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 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 and 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 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 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 .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 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 (O‡E) red stained areas divided by the sum of all red and and control groups. The O (n ˆ 5) group underwent blue stained areas which represented the ratio of bilateral oophorectomy alone. The O‡E(n ˆ 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 ˆ 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. Labeled estradiol competes with estradiol in was comparable to the control group (P > 0.05; the sample for antibody sites. The quantity of Figure 1). However, peak vaginal blood ¯ow during estradiol in the sample is determined by comparing nerve stimulation-induced vaginal engorgement the counts to a calibration curve. (28.9 Æ 16.3 ml=min=100 g tissue) was signi®cantly less than peak vaginal mucosal blood ¯ow in the control group (P < 0.05). Both basal vaginal mucosal Statistical analysis blood ¯ow in the quiescent state and peak vaginal mucosal blood ¯ow during nerve stimulation in- duced engorgement in the O‡E group (20.4 Æ 8.4 The data is expressed as mean Æ standard deviation and 48.7 Æ 12.2 ml=min=100 g tissue, respectively) of the mean. Paired Student's t-test was utilized to were comparable to those recorded in the control compare changes within the group. ANOVA was group (P > 0.05; Figure 1). utilized for comparison of data among the groups. Statistically signi®cance was determined at P < 0.05.

Results Decreased circulating levels of estrogen and hemodynamic changes during clitoral engorgement

Alterations in the circulating levels of estrogen In the control group, clitoral intracavernosal blood ¯ow signi®cantly increased during nerve stimula- The mean serum level of 17b-estradiol was signi®- tion-induced clitoral engorgement from a basal level cantly decreased in the O group (25.4 Æ 5.1 pg=ml) of 3.8 Æ 1.6 ml=min=100 g tissue to a peak level of compared with the control group (38.5 Æ 7.6 pg=ml; 11.0 Æ 2.4 ml=min=100 g tissue (Figure 2). In the O PK < 0.05). In the O‡E group, however, the mean group, basal clitoral intracavernosal blood ¯ow serum level of 17b-estradiol (115.9 Æ 57.3 pg=ml) (3.5 Æ 0.3 ml=min=100 g tissue) was comparable was signi®cantly greater than the control group to the control group (P > 0.05). However, peak (P < 0.05). clitoral intracavernosal blood ¯ow during nerve stimulation-induced clitoral engorgement (6.1 Æ 1.4 ml=min=100 g tissue) was signi®cantly less than Decreased circulating levels of estrogen and the control group (P < 0.05; Figure 2). In the O‡E hemodynamic changes during vaginal engorgement group, both basal clitoral intracavernosal blood ¯ow (4.7 Æ 1.4 ml=min=100 g tissue) and peak clitoral intracavernosal blood ¯ow during engorgement Oophorectomy alone and oophorectomy followed (10.1 Æ 2.8 ml=min=100 g tissue) were comparable by estrogen replacement therapy did not affect to the control group (P < 0.05; Figure 2).

Table 1 Effect of pelvic nerve stimulation (PNS) among control, bilateral oophorectomy and bilateral oophorectomy animals treated with estrogen groups

Control Bilateral oophorectomy Bilateral oophorectomy treated group group with estrogen group

Before PNS After PNS Before PNS After PNS Before PNS After PNS

Systolic pressure (mmHg) 108 Æ 12.7 112.8 Æ 12.1 109 Æ 11.2 115.3 Æ 13.9 102.5 Æ 10.7 110 Æ 11.2 Diastolic pressure (mmHg) 83.2 Æ 10.2 86.2 Æ 10.3 80.9 Æ 13.1 86.6 Æ 15.7 82.2 Æ 11.2 87.7 Æ 9.3 Vaginal blood ¯ow (ml=min=100 g tissue) 15.0 Æ 6.4 48.9 Æ 6.5* 16.7 Æ 11.8 28.9 Æ 16.3*,** 20.4 Æ 8.4 48.7 Æ 12.2* Clitoral blood ¯ow (ml=min=100 g tissue) 3.8 Æ 1.6 11.0 Æ 2.4* 3.5 Æ 0.3 6.1 Æ 1.4*,** 4.7 Æ 1.4 10.1 Æ 2.8*

Data are presented as mean Æ standard deviation. *Statistically signi®cant (P < 0.05) difference comparing parameters before and after PNS in the same group. **Statistically signi®cant (P < 0.05) difference between control and bilateral oophorectomy group.

