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Menstrual Cycle‐Related Morphometric and Vascular Modifications of the Clitoris
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ORIGINAL RESEARCH—ANATOMY/PHYSIOLOGY Menstrual Cycle-Related Morphometric and Vascular Modifications of the Clitoris Cesare Battaglia, MD, PhD,* Rossella Elena Nappi, MD,† Fulvia Mancini, MD, PhD,* Arianna Cianciosi, MD,* Nicola Persico, MD,* Paolo Busacchi, MD,* Fabio Facchinetti, MD,‡ and Domenico de Aloysio, MD* *Department of Obstetrics and Gynaecology, Alma Mater Studiorum—University of Bologna., Bologna, Italy; †Research Centre for Reproductive Medicine, University of Pavia, Pavia, Italy; ‡Department of Obstetrics and Gynaecology, University of Modena and Reggio Emilia, Modena, Italy DOI: 10.1111/j.1743-6109.2008.00972.x
ABSTRACT
Introduction. The evaluation of clitoral anatomy and function is of paramount importance to understand the physiology and pathology of clitoral function. Aim. To prospectively evaluate the clitoral volumetric and vascular modifications during the menstrual cycle, and analyze their relationship with circulating hormones and nitric oxide levels. Methods. Thirty healthy eumenorrheic women were studied in different phases of the menstrual cycle (day 3, 10, 14, 20, and 27). They were submitted to ultrasonographic (US) and Doppler analyses, and to hormonal and biochemical evaluations. Main Outcome Measures. Transvaginal US evaluation of uterus, ovaries, and clitoris; Doppler analysis of uterine and dorsal clitoral arteries; and measurement of plasma luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol, androstenedione, testosterone, and nitrites/nitrates concentration. Sex hormone binding globulin was assayed, and free androgen index was calculated. Results. During the menstrual cycle, FSH, LH, and estradiol changed as expected, whereas androgens did not show any significant change. The US assessment of the clitoral body volume evidenced a progressive increase with significant modifications during the periovulatory phase, after which it remained stable until day 20. Subsequently, the clitoral body volume decreased into the premenstrual phase (day 27), reaching values similar to those observed on cycle day 3. A comparable trend was observed in the nitrite/nitrate circulating values. The uterine and clitoral arteries presented significant modifications with reduced resistances in the periovulatory period. Estradiol levels resulted positively correlated with the clitoral body volume and inversely correlated with the dorsal clitoral artery pulsatility index (PI). Furthermore, the dorsal clitoral artery PI was inversely and significantly correlated with the nitrite/nitrate circulating values and the clitoral body volume. Conclusions. Clitoral anatomic and vascular modifications are observable during the normal menstrual cycle. Battaglia C, Nappi RE, Mancini F, Cianciosi A, Persico N, Busacchi P, Facchinetti F, and de Aloysio D. Menstrual cycle-related morphometric and vascular modifications of the clitoris. J Sex Med 2008;5:2853– 2861. Key Words. Clitoris; Ultrasound; MRI; Menstrual Cycle; Hormones
Introduction
I
n the lower mammals, there is a strict relationship between ovarian cycle and sexual behavior, such that virtually all sexual activities take place © 2008 International Society for Sexual Medicine
around the estrous (the time of maximal fertility). In nonhuman primates, this relationship is less distinct; nevertheless, sexual activity is at its highest level during the late follicular and periovulatory period of the menstrual cycle. A more J Sex Med 2008;5:2853–2861
2854 tenuous association has been found between the sexuality of women and the menstrual cycle. In fact, although Stanislaw and Rice [1] reported that sexual desire is generally experienced a few days before the basal body temperature shift (around the expected ovulation date), Meuwissen and Over [2] evidenced that the female sexual response does not vary during the menstrual cycle. Until a few years ago, the anatomy of the erogenous external genital structures (clitoris, labia majora and minora, vaginal introitus, and anus) was based on cadaveric studies; it lacked in details and included evident inaccuracies. Nuclear magnetic resonance [3–6], with three-dimensional sectional anatomy reconstruction, allowed a multiplanar evaluation of clitoral–vaginal anatomy (including the neurovascular structures) in the live state. More recently, two-dimensional and three-dimensional ultrasonography have been suggested as a less expensive, equally accurate, and noninvasive technique to analyze the clitoral-vaginal structures [7–9]. Sex involves a successful integration between an intact neural, vascular, and muscular circuitry; complex interactions between multiple neurotransmitter systems; and critical modulating influences from the endocrine system. One of the earliest signs of changes in the female sexual excitation is an increase in the vulval, clitoral, and vaginal blood flow. Modifications in the clitoral blood flow may be objectively measured by photopletismography, oxygenation temperature method, laser Doppler perfusion imaging, or Doppler ultrasonography [10–14]. The aim of the present study was to prospectively evaluate in a basal state, independently from sexual stimulation, the clitoral volumetric and vascular modifications during the menstrual cycle, and analyze their relationship with circulating hormones and nitric oxide (NO) levels. Patients and Methods
Between January and June 2007, 39 Caucasian (native Italians), adult (18–35 years old), eumenorrheic (menstrual cycle of 25–35 days) women, who were referred to our clinic for contraceptive necessities, were recruited into the study (by F. Mancini). All patients had regular ovulatory cycles, which were documented by ultrasonography, and serum midluteal progesterone levels >15 nmol/L in the previous cycle. Ovulation was similarly checked in the studied cycle. The subjects were all para 0 and, prior to the enrollment, undertook a J Sex Med 2008;5:2853–2861
Battaglia et al. pregnancy test, which resulted negative. Their body mass index (BMI) was normal (weight in kg/weight in m2; BMI = 19–25), and they were all in stable heterosexual relationships (>1 year) and without sexual dysfunction (as resulted from the two-factor Italian McCoy female sexuality questionnaire: MFSQ = ⱖ35) [10]. An informed consent was obtained from all women who participated in the study. The study protocol was in accordance with the Helsinki II declaration and was approved by the hospital research review committee (S. Orsola-Malpighi Review Committee). All subjects were nonsmokers; made no use of psychoactive drugs, recreational substances, or alcohol; did not exercise intensely on a regular basis; and had not received any hormonal therapy for at least 6 months prior to the study. In addition, women with neurological, psychiatric, cardiovascular, and endocrine disorders, hypertension (systolic blood pressure >140 mm Hg and/or diastolic pressure >90 mm Hg), hirsutism, diabetes, and renal or hepatic illnesses were excluded from the study. Further exclusion criteria were uterine malformations, dyspareunia, endometriosis, polycystic ovaries, ovarian functional cyst, unilateral ovarian resection or ovariectomy, urologic and proctologic diseases, and a history of perineal surgery or trauma. Thirty patients fulfilled the inclusion criteria and undertook, in five different phases of their menstrual cycle (early follicular, day 3; late follicular, day 10; periovulatory, day 14; luteal, day 20; and premenstrual, day 27), the utero-ovarian and clitoral ultrasonographic (US) analysis, and the color Doppler evaluation of the uterine and dorsal clitoral arteries. Contemporary, fasting blood samples were drawn to test hormonal parameters. Plasma concentrations of nitrites/nitrates (NO2-/ NO3-) were also assayed. On day 20, to assess the ovulatory cycles, we evaluated, by ultrasonography, the presence or absence of a corpus luteum and the midluteal serum progesterone rise (>15 nmol/mL).
