical IVF practice for similar short-term use of other "category X" medications, with no reported adverse maternal/fetal health consequences. For example, pituitary desensitization with a gonadotropin-releasing hormone agonist is common before initiating ovulation induction with gonadotropins, yet both groups of pharmaceuticals are classified as contraindicated in pregnancy. Indeed, the terminal elimination half-life of simvastatin is comparable to leuprolide,42and the latter agent is routinely administered alongside gonadotropins during the follicular recruitment phase in IVF. Considering the fact that we confined statin dosing only to the period up to periovulatory hCG administration (ie, no gestational exposure), the actual bioactivity of simvastatin at or near the time of blastocyst nidation was likely negligible. These data provide helpful evidence regarding a beneficial role of simvastatin on cardiovascular risk factors in this young but at-risk population of patients with PCOS undergoing IVF. How simvastatin might modify lipid parameters and/or markers of vascular inflammation among heavier, older patients with PCOS at even higher baseline risk for cardiovascular disease awaits future study. Our findings align with previous research on how simvastatin affects serum testosterone in women with PCOS and show how simvastatin can be used with IVF to attenuate inflammatory markers that predict cardiovascular sequela. Further prospective, randomized, double-blind, placebo-controlled clinical trials of simvastatin in IVF are needed to confirm these initial findings.