International Journal of Impotence Research Estrogen deprivation and pathophysiological changes in the vagina and clitoris KParket al 119

Figure 1 The patterns of nerve stimulation-induced increase of vaginal blood ¯ow in animals of the control (A), O (B) and O‡E (C) groups. The peak vaginal blood ¯ow during vaginal engorgement is signi®cantly decreased in the O group compared with the control and O‡E groups. The peak vaginal blood ¯ow in the O‡E group is comparable to the control group.

Decreased levels of circulating estrogen and prominent venous channels (Figure 3). The histo- structural changes in the vagina and clitoris logy of clitoral cross sections in the control group showed normal expression of clitoral cavernosal Histologically, cross sections of the vaginal wall in smooth muscle and (Figure 4). In the control group showed a normal mucosal layer, the O group, histology of vaginal tissue showed normal distribution of smooth muscle and connec- atrophic changes in the , distortion of the tive tissue, and normal microvasculature with mucosal layer and remarkable decrease in vaginal

International Journal of Impotence Research Estrogen deprivation and pathophysiological changes in the vagina and clitoris K Park et al 120

Figure 2 The patterns of nerve stimulation-induced increase of clitoral intracavernosal blood ¯ow in animals of the control (A), O (B) and O‡E (C) groups. The peak clitoral intracavernosal blood ¯ow during clitoral engorgement is signi®cantly decreased in the O group compared with the control and O‡E groups. The peak clitoral intracavernosal blood ¯ow in the O‡E group is comparable to the control group.

submucosal microvasculatures (Figure 3). The cli- Histomorphometeric image analysis revealed a sig- toral cavernosal tissue in the O group showed ni®cant decrease in the percentage of clitoral diffuse ®brosis and decreased smooth muscle ®bers cavernosal smooth muscle (mean Æ s.d.) in the O compared with the control group (Figure 4). The group (49.66.2) compared with the control overall histology of the vagina and clitoris in the (56.8 Æ 2.6) and O‡E (58.2 Æ 3.0) groups (P < 0.05; O‡E group was comparable to the control group. Figure 5).

International Journal of Impotence Research Estrogen deprivation and pathophysiological changes in the vagina and clitoris KParket al 121

Figure 3 Trichrome staining of vaginal tissue from animals of the control (A), O (B) and O‡E (C) groups. A and C show normal mucosa and normal distribution of smooth muscle and connective tissue and normal microvasculatures. B shows thinned vaginal epithelial layers and decreased vaginal submucosal vasculatures in the oophorectomized animal (Â90).

Figure 4 Trichrome staining of clitoral cavernosal tissue from animals of the control (A), O (B) and O‡E (C) groups. A and C show normal cavernosal trabecular structure and normal distribution of smooth muscle and connective tissue and normal microvasculatures. B shows diffuse ®brosis characterized by decreased smooth muscle and increased bundles of collagen (Â90).

Discussion pause. It has been reported that female sexual arousal function correlates negatively with meno- pausal symptoms such as hot ¯ushes.9 Menopause Decline in the circulating levels of estrogen is a related ovarian atrophy and decreased levels of major physiologic change during natural meno- estrogen have been shown to cause marked decrease

