Women's Sexual Function and Dysfunction. Irwin Goldstein MD

Animal models in the investigation of female sexual function and dysfunction

Kwangsung Park

Introduction

Female sexuality has many components, including physiologic, psychologic, social, and emotional factors. Female sexual dysfunction is an important health issue that affects the quality of life of many women. There have been, however, a limited number of investigations studying the physiology of female sexual function and dysfunction, primarily because of a lack of reliable experimental models and tools. An historical difficulty was the notion that female sexual dysfunction consists primarily of problems related to orgasm, and thus the only appropriate model for study was the human.1 Recently, several experimental models have been introduced to assess genital hemodynamics,2,3 genital smooth muscle contractility,4,5 vaginal lubrication,6,7 and the pharmacology of female genital sexual response.8,9

Female sexual dysfunction is defined as a disorder of sexual desire, arousal, orgasm, and/or sexual pain, and it is estimated to affect 20-50% of women.10-12 The prevalence of female sexual dysfunction has been shown to increase with age and be associated with the presence of vascular risk factors (e.g. diabetes mellitus, atherosclerosis) and the development of menopause.13 In addition, traumatic injury to the iliohypogastric/pudendal arterial bed from pelvic fractures or blunt trauma can result in sexual dysfunction.14-16 Investigation of female sexual dysfunction requires establishment of appropriate animal models to elucidate the pathophysiologic mechanisms. Recently, several animal models have been introduced to evaluate sexual dysfunction associated with menopause, atherosclerosis, and diabetes mellitus. This chapter will review animal models that help to elucidate female sexual function and dysfunction.

Models for female sexual arousal response

In vivo studies

Female sexual arousal is characterized by genital swelling and vaginal lubrication responses resulting from increased clitoral and vaginal blood flow. Animal models are valuable in investigating vaginal and clitoral hemodynamics, as they are able to mimic genital arousal in response to sexual stimulation. Recently, several objective methods were introduced that use experimental models to measure clitoral and vaginal blood flow. A rabbit model was first introduced by Park et al.2 to investigate the physiology and pathophysiology of female sexual arousal. In the New Zealand white female rabbit, vaginal wall blood flow and clitoral intracavernosal blood flow can be measured by laser Doppler flow probes that are placed in the vaginal wall and the clitoral corporal bodies, respectively (Fig. 5.6.1).2 The laser Doppler flowmeter (Transonic Systems, Ithaca, NY, USA) is calibrated against an internal standard to yield flow in units of ml/min per 100 g tissue. The branches of the pelvic nerve that serve the vagina and clitoris can be identified under the perivesical fat on the posterolateral aspect of the vagina as the nerve emerges from its associations with the rectum, bladder, and uterus.2 Pelvic nerve stimulation causes an increase in vaginal blood flow, vaginal wall pressure, and vaginal length, as well as clitoral blood flow and clitoral intracavernosal pressure. One of the drawbacks of laser Doppler flowmetry is its sensitivity to motion artifacts. As an alternative approach, Min et al.17 introduced laser oximetry, which can measure genital blood flow in the rabbit. As laser oximetry measures tissue hemoglobin content rather than rate of blood flow, it may be useful for measuring tissue engorgement during sexual arousal.

Figure 5.6.1. Schematic diagram showing female rabbit genitalia and experimental model. The symphysis pubis is dissected to expose the vaginal tube and uterus. Experimental setup for the measurement of vaginal and clitoral blood flow and pressure is shown within the box. Reproduced from Park et al.2 with permission.

