Three-dimensional power Doppler imaging of ovarian stromal blood flow in women with endometriosis undergoing in vitro fertilization

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1 Ultrasound Obstet Gynecol 2003; 21: Published online in Wiley InterScience ( DOI: /uog.113 Three-dimensional power Doppler imaging of ovarian stromal blood flow in women with endometriosis undergoing in vitro fertilization M.-H. WU*, S.-J. TSAI, H.-A. PAN*, K.-Y. HSIAO and F.-M. CHANG* *Department of Obstetrics and Gynecology, National Cheng-Kung University Hospital, Institute of Clinical Medicine and Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China KEYWORDS: endometriosis; in vitro fertilization; leptin; ovary; three-dimensional power Doppler ultrasonography ABSTRACT INTRODUCTION Objective The aims of this retrospective study were to investigate whether the quantification of ovarian stromal blood flow and/or leptin concentration are predictive of in vitro fertilization (IVF) outcomes in women after laparoscopic ovarian cystectomy for large endometriomas. Methods Twenty-two women undergoing IVF after laparoscopic surgery for ovarian endometriomas (> 6cm) comprised the study group. Twenty-six women with tubal factor infertility constituted the control group. Ovarian stromal blood flow was evaluated by threedimensional (3D) power Doppler ultrasound imaging using virtual organ computer-aided analysis (VOCAL TM ). Serum and follicular fluid (FF) leptin concentrations were quantified using an enzyme-linked immunosorbent assay kit. Results There were significantly decreased ovarian stromal blood flow parameters (including vascularization index, flow index (FI), and vascularization flow index) in the endometriosis group without an evident difference in total ovarian volume on the day of human chorionic gonadotropin. The value of FF leptin demonstrated a negative correlation with ovarian stromal FI in the control group, but there was a loss of this effect in the endometriosis group. Conclusions Quantification of ovarian stromal blood flow by 3D power Doppler ultrasound in women with endometriosis may provide an important prognostic indicator in those undergoing IVF. Copyright 2003 ISUOG. Published by John Wiley & Sons, Ltd. Endometriosis is a disease in which the endometrium ectopically implants and grows outside the uterine cavity. Laparoscopic surgery is indicated for infertile women with ovarian endometriomas with preservation of the normal residual ovarian cortex. A reduced follicular response in natural and clomiphene-stimulated cycles has been shown in younger women after ovarian cystectomy for endometriomas but can be recovered with gonadotropin stimulation 1. In vitro fertilization (IVF) outcome is not affected in women with endometriosis after laparoscopic surgery but requires more ampules of gonadotropin to achieve the same ovarian response 2.However,inpatients with advanced stages of endometriosis a significantly poorer IVF outcome has been demonstrated as compared to those patients with tubal infertility 3. Ovarian volume measurement and reconstruction in three-dimensional (3D) ultrasound is straightforward, accurate, and highly reproducible 4. However, conventional color Doppler flow imaging is less sensitive to slow flow and small vessels. Recent advances in 3D power Doppler ultrasound, also known as color Doppler angio, have rendered it sensitive to low-velocity blood flow and alterations in vascular flow 5. The ovary is an ideal organ to evaluate cyclic changes accompanying neoangiogenesis, and dynamic vascular flow in the ovarian stroma can be more accurately quantified with the aid of 3D power Doppler equipment 6. Leptin, encoded by the obese (ob) gene, is secreted by adipocytes and may act as a metabolic signal to the reproductive system 7,8. Leptin has been implicated in ovarian function, and also has immunoregulatory, proinflammatory and angiogenic effects that may contribute to the progression of endometriosis 9,10. A recent report Correspondence to: Dr F.-M. Chang, Department of Obstetrics and Gynecology, College of Medicine, National Cheng Kung University, Tainan, 70428, Taiwan, Republic of China ( fchang@mail.ncku.edu.tw) Accepted: 17 January 2003 Copyright 2003 ISUOG. Published by John Wiley & Sons, Ltd. ORIGINAL PAPER