International Journal of Impotence Research Estrogen deprivation and pathophysiological changes in the vagina and clitoris K Park et al 122 tion of the vaginal=clitoral branch of the pelvic nerve resulted in a signi®cant increase in vaginal mucosal blood ¯ow to 48.9 Æ 6.5 ml=min=100 g lead- ing to the development of vaginal engorgement. In our previous studies we utilized a similar technique in the rabbit model and found that vaginal blood ¯ow in the muscular layer of the vaginal wall in the quiescent vaginal state was 5.9 Æ 2.6 ml=min=100 g tissue.7 We also found that blood ¯ow in the muscular layer of the vagina increased to 13.9 Æ 4.5 ml=min=100 g during nerve stimulation- induced vaginal engorgement.7 This would suggest that vaginal blood ¯ow in the mucosal layer of the vagina is much greater compared with blood ¯ow in the muscular layer in the quiescent vaginal state as well as during engorgement. Oophorectomy- Figure 5 Effect of oophorectomy alone (O) and oophorectomy induced decrease in the circulating levels of estro- followed by estrogen replacement therapy (O‡E) on clitoral gen adversely affected vaginal mucosal blood ¯ow. cavernosal smooth muscle and connective tissue content. Histo- The peak vaginal blood ¯ow during nerve stimula- morphometeric image analysis revealed a signi®cant decrease in tion-induced vaginal engorgement signi®cantly de- the percentage of clitoral cavernosal smooth muscle in the O creased in animals in the O group compared with group compared with the control (C) and O‡E groups (*P < 0.05). control group. Vaginal blood ¯ow restoration by estrogen replacement therapy further con®rms that oophorectomy-induced vaginal hemodynamic in vaginal lubrication.2 The reduced estrogen levels changes were related to a decline in the circulating in the menopausal state has also been associated levels of estrogen. The aforementioned decreased with lack of orgasm, delayed orgasm and painful vaginal blood ¯ow appears to be responsible for the intercourse.10 While the relationship between de- development of vaginal engorgement insuf®ciency creased estrogen and the development of sexual in animals in the O group. In addition to vaginal arousal dysfunction has been frequently reported, engorgement insuf®ciency, such a decrease in the impact of estrogen de®ciency on the hemody- vaginal blood ¯ow is likely to produce vaginal namic mechanism of clitoral and vaginal engorge- dryness, or decrease in the amount of vaginal ment has not been investigated previously. lubrication. In the present study we utilized a rabbit model of While the relationship between estrogen levels bilateral oophorectomy to determine the effects of and vaginal blood ¯ow and structure have been decreased levels of circulating estrogen on clitoral investigated to some extent, the effect of decreased and vaginal blood ¯ows before and then during levels of estrogen on the clitoral intracavernosal clitoral and vaginal engorgement. Several studies blood ¯ow and clitoral structure has not been have shown that lack of blood ¯ow alters the reported previously. In a previous study we found structure of a variety of organs including the penis.11 that clitoral engorgement following stimulation of We, therefore, attempted to study whether oophor- the vaginal=clitoral branch of the pelvic nerve ectomy-induced long-term hemodynamic changes depends on the increase of intracavernosal blood in the vagina and clitoris interfere with their ¯ow.7 We also found that clitoral arterial insuf®- structure. To examine prophylactic measures of ciency diminished nerve stimulation-induced cli- oophorectomy-induced functional and structural toral engorgement. In our present study, nerve changes in the vagina and clitoris, we utilized a stimulation signi®cantly increased clitoral intraca- second model that underwent oophorectomy and vernosal blood ¯ow in the control group. In the O received estrogen replacement therapy. These two group, however, nerve stimulation-induced peak models were studied in parallel with an age- clitoral intracavernosal blood ¯ow was signi®cantly matched control group that did not undergo less than the control group, suggesting impairment oophorectomy or estrogen treatment. We found of clitoral engorgement in the O group. Our studies that circulating levels of estrogen were signi®- in the O+E group showed that estrogen replacement cantly decreased after bilateral oophorectomy. therapy following oophorectomy was capable of Subcutaneous injection of estradiol was very preserving peak clitoral intracavernosal blood ¯ow effective in preserving the serum estrogen levels to the levels of the control group. Clinical studies after oophorectomy. have also demonstrated that older women had Hemodynamic studies in the control animals decreased baseline and post-stimulation clitoral showed that the mean vaginal mucosal blood ¯ow arterial blood ¯ow compared to younger women.12 in the quiescent vaginal state, prior to pelvic nerve The precise mechanisms by which aging and stimulation was 15.0 Æ 6.4 ml=min=100 g. Stimula- decreased levels of estrogen interfere with vaginal