Although the rabbit is a good model for hemodynamic studies of the vagina and clitoris, the limitation of this model is that rabbits, being induced ovulators, have endocrine physiology that is very different from human.18 The rabbit vagina is also anatomically different from the human. It has two distinct anatomic regions, a proximal region that represents 2/3 of the length of the vagina with a single layer of columnar epithelium, and a distal region that serves coital functions and is characterized by squamous epithelium.19

The female rat is another good model for the study of female sexual function and dysfunction. The female rat has a regular estrous cycle similar to women; in addition, the human and rat vaginas have a similar histologic structure. Vachon et al.20 first developed a rat model for the study of physiology, pharmacology, and sexual dysfunction relating to blood flow to clitoral and vaginal tissue. Recently, Kim et al.3 introduced an improved rat model to investigate female vaginal arousal. Kim et al. recommended a longer duration of pelvic nerve stimulation (30 s instead of 5 s). They also suggested that an alternative to determining peak vaginal blood flow magnitudes might be measurement of the area under the curve of flowmetry recordings. The area under the curve measures the perfusion inter- grated over time, to get a more accurate reflection of blood flow. In contrast to the rabbit, the pelvic nerve in female rats is buried in fragile fatty tissue overlying the lateral vaginal wall (Fig. 5.6.2). Therefore, careful dissection is recommended to avoid inadvertent injury to the nerve.3 Giuliano et al.21 also described a rat model of sexual arousal in which they assessed vaginal vascular events with oximetry and temperature measurements in addition to laser Doppler flowmetry. In this study, pelvic nerve stimulation induced reproducible increases in various vaginal parameters. However, concomitant electrical stimulation of the paravertebral sympathetic chain inhibited vaginal response induced by pelvic nerve stimulation.

Vaginal lubrication is one of the indicators of genital arousal and tissue integrity. In the basal state, the vaginal epithelium reabsorbs sodium from the submucosal capillary plasma transudate. During arousal, a dramatic increase in capillary inflow in the submucosa overwhelms sodium reabsorption, leading to 3-5 ml of vaginal transudate, and enhancing lubrication essential for pleasurable coitus.6 Vaginal lubrication can be measured by use of a preweighed, cotton-tipped swab in rabbits.After insertion of the cotton swab into the distal vaginal canal, the pelvic nerve is stimulated for 1 min, and the swab is left in place for an additional 5 min. Vaginal lubrication is assumed to be proportional to the weight of the fluid absorbed by the cotton swab.

Beharry et al.22 introduced a rat model of female sexual arousal response for centrally initiated genital vasocongestive engorgement, using a video monitoring system. Low doses of apo- morphine were administered to the conscious female rat, resulting in reproducibly induced behavioral and genital responses. They also demonstrated that the apomorphine-induced genital arousal responses were hormonally regulated in this model.

Animal models of sexual behavior have also been used to study human sexual behavior and its neurobiologic bases.23 Tong et al.24 used infrared-light-illuminated video recording to study behavioral aspects of the sexual activities of rats. This dark- cycle video recording method has been successfully applied to study sexual arousal and copulation-ejaculatory responses in rat. When a male rat encounters a female in estrous, it explores her genital area and initiates copulatory acts. During mounting, the female rat displays a concave dorsiflexion, extending her hind legs and moving the tail to the side (lordosis posture).23-24 Recently, Agmo and Ellingsen23 reviewed the relevance of nonhuman animal studies to the understanding of human sexuality. They commented that two striking similarities between humans and other mammals are the hormone dependency of sexual motivation and the bisexual potential of sexual behaviors. The specific gonadal hormone involved in sexual motivation varies among species, even though the general principles remain con- stant.23,25 There have been limited studies on sexual climax in female nonhuman primates. Behavioral and physiologic evidence of sexual climax was observed in the female stump-tailed macaque.26

Organ bath studies

Functional studies of isolated tissues in organ baths are necessary to understand various local regulatory mechanisms that modulate tone in the clitoral erectile tissue and vaginal smooth muscle. Female New Zealand white rabbits are usually chosen for these studies.7,27 Cellek and Moncada4 demonstrated that nitrergic neurotransmission mediates the nonadrenergic, noncholinergic relaxation responses in the clitoral corpus caver- nosum of the rabbit. Pretreatment of clitoral corpus cavernosum strips with sildenafil (100пм) enhanced the electrical field stimulation-induced relaxation both in magnitude and dura- tion.28 Therefore, the nitric oxide (NO) cyclic guanosine monophosphate pathway seems critical for smooth muscle relaxation in the clitoris. However, nonnitrergic, nonadrenergic, noncholinergic relaxation responses seem to be involved in the rabbit vaginal wall.29 Ziessen et al.29 showed that nonadrenergic, noncholinergic relaxation was partly mediated by nitric oxide in the rabbit vagina, and the remaining part may be mediated by a non-nitrergic component that was not associated with any known neuropeptides or purines.