2 3D ovarian blood flow in endometriosis 481 has shown that peritoneal fluid and serum levels of leptin are increased in patients with pelvic endometriosis 11.It is of interest to note that the presence of peritoneal but not ovarian endometriosis affects the leptin levels in the peritoneal fluid 12. Moreover, findings in a recent study of ours demonstrate that ectopic endometriotic cells express leptin mrna, with the decreased leptin receptor mrna exclusively associated with endometriosis 13. Although ovarian hyperstimulation by gonadotropins increases the plasma leptin concentration in women undergoing IVF, the relatively high serum leptin correlates with a reduced ovarian response based on the numbers of follicles observed and oocytes retrieved 14.Leptinis also considered an angiogenic factor 15. Therefore, leptin has the potential to improve the ovarian response and IVF outcome in patients with deficient intra-ovarian vasculature. It is possible that an increased leptin concentration may serve as a compensatory mechanism for reduction in ovarian reserve, and may be useful in improving ovarian blood flow after ovarian damage caused by surgery for endometriomas. Since leptin has biological effects on the ovary and may be involved in the etiology of endometriosis, we set out to investigate the relationship between leptin levels and ovarian stromal blood flow in women with previous ovarian endometriomas. METHODS Patients This retrospective study analyzed the clinical results of 48 women aged years (Table 1) undergoing IVF treatment at the National Cheng Kung University Hospital. Twenty-two women were undergoing IVF after laparoscopic surgery for endometriomas. Twentysix women had tubal infertility. A total of 60 IVF-embryo transfer procedures were performed. Based on their past history, the patients with endometriosis were classified according to revised American Society for Reproductive Medicine criteria during laparoscopic inspection 16. All patients received a laparoscopic ovarian cystectomy (enucleation) for large endometriomas (> 6 cm) but had failed to conceive after laparoscopic surgery. The control patients had been diagnosed with pure tubal factor infertility. The study protocol was approved by the Institutional Review Board of National Cheng Kung University Hospital. All patients provided informed consent. Stimulation protocol The protocol for ovarian stimulation was the standard regimen consisting of follicle-stimulating hormone (FSH) (urofollitropin, Metrodin-HP; Serono, Rome, Italy), human menopausal gonadotropin (Humegon; Organon, Oss, Holland) and gonadotropin-releasing hormone agonist (Supremon, Buserelin; Hoechst, Frankfurt, Germany). Transvaginal ultrasonography and serum unconjugated estriol (E 2 ) concentrations were monitored every 2 days until there were at least two follicles > 16 mm based on sonographic criteria. At that point, IU human chorionic gonadotropin (hcg) was administered. Oocyte retrieval was performed h after hcg (Profasi; Serono, Aubonne, Switzerland) administration. The embryos were transferred into the uterine cavity transcervically after 3 days of culture. The pregnancy rate was defined as the incidence of biochemical pregnancies (with the serum level of β-hcg more than 20 miu/ml)/oocyte retrievals. 3D transvaginal power Doppler ultrasonography Transvaginal pelvic ultrasound was performed as part of the routine assessment prior to the oocyte retrieval procedures. On the day of the hcg injection, all ultrasound examinations were performed using 3D transvaginal 7.5-MHz power Doppler ultrasound (Voluson 530D; Medison-Kretz, Seoul, Korea and Zipf, Austria) as previously described 6,17. The vascular color Doppler flow data of the uterine arteries were evaluated with a pulsatility index (PI) and a resistance index (RI). The vascularization index (VI), flow index (FI), and vascularization flow index (VFI) were calculated using color power Doppler angio and the VOCAL TM (virtual organ computer-aided analysis) software for histogram analysis once the total volume of the ovary was stored, excluding the peripheral supplying vessels. Hormonal assays Plasma samples for hormone determinations were drawn on the morning of hcg administration. Follicular fluid (FF) from the first dominant follicle was collected without blood or medium contamination through follicle aspiration via a transvaginal ultrasound-guided puncture. Samples were maintained at 20 C until assayed. Serum and FF leptin levels were measured in duplicate with a commercial enzyme-linked immunosorbent assay (ELISA) kit (DRG International, Pine Brook, NJ, USA) that has a sensitivity of 0.2 ng/ml with inter- and intraassay coefficients of variation below 10% precision. All leptin measurements were analyzed in the same assay. Immulite enzyme immunoassay was used to measure plasma E 2 (SI conversion factor ; sensitivity 10 pmol/l), FSH (SI conversion factor 78/549; sensitivity 0.5 miu/ml), and LH concentrations (SI conversion factor 80/552; sensitivity 0.5 miu/ml) with inter- and intra-assay coefficients of variation below 10% precision. Statistical analysis Results are reported as mean ± standard deviation (SD). The differences in the mean leptin level and other variables, except the pregnancy rate, between the two study groups were assessed by Student s unpaired twotailed t-test. Pregnancy rates and live birth rates between the two groups were tested by Chi-square analysis. Correlations of leptin levels with other variables, except