Assays Peripheral blood flow was obtained from all patients between 08:00 and 11:00 am, after an overnight fast, on the same day that the ultrasound and Doppler examinations took place, and different hormonal and biochemical parameters were analyzed. Plasma concentrations of LH, FSH, estradiol (E2), androstenedione (A), and testosterone (T) were assayed (by A. Cianciosi and P. Busacchi)
Hormones and Clitoral Modifications as previously described [15,16]. Sex hormone binding globulin (SHBG) was immunoassayed using Immulite 1000 (EURO/Diagnostic Products Corp. Ltd., Los Angeles, CA, USA). The free androgen index (FAI) was calculated using the equation FAI = T (nmol/L)/SHBG (nmol/L) ¥ 100. Results of hormonal values were converted to SI units using the following conversion factors: LH (IU/L) = mIU/mL ¥ 1.0; FSH (IU/L) = mIU/ mL ¥ 1.0; E2 (pmol/L) = pg/mL ¥ 3.761; T (nmol/ L) = ng/mL ¥ 3.467; and A (nmol/L) = ng/dL ¥ 0.0349. An aliquot of peripheral blood was immediately centrifuged, and its serum was stored at -70°C until the assays were performed. NO production was assessed by monitoring the serum levels of stable oxidation products of NO metabolism (nitrites/nitrates [NO2-/NO3-]). Because very little or no NO2- is normally found in serum, we did not attempt to differentiate between NO2- and NO3amounts, and therefore we have reported our results as NO2-/NO3-. The NO2-/NO3- were assayed at Modena-Reggio Emilia University (by F. Facchinetti and R.E. Nappi) with the Greiss reaction using procedures previously described [17,18]. The serum NO2-/NO3- values are expressed in mmol/L.
Ultrasound and Color Doppler Examination US examination of the uterine and ovarian morphology was performed with the use of a 6.0–8.0 multifrequency transvaginal transducer (Aspen, Acuson-Siemens, Milan, Italy). Doppler flow measurements of the uterine vessels were performed transvaginally with a multifrequency color Doppler system (Aspen color Doppler, Acuson-Siemens). To ensure standardized conditions, the patients rested in a waiting room for at least 15 minutes before being scanned. Furthermore, in order to minimize external effects on blood flow, all patients were scanned in a noiseless laboratory with constant heat and light. They rested in a recumbent position, and were evaluated between 08:00 and 11:00 am. to exclude the effects of hormonal and NO2-/NO3- circadian rhythmicity on blood flow. A 50-Hz filter was used to eliminate low-frequency signals originating from vessel wall movements. The maximum Doppler ultrasonography energy was <80 mW/cm2. This intensity is within the safety limits suggested by the American Institute for Ultrasound in Medicine. Color flow images of the ascending branches of the uterine arteries were sampled laterally to the cervix in a longitudinal plane. The angle of
2855 insonation was always adjusted to obtain maximum color intensity. When adequate signals were obtained, blood flow velocity waveforms were recorded by placing the sample volume across the vessel and activating the pulsed Doppler mode. The pulsatility index (PI), defined as the difference between the peak systolic and end-diastolic flow divided by the mean maximum flow velocity, was electronically calculated by the machine for the uterine arteries. The PI has been shown to reflect blood flow impedance and may be used when the end-diastolic frequency shift is absent or reversed. For each examination, the mean value of three consecutive waveforms was obtained. No significant differences between the PIs of the left and the right side for uterine arteries were observed, and therefore, the average value of both side arteries was used. The correlation between the PIs and heart rate was not tested. Immediately after the previously described US and Doppler scanning, the patients were evaluated, in the same noiseless laboratory with constant heat and light, to analyze the clitoral anatomy and the blood flow in the dorsal clitoral artery. After an abundant coupling gel was applied, a high-resolution ultrasound transducer (7- to 10MHz multifrequency linear array transducer, Aspen, Acuson-Siemens) was placed on the upper part of the vulva for the clitoral transverse and axial section, and on the labia majora for the sagittal section. To prevent artifacts, care was taken to avoid excessive pressure on the clitoris. The clitoris was studied where the paired corpora join in a single body that projects into the glans [5] (Figure 1). The clitoral body volume was calculated. Volumes were measured by reporting the length, width, and depth assuming the forms to be ellipsoid, using the formula based on a prolate ellipsoid: V = p/6 ¥ D1 ¥ D2 ¥ D3, where D1, D2, and D3 are the maximal longitudinal, anteroposterior, and transverse diameter. Color flow images of the dorsal clitoral artery were sampled, in a longitudinal plane, on the outer surface of the clitoral body [5,19] (Figure 2). The angle of insonation was always adjusted to obtain the maximum color intensity. When good signals were obtained, blood flow velocity waveforms were recorded by placing the sample volume across the vessel and activating the pulsed Doppler mode. The PI, peak systolic blood flow velocity (Vmax), and time-averaged maximum velocity (TAMX) were electronically calculated by the machine. For each examination, the mean value of three consecutive waveforms was obtained. J Sex Med 2008;5:2853–2861
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Figure 1 Sagittal view of the clitoris. The clitoris has been studied where the paired corpora join a single body that projects into the glans. In the picture, it is possible to observe the glans, the body, and the bulb of the clitoris. The anatomic structures result to lie under the pubic symphysis.
An indication of within-patient precision of the Doppler procedures was obtained by analyzing the flow velocity waveforms recorded on three occasions from the uterine and dorsal clitoral arteries at 1-minute intervals. An analysis of variance (anova) of the results from the first 10 patients gave a mean coefficient of variation of 5.6% for uterine and 6.0% for dorsal clitoral arteries, and showed no significant differences between the replicate analyses. The ultrasound and color Doppler analyses were performed by a single examiner (C. Battaglia). Statistical Analysis
Statistical analysis (SPSS 12.0 software, SPSS Inc., Chicago, IL, USA) was performed using the
within-subjects anova test with Bonferroni’s post hoc correction. The relationship between the parameters analyzed was assessed using the linear regression method. A P value of 0.05 was considered statistically significant. Data are presented as mean ⫾ SD, unless otherwise indicated. The statistical analysis was independently performed by two researchers (N. Persico and D. de Aloysio). Results
All 30 women completed the study. The patients had a mean age of 29.9 ⫾ 6.3 years and a mean BMI of 21.6 ⫾ 2.8. The mean age at menarche was 12.1 ⫾ 1.7 years. The plasma levels of LH, FSH, E2, T, and A, evaluated in different phases of the cycle, are
Figure 2 Color flow images of the dorsal clitoral artery. The artery was sampled on the outer surface of the clitoral body. (A) The Doppler flow analysis in the early follicular phase of the menstrual cycle (day 3) evidences elevated resistances. (B) The Doppler flow analysis in the periovulatory phase of the menstrual cycle (day 14) shows a reduction of the resistances with a decreased evidence of the protodiastolic notch.
