DRAFT—PLEASE TO NOT CITE—JUNE 23, 2002 Female Sexual Arousal Response Using Serial MR Imaging with Initial Comparisons to Vaginal Photoplethysmography: Overview and Evaluation Julia R. Heiman1, Ph.D, and Kenneth R. Maravilla2, M.D 1 Department of Radiology, University of Washington, Seattle, WA 2 Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, WA Correspondence and Reprints: Julia R. Heiman, Ph.D. University of Washington Outpatient Psychiatry Center 4225 Roosevelt Way, NE, Suite 306 Seattle, WA 98105 Phone: 206-598-7152, Fax: 206-598-7794, Email: jheiman@u.washington.edu Pfizer, Inc, Groton CT. and EPIX Medical, Inc., Cambridge, MA provided funding for this research 2 INTRODUCTION Although the sexual arousal and response of women has been empirically studied over the past four decades, measurement methods have been minimally available. This is particularly the case in understanding the physiology and psychophysiology of female sexual dysfunction (FSD), even though problems of sexual functioning may be as prevalent as 25-40% (Bancroft, et al, 2003; Laumann et al.1999). Attempts to test and validate differences between functional and dysfunctional sexual response, as well as treatments for FSD subtypes have been difficult due in part to the limited availability of physiological measures that are reliable, reproducible and relatively non-invasive. Recently we presented a new method for objectively monitoring female sexual arousal response using serial magnetic resonance imaging (MRI) combined with MS-325 (EPIX Medical, Inc.) an investigational new gadolinium-based blood pool agent (1). Another female sexual arousal measurement method, vaginal photoplethysmography (VPP), has been used as a relative arousal measurement method for over 3 decades (2, 3). VPP has been shown to be a sensitive and reliable indicator of female sexual arousal, but it remains limited due to its lack of absolute scaling and thus must be used in within-subject (crossover) designs. We have conducted initial studies to develop the MRI method and technique for female genital measurement. The present paper is intended to review these results and to evaluate the advantages and disadvantages of the MS-325 enhanced serial MRI technique and provide comments on the comparison of it to VPP. Both techniques also included subjective measures of arousal. MATERIALS AND METHODS USED In order to give specifics on the use of MRI and comparison to VPP, we will detail the methods used thus far. Participants This work will summarize the work on 28 healthy, sexually functional women. Premenopausal women between the ages of 23 and 38 postmenopausal women were between the ages of 53 and 66. The university Institutional Review Board approved the protocol and consent form, and subjects signed a written consent prior to study entry. 3 Subjects were recruited by the use of advertising in local newspapers and flyers placed on the university campus. Potential subjects were initially screened by telephone. Subjects were invited to the study site if they were healthy and had no sexual disorders. All subjects underwent a complete physical examination, including a pelvic examination and a Papanicolaou (Pap) smear. Blood samples were drawn to assess baseline chemistry, hematology, coagulation, iron, and endocrinology values; and a urine sample was obtained for microscopic urinalysis in all subjects. All results showed no significant abnormalities. Pregnancy was excluded in the premenopausal group at both the screening visit and on the day of the MR visit. Premenopausal females were scanned (and for those women in the comparison study, run in the VPP procedure with in 8 days of that window) between day 7 and day 21 inclusive of their menstrual cycle. The postmenopausal subjects had additional hormonal testing performed including estradiol, serum luteinizing hormone, and follicle stimulating hormone levels, which had to be within a specified laboratory range typical for postmenopausal women. Subjects with a history of previous vaginal surgery, hysterectomy, abnormal menstrual cycles, and/or having taken hormone replacement therapy or birth control pills within the preceding six months were excluded from the study. There was some weight limits due to the MRI procedure. Weights ranged between 47 and 90kg and height from 157cm to 180cm. Safety assessments, including vital signs, 12-lead electrocardiograms (ECG), blood samples, urine tests, and physical examination were documented at specified intervals up to 96 hours postinjection of MS-325. These are detailed in the first report on MRI results (Deliganis et al., 2002). Measures MRI Measures Contrast Dosing. MS-325 is a small (MW 957) gadolinium chelate that binds reversibly to albumin. While bound to albumin, the agent increases its relaxivity 4-10 fold, depending on field strength (13). The agent shows a biexponential clearance, with a terminal clearance phase half-life of 10 to 14 hours (14). MS-325 is currently in phase III clinical 4 trials