3 482 Wu et al. pregnancy rate, were examined by Pearson correlation coefficient analysis. The correlation between leptin and pregnancy rate was analyzed by ordinal regression, with leptin levels divided into four groups (< 25%, 26 50%, 51 75%, and > 75%). Values for P < 0.05 were considered significant. RESULTS Analysis of subject data Clinical data of the two groups of women are shown in Table 1. Endometriosis patients had lower body mass index (BMI) and LH concentrations on the day of hcg, fewer numbers of oocytes collected, and reduced pregnancy rate. Analysis of ultrasound data As shown in Table 2, there were no significant differences found in the ovarian volume after controlled ovarian stimulation or in the uterine artery RI and PI values as determined by Doppler analysis between the endometriosis and control groups throughout the treatment cycle. However, the ovaries from patients with endometriosis showed significantly lower intraovarian stromal vascular patterns: lower VI, FI, and VFI values (Table 2, Figure 1). Ovaries of women with endometriosis were considered in separate groups: the ovaries after endometrioma surgery Table 1 Characteristics and clinical data of the study groups during IVF treatment Variables Tubal factor Endometriosis P Patients (n) IVF cycle Age (years) ± ± 0.60 NS BMI (kg/m 2 ) ± ± 0.52 < FSH-day ± ± 0.72 NS (miu/ml) LH-day 2 (miu/ml) 2.35 ± ± 0.42 NS E 2 (pg/ml)athcg 1674 ± ± NS LH (miu/ml) at 3.71 ± ± 1.11 < 0.05 hcg FF leptin (ng/ml) ± ± 1.05 NS Serum leptin 5.36 ± ± 1.89 NS (ng/ml) FF leptin/bmi 0.65 ± ± 0.05 NS Serum leptin/bmi 0.29 ± ± 0.10 NS Number of oocyte ± ± 0.80 < pick-ups Fertilization rate 72 ± ± 5.1 NS (%) Pregnancy rate per ET (%) Live birth rate (%) NS All values are mean ± standard deviation unless otherwise indicated. P < 0.05 signifies statistical difference. BMI, body mass index; E 2, estradiol; ET, embryo transfer; FF, follicular fluid; FSH, follicle-stimulating hormone; hcg, human chorionic gonadotropin; IVF, in vitro fertilization; LH, luteinizing hormone; NS, not significant. and the contralateral normal ovaries (if present) (Table 3). There was no significant difference between the treated endometriomatous ovaries and the contralateral normal ovaries in 3D stromal blood flow parameters. However, ovarian reserve was decreased in both the ovaries operated on for endometriomas and the contralateral normal ovaries as compared with the results of the control group in Table 2. Correlation between ovarian stromal flow parameters and variables Although there were significant differences in ovarian power Doppler parameters between the two groups, there was no correlation with other variables. Analysis of leptin in FF and serum A comparison of serum and FF leptin levels between the study groups is shown in Figure 2. The patients with endometriosis had significantly higher FF leptin concentrations when compared with serum levels (FF leptin 15.7 ± vs. serum leptin ± ng/ml, P < 0.05); this difference remained significant in the controls (FF leptin ± vs. serum leptin ± Table 2 Three-dimensional ultrasound data of the two study groups Ultrasound variables Tubal factor Endometriosis P Ovarian volume (cm 3 ) ± ± 4.01 NS Pulsatility index 2.02 ± ± 0.12 NS Resistance index 0.81 ± ± 0.05 NS Mean grayness* ± ± 1.07 NS Vascularization index 1.69 ± ± 0.20 < 0.05 Flow index ± ± 1.83 < 0.05 Vascularization flow index 0.91 ± ± 0.10 < 0.05 All values are mean ± standard deviation unless otherwise indicated. P < 0.05 signifies statistical difference. *Mean gray value of the color histogram in the power Doppler index. Table 3 Ovarian parameters of three-dimensional power Doppler ultrasonography in women with large endometriomas after laparoscopic surgery. The ovaries were subdivided into the ovaries operated on for endometriomas and the contralateral normal ovaries (if present) Variables Treated ovary Contralateral normal ovary Ovaries (n) Follicles (>15 mm) (n) 2.50 ± ± 0.51 Oocyte pick-ups (n) 1.00 ± ± 0.51 Ovarian volume (cm 3 ) ± ± 5.37 Mean grayness* ± ± 2.22 Vascularization index 0.71 ± ± 0.48 Flow index ± ± 2.28 Vascularization flow index 0.35 ± ± 0.22 *Mean gray value of the color histogram in the power Doppler index.

