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Departments of Obstetrics and Gynecology, and Neurology (M.F., M.M., E.X., F.Q., T.W., M.S.), Columbia University College of Physicians and Surgeons, New York, New York 10032; and Abbott Laboratories (L.K.), Abbott Park, Illinois 60064
Address all correspondence and requests for reprints to: Dr. Michel Ferin, Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032. E-mail: mf8{at}columbia.edu.
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Abstract |
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After a 2-month control, a 12- to 15-month VPA monotherapy was initiated in 7 regularly cycling rhesus monkeys. Overall mean levels of VPA were 88.7 ± 4.0 (SE) µg/ml. Mean body weight increased progressively during VPA treatment from 8.5 ± 0.5 kg before treatment to 9.6 ± 0.7 kg in the last week of treatment (P < 0.05). Monkeys continued to have regular ovulatory menstrual cycles throughout VPA monotherapy. Length of the cycles was 28 ± 0.58 d in control and 28.4 ± 1.18 d in the last 3 months of VPA treatment. Follicular and luteal lengths and peak preovulatory estradiol and integrated luteal progesterone levels did not differ between control and treatment. Ovaries from VPA-treated monkeys showed histological evidence of ovulation, and none had characteristic features of PCOS. Endocrine PCOS markers, such as increased early follicular LH/FSH ratio and androgen levels were not different in control and VPA treatment cycles. LH and 17-hydroxyprogesterone responses to GnRH agonist challenges and the insulin response to glucose tolerance tests were similar in control and VPA groups. Lipid profiles were not affected by VPA treatment. The data indicate that a 12- to 15-month therapeutic exposure to VPA does not induce cyclic hormonal or morphological ovarian abnormalities or characteristics of the PCOS when administered to nonepileptic normally cycling nonhuman primates.
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Introduction |
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VPA is a branched fatty acid that has a broad spectrum of antiepileptic efficacy in the prevention of both partial and generalized seizures (15). Because this drug is an FDA-approved treatment for epilepsy, migraine, and bipolar disease, it is important to determine its potential for inducing reproductive disturbances. For this reason, we have studied the effect of a 12- to 15-month VPA treatment in normally cycling nonhuman primates. We have postulated that if VPA monotherapy were to promote abnormal endocrine and metabolic parameters characteristic of PCOS in these normally cycling rhesus monkeys, changes in cyclic patterns could be readily demonstrated. Alternatively, maintenance of normal reproductive parameters despite this long-term therapy would substantiate the observation that VPA itself does not affect reproductive endocrinology in the normal individual.
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Materials and Methods |
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The experimental protocol was approved by the Animal Care and Use Committee of Columbia University and was performed in accordance with the NIH guide for the care and use of laboratory animals. Ten adult female rhesus monkeys (Macaca mulatta), 11–16 yr of age, were selected for this experiment. The animals were housed in individual cages in temperature- and light-controlled (lights on from 0730–1930 h) rooms. They were fed Purina monkey chow (Ralston-Purina, St. Louis, MO) twice a day and fresh fruit or vegetables. Water was available at all times. The experiments were conducted between September 2000 and February 2002.
In a preliminary step, we investigated whether each animal had normal menstrual cycles. Blood samples were obtained daily by venipuncture (a process to which the animals had previously been habituated) for estradiol and progesterone measurements over a period of two menstrual cycles. Menstruation was determined by daily vaginal swabbing. Seven monkeys that showed two normal menstrual cycles, according to criteria established previously in our laboratory (see Experimental protocol), were then immediately included in the study. Before the study, these animals had a mean (±SE) body weight of 8.5 ± 0.5 kg, excluding one animal (no. 7) who weighted 14.7 kg.