In contrast, the rat vagina shows a different relaxation response from rabbit. Giraldi et al.30 reported that electrical field stimulation elicits nonadrenergic, noncholinergic relaxation of the vaginal smooth muscle tissue, and these relaxations were mediated by the nitrergic system, since they were blocked by inhibitors of the nitric oxide-cyclic guanosine monophosphate pathway. This finding is supported by the large number of nerves that contain nitric oxide synthase in the vaginal sphincter (the distal part of the vagina).30 Therefore, the role of the nitric oxide-cyclic guanosine monophosphate pathway in the vagina needs further investigation. Vasoactive intestinal peptide also induces vaginal smooth muscle relaxation, but its functional role remains to be determined.31,32

The smooth muscle contractility of the clitoris and vagina is regulated by the balance between vasoconstrictors and smooth muscle relaxants. Exogenously added norepinephrine caused a dose-dependent contraction of the vaginal and clitoral tissues, and this contraction was attenuated by alpha-1 and alpha-2 adrenergic receptor antagonists.27 This finding implies that adrenergic nerves mediate the contraction of clitoral and vaginal smooth muscle through alpha-adrenergic receptors. In addition, the Rho-kinase signaling pathway seems to be involved in the regulation of angiotensin II-induced contraction in the clitoral cavernosal smooth muscle. Park et al.33 suggested that the RhoA/Rho-kinase pathway acts in angiotensin II-induced contraction independently of the nitric oxide pathway in rabbit clitoral cavernosal smooth muscle. The role of other vasoconstrictors (e.g., endothelin or eicosanoids) in regulating the smooth muscle tone of the clitoris and vagina remains to be determined.

Animal models of female sexual dysfunction

Surgical menopausal models

Estrogen is thought to have a major role in the maintenance of the functional integrity of the female genitalia.34 The decline in the circulating level of estrogen is a major physiologic change during natural menopause. It has been reported that female sexual arousal function correlates negatively with menopausal symptoms such as hot flashes.35

Park et al.36 investigated the effects of estrogen deprivation and replacement on genital hemodynamics by laser Doppler flowmetry. They showed that the decline in circulating levels of estrogen adversely affected the hemodynamic mechanism of vaginal and clitoral engorgement in the rabbit. Decreased levels of circulating estrogen also produced marked changes in the structure of the vaginal and clitoral tissues, leading to thinning of the vaginal epithelial layers, decreased vaginal submucosal vasculature, and diffuse clitoral cavernosal fibrosis.

Using laser oximetry, Min et al.7 investigated the effects of ovariectomy and androgen and estrogen treatment on genital blood flow and vaginal lubrication. In contrast to the observations made by Park et al.,36 ovariectomy did not significantly affect genital blood flow in the rabbit model. The difference between these two studies may be the length of time after ovariectomy. Park et al.36 performed data collection 6 weeks after ovariectomy, whereas Min et al.7 collected data after 2 weeks. It is possible that the longer period of estrogen deprivation may have produced changes in the tissue structure of the clitoris and vagina. Because the female rabbit remains in continuous diestrus until mounted, serum estrogen levels are normally low (32-39 pg/ml), and ovariectomy produces only a small decrease (22-25 pg/ml).7,36

Estradiol or estradiol plus testosterone treatment of ovari- ectomized rabbits increased genital blood flow after pelvic nerve stimulation compared with controls.7,36 However, treatment of ovariectomized rabbits with testosterone alone did not result in increased genital blood flow. These results suggest that estrogens, but not androgens, modulate the vascular component of the female genital organs. Min et al.37 also showed that vaginal lubrication was reduced in ovariectomized animals, but returned to normal after estrogen treatment.

