Effect of pituitary down-regulation on the ovary before in vitro fertilization as measured using three-dimensional power Doppler ultrasound

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1 FERTILITY AND STERILITY VOL. 79, NO. 5, MAY 2003 Copyright 2003 American Society for Reproductive Medicine ublished by Elsevier Inc. rinted on acid-free paper in U.S.A. Effect of pituitary down-regulation on the ovary before in vitro fertilization as measured using three-dimensional power Doppler ultrasound Ilkka Y. Järvelä, M.D., a,b ovilas Sladkevicius, M.D., c Simon Kelly, M.D., d Kamal Ojha, M.D., b Stuart Campbell, M.D., b and Geeta Nargund, M.D. b St. George s Hospital Medical School, London, United Kingdom This research has been supported by a Marie Curie Fellowship (to I. Y. J.) of the European Community rogramme Quality of Life and Management of Living Resources, contract number QLRI-CT Reprint requests: Ilkka Y. Järvelä, M.D., Department of Obstetrics and Gynaecology, Oulu University Hospital, Oulu, Finland (FAX: ; ijarvela@ cc.oulu.fi ). Received May 23, 2002; revised and accepted September 5, a Department of Obstetrics and Gynaecology, Oulu University Hospital, Oulu, Finland. b Diana, rincess of Wales Centre for Reproductive Medicine, Academic Department of Obstetrics and Gynaecology, St. George s Hospital Medical School. c Department of Obstetrics and Gynaecology, Kvinnokliniken, Universitetsjukhuse MAS, Malmö, Sweden. d Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, Québec, Canada /03/$30.00 doi: /s (03) Objective: To evaluate the changes taking place in the ovaries during pituitary down-regulation. Design: rospective observational study of women undergoing IVF. Setting: A tertiary referral center for assisted reproduction. atient(s): Forty women who received the long buserelin acetate protocol. Intervention(s): Transvaginal three-dimensional power Doppler ultrasound examinations before and after pituitary down-regulation. Main Outcome Measure(s): Ovarian volume, number of follicles, vascularization index (VI), flow index (FI), vascularization flow index (VFI), and mean gray value (MG). Result(s): Before the pituitary down-regulation, the dominant ovary was larger in volume and had a lower MG than the nondominant ovary. After the down-regulation, there was a significant decrease in the volume and number of follicles and an increase in MG. After pituitary down-regulation, the dominant and nondominant ovaries did not differ from each other in any of the parameters. olycystic ovaries were larger than normal ones before and after the down-regulation, without any differences in MG, VI, FI, or VFI. Right and left ovaries did not differ from each other after the down-regulation. Conclusion(s): The differences observed between dominant and nondominant ovaries seem to disappear after pituitary down-regulation. In addition, polycystic ovaries were always larger than the normal ones, but no differences could be detected in the stromal brightness or vascularity either before or after the administration of GnRH agonist therapy. (Fertil Steril 2003;79: by American Society for Reproductive Medicine.) Key Words: GnRH agonist, three-dimensional ultrasound, transvaginal color Doppler, transvaginal ultrasound ituitary suppression with GnRH agonist is commonly used during IVF since it enhances pregnancy rates after IVF (1). Satisfactory pituitary down-regulation is assessed by measuring pituitary and ovarian hormones and by ultrasound findings of thin endometrium and ovaries with only small follicles. The ovarian response to pituitary desensitization with GnRH analog has been evaluated in detail using two-dimensional (2D) Doppler ultrasound (2 6). The stromal blood flow velocities and ovarian volumes seem to decrease; nevertheless, the findings are not totally consistent (2 6). In 2D Doppler ultrasound studies, the conclusions concerning the vascularization and blood perfusion in the ovaries are drawn from blood flow velocities (and indices) obtained from a subjectively chosen artery lying in a 2D plane that is also subjectively chosen. The latest technical achievement in the field of ultrasound is three-dimensional (3D) power Doppler ultrasound, which theoretically provides the possibility of assessing the volume, blood perfusion, and vascularization of the whole target organ. So far, this modality has been used in quantifying the power Doppler signal in polycystic ovaries (7), in adnexal masses (8), and in the endometrium (9, 10). The aim of our study was to evaluate the changes taking place in the ovaries during pi- 1129