Experimental protocol
The study was designed to investigate potential effects of long-term VPA treatment on cyclic ovarian function. The content of four sprinkle capsules (Depakote Sprinkle capsules, Abbott Laboratories, North Chicago, IL; 125 mg/capsule), mixed with a variety of jams, jellies, and syrups (changed weekly) and coated on various cookies or fruits, was given orally twice a day at 0900 and 1600 h; each animal was closely observed for ingestion. The individual doses as well as total daily dosing of VPA in this monkey study exceeded on a milligram per kilogram basis the maximum daily dose recommended in the human (60 mg/kg). However, because the conversion factor for equieffective individual doses of VPA in the monkey compared with the human has been reported to be three (16), the daily human therapeutic dose (20 mg/kg) was multiplied by a factor of three for an equieffective monkey dose of 60 mg/kg. This dose was administered twice daily for a total dose of 120 mg/kg·d.
VPA blood levels were measured biweekly throughout the experiment at 1100 h, 2 h after morning drug administration. If VPA blood levels were less than 60 µg/ml or if the animal failed to ingest the mixture completely, the mix into which the drug was administered was immediately changed to make it more palatable to that individual animal. The overall course of VPA treatment extended from 12.7–15.7 months.
To determine menstrual cyclicity, menstruation was monitored, and blood samples were obtained daily for the duration of the treatment. Hematocrits were verified at frequent intervals; no animals required supplemental iron therapy. Midcycle peak estradiol levels and daily levels of progesterone during the entire luteal phase were measured in each control and treatment cycle. Follicular and luteal phase lengths were recorded, and the integrated daily progesterone luteal concentrations were calculated. The rise of progesterone above 0.4 ng/ml (coupled to the preceding decrease in estradiol) was used to mark the beginning of the luteal phase. To determine whether endpoints relevant to the development of PCOS were influenced by the experimental therapy, LH/FSH ratios were determined at monthly intervals on d 1 of each control and treatment cycle. Ovarian androgens (androstenedione, total testosterone) were measured in each cycle at their cyclic peak, which paralleled the time of the estradiol peak, whereas the adrenal androgen dehydroepiandrosterone sulfate (DHEAS) was measured on d 2 of the cycle.
At the end of VPA treatment, two tests were performed. For the GnRH agonist stimulation test, Lupron (Abbott Laboratories, 1 mg) was injected sc on d 1–4 of the cycle. Blood samples were obtained at 30-min intervals for the first 4 h, then at 6 h and 22 h after injection. The LH and 17-hydroxyprogesterone responses were measured. The glucose tolerance test was initiated after a 16-h fasting period. Dextrose (Abbott Laboratories; 0.1 g/kg) was given iv on d 2–5 of the follicular phase, and blood samples were taken at +5, 10, 20, and 30 min for glucose and insulin level measurements. For comparison, seven normally cycling monkeys (9.5 ± 0.5 kg) from our colony, tested at the same time of the cycle, served as controls. The lipid profile of each animal was compared in the control period and in the last month of treatment.
After completion of treatment, all animals were ovariectomized. Both ovaries were placed in saline, examined grossly to identify corpora lutea and cystic changes, and photographed. Each ovary was then bisected. Half of the ovary was fixed in a 10% formaldehyde solution. The tissue was then dehydrated in alcohol, embedded in paraffin, sectioned at 5 µm, and mounted serially. The histological sections were stained with hematoxylin-eosin and examined under the microscope. The other half of the ovary was frozen and stored at -80 C for potential future analysis.
Assays and statistical analysis
Serum estradiol, progesterone, testosterone, insulin, and VPA were measured by commercial chemiluminescent immunoassays using the Immulite system (Diagnostic Products Corp., Los Angeles, CA). Intra- and interassay coefficients of variation (CV) were 9.3% and 10.5% for estradiol, 6.6% and 7.9% for progesterone, 7.4% and 9.8% for testosterone, 4.7% and 8.2% for insulin, and 3.8% and 9.5% for VPA, respectively. LH and FSH levels were measured by in-house recombinant homologous RIA (17, 18). Intra- and interassay CV were 7.9% and 13.1% for LH and 5.0% and 6.1% for FSH, respectively. Androstenedione and 17-hydroxyprogesterone were measured by ELISA (Diagnostic Systems Laboratories, Inc., Webster, TX). Intra- and interassay CV were 2.4% and 8.0% for androstenedione and 2.1% and 8.8% for 17-hydroxyprogesterone, respectively. DHEAS was measured by a commercial RIA (Coat-A-Count, Diagnostic Products Corp.), with an intra- and interassay CV of 5.9% and 8.2%, respectively. Glucose levels were measured by a blood glucose testing system (Precision G, Abbott Diagnostic Laboratories, Bedford, MA). Enzymatic tests (Roche Diagnostics, Indianapolis, IN) were used to measure serum total cholesterol, high-density lipoprotein cholesterol after precipitation, low-density lipoprotein cholesterol and triglycerides, the latter after lipase hydrolysis.