International Journal of Impotence Research Estrogen deprivation and pathophysiological changes in the vagina and clitoris KParket al 123 and clitoral intracavernosal blood ¯ows are un- shown that estrogen is a potent inhibitor of the known. One possible mechanism by which de- deposition of extracellular matrix proteins such as creased levels of estrogen impairs vaginal and collagen.18 Although functional receptors for estro- clitoral blood ¯ow may involve the nitric oxide gen exist in both vaginal and clitoral tissues, their role (NO) pathway. It is believed that increased vaginal in the regulation of vaginal and clitoral structure and wall and clitoral intracavernosal blood ¯ows in the function has not been thoroughly investigated.19,20 arousal state results from relaxation of clitoral Our present study, in conjunction with previous cavernosal and vaginal tissues and dilation of reports, suggests that physiologic levels of circulating microvasculature. Our recent studies in the organ estrogen may be of great importance in maintaining bath have shown that relaxation of the rabbit clitoral the normal hemodynamic necessary for vaginal and cavernosal smooth muscle is mediated by nitric clitoral engorgement. Physiologic levels of estrogen oxide NO.13 The vaginal tissue is also capable of also appear to play an important role in maintaining relaxing via the NO pathway.13 However, the the normal structure of the vaginal wall and clitoral primary mediator of the neurogenic relaxation of cavernosal tissue. vaginal tissue is not known. Nitric oxide synthase (NOS) subtypes have been localized in human clitoral cavernosal tissue.14 Hayashi et al reported Conclusion that the vasoprotective effect of estrogen may be mediated by increasing the expression and activity Our studies show that decline in the circulating of endothelial NOS.15 Studies with the rat model levels of estrogen adversely affect the hemodynamic have shown that oophorectomy-induced decrease in mechanism of vaginal and clitoral engorgement in circulating levels of estrogen downregulated NOS the rabbit. Decreased levels of circulating estrogen subtype expression in the vaginal tissue.8 also produced marked changes in the structure of Our histologic studies revealed that oophorec- vaginal and clitoral tissues leading to thinning of the tomy-induced decline in the circulating levels of vaginal epithelial layers, decreased vaginal submu- estrogen caused atrophic changes in the vaginal cosal vasculatures and diffuse clitoral cavernosal mucosal layer, decreased vaginal submucosal micro- ®brosis. These observations suggest that female vasculature and led to loss of clitoral cavernosal sexual arousal dysfunction in the menopausal state smooth muscle. Association of atrophic changes in may result, in part, from structural and functional the mucosa and decreased submucosal microvascu- changes in the vagina and clitoris as a consequence lature in our animal model may suggest the role of of decreased levels of estrogen. decreased arterial in¯ow in the development of structural changes in the vaginal mucosa. While prevention of structural damage by estrogen replace- Acknowledgement ment therapy supports this hypothesis, it also suggests that decreased levels of estrogen may also directly interfere with vaginal structure, possibly by This paper was supported by Fund (CUHRI-U- altering the expression of growth factors. This 98011) of Chonnam University Hospital Research possibility may be further supported by studies of Institute of Clinical Medicine. Hodgins et al demonstrating abundant estrogen receptors in the vaginal epithelium and smooth muscle.16 Previous studies with the rat model have References shown that estrogen withdrawal resulted in overall vaginal atrophy, diffuse intramural collagen accu- 8 1 Sarrel PM. Sexuality and menopause. Obstet Gynecol 1990; mulation and thickening of the perivascular wall. 75(Suppl 4): 26S ± 30S. In our studies with human clitoral cavernosal tissue 2 Sarrel PM. Ovarian hormones and vaginal blood ¯ow: using we found that the percentage clitoral cavernosal laser Doppler velocimetry to measure effects in a clinical trial smooth muscle decreased with age.6 Studies with of post-menopausal women. Int J Impot Res 1998; 10(Suppl 2): the rat model showed that decreased levels of S91 ± S93. 3 Nation B, Maclusky NJ, Leranth CZ. The cellular effects of estrogen lead to apoptosis in nerves, smooth muscle, estrogen on neuroendocrine tissue. J Steroid Biochem 1988; vascular and epithelium of the rat 30: 195 ± 207. vagina.8 4 Laumann EO, Paik A, Rosen RC. Sexual dysfunction in the The precise mechanisms by which decreased United States: prevalence and predictors. JAMA 1999; 281: 537 ± 544. levels of circulating estrogen lead to clitoral and 5 Bachmann GA. Sexual function in the perimenopause. OB vaginal structural changes are unknown. Evidence is Gyn Clin 1993; 20: 379 ± 389. accumulating from vascular tissue research that 6 Tarcan T et al. Histomorphometric analysis of age-related estrogen may directly modulate physiologic smooth structural changes in human clitoral cavernosal tissue. J Urol muscle cell growth.17,18 It has been reported that 1999; 161: 940 ± 944. 7 Park K et al. Vasculogenic female sexual dysfunction: the estrogen inhibits mitogen-induced proliferation of hemodynamic basis for vaginal engorgement insuf®ciency and vascular smooth muscle cells.18 It has also been clitoral erectile insuf®ciency. Int J Impot Res 1997; 9: 27 ± 37.