Estrogen plays an important role in regulating vaginal and clitoral nitric oxide synthase expression. Studies with the rat model have shown that the decrease in circulating levels of estrogen induced by ovariectomy downregulates nitric oxide synthase expression and increases apoptosis in nerves, smooth muscle, vascular endothelium, and epithelium of the rat vagina.38 Using an imaging analyzer, Dundar et al.39 reported the effect of estrogen-replacement therapy on clitoral- cavernosal tissue in oophorectomized rats. Although there was a tendency for the untreated group to have a higher collagen fiber content, no statistically significant difference was found among groups.

There have been discrepancies in data reported for nitric oxide synthase regulation by estrogen in the vagina, and these discrepancies may be due to differences either in species or in methods for assessment of nitric oxide synthase expression and activity.40 Vaginal nitric oxide synthase activity was significantly reduced by estrogen treatment in castrated rabbit.41,42 However, vaginal endothelial nitric oxide synthase and neural nitric oxide synthase expression increased significantly after estrogen replacement in castrated rat.38

Diabetic models

Diabetes mellitus causes female sexual dysfunction.43,44 Diabetic women may experience less sexual desire, less sexual arousal, inadequate lubrication, difficulty achieving orgasm, and dys- pareunia.43 To investigate the pathophysiologic mechanism of diabetes mellitus in female sexual dysfunction, Park et al.45 introduced a streptozotocin-induced diabetic rat model. They used a single high dose of intravenous streptozotocin (50 mg/kg) and found that 3 or 4 weeks was enough to develop a diabetic model.45 Giraldi et al.46 made a diabetic rat model by an intraperitoneal injection of streptozotocin (45 mg/kg), and then leaving the animals untreated for 8 weeks. The indicators that confirm the development of diabetes are weight loss, glucosuria, and increase in blood glucose levels. In diabetic animals, vaginal tissue revealed reduced epithelial layers and decreased vaginal submucosal vasculature, and also showed a dense and distorted arrangement of the collagen connective tissue compared with control animals (Fig. 5.6.3). In functional studies, diabetes interfered with adrenergic-, cholinergic-, and nonadrenergic, noncholinergic-neurotransmitter mechanisms in the smooth muscle of the rat vagina.46 In an insulin-controlled diabetic rat model, Park et al.47 reported that hyperglycemia causes alteration of vaginal blood flow and structure.

A diabetic rabbit model was established by an intravenous injection of alloxan hydrochloride (100mg/kg).48 However, diabetes developed in only 25% of cases, as determined by serum glucose greater than 200 mg/dl after 12 weeks. In this study, diabetes mellitus produced significant adverse effects on the hemodynamic mechanism of clitoral engorgement and led to diffuse clitoral cavernous fibrosis.

Atherosclerotic models

The prevalence of female sexual arousal disorders increases with age and vascular risk factors.13 A female New Zealand White rabbit model was developed to examine hemodynamic function in the presence of pelvic atherosclerotic vascular disease of the ilio-hypogastric-pudendal arterial bed.2 This model was induced by repeated aortofemoral balloon de-endothelialization followed by feeding the rabbits a 0.5% cholesterol diet for 16 weeks. In this study, atherosclerosis inhibited vaginal and clitoral engorgement and elongation, and led to diffuse vaginal wall and clitoral cavernosal smooth muscle fibrosis.