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Hormones and Clitoral Modifications
Table 1 Hormonal and biochemical profile during the menstrual cycle in 30 eumenorrheic women without sexual health problems Day 3
Day 10
Day 14
Day 20
Day 27
Significance
LH (IU/L) FSH (IU/L) Estradiol (pmol/L) Androstenedione (nmol/L) Testosterone (nmol/L) SHBG (nmol/L) FAI (%)
6.2 ⫾ 3.0 5.0 ⫾ 2.0 165 ⫾ 63* 8.7 ⫾ 2.2 1.17 ⫾ 0.72 55 ⫾ 24 2.5 ⫾ 1.8
9.9 ⫾ 7.2 7.3 ⫾ 4.8 473 ⫾ 262 10.1 ⫾ 3.3 1.31 ⫾ 0.69 51 ⫾ 19 2.7 ⫾ 1.5
14.2 ⫾ 9.6 11.1 ⫾ 4.5 660 ⫾ 441 10.7 ⫾ 3.7 1.31 ⫾ 0.73 55 ⫾ 21 2.5 ⫾ 1.4
13.4 ⫾ 15.2 5.0 ⫾ 1 < 1.2 536 ⫾ 349 12.3 ⫾ 3.9 1.42 ⫾ 0.81 58 ⫾ 20 2.6 ⫾ 1.5
7.2 ⫾ 4.3 3.8 ⫾ 1.7 435 ⫾ 312 9.4 ⫾ 3.8 1.24 ⫾ 0.83 61 ⫾ 25 2.2 ⫾ 1.6
†,‡
NO2-/NO3- (mmol/L)
21.4 ⫾ 10.7
33.9 ⫾ 10.1
38.5 ⫾ 15.7
29.4 ⫾ 14.1
24.8 ⫾ 13.3
§,¶, ** *,††,‡‡ NS NS NS NS §§,¶¶,
***
*The basal value of circulating estradiol was significantly (P ⱕ 0.0001) lower than all other values during the entire cycle; day 3 vs. day 14, P = 0.009; ‡day 14 vs. day 27, P = 0.005; §day 3 vs. day 14, P = 0.002; ¶day 14 vs. day 20, P < 0.001; **day 14 vs. day 27, P < 0.001; ††day 10 vs. day 14, P = 0.005; ‡‡day 14 vs. day 27, P = 0.008; §§day 3 vs. day 10, P = 0.030; ¶¶day 3 vs. day 14, P = 0.017; ***day 14 vs. day 27, P = 0.031. LH = luteinizing hormone; FSH = follicle stimulating hormone; SHBG = sex hormone binding globulin; FAI = free androgen index; NS = not significant. †
reported in Table 1. The SHBG values and the FAI resulted as reported in Table 1. In the midluteal phase (cycle day 20), all patients showed regular ovulatory cycles as documented by the US evidence of a corpus luteum and serum progesterone levels >15 nmol/mL. In these healthy, not sexually aroused women, the US assessment of the clitoral body volume evidenced a progressive increase with significant modifications during the periovulatory phase (Table 2). The volume remained stable up to day 20. Then, it decreased into the premenstrual phase (day 27), reaching values similar to those observed on cycle day 3 (Table 2). A comparable trend was observed in the NO2-/NO3- circulating values (Table 1). The uterine and clitoral arteries showed significant modifications as reported in Table 2. The mean duration of the US and Doppler procedures always lasted <20 minutes (mean 14 ⫾ 3 minutes). During the study, we observed a significant and progressive reduction of the time spent to perform the examinations (from 17 ⫾ 4 to 11 ⫾ 3 minutes, P = 0.033). No patient claimed any discomfort during the US and Doppler evaluations. The relationship between the different parameters (Table 3A and B) essentially evidenced that estradiol correlated positively with the clitoral
body volume (r = 0.340; P = 0.038) and negatively with the dorsal clitoral artery PI (r = -0.423; P = 0.003). Furthermore, the dorsal clitoral artery PI was inversely and significantly correlated with the NO2-/NO3- circulating values (r = -0.410; P = 0.007) and the clitoral body volume (r = -0.347; P = 0.036). Finally, the dorsal clitoral artery PI was positively and significantly correlated with the uterine artery PI (r = 0.331; P = 0.041). Discussion
Since the 16th century, prominent anatomists have described the clitoris structure. Nevertheless, their studies lacked in details and included evident inaccuracies [5]. A better knowledge of the clitoral anatomy is of paramount importance in understanding the physiology and pathology of the clitoral function. Recently, the magnetic resonance [3–6] and the two-dimensional and threedimensional ultrasonography have both been suggested as accurate and noninvasive analyses of the clitoral-vaginal structures [7–9]. The present study, in addition to confirming the role of ultrasonography as a noninvasive analysis of the clitoral structures [7,9], showed that the
Table 2 Ultrasonographic and Doppler flow modifications during the menstrual cycle in 30 eumenorrheic women without sexual health problems
Clitoral volume (mL) Clitoral artery PI Vmax (cm/second) TAMX (cm/second) Uterine artery PI
Day 3
Day 10
Day 14
Day 20
Day 27
Significance
0.67 ⫾ 0.22
0.69 ⫾ 0.17
0.78 ⫾ 0.25
0.78 ⫾ 0.19
0.64 ⫾ 0.19
*,†,‡,§
1.75 ⫾ 0.39 14 ⫾ 7 8⫾2 2.61 ⫾ 0.73
1.46 ⫾ 0.27 13 ⫾ 6 8⫾2 2.48 ⫾ 0.50
1.29 ⫾ 0.16 14 ⫾ 5 7⫾3 2.35 ⫾ 0.53
1.64 ⫾ 0.39 13 ⫾ 6 6⫾1 2.68 ⫾ 0.71
1.76 ⫾ 0.41 11 ⫾ 4 8⫾1 3.08 ⫾ 0.90
¶,
**,††,‡‡ NS NS §§,¶¶
*Day 3 vs. day 14, P = 0.039; †day 3 vs. day 20, P = 0.037; ‡day 14 vs. day 27, P = 0.033; §day 20 vs. day 27, P = 0.031; ¶day 3 vs. day 10, P = 0.009; **day 3 vs. day 14, P = 0.005; ††day 14 vs. day 20, P = 0.025; ‡‡day 14 vs. day 27, P = 0.020; §§day 3 vs. day 14, P = 0.042; ¶¶day 14 vs. day 27, P = 0.011. PI = pulsatility index; Vmax = peak systolic blood flow velocity; TAMX = time-averaged maximum velocity; NS = not significant.
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Table 3 Relationship between ultrasonographic, hormonal, and biochemical measures analyzed using the Pearson’s correlation Clitoral volume
Clitoral artery PI
Uterine artery PI
LH
FSH
A
r
P
r
P
r
P
r
P
r
P
Clitoral volume Clitoral artery PI Uterine artery LH FSH Estradiol Testosterone Androstenedione SHBG FAI NO2-/NO3-
1.000 -0.347 0.244 0.020 0.090 0.340 0.015 0.056 -0.086 0.079 0.022
NC 0.036 0.079 0.493 0.455 0.038 0.511 0.471 0.459 0.466 0.492
-0.347 1.000 0.331 -0.175 0.119 -0.423 -0.040 -0.109 0.091 -0.129 -0.410
0.036 NC 0.041 0.195 0.394 0.003 0.478 0.431 0.455 0.375 0.007
0.244 0.331 1.000 -0.107 0.136 -0.258 0.233 0.271 0.088 0.023 -0.198
0.079 0.041 NC 0.437 0.339 0.061 0.082 0.055 0.459 0.490 0.207
0.020 -0.175 -0.107 1.000 0.768 0.233 0.551 0.427 0.035 0.144 0.087
0.493 0.195 0.437 NC <0.001 0.082 <0.001 0.002 0.488 0.297 0.458
0.090 0.119 0.136 0.768 1.000 0.643 0.018 0.057 0.053 0.007 0.063
0.455 0.394 0.339 <0.001 NC <0.001 0.502 0.469 0.476 0.588 0.461
Estradiol
Testosterone
Androstenedione
SHBG
FAI
B
r
P
r
P
r
P
r
P
r
P
NO2-/NO3-
Clitoral volume Clitoral artery PI Uterine artery LH FSH Estradiol Testosterone Androstenedione SHBG FAI NO2-/NO3-
0.340 -0.423 -0.258 0.233 0.643 1.000 0.015 0.116 0.350 -0.073 0.022
0.038 0.003 0.061 0.082 <0.001 NC 0.511 0.428 0.034 0.464 0.490
0.015 -0.040 0.233 0.551 0.018 0.015 1.000 0.582 -0.051 0.779 0.072
0.511 0.478 0.082 <0.001 0.502 0.511 NC <0.001 0.475 <0.001 0.464
0.56 -0.109 0.271 0.427 0.057 0.116 0.582 1.000 -0.031 0.526 0.056
0.471 0.431 0.055 0.002 0.469 0.428 <0.001 NC 0.483 <0.001 0.471
-0.086 0.091 0.088 0.035 0.053 0.350 -0.051 -0.031 1.000 -0.520 -0.010
0.459 0.455 0.459 0.488 0.476 0.034 0.475 0.483 NC <0.001 0.523
0.079 -0.129 0.023 0.144 0.007 -0.073 0.779 0.526 -0.520 1.000 0.146
0.466 0.375 0.490 0.297 0.588 0.464 <0.001 <0.001 <0.001 NC 0.291
0.022 -0.410 -0.198 0.087 0.063 0.022 0.072 0.056 -0.010 0.146 1.000
0.492 0.007 0.207 0.458 0.461 0.490 0.464 0.471 0.523 0.291 NC
The results in bold are the significant values. PI = pulsatility index; LH = luteinizing hormone; FSH = follicle stimulating hormone; SHBG = sex hormone binding globulin; FAI = free androgen index; NC = not calculable.