for use in MR angiography. MS-325 is formulated at a concentration of 0.25mmol/mL. All twelve women received a dose of MS-325 calculated at 0.05mmol/kg of body weight. Contrast MS-325 was injected intravenously at a maximum rate of 1.5mL/seconds, followed by a 30mL normal saline flush. Videotaped material. The videotapes (the same videotapes as were used in the VPP when we compared the 2 methods) contained segments of neutral and erotic (sexually explicit) material. The videotape format included a 21-minute neutral section, a 15-minute erotic section, and another 9-minute neutral section. MR imaging was performed during the entire 45-minute videotape presentation. Subjects were able to see and hear the video material through a fiberoptic audiovisual display system (Avotec, Inc., Stuart, FL) while in the magnet bore. (Two subjects (one pre- and one post-menopausal female) were injected with MS-325 but saw only neutral video material, serving as controls.) Subjective Measurements. Subjective (self-report) sexual responses were measured using a 30 item Film Evaluation Scale (FES) that has been found to be a reliable and sensitive measure of sexual response and affect 4, 5 All subjects were asked to complete the FES at three time-points: prior to viewing the neutral video material, prior to viewing the erotic video material, and immediately after the MRI was completed (evaluating their responses during the erotic videotape). MR Imaging. MR images were obtained on a GE Signa 1.5 Tesla system (General Electric Medical Systems, Waukesha, WI). Images of the perineum were acquired using specially designed phased-array (PA) coils built by scientists at the University of Washington. A 10cm PA coil (15) was placed anterior to the pubic symphysis and a larger 2-coil PA receiver was positioned posterior to the pelvis. Subjects were imaged using a T1-weighted fast, three-dimensional (3D) spoiled gradient recalled echo (SPGR), acquired in an axial orientation (TR/TE/flip angle = 11/1.7/35°, 256x256, FOV = 24cm, 2.0mm slice thickness, 1 NEX). Thus, voxel size was very small, measuring approximately 0.9 x 0.9 x 2 mm for a total voxel volume of 1.6 mm3. This voxel size was felt to be warranted since the clitoral structures are small and require fine detail imaging for accurate measurement. The use of local PA coils boosted the signal-to-noise ratio to enable detailed imaging of the external genitalia and to provide both reliable anatomical structure outlines and measures of signal intensity (SI) within small regions of interest 5 (ROI). Shim values, transmitter gain, and receiver gain settings were kept constant for the entire 45 minute imaging session to provide accurate time versus signal intensity plots for calculation of regional blood volume changes. One 3D-volume series was obtained prior to contrast administration. Intravenous contrast injection was followed by a 3minute delay for contrast level equilibration. MR images were obtained on a GE Signa Horizon Echo Speed 1.5 Tesla system (General Electric Medical Systems, Waukesha, WI). Images of the perineum were acquired using specially designed phased-array (PA) coils built by scientists at the University of Washington. A 10cm PA coil (17) was placed anterior to the pubic symphysis and a larger 2-coil PA receiver was positioned posterior to the pelvis. Subjects were imaged using a T1-weighted fast, three-dimensional (3D) spoiled gradient recalled echo (SPGR), acquired in an axial orientation (TR/TE/flip angle = 11/1.7/35°, 256x256, FOV = 24cm, 2.0mm partitions, 1 NEX). Thus, voxel size was very small, measuring approximately 0.9 x 0.9 x 2 mm for a total voxel volume of 1.6 mm 3. This voxel size was selected because the clitoral structures are small and require fine, detailed imaging for accurate measurement. The use of local PA coils boosted the signal-to-noise ratio to enable detailed imaging of the external genitalia and to provide both reliable anatomical structure outlines and measures of signal intensity (SI) within small regions of interest (ROI). Fat saturation was not used. Shim values, transmitter gain, and receiver gain settings were kept constant for the entire 45 minute imaging session to provide accurate time versus signal intensity plots for calculation of regional blood volume changes. One 3D-volume series was obtained prior to contrast administration. Intravenous contrast injection was followed by a 3-minute delay for contrast level equilibration. Postcontrast images were then acquired every three minutes while subjects simultaneously viewed the video material. (Include flow chart?) VPP Measures A vaginal photoplethysmograph (Behavioral Technology Instruments, Salt Lake City, Utah) was used to measure vaginal pulse amplitude (VPA) and vaginal blood volume (VBV) responses. The vaginal photoplethysmograph has been shown to be a valid and reliable measure of sexual arousal in women. The software program AcqKnowledge III, 6 version 3.3 (BIOPAC Systems, Inc, Santa Barbara, Calif) and