Model for neurogenic female sexual dysfunction

Central nervous system disorders have a significant impact on female sexuality and sexual response.49 There have been a few animal models for the study of the central nervous system, including spinal cord lesion. McKenna et al.50 introduced a model for experimental study of the neural mechanisms of sexual function. They measured the urethrogenital reflex, which is produced by a spinal pattern generator and is under tonic descending inhibition from the brainstem. This model has been used to study sexually relevant genital responses in females. The role of the hypothalamus has been extensively studied for sexual behavior with particular attention to lordosis in the female rat. The medial preoptic area has been shown to be involved in the expression of lordosis. Furthermore, the medial preoptic area may have an important role in the control of the female sexual response. Giuliano et al.21,51 showed that electrical stimulation of the medial preoptic area induced a significant increase in vaginal blood flow and a corresponding decrease in vaginal vascular resistance in female rats, suggesting that sexual genital arousal may be triggered by medial preoptic area activation.

Future research directions

Animal model studies have provided a better understanding of the physiology and pharmacology of both central and peripheral (vaginal and clitoral) female sexual arousal by allowing assessments of genital hemodynamics, vaginal lubrication, and regulation of genital smooth muscle contractility. However, research on women’s sexual health is still in its early phases compared with that of men’s sexual health. The role of neurotransmitters and their signaling mechanisms in the clitoris and vagina needs further investigation. Although the nitric oxide-cyclic guano- sine monophosphate system seems to have a key role in the regulation of clitoral cavernosal smooth muscle relaxation, there is still a lack of knowledge about the neurotransmitters regulating vaginal smooth muscle contractility. Steroid sex hormones significantly influence female sexual function and dysfunction. Although the effect of androgens on sexual desire is widely acknowledged, the role of steroid sex hormones on genital sexual arousal is not well understood. Animal models such as rat and rabbit have some limitations in the investigation of sexual desire and orgasmic function. Nevertheless, animal models of sexual behavior have proven to be useful for studying human sexual behavior and its neurobiologic bases. Investigation of female sexual dysfunction requires establishment of appropriate animal models to elucidate the pathophysiologic mechanisms. Recently, several animal models have been introduced to evaluate the pathologic mechanisms of menopause, atherosclerosis, and diabetes mellitus. However, further efforts are needed to develop a model for hypercholesterolemia, smoking, and other cardiovascular risk factors. In addition, future research is needed to develop an animal model to determine the impact of various central nervous system disorders and neuroanatomic sites of injury on sexual function.

References

1. Levin R. The mechanisms of human female sexual arousal. Annu Rev Sex Res 1992; 3: 1-48.

2. Park K, Goldstein I, Andry C et al. Vasculogenic female sexual dysfunction: the hemodynamic basis for vaginal engorgement insufficiency and clitoral erectile insufficiency. Int Umbbot res 1997; 9: 27-37.

3. Kim SW, Jeong SJ, Munarriz R et al. An in vivo rat model to investigate female vaginal arousal response. J Urol 2004; 171: 1357-61.

4. Cellek S, Moncada S. Nitrergic neurotransmission mediates the non-adrenergic non-cholinergic responses in the clitoral corpus cavernosum of the rabbit. br j. pharmacol

5. Munarriz R, Kim SW, Kim NN et al. A review of the physiology and pharmacology of peripheral (vaginal and clitoral) female genital arousal in the animal model. 

6. Munarriz R, Kim NN, Goldstein I et al. Biology of female sexual function. Urol clim north am 2002; 29: 685-93.

7. Min K, Munarriz R, Kim NN et al. Effects of ovariectomy and estrogen and androgen treatment on sildenafil-mediated changes in female genital blood flow and vaginal lubrication in the animal model. Am J Obstet Gynecol 2002; 187: 1370-6.

8. Tarcan T, Siroky MB, Park K et al. Systemic administration of apomorphine improves the hemodynamic mechanism of clitoral and vaginal engorgement in the rabbit. Intj imbot Res 2000; 12: 235-40.

9. Min K, Kim NN, McAuley I et al. Sildenafil augments pelvic nerve-mediated female genital sexual arousal in the anesthetized rabbit. Int j. imbot tRes 2000; 12 (Suppl 3): S32-9.

10. Laumann EO, Paik A, Rosen RC. Sexual dysfunction in the United States: prevalence and predictors. JAMA 1999; 281: 537-44.