technique, after a very short learning phase, is easy, not time-consuming, and well accepted by the patients. Moreover, although clitoral ultrasonography depends on free-hand placement and manipulation of the ultrasound probe on the clitoris, we believe that the extreme care we used in avoiding any pressure on the studied structures allowed us to prevent any inadvertent increase of the clitoral artery blood flow and to obtain reproducible results. To the best of our knowledge, we report, for the first time in literature, the clitoral morphofunctional modifications during the menstrual cycle in women who are not aroused sexually. The clitoral body volume showed a progressive increase up to the periovulatory period. This was associated with an equally significant increase in the NO2-/NO3circulating values and with a progressive decrease in the resistances at the dorsal clitoral artery level. In the luteal phase and into the premenstrual period, the registered parameters slowly decreased back to basal values. These phenomena were associated with estradiol variations. The clitoris is highly vascularized, and many vessels branch out in each clitoral artery structure. In contrast with previous studies where the J Sex Med 2008;5:2853–2861
position of the sampled vessels was not anatomically specified and described (thus making the data poorly reproducible) [10–12,14], we analyzed the dorsal clitoral artery at the outer surface level of the clitoral body [19]. In this area, the course of the artery is always detectable in the clitoral neurovascular bundle. We think that a precise description of the vessel sampling procedure is of paramount importance in order to standardize the examination of the clitoral vascularization. In the present study, to analyze by color Doppler the clitoral arteries, we chose to consider the PI instead of the resistance index, because the PI adequately reflects blood flow impedance and may be used when the diastolic frequency shift is absent or reversed. Some studies underlined the importance of ancillary Doppler flow parameters (i.e., peak systolic and end diastolic velocities) [10–12,14] for the evaluation of clitoral hemodynamic changes during the menstrual cycle [14] and sexual arousal [11]. However, our data, registered in the basal state and independently from sexual stimulation, did not show any modification of the Vmax and TAMX during the menstrual cycle. We speculated that in the absence of significant
Hormones and Clitoral Modifications changes in the heart rate or in the vessel diameter, the observed values express no resistance differences in the small capillaries distal from the dorsal clitoral artery. The role of estrogens in the regulation of female sexual function has been frequently reported. Estrogens are known to be vasoprotective and to regulate the vascular smooth muscle tone [20]. Studies with rat models demonstrate that in the clitoris, smooth muscle decreases with age, and that decreased levels of estrogens lead, in the rat’s vagina, to apoptosis in the nerves, smooth muscle, and vascular endothelium [21]. Furthermore, clinical studies on humans have shown that the uterine, vaginal, clitoral, and urethral blood flow, and vaginal lubrication significantly decrease during menopause with a decline in circulating ovarian hormones. On the contrary, estrogen replacement therapy enhances pelvic blood flow in postmenopausal women [22–24]. Moreover, Dennerstein et al. [25], recently demonstrated that “sexual responsivity” (a measure of interest, arousal, enjoyment, and orgasm) improves when, in a spontaneous cycle, estradiol circulating levels are comprised between 650 and 759 pmol/L. These values match those we observed in our patients during the periovulatory period, and the result associated with the highest value of clitoral volume and NO2-/NO3- circulating values, and with the lowest resistances of the uterine and dorsal clitoral arteries. This observation emphasizes that uterine and clitoral vascularization is equally sensitive to estrogen modifications. In addition, our findings agree with Hayashi et al.[26] who reported that estrogens may increase NO production by boosting the expression and activity of endothelial nitric oxide synthase (NOS). We speculated, as did Gragasin et al. [27], that NO, produced by the estrogen-mediated action of NOS on L-arginine, induces a rise of cyclic guanosine monophosphate in postjunctional cells and causes a calcium influx to the clitoral vascular smooth muscle. This causes vasodilatation, relaxation of the clitoral cavernosal tissue, and increase in the clitoral size. In addition, estradiol stimulates neuronal-NOS expression in the preoptic area and exerts an adjuvant influence on NO-producing neurons in the amygdala. Thus, the periovulatory surge of NO2-/NO3- circulating levels may also be involved in the central nervous system activity of the amygdala [28]. The latter is intimately involved in sex and sexuality, and has an important role in the coordination of the endocrine system and in pregnancy. Its stimulation produces sexual
2859 sensations, memories of intercourse, and orgasm. Furthermore, the corticomedial amygdala seems to be able to modulate the activity of gonadotropin releasing hormone neurons and to induce the LH surge and ovulation. Although it has been reported that subjective sexual and genital arousal, and sexual desire do not vary significantly with the phase of the menstrual cycle [2], the intimate relationship between central nervous system, hormonal modulation, and erogenous external genital structures underline that, in nature, sexuality may be conditioned by processes that regulate the menstrual cycle, and probably, the maximal arousability is reached in the periovulatory period [1,29]. In our study, we observed a progressive decrease of the clitoral engorgement during the middle and late luteal phase of the cycle. This finding agrees with Slob et al. [30] who found that objective (increased labium minus temperature) and subjective (semantic scales) changes in sexual arousability to erotic video stimulation are higher in the late follicular period when compared with the luteal phase of the menstrual cycle. We think that the progesterone rise may counteract the positive estrogen effects on clitoral vascularization and engorgement [12] by (i) inducing a moderate decrease of NO2-/NO3- circulating levels; (ii) increasing the b-adrenergic receptor formation; and (iii) enhancing the anticholinergic effect at clitoris level. This may be associated with a fall of the clitoral volume and vascularization, and with the reduction of the vaginal lubrication. These processes may, in lower mammals, be responsible for the sex refusal during the post-estrous period. In humans, the correlation is certainly more tenuous. However, the action mediated by progesterone may, by lowering the libido and the spontaneous arousability, diminish the frequency of sexual intercourse. This could be associated with a decline in oxytocin production [29], causing a decrease in uterine contractility, which could favor the endometrial blastocyst penetration. The exact physiological and biochemical role of androgens in the female sexual function remains controversial and poorly understood [20,31,32]. Whether the effects of androgens are mediated by a direct action on the nervous system or by an effect on the genital system, or both, has yet to be established. In women, the role of androgens is confounded by the fact that their synthesis and metabolism take place in the ovaries, the adrenal glands, and the peripheral tissue. Furthermore, active androgens may be synthesized on demand in J Sex Med 2008;5:2853–2861
2860 target tissues [31–33]. Thus, although Salonia et al. [34] recently provided the clinically applicable ranges for androgens throughout the menstrual cycle, we think, in agreement with Labrie et al. [35], that the levels of circulating androgens may not give the correct information on the bioavailability of their metabolites. Davis and Tran [36] have suggested that androgen insufficiency is associated with impaired sexual function. However, two large recent studies have failed to find any correlation between sexual function and circulating androgen levels measured as total testosterone and FAI in 2,900 pre- and perimenopausal North American women [37], and as free and total testosterone in 1,021 Australian women [38]. It was suggested that androgens have no effect on sexual arousal “per se,” but may influence other aspects of sexual desire such as thoughts and fantasies. Our data showed that throughout the menstrual cycle, there are no significant modifications in the concentrations of circulating androgens. We speculated that, although androgens have a well-known midcycle elevation, their circadian variations and their local tissue concentration (correlated with local peripheral conversion) mediate the tissue-specific response and inhibit defining properly which metabolite regulates the physiology of central and peripheral tissues involved in sexual function. Conclusions
During the normal menstrual cycle, clitoral anatomic and vascular modifications are highly and cyclically influenced by steroid hormones and neuromodulators. How this may be integrated into the sexual function of all women will necessitate further, broader, and prospective studies. Corresponding Author: Cesare Battaglia, MD, PhD, Department of Obstetrics and Gynaecology, Alma Mater Studiorum—University of Bologna, Via Massarenti, 13-40138 Bologna, Italy. Tel: 39-051-6364377; Fax: 39-051-6364377; E-mail: [email protected] Conflict of Interest: None declared.