data acquisition unit (model MP100WS, BIOPAC Systems, Inc) were used with a personal computer (Power Macintosh 6100/70, Apple, Cupertino, Calif) to collect, convert (from analog to digital) and transform data. Subjective Measurements. Subjective (self-report) sexual responses were measured using the 30 item Film Evaluation Scale (FES). A shortened 7 item of the version of the FES was given before presentation of a neutral video (after the subject had inserted the photoplethysmograph) and immediately after the presentation of the neutral videotaped the material. The full length FES was completed immediately after the termination of erotic segment (before removal of the photoplethysmograph). VPP Data Sampling and Reduction Vaginal responses to neutral and erotic stimuli were measured during the initial adaptation period and throughout the presentation of the videotaped material. Physiological responses were sampled at a rate of 60 samples per second throughout the adaptation (baseline phase), the entire 10 minutes of neutral film, and 14 minutes of erotic film. The BIOPAC software allowed for an automated transformation of raw data into VPA and VBV scores that were ultimately used in the statistical analysis. Data Analysis The images were subjectively evaluated for pre- and postcontrast image quality and for visualization of the anatomy of the genital tract by two experienced radiologists. Signal intensities were measured by selection of ROI's positioned within the vaginal wall, vaginal mucosa, common femoral vein, muscle, clitoris, and in air outside the subject to provide a measure of background noise. The size of the ROI varied depending on the size of the structure being analyzed. For example, across all subjects the measurements of the vaginal wall ROI ranged in size between 4 mm2 and 22mm2, while the clitoral body ROI was between 7 mm2 and 70 mm2. Within each subject, the size of the ROI was kept consistent at each time point throughout the neutral and erotic segments. Image volumes for each time point within a given subject were analyzed in random order to attempt to reduce bias. However, all volumes with a given subject were analyzed together. 7 Changes in Blood Volume Relative regional blood volume was estimated from signal intensity versus time curves derived from ROI measurements of the vaginal wall and clitoris using the following equation: rRBVstructure = SIstructure(t) - SIstructure (t0) [Equation 1] SIfemoral (t) - SIfemoral (t0) Where rRBV is the relative regional blood volume and SI (t) refers to the signal intensity measurements from the region of interest taken at the various time points, and t0 refers to the precontrast scan. The denominator compensates for the slow clearance of MS-325 over the time of the study. In order to smooth statistical fluctuations in SI measurements of the femoral vein, the femoral signal intensity data was fit to a bi-exponential clearance, and then the fit values were used in Equation 1. ROI measurements were taken separately over the body and glans of the clitoris. From these signal intensity measurements, SI versus time curves were generated and rRBV measurements over time were calculated using femoral vein SI's corrected for the bi-exponential decay of MS-325 over time. To determine whether erotic video material differentially changed estimated rRBV, the estimated rRBV changes were fit as a function of time to the following three-parameter model (A, B, and C) to account for these changes: rRBV(t) = A + B t + C video(t), [Equation 2] where (t) is the time, video(t) equals 1 during erotic video and 0 during neutral video corresponding to the last eleven image data sets acquired. Typically, this represents 18 minutes of neutral video material, followed by 15 minutes of erotic video material and 6 minutes of neutral video material. The "A" coefficient depends on the initial blood volume and "B" reflects both slow extravasation of the agent over time as well as potential incomplete return of blood volume to a baseline state after the erotic video. "C" reflects the video-dependent changes in rRBV. The presence of a significant influence of the C term in Equation 2 was tested using the t-statistic in the regression, and the relative change 8 in rRBV associated with the video segments were estimated and tabulated as C/A on a subject-by-subject basis for clitoral body and glans clitoris measurements. Changes in Clitoral Volume The various structures comprising the clitoris, including the crura, body, and glans, were identified and outlined from each slice containing clitoral tissue. These areas were summed and the total volume was measured at each time point using a planimetric method, with volumes reported in cubic centimeters (cc). If there was difficulty defining the precise boundary between the clitoris and adjacent mucosal or glandular tissue, all of the enhancing tissues were included. This was often the case in the area of the clitoral body, but this was not felt to cause a problem