Cycle parameters, such as the length of the follicular and luteal phases, hormone concentrations, as well as luteal function as reflected by integrated progesterone values in the luteal phase, were compared in the control, first and last trimester of treatment. For luteal progesterone evaluation, the areas under the luteal phase progesterone curves (from the day of LH surge +1 to the day of menstruation -1) were calculated by trapezoidal analysis. Insulin sensitivity after long-term VPA treatment was evaluated using fasting glucose and insulin levels by the quantitative insulin sensitivity check index method (19) as follows: 1/[log(insulin µU/ml) + log(glucose mg/dl)].
Comparisons were made by multiple ANOVA followed by the Tukey test. Paired Student’s t tests were used for the comparison of body weights before and after VPA treatment. The level of significance was established at P less than 0.05.
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Results |
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Mean (±SE) body weight in six of the seven animals increased progressively during treatment with VPA from 8.5 ± 0.5 kg before treatment began to 8.5 ± 1.0 after 3 months of treatment, 9.2 ± 0.6 after 9 months, and 9.6 ± 0.7 kg in the last week of treatment (P < 0.05 vs. pretreatment). Body weight in monkey 7 was 14.2 kg in the last week of treatment vs. 14.7 kg before treatment.
Menstrual cyclicity
All monkeys, except one, continued to have regular ovulatory menstrual cycles throughout VPA monotherapy. One exception to that pattern was monkey 4JU who, after three regular menstrual cycles during the first 3 months of treatment, had a 102-d amenorrheic period that terminated in a normal ovulatory cycle. After this period, she resumed normal-length cycles. Overall mean (±SE) length of menstrual cycles was 28 ± 0.58 d in control cycles, 28.1 ± 0.54 d in the first 3 months of treatment, and 28.4 ± 1.18 d in the last 3 months of treatment. Mean follicular and luteal length did not differ between the control period and the first and last trimesters of the treatment (Table 1
). Similarly, peak preovulatory estradiol levels and integrated luteal progesterone were similar in all groups (Table 1). One monkey (no. 7) had a significant decrease in integrated luteal progesterone levels during the months of May to September only (33.3 ± 1.5 vs. 48.3 ± 2.3 ng/ml), a phenomenon that occurs spontaneously in about 10–15% of the animals in our colony during the summer months. Early follicular phase LH/FSH ratios were similar in control and treatment cycles (Table 1). Figure 1 illustrates daily follicular estradiol and luteal progesterone levels in successive control (September and October 2000) and treatment cycles (November 2000 to February 2002) in an individual monkey.
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Figure 2 compares mean LH levels on d 2 of the cycle and mean testosterone levels measured on the day of the preovulatory estradiol peak in control cycles and in the first, second, and last trimesters of VPA treatment. No significant difference was observed. Figure 3 compares mean DHEAS and androstenedione levels before and during VPA treatment. Although there was a tendency for the levels to decrease with treatment, the decrease was not significant. Figure 4 compares the mean LH (top left panel) and 17-hydroxyprogesterone (top right panel) responses to a GnRH agonist challenge test in control and VPA-treated monkeys. Although LH was significantly increased 22 h after the agonist administration, there was no difference between the control and VPA-treated groups. No significant differences were observed in regard to 17-hydroxyprogesterone. Figure 4 (bottom) illustrates glucose levels (left panel) and the insulin response (right panel) to a glucose tolerance test. No significant differences were observed between VPA-treated and control groups. Quantitative insulin sensitivity check index values were 0.3057 ± 0.0037 and 0.3050 ± 0.004 in control and VPA-treated animals, respectively, and were not significantly different. Mean (± SE) total cholesterol was 204.9 ± 9.4 and 188.4 ± 10.3 mg/dl before and at the end of VPA treatment [not significant (NS)], respectively; high-density lipoprotein cholesterol was 88.0 ± 6.3 and 88.2 ± 4.3 mg/dl (NS); low-density lipoprotein cholesterol was 100.6 ± 6.2 and 88.1 ± 9.5 mg/dl (NS); and triglycerides were 81.0 ± 22.6 and 93.3 ± 16.5 ng/dl (NS).
