Improvements of vaginal atrophy without systemic side effects after topical application of Pueraria mirifica, a phytoestrogen-rich herb, in postmenopausal cynomolgus macaques

Abstract

The estrogenic efficacy of topical vaginal application of Pueraria mirifica extract (PM) on the restoration of vaginal atrophy, and the presence of any systemic side effects, were investigated in postmenopausal cynomolgus macaques. Twelve postmenopausal cynomolgus macaques, with complete cessation of menstruation for at least 5 years before start of this experiment, were divided into three groups. They received a topical vaginal application daily of 0.1 or 1% (w/w) PM cream or a conjugated equine estrogen (CEE) cream (a mixture of estrone, equilin, 17β-dihydroequilin, 17α-estradiol and 17α-dihydroequilin at 0.625 mg total estrogen/g cream) for 28 days. Estrogenic efficacy was assessed weekly by vaginal cytology assay and vaginal pH measurement, whilst the plasma luteinizing hormone (LH) and sex skin coloration levels were determined at the end of each treatment period to evaluate the systemic side effects. PM significantly increased the proportion of superficial cells in a dose-dependent manner, with a similar efficacy between 1% (w/w) PM and CEE. Together with increased vaginal maturation, PM decreased the vaginal pH to acidic levels, as observed in the CEE group. PM induced no detected systemic side effects, whilst CEE decreased the plasma LH level and increased the reddish color of the sex skin during the posttreatment period. Topical vaginal treatment with PM stimulated the maturation of the vaginal epithelium without causing systemic side effects in postmenopausal monkeys. The implication is that PM could be a safer alternative to treat vaginal atrophy in postmenopausal women.

Fig. 1.

Approximate location of the four areas of the sex skin around the vagina where the color was measured in each postmenopausal monkey: left side upper ischial callosity (LU), right side upper ischial callosity (RU), left side lower ischial callosity (LW), and right side lower ischial callosity (RW). A, V and the grey areas indicate the anus, vagina and ischial callosities, respectively.

Fig. 2.

HPLC chromatogram of the P. mirifica extract revealing its principal isoflavone contents as puerarin, daidzin, genistin, daidzein and genistein.

Fig. 3.

Vaginal cytology for postmenopausal macaques before and after treatment with 0.1 or 1% (w/w) P. mirifica (PM) or conjugated equine estrogens (CEE) vaginal cream for 28 days. Pretreatment vaginal cytology: a low proportion of superficial cells (S; orange-red-stained cytoplasm), a high proportion of intermediate cells (I; pale blue-stained cytoplasm) and very few parabasal cells (P; a nuclear diameter (arrow) of greater than one-third the diameter of the cell). Posttreatment vaginal cytology: an increased proportion of superficial cells (S) in all treatment groups. Images shown are representative fields of vaginal smears from the four monkeys. 200 × magnification, Papanicolau’s stain.

Fig. 4.

The proportion (%) of vaginal (A) superficial cells and (B) intermediate cells in postmenopausal monkeys topically treated daily with 0.1 or 1% (w/w) P. mirifica (PM) or conjugated equine estrogens (CEE) vaginal cream during the pretreatment (day 0, 7, 14, 21, and 28), treatment (grey area; day 7, 14, 21, and 28) and posttreatment (day 7, 14, 21, and 28) periods. Data are shown as the mean ± 1 SE of the four animals in each treatment. *P< 0.05 vs. pretreatment period.

Fig. 5.

The vaginal pH in postmenopausal monkeys topically treated daily with 0.1 or 1% (w/w) P. mirifica (PM) or conjugated equine estrogen (CEE) vaginal cream during the pretreatment (day 0, 7, 14, 21, and 28), treatment (grey area; day 7, 14, 21, and 28) and posttreatment (day 7, 14, 21, and 28) periods. Data are shown as the mean ± 1 SE of four animals. *P< 0.05 vs. pretreatment period.

Fig. 6.

The levels (A) and (B) change (%) in plasma LH and (C) a* values (the hue of green (–60) to red (+60)) for the sex skin color in postmenopausal monkeys topically treated daily with 0.1 or 1% (w/w) P. mirifica (PM) or conjugated equine estrogen (CEE) vaginal cream at the end of the 28-day pretreatment, treatment and posttreatment periods. The values for sex skin color were expressed only for areas below the ischial callosities. Data are shown as the mean ± 1 SE of four animals. **P< 0.01 vs. pretreatment period. ^†,†† P< 0.05 and 0.01 vs. treatment period, respectively.