with comparative measurements within a given subject since the same anatomical areas were outlined at each time point. However, this method may have resulted in a slight over-estimation of the true clitoral volume in some subjects. Data analysis and graphic presentation was done using a two-tailed t-test with commercially available spreadsheet and graphing programs. Design and Procedures Protocols were carried out at two separate locations at the University of Washington Medical Center 1) MR imaging at the Imaging Center of the Hospital and 2) Vaginal photoplethysmography at the Reproductive and Sexual Medicine Clinic of the outpatient clinics. The same neutral and erotic videos were used at both sites, but were presented in counterbalanced order. A familiarization visit conducted at each site prior to the experimental session ensured that women were comfortable with the procedures. The same FES scale was used at both sites to measure sexual arousal and affect. VPP. During the VPP experimental sessions, the female participant inserted the vaginal probe in the privacy of the experimental room. She then watched a 10 minute neutral and 14 minute erotic film segment. The data were collected on a computer located in a separate room and an intercom allowed for communication between the subject and experimenter. Subjective data were collected before presentation of neutral video material but after the subject had inserted the photoplethysmograph, immediately after 9 the presentation of the neutral videotaped material, and after the termination of the erotic segment, but before removal of the photoplethysmograph. RESULTS 1. From Deliganis et al. 2002 MRI response. There was an increase in both degree of enhancement and overall size of the clitoris during the erotic video segment as compared to the first neutral segment. However when ROI measurements of the vaginal wall were analyzed, there was very low contrast enhancement and no significant trends were observed in the vaginal wall blood volume curves. Vaginal mucosa was not well visualized as a separate structure and attempts to generate measurements of this very thin structure proved unfeasible. rRBV. For the glans clitoris, rRBV changed 40 + 10% (SEM) in the subjects viewing the erotic video versus -3% + 5% for the control group. In the clitoral body, we found rRBV increased 24% + 8% in the group shown erotic videos versus 3% + 8% for the neutral only control group. No significant differences were observed for the pre- and post-menopausal groups, although average rRBV increases were slightly greater in the post-menopausal group in the clitoral body (pre-menopausal, 9% + 4%, post-menopausal, 39% + 12%). Clitoral volume. Quantitative changes in clitoral volume over time proved more robust than rRBV measures. The average clitoral volume increased from 10.74cc at baseline (during the first neutral segment) to 21.17cc while viewing the erotic material and then decreased to 15.42cc while viewing the second neutral segment. There was no significant difference in clitoral volume changes between pre-menopausal subjects and post-menopausal subjects. Clitoral volume measurements compared between the first neutral and erotic segments in the pre-menopausal group showed an average increase of 107% + 26% (range of 45 - 181%), while the post-menopausal group averaged 129% + 43% (range of 55 - 280%). No significant change in clitoral volume was measured in the control subjects that viewed only neutral material. 10 Subjective sexual arousal. A review of the subjective arousal questionnaire showed that all of the 12 study subjects reported no feelings of sexual arousal on the first two queries administered prior to viewing the initial neutral and again prior to viewing the erotic videotape segments (average score = 1.0). After viewing the erotic material, all subjects reported sexual arousal with an average score for all subjects of 3.87 (range of 2 - 6). Adverse events. Given the Phase I testing of the MS-325, a review of adverse events was required and provides additional information on the MRI lab context. Subjects tolerated the procedure well and there were no serious adverse events reported. A total of 40 adverse events of mild to moderate severity were noted. Of the adverse events reported, 17 were considered unlikely related to study drug, while 5 were considered possibly and 18 were considered probably related to the use of the study drug, MS-325. All of the 23 adverse events considered possibly or probably related to study drug resolved spontaneously without medical intervention. A breakdown of those adverse events revealed 14 reports of groin itching, burning, or tingling in the perineum. There were also 2 reports of nausea, 2 reports of general malaise or fatigue, and 1 report each of metallic taste, scalp itch, lightheadedness, and a thick, tingling tongue. One subject demonstrated a transient increase in urine white blood cells. No other clinically significant laboratory changes were reported. There were no clinically significant changes seen in the electrocardiograms, vital signs, or physical examination findings. 