4 3D ovarian blood flow in endometriosis 483 concentrations in the patients with endometriosis when compared with those in the control group (Table 1). In addition, there was a strong association between FF and serum leptin levels in all patients (r = 0.551, 95% CI , P (two-tailed) = ). Correlation between leptin concentration and other variables b Figure 1 Three-dimensional power Doppler ultrasonographic pictures using the VOCAL TM technique. The intraovarian power Doppler signals in the endometriosis group (a) were lower than in the tubal group (b). Leptin (ng/ml) NS P < 0.05 NS P < 0.05 Serum FF Serum FF Tubal factor Endometriosis Figure 2 Comparisons of mean concentrations of leptin in serum and follicular fluid (FF) from women undergoing IVF in the study groups, as determined by ELISA. P < 0.05 signifies statistical difference. NS, not significant ng/ml, P < 0.05). Although there were slightly increased serum leptin values in the endometriosis group, there were no significant differences in FF or serum leptin In the tubal factor group there was no correlation between serum leptin levels and other parameters. In contrast, FF leptin showed a significantly positive correlation with BMI (r = 0.67, P < 0.05) and a negative correlation with ovarian stromal FI (r = 0.62, P < 0.05). Furthermore, FF leptin/bmi positively correlated with LH levels on the day of hcg (r = 0.52, P < 0.05) in the control group. A correlation between serum leptin concentration and age was seen in the endometriosis group (r = 0.55, P < 0.05). A positive correlation between pregnancy outcome and serum leptin/bmi was observed (r = 0.56, P < 0.05). There was no significant correlation between FF leptin and ovarian stromal flow, including VI, FI and VFI in the endometriosis group. A positive correlation between FF leptin/bmi and E 2 on the day of hcg administration in women with endometriosis was observed (r = 0.46, P < 0.05). After adjustment for age, FF leptin still demonstrated a positive correlation with BMI in the tubal factor group (r = 0.66, P < 0.05). However, in the endometriosis group, both serum and FF leptin values showed positive correlations with BMI only after adjustment for age (r = 0.53 and 0.53, respectively, P < 0.05). DISCUSSION Clinical applications of Doppler and 3D ultrasound have been described in the field of obstetrics and gynecology, including IVF 18,19. This has facilitated objective assessment of ovarian stromal volume and blood flow. The 3D volume measurement using VOCAL software is more convenient and rapid than previously described methods 4,20. Moreover, conventional color Doppler flow imaging is less sensitive to slow flow and small vessels in detecting ovarian vascularity. Color amplitude imaging and/or power Doppler can provide information on the quantity of moving blood cells per volume and thus is more sensitive in demonstrating the real vascularity of the ovary 5.Asin the formula defined by Pairleitner et al. 21, the VI indicates the proportion of flow signal (vessel density) detected within the total ovary. The FI describes the average value of the flow intensity inside the whole ovary (an estimate of total ovarian blood flow). The VFI demonstrates both vessel and flow intensity derived by multiplying the VI and FI. During the operation and process of 3D power Doppler ultrasound one needs to pay attention to several procedures to increase the data accuracy in the assessment of ovarian stromal blood flow 22. 3D power Doppler ultrasound may enhance our ability to diagnose benign and malignant ovarian lesions 21,23.