Statement of Authorship
Category 1 (a) Conception and Design Cesare Battaglia (b) Acquisition of Data Fulvia Mancini; Arianna Cianciosi; Paolo Busacchi; Fabio Facchinetti; Rossella Elena Nappi; Cesare Battaglia J Sex Med 2008;5:2853–2861
Battaglia et al. (c) Analysis and Interpretation of Data Nicola Persico; Domenico de Aloysio
Category 2 (a) Drafting the Article Cesare Battaglia (b) Revising It for Intellectual Content Fulvia Mancini
Category 3 (a) Final Approval of the Completed Article Domenico de Aloysio References
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ORIGINAL RESEARCH—ANATOMY/PHYSIOLOGY Menstrual Cycle-Related Morphometric and Vascular Modifications of the Clitoris Cesare Battaglia, MD, PhD,* Rossella Elena Nappi, MD,† Fulvia Mancini, MD, PhD,* Arianna Cianciosi, MD,* Nicola Persico, MD,* Paolo Busacchi, MD,* Fabio Facchinetti, MD,‡ and Domenico de Aloysio, MD* *Department of Obstetrics and Gynaecology, Alma Mater Studiorum—University of Bologna., Bologna, Italy; †Research Centre for Reproductive Medicine, University of Pavia, Pavia, Italy; ‡Department of Obstetrics and Gynaecology, University of Modena and Reggio Emilia, Modena, Italy DOI: 10.1111/j.1743-6109.2008.00972.x
ABSTRACT
Introduction. The evaluation of clitoral anatomy and function is of paramount importance to understand the physiology and pathology of clitoral function. Aim. To prospectively evaluate the clitoral volumetric and vascular modifications during the menstrual cycle, and analyze their relationship with circulating hormones and nitric oxide levels. Methods. Thirty healthy eumenorrheic women were studied in different phases of the menstrual cycle (day 3, 10, 14, 20, and 27). They were submitted to ultrasonographic (US) and Doppler analyses, and to hormonal and biochemical evaluations. Main Outcome Measures. Transvaginal US evaluation of uterus, ovaries, and clitoris; Doppler analysis of uterine and dorsal clitoral arteries; and measurement of plasma luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol, androstenedione, testosterone, and nitrites/nitrates concentration. Sex hormone binding globulin was assayed, and free androgen index was calculated. Results. During the menstrual cycle, FSH, LH, and estradiol changed as expected, whereas androgens did not show any significant change. The US assessment of the clitoral body volume evidenced a progressive increase with significant modifications during the periovulatory phase, after which it remained stable until day 20. Subsequently, the clitoral body volume decreased into the premenstrual phase (day 27), reaching values similar to those observed on cycle day 3. A comparable trend was observed in the nitrite/nitrate circulating values. The uterine and clitoral arteries presented significant modifications with reduced resistances in the periovulatory period. Estradiol levels resulted positively correlated with the clitoral body volume and inversely correlated with the dorsal clitoral artery pulsatility index (PI). Furthermore, the dorsal clitoral artery PI was inversely and significantly correlated with the nitrite/nitrate circulating values and the clitoral body volume. Conclusions. Clitoral anatomic and vascular modifications are observable during the normal menstrual cycle. Battaglia C, Nappi RE, Mancini F, Cianciosi A, Persico N, Busacchi P, Facchinetti F, and de Aloysio D. Menstrual cycle-related morphometric and vascular modifications of the clitoris. J Sex Med 2008;5:2853– 2861. Key Words. Clitoris; Ultrasound; MRI; Menstrual Cycle; Hormones
Introduction
I
n the lower mammals, there is a strict relationship between ovarian cycle and sexual behavior, such that virtually all sexual activities take place © 2008 International Society for Sexual Medicine
around the estrous (the time of maximal fertility). In nonhuman primates, this relationship is less distinct; nevertheless, sexual activity is at its highest level during the late follicular and periovulatory period of the menstrual cycle. A more J Sex Med 2008;5:2853–2861
2854 tenuous association has been found between the sexuality of women and the menstrual cycle. In fact, although Stanislaw and Rice [1] reported that sexual desire is generally experienced a few days before the basal body temperature shift (around the expected ovulation date), Meuwissen and Over [2] evidenced that the female sexual response does not vary during the menstrual cycle. Until a few years ago, the anatomy of the erogenous external genital structures (clitoris, labia majora and minora, vaginal introitus, and anus) was based on cadaveric studies; it lacked in details and included evident inaccuracies. Nuclear magnetic resonance [3–6], with three-dimensional sectional anatomy reconstruction, allowed a multiplanar evaluation of clitoral–vaginal anatomy (including the neurovascular structures) in the live state. More recently, two-dimensional and three-dimensional ultrasonography have been suggested as a less expensive, equally accurate, and noninvasive technique to analyze the clitoral-vaginal structures [7–9]. Sex involves a successful integration between an intact neural, vascular, and muscular circuitry; complex interactions between multiple neurotransmitter systems; and critical modulating influences from the endocrine system. One of the earliest signs of changes in the female sexual excitation is an increase in the vulval, clitoral, and vaginal blood flow. Modifications in the clitoral blood flow may be objectively measured by photopletismography, oxygenation temperature method, laser Doppler perfusion imaging, or Doppler ultrasonography [10–14]. The aim of the present study was to prospectively evaluate in a basal state, independently from sexual stimulation, the clitoral volumetric and vascular modifications during the menstrual cycle, and analyze their relationship with circulating hormones and nitric oxide (NO) levels. Patients and Methods
Between January and June 2007, 39 Caucasian (native Italians), adult (18–35 years old), eumenorrheic (menstrual cycle of 25–35 days) women, who were referred to our clinic for contraceptive necessities, were recruited into the study (by F. Mancini). All patients had regular ovulatory cycles, which were documented by ultrasonography, and serum midluteal progesterone levels >15 nmol/L in the previous cycle. Ovulation was similarly checked in the studied cycle. The subjects were all para 0 and, prior to the enrollment, undertook a J Sex Med 2008;5:2853–2861
Battaglia et al. pregnancy test, which resulted negative. Their body mass index (BMI) was normal (weight in kg/weight in m2; BMI = 19–25), and they were all in stable heterosexual relationships (>1 year) and without sexual dysfunction (as resulted from the two-factor Italian McCoy female sexuality questionnaire: MFSQ = ⱖ35) [10]. An informed consent was obtained from all women who participated in the study. The study protocol was in accordance with the Helsinki II declaration and was approved by the hospital research review committee (S. Orsola-Malpighi Review Committee). All subjects were nonsmokers; made no use of psychoactive drugs, recreational substances, or alcohol; did not exercise intensely on a regular basis; and had not received any hormonal therapy for at least 6 months prior to the study. In addition, women with neurological, psychiatric, cardiovascular, and endocrine disorders, hypertension (systolic blood pressure >140 mm Hg and/or diastolic pressure >90 mm Hg), hirsutism, diabetes, and renal or hepatic illnesses were excluded from the study. Further exclusion criteria were uterine malformations, dyspareunia, endometriosis, polycystic ovaries, ovarian functional cyst, unilateral ovarian resection or ovariectomy, urologic and proctologic diseases, and a history of perineal surgery or trauma. Thirty patients fulfilled the inclusion criteria and undertook, in five different phases of their menstrual cycle (early follicular, day 3; late follicular, day 10; periovulatory, day 14; luteal, day 20; and premenstrual, day 27), the utero-ovarian and clitoral ultrasonographic (US) analysis, and the color Doppler evaluation of the uterine and dorsal clitoral arteries. Contemporary, fasting blood samples were drawn to test hormonal parameters. Plasma concentrations of nitrites/nitrates (NO2-/ NO3-) were also assayed. On day 20, to assess the ovulatory cycles, we evaluated, by ultrasonography, the presence or absence of a corpus luteum and the midluteal serum progesterone rise (>15 nmol/mL).