2. From Maravilla et al, in press The purpose of this study was to see if the MRI responses in healthy women could be replicated across two separate sessions. The two sessions occurred 45 min apart Subjective sexual arousal. In Session 1, the mean score across all subjects taken after the sexually-explicit video material was shown was 4.2 (range 2.33 – 5.33); while in Session 2 the same score was 4.3 (range 2.0 – 6.33). Arousal scores were tabulated for each video to determine if one scored significantly different than the other. Figure 1 shows the correlation curve of the arousal scores among all subjects between Session 1 and Session 2. The r2-value was 0.52. The average arousal scores were 4.3 (range 2.0 – 5.33) for 11 Video A and 4.2 (range 2.0 – 6.33) for Video B. Thus, there was no significant difference noted in the arousal response between Session 1 and Session 2, nor was there a significant difference between the two stimulus videos that were used. Signal intensity. The average percent change in signal intensity from baseline to arousal increased approximately 90% in Session 1 and 32% in Session 2, The combined percent change of both sessions was over 60%, with a range between 13% and 177%. Although there appears to be a decrease in the percent change in clitoral signal intensity with arousal in Session 2 versus Session 1, this is likely related to residual engorgement from the arousal response during the initial imaging session. Due to MR tuning changes required prior to Session 2, it was felt that baseline signal intensity values could not be reliably extrapolated from Session 1 to Session 2 in order to normalize signal intensity measurements for Session 2. For this reason, the SI∆ data was not felt to be as reliable as the anatomic clitoral volume analysis. Clitoral volume. Average increase in clitoral volume from baseline to arousal increased approximately 107% in Session 1 and 110% in Session 2. For all subjects, the combined percent change of both sessions was over 108%, with a range between 16% and 260%, There was an increase in baseline volume for Session 2 present in varying degrees in 8 out of 9 subjects and likely represents residual clitoral engorgement following the sexually-explicit video presentation during Session 1. The remaining subject showed no change in baseline volume between the two sessions. The best fit line plotting the percent change in clitoral volumes for all subjects between Session 1 and Session 2 are shown in Figure xx. There was excellent correlation between the two imaging sessions, as indicated by the 0.95 r2-value. Analysis of both pre- and postmenopausal subject groups also showed good correlation for each group with calculated r2-values of 0.94 and 0.98, respectively, in the percent clitoral volume change between Session 1 and Session 2. 3. Heiman et al, submitted This study compared the use of MRI and VPP in the same women. 12 VPP Results. VPA = 61-70% increase during erotic video. VBV = 9-25%) Subjective sexual arousal. Self-reported arousal to erotic video (7-point scale). Mean (SD) = 4.1 (1.60) to 4.6 (1.21), depending on the video. Correlations between subjective and MRI and VPP measures. Change Change Change Change During the tape, I felt VPA VBV AVW BVW (1) SEXUALLY AROUSED (2) ANY genital feeling 0.65* 0.47 0.50 0.51 0.73* 0.54 0.45 0.42 (3) GENITAL PULSE THROB 0.59* 0.47 0.54 0.55 0.10 0.09 0.43 0.34 (4) GENITAL WETNESS/ LUBRICATION Significant at p<.05 MRI-VPP measurement correlations. There were no significant correlations between MRI and VPP measures. DISCUSSION Data on small sample, thus preliminary 1. MRI reveals important data on clitoral blood volume and anatomical volume. This is the first time précised images have been available in a less intrusive measurement. Also the absolute measurement of MRI is an advantage. 2. MRI shows good test-retest reliability. 3. VPP, especially VPA measure, shows good correlations with subjective report. 4. VPP and MRI may be measuring somewhat independent system responses. However, it is also the case that the MRI method was not scanning the same level of tissue as that of the VPP recording. 13 5. Data on small samples and are thus preliminary and need further replication by other sites. More discussion and critique in final draft. Including use of contrast agent And a number of figures will be included to illustrate findings. References (very rough, unfinished) 1. Deliganis, A, Maravilla, K, Heiman, J., et al. Dynamic MR imaging of the female genitalia using MS-325: Initial Experience Evaluating the Female Sexual Response. Radiology, 2002. 2. Laan E, Everaerd W, Evers A. Assessment of female sexual arousal: Response specificity and construct validity. Psychophysiology. 1995; 32:476-485. 3. Heiman, J.R. (1998). Psychophysiological models of female sexual response. 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