11. Rosen RC, Taylor JF, Leiblum SR et al. Prevalence of sexual dysfunction in women: results of a survey study of 329 women in an outpatient gynecological clinic. J Sex Marital Ther 1993; 19: 171-88.

12. Basson R, Berman J, Burnett A et al. Report of the international consensus development conference on female sexual dysfunction: definitions and classifications.

13. Sadeghi-Nejad HMR, Traish A, Azadzoi K et al. Impotence is a couple’s disease: studies in female sexual dysfunction. J Urol 1996; 155: 677A.

14. Berman JR, Bassuk J. Physiology and pathophysiology of female sexual function and dysfunction. World J Urol 2002; 20: 111-18.

15. Goldstein MK, Teng NN. Gynecologic factors in sexual dysfunction of the older woman. Clin Geriatr Med 1991; 7: 41-61.

16. Slob AK, Koster J, Radder JK et al. Sexuality and psychophysiological functioning in women with diabetes mellitus. J Sex Marital Ther 1990; 16: 59-69.

17. Min K, O’Connell L, Munarriz R et al. Experimental models for the investigation of female sexual function and dysfunction. Int J Impot Res 2001; 13: 151-6.

18. Hilliard J, Eaton LW Jr. Estradiol-17 beta, progesterone and 20-alpha-hydroxypregn-4-en-3-one in rabbit ovarian venous plasma. II. From mating through implantation. Endocrinology 1971; 89: 522-7.

19. Barberini F, Correr S, De Santis F et al. The epithelium of the rabbit vagina: a microtopographical study by light, transmission and scanning electron microscopy. Arch Histol Cytol 1991; 54: 365-78.

20. Vachon P, Simmerman N, Zahran AR et al. Increases in clitoral and vaginal blood flow following clitoral and pelvic plexus nerve stimulations in the female rat. Intj imbot Res 2000; 12: 53-7.

21. Giuliano F, Allard J, Compagnie S et al. Vaginal physiological changes in a model of sexual arousal in anesthetized rats. Am J Physiol Regul Integr Comp Physiol 2001; 281: R140-9.

22. Beharry RK, Hale TM, Wilson EA et al. Evidence for centrally initiated genital vasocongestive engorgement in the female rat: findings from a new model of female sexual arousal response. Int J Impot Res 2003; 15: 122-8.

23. Agmo A, Ellingsen E. Relevance of non-human animal studies to the understanding of human sexuality. Scand J Psychol 2003; 44: 293-301.

24. Tong YC, Hung YC, Lin SN et al. Dark-cycle video surveillance of sexual performances of normal and diabetic rats. Urol Int 1996; 56: 207-10.

25. Sherwin BB, Gelfand MM, Brender W. Androgen enhances sexual motivation in females: a prospective, crossover study of sex steroid administration in the surgical menopause. Psychosom Med 1985; 47: 339-51.

26. Goldfoot DA, Westerborg-van Loon H, Groeneveld W et al. Behavioral and physiological evidence of sexual climax in the female stump-tailed macaque (Macaca arctoides). Science 1980; 208(4451): 1477-9.

27. Kim NN, Min K, Huang YH et al. Biochemical and functional characterization of alpha-adrenergic receptors in the rabbit vagina. Life Sci 2002; 71: 2909-20.

28. Vemulapalli S, Kurowski S. Sildenafil relaxes rabbit clitoral corpus cavernosum. LfSi 2000; 67: 23-9.

29. Ziessen T, Moncada S, Cellek S. Characterization of the non-nitr- ergic NANC relaxation responses in the rabbit vaginal wall. Br J Pharmacol 2002; 135: 546-54.

30. Giraldi A, Alm P, Werkstrom V et al. Morphological and functional characterization of a rat vaginal smooth muscle sphincter. Int J Impot Res 2002; 14: 271-82.

31. Ottesen B, Ulrichsen H, Fahrenkrug J et al. Vasoactive intestinal polypeptide and the female genital tract: relationship to reproductive phase and delivery. Am J Obstet Gynecol 1982; 143: 414-20.