International Journal of Impotence Research Estrogen deprivation and pathophysiological changes in the vagina and clitoris K Park et al 124 8 Berman JR, McCarthy M, Kyprianou N. Effect of estrogen 15 Hayashi T et al. Effect of estogen on isoforms of nitric oxide withdrawal on nitric oxide synthase xpression and apoptosis synthase; possible mechanism of anti-atherosclerotic effects of in the rat vagina. Urology 1998; 51: 650 ± 656. estrogen. Gerontology 1997; 43(Suppl 1): 24 ± 34. 9 McCoy N, Cutler W, Davidson J. Relationships among sexual 16 Hodgins MB, Spike RC, Mackie RM, MacLean AB. An behavior, hot ¯ashes and hormone levels in perimenopausal immunohistochemical study of androgen, oestrogen and women. Arch Sex Behav 1985; 14: 385 ± 394. progensterone receptors in the and vagina. Br J Obstet 10 Diokno AC, Brown MB, Herzog AR. Sexual function in the G 1998; 105: 216 ± 222. elderly. Arch Intern Med 1990; 150: 197 ± 200. 17 Krasinski K et al. Estradiol accelerates functional endothelial 11 Azadzoi KM, Goldstein I, Siroky MB. Relationship between recovery after arterial injury. Circulation 1997; 95: 1768 ± 1772. cavernosal ischemia and corporal veno-occlusive dysfunction 18 Rosselli M et al. Estradiol inhibits mitogen-induced prolifera- in an animal model. J Urol 1997; 157: 1011 ± 1017. tion and migration of human aortic smooth muscle cells. 12 Werbin T et al. Effect of sexual stimulation and age on genital Circulation 1994; 90: I ± 87 (461). blood ¯ow in women with sexual arousal disorder. J Urol 19 Sadeghi-Nejad H et al. Preliminary report on the development 1999; 161: 178 (688). and characterization of rabbit clitoral smooth muscle cell 13 Azadzoi KM et al. Mechanism of clitoral cavernosal and culture. Int J Impot Res 1998; 10: 165 ± 169. vaginal smooth muscle contractility in the rabbit. J Urol 1999; 20 Chen GD et al. Estrogen receptor alpha and beta expression in 161: 216 (834). the vaginal walls and uterosacral of premenopausal 14 Burnett AL et al. Immunohistochemical description of nitric and postmenopausal women. Fertil Steril 1998; 71: 1099 ± oxide synthase isoforms in human clitoris. J Urol 1997; 158: 1102. 75 ± 78.

International Journal of Impotence Research