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All 14 ovaries from the 7 VPA-treated monkeys were reviewed on gross and microscopic examinations. Ovaries measured from 1.0 x 0.7 x 0.5 cm to 2.2 x 0.8 x 0.7 cm. The characteristic histological features of PCOS in humans (multiple superficial cortical cysts and a region of subcortical stromal fibrosis resulting in the appearance of a white capsule) were not identified in any of the ovaries (Fig. 5). In addition, in humans with PCOS, the central portion of the ovarian cortex usually consists of a homogenous stroma that lacks stigmata of ovulation, such as corpora lutea or corpora albicantia. All 14 ovaries from the VPA-treated monkeys showed histological evidence of ovulation. Table 2 illustrates histological landmarks in each ovary as well as the hormonal background at that time. Recent corpora lutea (RCL) were identified in five of seven monkeys, whereas degenerating corpora lutea (DCL) and/or corpora albicantia were seen in all ovaries. Numerous primordial follicles were present in all ovaries. Developing follicles, including preantral and antral follicles, were identified in all ovaries. Figure 6 shows a section of the whole ovary in monkey 53-124 treated with VPA for 477 d. Note the recently matured corpus luteum and developing follicles at different stages present in the background. No hyperthecosis was observed.
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Discussion |
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Disorders of the menstrual cycle, including menstrual cycle irregularities, ovulatory dysfunction, and amenorrhea, are the most common finding associated with PCOS (21). These have also been described in women with epilepsy, with the highest frequency of anovulatory cycles in women with primary (idiopathic) generalized epilepsy receiving VPA. Hyperandrogenemia has also been consistently described in women with epilepsy receiving VPA for epilepsy as well as bipolar disease (5, 8, 9, 11, 25). However, our data clearly demonstrate that prolonged VPA treatment in normal monkeys with regular menstrual cycles neither induces cyclic irregularities nor results in anovulation or amenorrhea. Regular cycles and appropriate hormone levels were monitored on a daily basis over the entire control and treatment periods, which lasted from 12.7–15.7 months. The estradiol profiles characteristic of normal follicular development, as well as progesterone secretion representative of the normal corpus luteum, remained cyclic and within normal limits.
PCOS is also characterized by major hormonal dysfunction. In a majority of patients, PCOS is accompanied by an increase in GnRH pulse frequency (26, 27), and, because the increased GnRH pulse generator frequency enhances the synthesis of LH over that of FSH (28), PCOS women preferentially secrete LH over FSH and show a typical increased LH/FSH ratio (29). Although experiments have shown that an acceleration of GnRH pulse frequency readily increases the LH/FSH ratio in the monkey (30), no significant increase in this ratio was observed in our animals treated with VPA. Furthermore, the increase in LH after GnRH agonist stimulation was similar in the control and VPA-treated groups, in contrast to the response in women with PCOS where it is enhanced (31). A prime characteristic of PCOS is hyperandrogenism (22). Measurements of testosterone were performed at the time of the preovulatory estradiol peak in each control and treatment cycle, but there were no significant changes between pretreatment and early or late treatment samples. Functional ovarian hyperandrogenism in PCOS women is also characterized by 17-hydroxyprogesterone and androstenedione hyperresponsiveness to GnRH stimulation (32, 33). However, the response in the VPA-treated animals was not different from that in controls. Although not significant, there was a tendency for DHEAS, an androgen produced by the adrenals, to decrease over time during treatment. A similar tendency was seen for androstenedione. Previous reports have indicated a significant decrease in DHEAS within 3 months of VPA treatment in female but, not in male, epileptic patients (34, 35).