2 tuitary down-regulation. In this study, the ovarian volumes, quantification of the power Doppler signal, and tissue echogenity (brightness) were assessed using 3D power Doppler ultrasound. ATIENTS AND METHODS The patients were women attending the Assisted Conception Unit at St. George s Hospital Medical School for IVF. Approval was obtained from the Ethics Committee of the Medical School, and each subject gave a written informed consent before participating in the study. Women with uterine fibroids, endometriosis or endometrioma, single ovary, salpingectomy, ovarian cystectomy, or known contraindications for the use of GnRH agonists and hmg were excluded from the study. We reported on the differences between dominant and nondominant ovaries (11) and between normal and polycystic ovaries (12), having taken measurements during the late follicular, as in this study. Some of the patients are the same, and therefore the analysis here focuses on the effect of pituitary suppression. Each patient had their FSH and LH levels measured between days 1 and 5 of their cycles during the previous 6 months. The first ultrasound examination took place during cycle days 8 16 in the cycle before beginning drug therapy. Where a corpus luteum was visible by ultrasound, the patient was excluded from the study. The ovaries were categorized as either normal or polycystic according to the following criteria: if eight or more subcapsular follicles of 2 8 mm in diameter in one plane were detected in either of the ovaries, the patient was categorized as having polycystic ovaries (CO; Fig. 1A); otherwise, the ovaries were considered normal (Fig. 1B). Ovarian volume, stromal volume, and stromal echogenity were not used as criteria, since neither the Adams et al. (13) study nor any of the other available definitions (14) provide objective criteria or precise cutoff values to determine them (15). Because the COs in patients with and without polycystic ovarian syndrome (COS) do not seem to differ from each other in terms of volume (16) or stromal color Doppler characteristics (17 19), no subdivision according to endocrine or clinical manifestations was performed in the CO group. In cases in which no pathology in the uterus or ovaries could be observed in the first examination, GnRH agonist (buserelin acetate 0.5 mg SC per day) was started on cycle day 21. Two weeks later, the patient was scanned, and if an endometrium under 4 mm and ovaries with only small antral follicles were detected, the stimulation of ovaries using FSH was initiated. Otherwise the patient continued using GnRH agonist and was scanned again 1 week later. The 3D power Doppler ultrasound examinations were performed using Kretz Combison 530D Voluson (Kretztechnik-Medison, Zipf, Austria). It was equipped with a transvaginal 3- to 7-MHz volume transducer, which has a 100 field of view. Identical preinstalled instrument settings (color gain, 45.6; pulse repetition frequency, 0.5; color power, 2; wall motion filter, 72; frame rate, 4 6) were applied in all patients. The ultrasound examinations were performed in a manner similar to that in the earlier studies (11, 12). Briefly, after the 3D volume acquisition, the stored volumes were transferred to a personal computer using a DICOM (Digital Imaging and Communications in Medicine) connection for subsequent analysis. The analysis was performed using the VOCAL - Imaging program (Virtual Organ Computer-aided AnaLysis) version 4.0, which is integrated in Kretztechnik s Voluson 530D sonography system (Kretztechnik-Medison, Zipf, Austria). The contour of the ovary was traced manually in six different planes using VOCAL. Once the volume of the ovary was obtained, the program automatically calculated gray-scale and color-scale indices for the ovary. The stored volume data obtained using 3D power Doppler sonography is defined by voxels (smallest unit of volume). Gray-scale voxels contain all 3D information from black to white, the lowest value (intensity) being 0 and the highest being 100 (g0...g100). A similar scale was used for color values (c0...c100). According to these values, four indices were calculated: mean gray value (MG), vascularization index (VI), flow index (FI), and vascularization flow index (VFI). MG expresses the mean echogenity or brightness of the defined volume (range, voxels). VI measures the ratio of