5 484 Wu et al. In our recent study, 3D power Doppler ultrasound readily demonstrates the signals in ovarian stroma 6.The presence of increased VI, FI, and VFI were noted in the ovarian stromal Doppler signals in polycystic ovarian syndrome. Pellicer et al. introduced 3D ultrasound in the evaluation of low IVF responders to explore ovarian reserve 24. Their results show that young low responders have a decreased number of antral follicles without evident changes in ovarian volume, which is similar to our present result with the endometriosis group. With the advance of 3D power Doppler ultrasound, its parameters will provide additional predictive information to more accurately assess potential ovarian response for IVF. Our study demonstrated significantly lower indices of intraovarian blood flow in endometriosis patients during IVF using power Doppler ultrasound analysis. The decreased ovarian vascularity in the endometriosis group may be ascribed to operative ovarian damage. Engmann et al. found that the value of ovarian stromal blood flow velocity after pituitary suppression is an important independent predictor of ovarian response and IVF outcome 25. They also demonstrated that deficient intraovarian vascularity potentially is an initial marker of reduced ovarian reserve before the increase of FSH level and the reduction of ovarian volume. These results are consistent with our present data that decreased ovarian stroma FI and lower pregnancy rate were noted in the endometriosis group without changes in baseline FSH level and ovarian volume after gonadotropin stimulation. There are positive correlations between perifollicular blood flow, follicular oxygen content, and the developmental capacity of the corresponding oocyteembryo 26. In a study of perifollicular vascularity, women who received embryos derived from oocytes grown in more well-vascularized follicles had a statistically higher pregnancy rate and a better IVF outcome 27. It is interesting to note in our study (Table 3) that the contralateral ovary had similarly reduced stromal flow as the treated ovary in the endometriosis group. Further studies are needed to clarify the mechanism. Follicular fluid concentrations of leptin reportedly reflect serum leptin 14,28,29. Our data also demonstrate a positive correlation between FF and serum leptin levels in all patients. However, in our study leptin levels in FF were significantly higher than the serum levels of leptin in both groups. Physiological leptin levels in FF induce the phosphorylation of STAT3 in mouse MII-stage oocytes, suggesting a role of leptin in oocyte maturation 30. Higher leptin levels will interfere with dominant follicle formation and suppress E 2 production in follicles under IGF-1 augmentation, which lead to insufficient LH surge and immature follicular development 29. If there is any physiological importance in the difference between circulating leptin and FF leptin concentrations, further studies to elucidate the mechanisms are needed. In the present study we focused on ovarian stromal power Doppler blood flow and its relationship with leptin levels in women with endometriomas having undergone laparoscopic surgery. It is interesting that there was a negative correlation between the FF leptin levels and the power Doppler FI in our control group. Leptin is an angiogenic factor and FF leptin is a marker of follicular hypoxia 30. Reduced blood flow (decreased FI) through the ovarian stroma may lead to a follicular hypoxia that in turn induces the secretion of several angiogenic factors, such as leptin, in ovarian follicles. The increase of FF leptin for the stimulation of angiogenesis may be modulated under physiological and pathophysiological conditions 31. Loss of this negative correlation between FF leptin and FI in the endometriosis group may represent deficient angiogenic responses in ovarian cortices after surgery. In this study 3D power Doppler was used to demonstrate the dynamic vascularity of the IVF process; its relationship with leptin levels may provide additional data for conventional endocrine and ultrasound diagnostic methods in IVF. It may also provide an indirect index of oocyte quality and quantity and therefore serve as a prognostic marker of embryo viability and implantation rate. We expect that high blood flow (FI) may be a predictor for the dynamic changes during IVF. Kupesic and Kurjak also demonstrated that ovarian stromal FI obtained by 3D power Doppler ultrasound after pituitary suppression is one of the useful predictors of IVF outcome 32. Their data showed that patients with higher stromal blood flow (FI) had more favorable IVF outcomes due to higher antral follicle and oocyte retrieval numbers. Further studies are needed to determine whether the quantitative assessment of ovarian blood flow by 3D power Doppler ultrasound may play a role in assessing IVF cycles likely to result in pregnancy. In conclusion, the power Doppler parameters using 3D ultrasound can measure blood flow in total ovarian volume accurately and rapidly. The combination of FF leptin and the quantification of ovarian stromal blood flow by 3D power Doppler ultrasound on the day of hcg may provide a prognostic tool to achieve a predictive measure of the ovarian reserve and better IVF outcomes. Further clinical studies in an IVF setting are needed to clarify the underlying mechanisms controlling ovarian stromal blood flow and leptin accumulation. Confirmation of these relationships may assist clinicians in counseling patients and has the potential to improve the success rate of assisted reproduction techniques. ACKNOWLEDGMENTS The authors are grateful for the financial support provided by grants from the National Science Council, ROC (NSC B ), and National Cheng Kung University Hospital (NCKUH-90-44). The authors thank Kuei-Hsiang Huang and Li-Hsiang Wu for their assistance with data collection, Mei-Feng Huang for assistance with ELISA analysis, and Yueh-Chin Cheng and Yi-Jen Wang for assistance with ultrasound examination. We also thank Kathleen Burns and David Miner for critical review of the manuscript.

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