Assays Peripheral blood flow was obtained from all patients between 08:00 and 11:00 am, after an overnight fast, on the same day that the ultrasound and Doppler examinations took place, and different hormonal and biochemical parameters were analyzed. Plasma concentrations of LH, FSH, estradiol (E2), androstenedione (A), and testosterone (T) were assayed (by A. Cianciosi and P. Busacchi)
Hormones and Clitoral Modifications as previously described [15,16]. Sex hormone binding globulin (SHBG) was immunoassayed using Immulite 1000 (EURO/Diagnostic Products Corp. Ltd., Los Angeles, CA, USA). The free androgen index (FAI) was calculated using the equation FAI = T (nmol/L)/SHBG (nmol/L) ¥ 100. Results of hormonal values were converted to SI units using the following conversion factors: LH (IU/L) = mIU/mL ¥ 1.0; FSH (IU/L) = mIU/ mL ¥ 1.0; E2 (pmol/L) = pg/mL ¥ 3.761; T (nmol/ L) = ng/mL ¥ 3.467; and A (nmol/L) = ng/dL ¥ 0.0349. An aliquot of peripheral blood was immediately centrifuged, and its serum was stored at -70°C until the assays were performed. NO production was assessed by monitoring the serum levels of stable oxidation products of NO metabolism (nitrites/nitrates [NO2-/NO3-]). Because very little or no NO2- is normally found in serum, we did not attempt to differentiate between NO2- and NO3amounts, and therefore we have reported our results as NO2-/NO3-. The NO2-/NO3- were assayed at Modena-Reggio Emilia University (by F. Facchinetti and R.E. Nappi) with the Greiss reaction using procedures previously described [17,18]. The serum NO2-/NO3- values are expressed in mmol/L.
Ultrasound and Color Doppler Examination US examination of the uterine and ovarian morphology was performed with the use of a 6.0–8.0 multifrequency transvaginal transducer (Aspen, Acuson-Siemens, Milan, Italy). Doppler flow measurements of the uterine vessels were performed transvaginally with a multifrequency color Doppler system (Aspen color Doppler, Acuson-Siemens). To ensure standardized conditions, the patients rested in a waiting room for at least 15 minutes before being scanned. Furthermore, in order to minimize external effects on blood flow, all patients were scanned in a noiseless laboratory with constant heat and light. They rested in a recumbent position, and were evaluated between 08:00 and 11:00 am. to exclude the effects of hormonal and NO2-/NO3- circadian rhythmicity on blood flow. A 50-Hz filter was used to eliminate low-frequency signals originating from vessel wall movements. The maximum Doppler ultrasonography energy was <80 mW/cm2. This intensity is within the safety limits suggested by the American Institute for Ultrasound in Medicine. Color flow images of the ascending branches of the uterine arteries were sampled laterally to the cervix in a longitudinal plane. The angle of
2855 insonation was always adjusted to obtain maximum color intensity. When adequate signals were obtained, blood flow velocity waveforms were recorded by placing the sample volume across the vessel and activating the pulsed Doppler mode. The pulsatility index (PI), defined as the difference between the peak systolic and end-diastolic flow divided by the mean maximum flow velocity, was electronically calculated by the machine for the uterine arteries. The PI has been shown to reflect blood flow impedance and may be used when the end-diastolic frequency shift is absent or reversed. For each examination, the mean value of three consecutive waveforms was obtained. No significant differences between the PIs of the left and the right side for uterine arteries were observed, and therefore, the average value of both side arteries was used. The correlation between the PIs and heart rate was not tested. Immediately after the previously described US and Doppler scanning, the patients were evaluated, in the same noiseless laboratory with constant heat and light, to analyze the clitoral anatomy and the blood flow in the dorsal clitoral artery. After an abundant coupling gel was applied, a high-resolution ultrasound transducer (7- to 10MHz multifrequency linear array transducer, Aspen, Acuson-Siemens) was placed on the upper part of the vulva for the clitoral transverse and axial section, and on the labia majora for the sagittal section. To prevent artifacts, care was taken to avoid excessive pressure on the clitoris. The clitoris was studied where the paired corpora join in a single body that projects into the glans [5] (Figure 1). The clitoral body volume was calculated. Volumes were measured by reporting the length, width, and depth assuming the forms to be ellipsoid, using the formula based on a prolate ellipsoid: V = p/6 ¥ D1 ¥ D2 ¥ D3, where D1, D2, and D3 are the maximal longitudinal, anteroposterior, and transverse diameter. Color flow images of the dorsal clitoral artery were sampled, in a longitudinal plane, on the outer surface of the clitoral body [5,19] (Figure 2). The angle of insonation was always adjusted to obtain the maximum color intensity. When good signals were obtained, blood flow velocity waveforms were recorded by placing the sample volume across the vessel and activating the pulsed Doppler mode. The PI, peak systolic blood flow velocity (Vmax), and time-averaged maximum velocity (TAMX) were electronically calculated by the machine. For each examination, the mean value of three consecutive waveforms was obtained. J Sex Med 2008;5:2853–2861
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Figure 1 Sagittal view of the clitoris. The clitoris has been studied where the paired corpora join a single body that projects into the glans. In the picture, it is possible to observe the glans, the body, and the bulb of the clitoris. The anatomic structures result to lie under the pubic symphysis.
An indication of within-patient precision of the Doppler procedures was obtained by analyzing the flow velocity waveforms recorded on three occasions from the uterine and dorsal clitoral arteries at 1-minute intervals. An analysis of variance (anova) of the results from the first 10 patients gave a mean coefficient of variation of 5.6% for uterine and 6.0% for dorsal clitoral arteries, and showed no significant differences between the replicate analyses. The ultrasound and color Doppler analyses were performed by a single examiner (C. Battaglia). Statistical Analysis
Statistical analysis (SPSS 12.0 software, SPSS Inc., Chicago, IL, USA) was performed using the
within-subjects anova test with Bonferroni’s post hoc correction. The relationship between the parameters analyzed was assessed using the linear regression method. A P value of 0.05 was considered statistically significant. Data are presented as mean ⫾ SD, unless otherwise indicated. The statistical analysis was independently performed by two researchers (N. Persico and D. de Aloysio). Results
All 30 women completed the study. The patients had a mean age of 29.9 ⫾ 6.3 years and a mean BMI of 21.6 ⫾ 2.8. The mean age at menarche was 12.1 ⫾ 1.7 years. The plasma levels of LH, FSH, E2, T, and A, evaluated in different phases of the cycle, are
Figure 2 Color flow images of the dorsal clitoral artery. The artery was sampled on the outer surface of the clitoral body. (A) The Doppler flow analysis in the early follicular phase of the menstrual cycle (day 3) evidences elevated resistances. (B) The Doppler flow analysis in the periovulatory phase of the menstrual cycle (day 14) shows a reduction of the resistances with a decreased evidence of the protodiastolic notch.