color voxels to all the voxels in the defined volume and represents the vessels in the tissue and is expressed as a percentage (%). FI, the mean value of the color voxels, represents the average intensity of flow (range, ). VFI is the mean color value in all the voxels in the defined volume and is a feature of both vascularization and flow (range, ). In addition to this, the total number of follicles was calculated in each ovary. We have earlier assessed the intra-observer and interobserver variability in ovarian volume, color, and grayness index measurements using transvaginal 3D power Doppler ultrasound (20). According to our results, it seems that the measurement of the indices is reproducible and allows their use in clinical research. Statistical analyses were performed using the SSS program (SSS Inc., Chicago, IL). Departure from a normal distribution was assessed using the Kolmogorov-Smirnov test. aired t-tests were used for normally distributed data, and the Wilcoxon test for skewed data..05 was considered statistically significant. All values given are means ( SD). RESULTS We examined the ovaries of 40 women using 3D ultrasound. According to ultrasound criteria, 29 of the patients 1130 Järvelä et al. GnRH effect on ovary in 3D ultrasound Vol. 79, No. 5, May 2003

3 FIGURE 1 olycystic ovary (A) and normal ovary (B) after pituitary suppression. The volume of the polycystic ovary is 19.3 cm 3, and it contains 26 small follicles. The volume of the normal ovary is 4.2 cm 3, and it contains 11 small follicles. The histogram presents the data for vascularization index (VI), flow index (FI), vascularization flow index (VFI), and mean gray value (MG). FERTILITY & STERILITY 1131

4 TABLE 1 The effect of GnRH agonist therapy on right and left ovaries (n 40). TABLE 2 The effect of GnRH agonist therapy on ovaries in ovulatory cycles (n 28). Right ovary Volume, cm Vascularization index Flow index a Vascularization flow index Mean gray value Follicles Left ovary Volume, cm Vascularization index Flow index a Vascularization flow index Mean gray value Follicles a.019 for the left and right side. had normal and 11 had COs. The LH/FSH ratio was higher in patients with COs, (1.04, SD 0.50) in comparison with patients with normal ovaries (0.59, SD 0.26;.027). Leading follicles during the late follicular could be detected in 28 out of 40 cycles, in 23 out of 29 patients with normal ovaries (79.3%), and in five out of 11 patients with COs (45.5%). The duration of GnRH agonist therapy ranged from 14 to 33 days (mean, 16 days). The results of analysis of the 3D images acquired during the late follicular revealed that when comparing left and right ovaries (n 40) there was no difference in any parameter other than a higher FI on the left side (Table 1). The ovary with the dominant follicle was larger and had lower MG in comparison with the nondominant ovary, but no differences were detected in VI, FI, or VFI (Table 2). The mean volume (SD) of the leading follicle was 2.1 cm 3 (1.7). The right CO was larger than the left one before the initiation of GnRH agonist therapy (Table 4). COs were larger than the normal ones on both the right (.001) and left (.001) sides, without any differences in VI, FI, VFI, or MG. The total follicle count was also higher in the polycystic than in the normal ovaries on both sides. After the administration of GnRH agonist, there was an overall decrease in the ovarian volume and total follicle count and a rise in MG (Tables 1, 2, 3, and 4). The color indices, like ovarian volumes and MG, corresponded between the right and left sides (Table 1). There were no longer any differences between the dominant and nondominant ovaries of the preceding cycle (Table 2). In addition to a higher follicle count, COs were larger than the normal ones Dominant ovary Volume, cm a Vascularization index Flow index Vascularization flow index Mean gray value b Nondominant ovary Volume, cm a Vascularization index Flow index Vascularization flow index Mean gray value b a.001. b.001 for the dominant and nondominant ovary. on both sides (right.001; left.001), but no other differences could be detected after the pituitary suppression. The volumes of the right and left CO also no longer differed from each other (Table 4). DISCUSSION The aim of the study was to evaluate the ovarian response to pituitary down-regulation using 3D power Doppler ultrasound. GnRH-induced pituitary down-regulation was fol- TABLE 3 The effect of GnRH agonist therapy on normal ovaries (n 29). Right ovary Volume, cm Vascularization index Flow index a Vascularization flow index Mean gray value Follicles Left ovary Volume, cm Vascularization index Flow index a Vascularization flow index Mean gray value Follicles a.021 for the left and right side Järvelä et al. GnRH effect on ovary in 3D ultrasound Vol. 79, No. 5, May 2003

