International Journal of Impotence Research (2001) 13, 116±124 ß 2001 Nature Publishing Group All rights reserved 0955-9930/01 $15.00 www.nature.com/ijir Decreased circulating levels of estrogen alter vaginal and clitoral blood ¯ow and structure in the rabbit KPark1*, K Ahn2, S Lee2, S Ryu1, Y Park1 and KM Azadzoi3 1Department of Urology, Chonnam National University Medical School, Kwangju, Korea; 2Department of Anatomy, Chonnam National University Medical School, Kwangju, Korea; and 3Departments of Urology, Boston University School of Medicine, Boston, Massachusetts, USA Aging and menopause related decline in circulating levels of estrogen has been shown to adversely affect female sexual arousal function. Our aim was to study the effects of circulating levels of estrogen on the hemodynamic mechanism of vaginal and clitoral engorgement and on the structure of the vaginal and clitoral cavernosal tissue in the rabbit. New Zealand White female rabbits (3.5 ± 4 kg) were randomly divided into three groups with ®ve rabbits in each group: control; bilateral oophorectomy; bilateral oophorectomy undergoing subcutaneous injection of estrogen (40 mg=kg=day). After 6 weeks, the serum levels of 17 b-estradiol were measured and systemic blood pressure was monitored. Vaginal and clitoral cavernosal blood ¯ows were measured with laser Doppler ¯owmeter before and after pelvic nerve stimulation. Cross sections of the clitoris and vagina were processed for histologic examination and histomorphometric image analysis. Serum level of 17 b-estradiol (pg=ml; mean Æ s.d.) revealed a signi®cant decrease in the oophorectomy group (25.4 Æ 5.1) compared with the control (38.5 Æ 7.6) and estrogen replacement (115.9 Æ 57.3) groups (P < 0.05). Nerve stimulation-induced peak vaginal and clitoral intracaver- nosal blood ¯ows in the oophorectomy group (28.9 Æ 16.3 and 6.1 Æ 1.4, respectively) were signi®cantly less than those recorded in the control (48.9 Æ 6.5 and 11.0 Æ 2.4, respectively) or estrogen replacement (48.7 Æ 12.2 and 10.1 Æ 2.8, respectively) group (P < 0.05). In histology, marked thinning of the vaginal epithelial layers, decreased vaginal submucosal microvasculature, and diffuse clitoral cavernosal ®brosis were evident in the oophorectomy group but not in the estrogen supplement and control groups. In histomorphometry, the percentage of clitoral cavernosal smooth muscle in the oophorectomy group (49.6 Æ 6.2) was signi®cantly decreased compared with the control (56.8 Æ 2.6) and estrogen replacement (58 Æ 3.0) groups (P < 0.05). Our studies show that decline in circulating levels of estrogen impairs the hemodynamic mechanism of vaginal and clitoral engorgement and leads to histopathologic changes in the vagina and clitoral cavernosal tissue. These observations suggest that decreased circulating levels of estrogen, a physiologic change in the menopausal state, may play a role in the development of female sexual arousal dysfunction. International Journal of Impotence Research (2001) 13, 116±124. Keywords: estrogen; vagina; clitoris; ischemia; ®brosis Introduction the vagina, clitoris and urethra has been well documented.1,2 It has been shown that estrogen replacement therapy in post-menopausal women The role of estrogen in the regulation of female restores clitoral and vaginal vibration and pressure sexual arousal function has been frequently re- threshold to the levels of pre-menopausal women.1 ported.1±3 Estrogens are known to be vasoprotective Studies with animal models have shown that and regulate vascular smooth muscle tone.1,2 In the treatment with estradiol increases the touch recep- clinical studies, correlation between circulating tor zone along the pudendal nerve and improves levels of estrogen and the status of blood ¯ow in sensory threshold.3 It was recently reported that approximately 43% *Correspondence: K Park, Department of Urology, Chonnam of women complain about sexual dissatisfaction.4 National University Medical School, 8, Hakdong Dongku, The major complaints include loss of desire, painful Kwangju #501-747, Korea. E-mail: [email protected] intercourse, dif®culty achieving orgasm and de- Received 7 June 2000; revised 16 October 2000; creased frequency of sexual activity.4 Several stu- accepted 4 December 2000 dies have shown that female sexual arousal Estrogen deprivation and pathophysiological changes in the vagina and clitoris KParket al 117 disorders increase with age and menopause and venous injections of pentobarbital (25 mg=kg) as correlate with the decline in circulating levels of needed. A 20-gauge angiocatheter was placed into estrogen.1,2,5 Our studies with human clitoris the femoral artery for measurement of arterial blood showed that aging women undergo histological pressure. A midline abdominal incision was made changes in the clitoral erectile tissue.6 Clinical and the vaginal=clitoral branch of the pelvic nerve studies have shown that decrease in vaginal blood was exposed. The clitoris and corresponding cor- ¯ow and diminished vaginal secretion during poral bodies were exposed via a dorsal midline menopause