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Table 1 Hormonal and biochemical profile during the menstrual cycle in 30 eumenorrheic women without sexual health problems Day 3
Day 10
Day 14
Day 20
Day 27
Significance
LH (IU/L) FSH (IU/L) Estradiol (pmol/L) Androstenedione (nmol/L) Testosterone (nmol/L) SHBG (nmol/L) FAI (%)
6.2 ⫾ 3.0 5.0 ⫾ 2.0 165 ⫾ 63* 8.7 ⫾ 2.2 1.17 ⫾ 0.72 55 ⫾ 24 2.5 ⫾ 1.8
9.9 ⫾ 7.2 7.3 ⫾ 4.8 473 ⫾ 262 10.1 ⫾ 3.3 1.31 ⫾ 0.69 51 ⫾ 19 2.7 ⫾ 1.5
14.2 ⫾ 9.6 11.1 ⫾ 4.5 660 ⫾ 441 10.7 ⫾ 3.7 1.31 ⫾ 0.73 55 ⫾ 21 2.5 ⫾ 1.4
13.4 ⫾ 15.2 5.0 ⫾ 1 < 1.2 536 ⫾ 349 12.3 ⫾ 3.9 1.42 ⫾ 0.81 58 ⫾ 20 2.6 ⫾ 1.5
7.2 ⫾ 4.3 3.8 ⫾ 1.7 435 ⫾ 312 9.4 ⫾ 3.8 1.24 ⫾ 0.83 61 ⫾ 25 2.2 ⫾ 1.6
†,‡
NO2-/NO3- (mmol/L)
21.4 ⫾ 10.7
33.9 ⫾ 10.1
38.5 ⫾ 15.7
29.4 ⫾ 14.1
24.8 ⫾ 13.3
§,¶, ** *,††,‡‡ NS NS NS NS §§,¶¶,
***
*The basal value of circulating estradiol was significantly (P ⱕ 0.0001) lower than all other values during the entire cycle; day 3 vs. day 14, P = 0.009; ‡day 14 vs. day 27, P = 0.005; §day 3 vs. day 14, P = 0.002; ¶day 14 vs. day 20, P < 0.001; **day 14 vs. day 27, P < 0.001; ††day 10 vs. day 14, P = 0.005; ‡‡day 14 vs. day 27, P = 0.008; §§day 3 vs. day 10, P = 0.030; ¶¶day 3 vs. day 14, P = 0.017; ***day 14 vs. day 27, P = 0.031. LH = luteinizing hormone; FSH = follicle stimulating hormone; SHBG = sex hormone binding globulin; FAI = free androgen index; NS = not significant. †
reported in Table 1. The SHBG values and the FAI resulted as reported in Table 1. In the midluteal phase (cycle day 20), all patients showed regular ovulatory cycles as documented by the US evidence of a corpus luteum and serum progesterone levels >15 nmol/mL. In these healthy, not sexually aroused women, the US assessment of the clitoral body volume evidenced a progressive increase with significant modifications during the periovulatory phase (Table 2). The volume remained stable up to day 20. Then, it decreased into the premenstrual phase (day 27), reaching values similar to those observed on cycle day 3 (Table 2). A comparable trend was observed in the NO2-/NO3- circulating values (Table 1). The uterine and clitoral arteries showed significant modifications as reported in Table 2. The mean duration of the US and Doppler procedures always lasted <20 minutes (mean 14 ⫾ 3 minutes). During the study, we observed a significant and progressive reduction of the time spent to perform the examinations (from 17 ⫾ 4 to 11 ⫾ 3 minutes, P = 0.033). No patient claimed any discomfort during the US and Doppler evaluations. The relationship between the different parameters (Table 3A and B) essentially evidenced that estradiol correlated positively with the clitoral
body volume (r = 0.340; P = 0.038) and negatively with the dorsal clitoral artery PI (r = -0.423; P = 0.003). Furthermore, the dorsal clitoral artery PI was inversely and significantly correlated with the NO2-/NO3- circulating values (r = -0.410; P = 0.007) and the clitoral body volume (r = -0.347; P = 0.036). Finally, the dorsal clitoral artery PI was positively and significantly correlated with the uterine artery PI (r = 0.331; P = 0.041). Discussion
Since the 16th century, prominent anatomists have described the clitoris structure. Nevertheless, their studies lacked in details and included evident inaccuracies [5]. A better knowledge of the clitoral anatomy is of paramount importance in understanding the physiology and pathology of the clitoral function. Recently, the magnetic resonance [3–6] and the two-dimensional and threedimensional ultrasonography have both been suggested as accurate and noninvasive analyses of the clitoral-vaginal structures [7–9]. The present study, in addition to confirming the role of ultrasonography as a noninvasive analysis of the clitoral structures [7,9], showed that the
Table 2 Ultrasonographic and Doppler flow modifications during the menstrual cycle in 30 eumenorrheic women without sexual health problems
Clitoral volume (mL) Clitoral artery PI Vmax (cm/second) TAMX (cm/second) Uterine artery PI
Day 3
Day 10
Day 14
Day 20
Day 27
Significance
0.67 ⫾ 0.22
0.69 ⫾ 0.17
0.78 ⫾ 0.25
0.78 ⫾ 0.19
0.64 ⫾ 0.19
*,†,‡,§
1.75 ⫾ 0.39 14 ⫾ 7 8⫾2 2.61 ⫾ 0.73
1.46 ⫾ 0.27 13 ⫾ 6 8⫾2 2.48 ⫾ 0.50
1.29 ⫾ 0.16 14 ⫾ 5 7⫾3 2.35 ⫾ 0.53
1.64 ⫾ 0.39 13 ⫾ 6 6⫾1 2.68 ⫾ 0.71
1.76 ⫾ 0.41 11 ⫾ 4 8⫾1 3.08 ⫾ 0.90
¶,
**,††,‡‡ NS NS §§,¶¶
*Day 3 vs. day 14, P = 0.039; †day 3 vs. day 20, P = 0.037; ‡day 14 vs. day 27, P = 0.033; §day 20 vs. day 27, P = 0.031; ¶day 3 vs. day 10, P = 0.009; **day 3 vs. day 14, P = 0.005; ††day 14 vs. day 20, P = 0.025; ‡‡day 14 vs. day 27, P = 0.020; §§day 3 vs. day 14, P = 0.042; ¶¶day 14 vs. day 27, P = 0.011. PI = pulsatility index; Vmax = peak systolic blood flow velocity; TAMX = time-averaged maximum velocity; NS = not significant.
J Sex Med 2008;5:2853–2861
2858
Battaglia et al.
Table 3 Relationship between ultrasonographic, hormonal, and biochemical measures analyzed using the Pearson’s correlation Clitoral volume
Clitoral artery PI
Uterine artery PI
LH
FSH
A
r
P
r
P
r
P
r
P
r
P
Clitoral volume Clitoral artery PI Uterine artery LH FSH Estradiol Testosterone Androstenedione SHBG FAI NO2-/NO3-
1.000 -0.347 0.244 0.020 0.090 0.340 0.015 0.056 -0.086 0.079 0.022
NC 0.036 0.079 0.493 0.455 0.038 0.511 0.471 0.459 0.466 0.492
-0.347 1.000 0.331 -0.175 0.119 -0.423 -0.040 -0.109 0.091 -0.129 -0.410
0.036 NC 0.041 0.195 0.394 0.003 0.478 0.431 0.455 0.375 0.007
0.244 0.331 1.000 -0.107 0.136 -0.258 0.233 0.271 0.088 0.023 -0.198
0.079 0.041 NC 0.437 0.339 0.061 0.082 0.055 0.459 0.490 0.207
0.020 -0.175 -0.107 1.000 0.768 0.233 0.551 0.427 0.035 0.144 0.087
0.493 0.195 0.437 NC <0.001 0.082 <0.001 0.002 0.488 0.297 0.458
0.090 0.119 0.136 0.768 1.000 0.643 0.018 0.057 0.053 0.007 0.063
0.455 0.394 0.339 <0.001 NC <0.001 0.502 0.469 0.476 0.588 0.461
Estradiol
Testosterone
Androstenedione
SHBG
FAI
B
r
P
r
P
r
P
r
P
r
P
NO2-/NO3-
Clitoral volume Clitoral artery PI Uterine artery LH FSH Estradiol Testosterone Androstenedione SHBG FAI NO2-/NO3-
0.340 -0.423 -0.258 0.233 0.643 1.000 0.015 0.116 0.350 -0.073 0.022
0.038 0.003 0.061 0.082 <0.001 NC 0.511 0.428 0.034 0.464 0.490
0.015 -0.040 0.233 0.551 0.018 0.015 1.000 0.582 -0.051 0.779 0.072
0.511 0.478 0.082 <0.001 0.502 0.511 NC <0.001 0.475 <0.001 0.464
0.56 -0.109 0.271 0.427 0.057 0.116 0.582 1.000 -0.031 0.526 0.056
0.471 0.431 0.055 0.002 0.469 0.428 <0.001 NC 0.483 <0.001 0.471
-0.086 0.091 0.088 0.035 0.053 0.350 -0.051 -0.031 1.000 -0.520 -0.010
0.459 0.455 0.459 0.488 0.476 0.034 0.475 0.483 NC <0.001 0.523
0.079 -0.129 0.023 0.144 0.007 -0.073 0.779 0.526 -0.520 1.000 0.146
0.466 0.375 0.490 0.297 0.588 0.464 <0.001 <0.001 <0.001 NC 0.291
0.022 -0.410 -0.198 0.087 0.063 0.022 0.072 0.056 -0.010 0.146 1.000
0.492 0.007 0.207 0.458 0.461 0.490 0.464 0.471 0.523 0.291 NC
The results in bold are the significant values. PI = pulsatility index; LH = luteinizing hormone; FSH = follicle stimulating hormone; SHBG = sex hormone binding globulin; FAI = free androgen index; NC = not calculable.