5 TABLE 4 The effect of GnRH agonist therapy on polycystic ovaries (n 11). Right ovary Volume, cm a Vascularization index Flow index Vascularization flow index Mean gray value Follicles Left ovary Volume, cm a Vascularization index Flow index Vascularization flow index Mean gray index Follicles a.007 for the left and right side. lowed by a significant decrease in the ovarian volumes and follicle count and by an increase in MG, without any changes in the color indices VI, FI, or VFI. The dominant and nondominant ovary no longer differed from each other after the down-regulation. In addition, COs were larger in volume and contained more follicles than the normal ones, but no other differences could be detected between them. We have reported earlier about the differences between dominant and nondominant ovaries (11) and about the differences between normal and COs during the late follicular (12) using the same 3D ultrasound equipment. Because the patient group is partly the same and the circumstances during the late follicular are discussed in detail in those manuscripts (11, 12), we focus here on the effect of GnRH therapy. The effect of GnRH agonist therapy on ovarian volume has been assessed in two earlier studies using 2D ultrasound (4, 6). Sharara et al. used oral contraceptives 3 6 weeks before commencement of GnRH agonist therapy. According to them, pituitary desensitization with leuprolide acetate for 21 days had no effect on overall ovarian volume measurements (4). Dada et al. used either intranasal nafarelin or SC buserelin acetate from the first day of the menstrual cycle for 21 days. They discovered a decrease of almost 30% in the ovarian volume on both sides and no difference between the two GnRH agonists used (6). In our study, GnRH therapy was associated with a decrease in the ovarian volumes, which were most prominent in the dominant ovaries; nevertheless, there was a trend toward a decrease in the nondominant ovaries too. During the late follicular, the dominant ovary was larger and had a lower MG than the nondominant one, but after the GnRH agonist therapy there was no longer any discernable difference. An increase in MG was detected in both the dominant and nondominant ovary. For the most part, the changes in the volume and MG are probably caused by the disappearence of the dominant follicle. The dominant follicle contains fluid, which has low echogenity in ultrasound. The disappearance of the follicle decreases the whole ovarian volume, while the average ovarian echogenity increases. The overall decrease in the number of follicles after pituitary suppression probably also explains the increase in MG in the nondominant ovaries. Another option is loss of stromal fluid, which would increase the mean ovarian echogenity. The connection between fluid and ovarian echogenity has been observed in massive ovarian edema (21 24), where the accumulation of interstitial fluid within the stroma expands the ovary and diminishes its echogenity. The color indices in our study were similar in the dominant and nondominant ovary at the late follicular, and they remained constant after the GnRH agonist therapy. In earlier studies using 2D color Doppler, it was observed that the perifollicular blood flow velocities rise gradually during the periovulatory period, suggesting a marked increase in blood flow at that time (25, 26). The effect of GnRH agonist on the ovarian blood flow has also been assessed by several authors (2, 3, 5, 6). In study by Dada et al., the ovarian artery resistance index (RI) and pulsatility index (I) increased and the peak systolic velocity (SV) and time-averaged maximum velocity (TAMX) decreased during the GnRH agonist therapy. On the contrary, inkas et al. could not detect any change in ovarian artery RI or I; nevertheless, in their study, hmg was started just 3 days after the commencement of GnRH therapy. Engmann et al. discovered a decrease in stromal artery SV after 2 3 weeks of GnRH agonist therapy but no effect on stromal artery I. Vrtacnik-Bokal et al. used a protocol like ours, starting the GnRH agonist on cycle day 