signi®cantly correlates with the decline incision. A Harvard subminiature electrode was in circulating ovarian hormones.2 It is also reported placed around the vaginal=clitoral branch of the that estrogen replacement therapy enhances pelvic pelvic nerve. Unilateral nerve stimulation was blood ¯ow in post-menopausal women.2 performed at 10 V, 8 msec and 16 Hz delivered for In our previous studies with the rabbit model, we 20 sec. found that the development of vaginal and clitoral engorgement depends on an adequate increase in the vaginal wall and clitoral intracavernosal blood ¯ows.7 We also found that occlusive disease of the Measurement of vaginal and clitoral intracavernosal iliac and pudendal arteries reduced arterial in¯ow blood ¯ows to the vagina and clitoris and impaired vaginal and clitoral engorgement.7 Studies with a rat model showed that estrogen withdrawal resulted in vaginal Vaginal mucosal blood ¯ow was measured by a atrophy, intramural collagen accumulation and surface type laser Doppler ¯ow probe placed on the structural changes in vaginal wall microvascula- vaginal mucosa 2 ± 3 cm proximal to the vaginal introitus. Clitoral intracavernosal blood ¯ow was ture.8 These structural changes were shown to measured with a needle type laser Doppler ¯ow diminish by estrogen replacement therapy.8 Despite evidence of female sexual arousal dysfunction in probe placed directly into the cavernosal tissue. The conjunction with hormonal changes during meno- ¯ow probes were connected to a dual channel laser pause, the precise mechanisms by which sex Doppler ¯owmeter (Transonic Systems, Inc.) which hormone insuf®ciency interfere with female sexual was calibrated against an internal standard reading arousal function are unknown. ¯ow in units of ml=min=100 g of tissue. Our aim was to study the mechanisms by which decreased levels of circulating estrogen impair the hemodynamics of vaginal and clitoral engorgement Histology and histomorphometric image analysis in the rabbit. We also examined whether decreased circulating levels of estrogen lead to structural damage in the vaginal wall and clitoral cavernosal The lower third of vaginal tissue was dissected from tissues. Another aim was to determine whether both lateral walls. The clitoris was removed and estrogen replacement therapy could prevent oophor- the corpus cavernosum tissue was dissected. The ectomy-induced hemodynamic and structural tissues were placed in phosphate buffered parafor- changes in the vaginal and clitoral cavernosal maldehyde for 3 h and then processed for washing tissues. and dehydration. The tissues were routinely em- bedded in paraf®n and 5 mm sections were prepared. Tissues were stained with hematoxylin and eosin or Materials and methods Masson's trichrome. Percentage of smooth muscle in clitoral cavernosal tissue was assessed by computer- assisted histomorphometric image analysis of Mas- Experimental model son's trichrome stained slides. Fifteen to twenty different high power ®elds (Â200 magni®cation) were randomly selected and analyzed for each The New Zealand White female rabbits (3.5 ± 4 kg) specimen. The percentage of smooth muscle was were randomly divided into oophorectomy (O), calculated for every high power ®eld as the sum of oophorectomy with estrogen replacement (OE) red stained areas divided by the sum of all red and and control groups. The O (n 5) group underwent blue stained areas which represented the ratio of bilateral oophorectomy alone. The OE(n 5) smooth muscle:total cavernosal tissue. During high group underwent bilateral oophorectomy followed power ®eld selection, care was taken not to include by treatment with subcutaneous estradiol on a daily clitoral tunica albuginea, intracavernosal septum or basis (40 mg=kg). The control group (n 5) did not cavernosal artery or vein in the examined area. undergo oophorectomy or estrogen treatment. Because of the unique histology of the vaginal After 6 weeks, the animals were anesthetized tissue, histomorphometry of the vaginal wall was with intramuscular injections of ketamine not possible with our existing image analysis (50 mg=kg). Anesthesia was maintained with intra- system. International Journal of Impotence Research Estrogen deprivation and pathophysiological changes in the vagina and clitoris K Park et al 118 Measurement of circulating levels of estrogen systemic arterial blood pressure (Table 1). In the control group, basal vaginal mucosal blood ¯ow in the quiescent state was 15.0 Æ 6.4 ml=min=100 g Before hemodynamic studies, a blood sample (2 ml) tissue. During nerve stimulation-induced vaginal was obtained from the femoral artery. The serum engorgement, vaginal mucosal blood ¯ow signi®- concentration of 17b-estradiol was determined by cantly increased to a peak level of 48.9 Æ radioimmunoassay using Coat-A-Count Estradiol kit 6.5 ml=min=100 g tissue (P < 0.05; Figure 1). In the (Diagnostic Products Co, CA). The Coat-A-Count O group, basal vaginal mucosal blood ¯ow in the Estradiol procedure is based on antibody-coated quiescent state (16.7 Æ 11.8 ml=min=100 g tissue) tubes.