32. Ottesen B. Vasoactive intestinal polypeptide as a neurotransmitter in the female genital tract. Am J Obstet Gynecol 1983; 147: 208-24.

33. Park JK, Lee SO, Kim YG et al. Role of rho-kinase activity in angiotensin II-induced contraction of rabbit clitoral cavernosum smooth muscle. Int J imbot Res 2002; 14: 472-7.

34. Laan E, van Lunsen RH. Hormones and sexuality in postmenopausal women: a psychophysiological study. J Psychosom Obstet Gynaecol 1997; 18: 126-33.

35. McCoy N, Culter W, Davidson JM. Relationships among sexual behavior, hot flashes, and hormone levels in perimenopausal women. Arch Sex Behav 1985; 14: 385-94.

36. Park K, Ahn K, Lee S et al. Decreased circulating levels of estrogen alter vaginal and clitoral blood flow and structure in the rabbit. Lnt Jimbot Res 2001; 13: 116-24.

37. Min K, Munarriz R, Kim NN et al. Effects of ovariectomy and estrogen replacement on basal and pelvic nerve stimulated vaginal lubrication in an animal model. J Sex Marital Ther 2003; 29 (Suppl 1): 77-84.

38. Berman JR, McCarthy MM, Kyprianou N. Effect of estrogen withdrawal on nitric oxide synthase expression and apoptosis in the rat vagina. Urology 1998; 51: 650-6.

39. Dundar M, Kocak I, Erkus M et al. The effect of estrogen-replacement therapy on clitoral-cavernosal tissue in oophorectomized rats: a histo-quantitative study by image analyzer. Urol Res 2001; 29: 317-20.

40. Traish AM, Kim NN, Huang YH et al. Sex steroid hormones differentially regulate nitric oxide synthase and arginase activities in the proximal and distal rabbit vagina. Int J Impot Res 2003; 15: 397-404.

41. Yoon HN, Chung WS, Park YY et al. Effects of estrogen on nitric oxide synthase and histological composition in the rabbit clitoris and vagina. Int J Impot Res 2001; 13: 205-11.

42. Al-Hijji J, Batra S. Downregulation by estrogen of nitric oxide synthase activity in the female rabbit lower urinary tract. Urology 1999; 53: 637-41.

43. Enzlin P, Mathieu C, Vanderschueren D et al. Diabetes mellitus and female sexuality: a review of 25 years’ research. Diabet Med 1998; 15: 809-15.

44. Meeking D, Fosbury J, Cradock S. Assessing the impact of diabetes on female sexuality. Community Nurse 1997; 3: 50-2.

45. Park K, Ryu SB, Park YI et al. Diabetes mellitus induces vaginal tissue fibrosis by TGF-beta 1 expression in the rat model. J Sex Marital Ther 2001; 27: 577-87.

46. Giraldi A, Persson K, Werkstrom V et al. Effects of diabetes on neurotransmission in rat vaginal smooth muscle. Int J Impot Res 2001; 13: 58-66.

47. Park K, Ahn K, Kim MK et al. Effect of hyperglycemia on vaginal blood flow and structure in the streptozotocin-induced diabetic rat. J Urol 2003; 169: 310S.

48. Park K, Ahn K, Chang JS et al. Diabetes induced alteration of clitoral hemodynamics and structure in the rabbit. J Urol 2002; 168: 1269-72.

49. Sipski ML. Central nervous system based neurogenic female sexual dysfunction: current status and future trends. Arch Sex Behav 2002; 31: 421-4.

50. McKenna KE, Chung SK, McVary KT. A model for the study of sexual function in anesthetized male and female rats. Am J Physiol 1991; 261(5 Pt 2): R1276-85.

51. Giuliano F, Rampin O, Allard J. Neurophysiology and pharmacology of female genital sexual response. J Sex Marital Ther 2002; 28 (Suppl 1): 101-21.