22 of the preceeding cycle. They measured the arterial flow in the hilum of the ovary and discovered an increase in RI in the active (dominant) ovary but no change in the inactive ovary. Despite changes detected in the vascular impedance and blood flow velocities in single ovarian arteries, it may be that the mean vascularization and perfusion within the entire ovary is not affected enough by the dominant follicle or GnRH agonist therapy, since we could not demonstrate any differences or changes in the color indices either in the dominant or in the nondominant ovary. The comparison between normal and COs revealed that the COs were larger and contained more follicles than the normal ones at both measurements. Increased ovarian volume is considered one of the ultrasound criteria used for the diagnosis of COs (27, 28). Surprisingly, we observed that the COs on the right side were larger than the left ones FERTILITY & STERILITY 1133

6 during late follicular ; nevertheless, the existence of dominant follicles in five of the ovaries may have distorted the volumes. After pituitary suppression, the volumes no longer differed. Wu et al. (29) have also established, using 3D ultrasound, that the right CO is larger than the left one, even though their latest study reports that there is no difference in the volumes between right and left CO during cycle days 2 3 (7). The of cycle in which the measurements were taken in the first study was not reported (29). The mean volumes of COs ranged from 10.4 to 12.9 cm 3 in their studies (7, 29), which are similar to those observed here. According to earlier studies using 2D transvaginal color Doppler ultrasound, stromal blood flow characteristics in COs seem to differ from those in normal ovaries (2, 17, 18, 30). SV and TAMX have been higher (5, 17, 18), and I and RI have been either at the same level or lower (2, 5, 18, 30, 31) in COs, suggesting that the stroma in COs is highly vascularized. In our study VI, FI, and VFI were equal in normal and COs, both before (12) and after pituitary suppression. an et al. have used similar 3D power Doppler equipment to compare normal ovaries to ovaries in patients with true COS. The measurements were performed on day 2 or 3 of the menstrual cycle. According to them, VI, FI, and VFI were higher in the COs than in the normal ovaries. The mean difference between the CO and normal ovary was 2.45% for VI, 5.92 for FI, and 1.30 for VFI (7). The patients with normal ovaries were significantly older than the patients with COs, which may have affected the ovarian blood flow characteristics (7). In addition, in their IVF protocol the pituitary down-regulation was initiated after the scan, whereas in our protocol the patients were already downregulated, which may partly explain the discordance between the results. Differences in MG were not evaluated (7). Increased stromal volume and increased echogenity has been considered typical for COs (27, 28). In a recent study by Buckett et al., the stromal volume in COs was found to be higher than the one in normal ovaries, but the stromal echogenity did not differ from that in normal ovaries. The mean echogenity in the area of interest was defined from a single 2D plane, and surprisingly the mean total ovarian echogenity was lower in COs than in normal ovaries (32). In a larger patient group, we earlier established that there is no difference in the stromal echogenity between normal and COs during the late follicular (12). Despite the fact that there was an overall increase in the ovarian MG, we could not detect any difference in MG value between normal and COs after pituitary down-regulation. In this study, we have evaluated the changes taking place in the ovary after pituitary down-regulation using 3D power Doppler ultrasound. The dominant and nondominant ovaries in the preceeding cycle showed no differences between each other after GnRH analog therapy, which was also the case when comparing the right and left ovary. COs were larger than the normal ones, but no differences could be detected in the stromal brightness or vascularity, which is not in accordance with the earlier studies. As a method, 3D power Doppler ultrasound provides a new aspect to examine the changes taking place in ovarian vascularization and blood flow. The clinical usefulness of the method remains to be clarified. References 1. Serafini, Stone B, Kerin J, Batzofin J, Quinn, Marrs R. 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