Ovarian morphology in PCOS is characterized by cortical thickening, subcapsular follicular cysts, hyperplastic theca interna, and stromal hyperplasia (21). In our animals, the ovaries were removed at the end of the 12.7- to 15.7-month VPA therapy, and no such changes were observed in any of the 14 ovaries that were removed. In fact, all ovaries demonstrated normal cyclic histological features, including antral and mature follicles and active corpora lutea, in accord with the stage of the cycle when they were removed (36). This is notwithstanding previous observations that polycystic ovarian characteristics can readily be induced in the rhesus monkey experimentally, for instance after prenatal androgen treatment (37) or after active immunization to estradiol (38). In contrast to the nonhuman primate, a 3-month VPA treatment in the rat was reported to result in a dose-dependent increase in the number of ovarian follicular cysts and reduction in the number of corpora lutea (39). However, the absence of concomitant increases in LH, androgens, and insulin in these animals suggests a different mechanism of action than that responsible for PCOS and a possible direct effect of VPA at higher doses on the ovary in this species (7, 40). Such a direct effect on the ovary was not observed in our animals.
A side effect of VPA therapy observed in six of our seven animals was a significant increase in body weight, in the presence of normal glucose tolerance tests. This increase became significant after 9 months of VPA therapy. This change in body weight may not be surprising because the use of VPA has been previously reported to induce weight gain (41, 42). The mechanisms leading to weight gain during VPA medication are, however, still unknown. In women with epilepsy, this VPA-related weight gain has been associated with hyperinsulinemia (11).
Although the metabolic fate of VPA is similar in monkeys compared with humans (43), there are several pharmacokinetic differences that need to be taken into account in this study. VPA is eliminated more rapidly in the monkey, by a 10-fold higher metabolic clearance rate (19, 44). However, monkeys have less drug-binding activity (less protein bound) and higher free (active) drug levels compared with the human at similar total plasma concentrations (10–15% in humans vs. 46% in monkeys; Ref. 20). Peak plasma levels were repeatedly measured at 2 h post dose and determined to reach the therapeutic range. However, additional time points throughout the day were not examined. A pharmacokinetic simulation suggested that levels were not likely maintained within the therapeutic range throughout the 24-h period. However, due to the use of equieffective dosing (16) and decreased drug-binding capacity, it is likely that daily exposure to free VPA concentrations in our monkeys was comparable to human daily exposure from therapeutic doses.
In conclusion, our data suggest that, although effects on body weight were observed, a long period of therapeutic exposure to VPA did not induce cyclic hormonal or morphological abnormalities or hormonal characteristics of the PCOS when administered to nonepileptic normally cycling nonhuman primates. These results do not support the hypothesis that VPA treatment per se, at least of this duration, is entirely responsible for the induction of PCOS in some women with epilepsy and bipolar disease. This conclusion is evidently limited by the 12- to 15-month length of treatment with VPA, compared with chronic treatment in humans with epilepsy, and the fact that although VPA levels reached the therapeutic range, these levels were not likely maintained within the therapeutic range throughout each 24-h period. Overall, the data suggest that VPA treatment by itself may not be associated with the development of PCOS, but rather the increased incidence of this syndrome in women with epilepsy and bipolar disease may represent either a result of the disorder or an interaction between a brain disorder and VPA, which predisposes susceptible individuals to this syndrome.
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Acknowledgments |
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Footnotes |
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Abbreviations: CV, Coefficient(s) of variation; DCL, degenerating corpora lutea; DHEAS, dehydroepiandrosterone sulfate; NS, not significant; PCOS, polycystic ovarian syndrome; RCL, recent corpora lutea; VPA, valproate.
Received October 17, 2002.
Accepted February 25, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit secretion in women with hyperandrogenic anovulation. J Clin Endocrinol Metab 77:895–901[Abstract]
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