technique, after a very short learning phase, is easy, not time-consuming, and well accepted by the patients. Moreover, although clitoral ultrasonography depends on free-hand placement and manipulation of the ultrasound probe on the clitoris, we believe that the extreme care we used in avoiding any pressure on the studied structures allowed us to prevent any inadvertent increase of the clitoral artery blood flow and to obtain reproducible results. To the best of our knowledge, we report, for the first time in literature, the clitoral morphofunctional modifications during the menstrual cycle in women who are not aroused sexually. The clitoral body volume showed a progressive increase up to the periovulatory period. This was associated with an equally significant increase in the NO2-/NO3circulating values and with a progressive decrease in the resistances at the dorsal clitoral artery level. In the luteal phase and into the premenstrual period, the registered parameters slowly decreased back to basal values. These phenomena were associated with estradiol variations. The clitoris is highly vascularized, and many vessels branch out in each clitoral artery structure. In contrast with previous studies where the J Sex Med 2008;5:2853–2861
position of the sampled vessels was not anatomically specified and described (thus making the data poorly reproducible) [10–12,14], we analyzed the dorsal clitoral artery at the outer surface level of the clitoral body [19]. In this area, the course of the artery is always detectable in the clitoral neurovascular bundle. We think that a precise description of the vessel sampling procedure is of paramount importance in order to standardize the examination of the clitoral vascularization. In the present study, to analyze by color Doppler the clitoral arteries, we chose to consider the PI instead of the resistance index, because the PI adequately reflects blood flow impedance and may be used when the diastolic frequency shift is absent or reversed. Some studies underlined the importance of ancillary Doppler flow parameters (i.e., peak systolic and end diastolic velocities) [10–12,14] for the evaluation of clitoral hemodynamic changes during the menstrual cycle [14] and sexual arousal [11]. However, our data, registered in the basal state and independently from sexual stimulation, did not show any modification of the Vmax and TAMX during the menstrual cycle. We speculated that in the absence of significant
Hormones and Clitoral Modifications changes in the heart rate or in the vessel diameter, the observed values express no resistance differences in the small capillaries distal from the dorsal clitoral artery. The role of estrogens in the regulation of female sexual function has been frequently reported. Estrogens are known to be vasoprotective and to regulate the vascular smooth muscle tone [20]. Studies with rat models demonstrate that in the clitoris, smooth muscle decreases with age, and that decreased levels of estrogens lead, in the rat’s vagina, to apoptosis in the nerves, smooth muscle, and vascular endothelium [21]. Furthermore, clinical studies on humans have shown that the uterine, vaginal, clitoral, and urethral blood flow, and vaginal lubrication significantly decrease during menopause with a decline in circulating ovarian hormones. On the contrary, estrogen replacement therapy enhances pelvic blood flow in postmenopausal women [22–24]. Moreover, Dennerstein et al. [25], recently demonstrated that “sexual responsivity” (a measure of interest, arousal, enjoyment, and orgasm) improves when, in a spontaneous cycle, estradiol circulating levels are comprised between 650 and 759 pmol/L. These values match those we observed in our patients during the periovulatory period, and the result associated with the highest value of clitoral volume and NO2-/NO3- circulating values, and with the lowest resistances of the uterine and dorsal clitoral arteries. This observation emphasizes that uterine and clitoral vascularization is equally sensitive to estrogen modifications. In addition, our findings agree with Hayashi et al.[26] who reported that estrogens may increase NO production by boosting the expression and activity of endothelial nitric oxide synthase (NOS). We speculated, as did Gragasin et al. [27], that NO, produced by the estrogen-mediated action of NOS on L-arginine, induces a rise of cyclic guanosine monophosphate in postjunctional cells and causes a calcium influx to the clitoral vascular smooth muscle. This causes vasodilatation, relaxation of the clitoral cavernosal tissue, and increase in the clitoral size. In addition, estradiol stimulates neuronal-NOS expression in the preoptic area and exerts an adjuvant influence on NO-producing neurons in the amygdala. Thus, the periovulatory surge of NO2-/NO3- circulating levels may also be involved in the central nervous system activity of the amygdala [28]. The latter is intimately involved in sex and sexuality, and has an important role in the coordination of the endocrine system and in pregnancy. Its stimulation produces sexual
2859 sensations, memories of intercourse, and orgasm. Furthermore, the corticomedial amygdala seems to be able to modulate the activity of gonadotropin releasing hormone neurons and to induce the LH surge and ovulation. Although it has been reported that subjective sexual and genital arousal, and sexual desire do not vary significantly with the phase of the menstrual cycle [2], the intimate relationship between central nervous system, hormonal modulation, and erogenous external genital structures underline that, in nature, sexuality may be conditioned by processes that regulate the menstrual cycle, and probably, the maximal arousability is reached in the periovulatory period [1,29]. In our study, we observed a progressive decrease of the clitoral engorgement during the middle and late luteal phase of the cycle. This finding agrees with Slob et al. [30] who found that objective (increased labium minus temperature) and subjective (semantic scales) changes in sexual arousability to erotic video stimulation are higher in the late follicular period when compared with the luteal phase of the menstrual cycle. We think that the progesterone rise may counteract the positive estrogen effects on clitoral vascularization and engorgement [12] by (i) inducing a moderate decrease of NO2-/NO3- circulating levels; (ii) increasing the b-adrenergic receptor formation; and (iii) enhancing the anticholinergic effect at clitoris level. This may be associated with a fall of the clitoral volume and vascularization, and with the reduction of the vaginal lubrication. These processes may, in lower mammals, be responsible for the sex refusal during the post-estrous period. In humans, the correlation is certainly more tenuous. However, the action mediated by progesterone may, by lowering the libido and the spontaneous arousability, diminish the frequency of sexual intercourse. This could be associated with a decline in oxytocin production [29], causing a decrease in uterine contractility, which could favor the endometrial blastocyst penetration. The exact physiological and biochemical role of androgens in the female sexual function remains controversial and poorly understood [20,31,32]. Whether the effects of androgens are mediated by a direct action on the nervous system or by an effect on the genital system, or both, has yet to be established. In women, the role of androgens is confounded by the fact that their synthesis and metabolism take place in the ovaries, the adrenal glands, and the peripheral tissue. Furthermore, active androgens may be synthesized on demand in J Sex Med 2008;5:2853–2861
2860 target tissues [31–33]. Thus, although Salonia et al. [34] recently provided the clinically applicable ranges for androgens throughout the menstrual cycle, we think, in agreement with Labrie et al. [35], that the levels of circulating androgens may not give the correct information on the bioavailability of their metabolites. Davis and Tran [36] have suggested that androgen insufficiency is associated with impaired sexual function. However, two large recent studies have failed to find any correlation between sexual function and circulating androgen levels measured as total testosterone and FAI in 2,900 pre- and perimenopausal North American women [37], and as free and total testosterone in 1,021 Australian women [38]. It was suggested that androgens have no effect on sexual arousal “per se,” but may influence other aspects of sexual desire such as thoughts and fantasies. Our data showed that throughout the menstrual cycle, there are no significant modifications in the concentrations of circulating androgens. We speculated that, although androgens have a well-known midcycle elevation, their circadian variations and their local tissue concentration (correlated with local peripheral conversion) mediate the tissue-specific response and inhibit defining properly which metabolite regulates the physiology of central and peripheral tissues involved in sexual function. Conclusions
During the normal menstrual cycle, clitoral anatomic and vascular modifications are highly and cyclically influenced by steroid hormones and neuromodulators. How this may be integrated into the sexual function of all women will necessitate further, broader, and prospective studies. Corresponding Author: Cesare Battaglia, MD, PhD, Department of Obstetrics and Gynaecology, Alma Mater Studiorum—University of Bologna, Via Massarenti, 13-40138 Bologna, Italy. Tel: 39-051-6364377; Fax: 39-051-6364377; E-mail: [email protected] Conflict of Interest: None declared.
Statement of Authorship
Category 1 (a) Conception and Design Cesare Battaglia (b) Acquisition of Data Fulvia Mancini; Arianna Cianciosi; Paolo Busacchi; Fabio Facchinetti; Rossella Elena Nappi; Cesare Battaglia J Sex Med 2008;5:2853–2861
Battaglia et al. (c) Analysis and Interpretation of Data Nicola Persico; Domenico de Aloysio
Category 2 (a) Drafting the Article Cesare Battaglia (b) Revising It for Intellectual Content Fulvia Mancini
Category 3 (a) Final Approval of the Completed Article Domenico de Aloysio References
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