Article Ovarian stromal vascularity is not predictive of ovarian response and pregnancy

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1 RBMOnline - Vol 12. No Reproductive BioMedicine Online; on web 24 November 2005 Article Ovarian stromal vascularity is not predictive of ovarian response and pregnancy Dr Ernest Ng is an Associate Professor at the Department of Obstetrics and Gynaecology, The University of Hong Kong. His main research interests are assisted reproduction technology, assessment of ovarian reserve and three-dimensional ultrasound in reproductive medicine. He has published about 100 papers in refereed journals. Dr Ernest Hung Yu Ng Ernest Hung Yu Ng 1, Oi Shan Tang, Carina Chi Wai Chan, Pak Chung Ho Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong Special Administrative Region, People s Republic of China 1 Correspondence: Department of Obstetrics and Gynaecology, The University of Hong Kong, 6/F, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong. Tel: ; Fax: ; nghye@hkucc. hku.hk Abstract The role of ovarian stromal vascularity in the prediction of the ovarian response and pregnancy in infertile women was evaluated by comparing age of women, body mass index (BMI), basal FSH concentration, antral follicle count (AFC) and ovarian stromal vascularity indices measured by three-dimensional power Doppler ultrasound. A total of 111 women in their first IVF cycle were analysed. They were aged <40 years with basal FSH concentration <10 IU/l on recruitment for IVF treatment. AFC, mean ovarian volume and mean ovarian 3D power Doppler flow indices were determined on day 2 of the treatment cycle prior to a standard regimen of ovarian stimulation. Ovarian response was represented by the number of oocytes, serum oestradiol, the duration and dosage of gonadotrophins. AFC achieved the best predictive value in relation to the number of oocytes obtained, followed by age of women and BMI. Basal FSH concentration was the only predictive factor for the duration and dosage of gonadotrophin used. Mean ovarian 3D power Doppler flow indices were not predictive of pregnancy in a multiple logistic regression analysis. Ovarian 3D power Doppler flow indices measured after pituitary downregulation were not predictive of the ovarian response and pregnancy in the IVF treatment. Keywords: ovarian response; ovarian stromal blood fl ow; three-dimensional power Doppler; ultrasound Introduction Development of multiple follicles in response to gonadotrophin stimulation is the key factor leading to a successful outcome of IVF treatment. Poor ovarian response may be associated with poor pregnancy rates, and many of these cycles are cancelled without proceeding to oocyte retrieval (Keay et al., 1997; Tarlatzis et al., 2003). On the other hand, exaggerated ovarian response leads to an increased risk of ovarian hyperstimulation syndrome (OHSS) (Aboulghar and Mansour, 2003) and the resulting high serum oestradiol concentrations may adversely affect the outcomes of the IVF treatment (Ng et al., 2000a). Prediction of ovarian responses prior to stimulation is useful in counselling patients, and may be helpful in tailoring the dosage of gonadotrophin to individual patients (Ubaldi et al., 2005). Ultrasound is essential during the IVF treatment for monitoring ovarian response, assessing endometrial receptivity, guiding transvaginal aspiration of oocytes and subsequent transcervical transfer of embryos to the uterus. Several ultrasound parameters have been employed to predict the ovarian response to gonadotrophins, including ovarian volume (Lass et al., 1997; Syrop et al., 1999), antral follicle count (Tomás et al., 1997; Chang et al., 1998; Ng et al., 2000b; Bancsi et al., 2002; Loverro et al., 2003) and ovarian stromal vascularity (Zaidi et al., 1996; Bassil et al., 1997; Engmann et al., 1999; Kupesic and Kurjak, 2002; Kupesic et al., 2003; Popovic-Todorovic et al., 2003; Ng et al., 2005b). Hormonal markers such as early follicular serum FSH (Scott and Hofmann et al., 1995; Sharara et al., 1998), serum inhibin B (Seifer et al., 1997; Tinkanen et al., 1999; Dzik et al., 2000), serum anti-müllerian hormone 43

2 44 (Seifer et al., 2002; van Rooij et al., 2002; Fanchin et al., 2003) are also shown to be predictive of ovarian response. Folliculogenesis in the human ovary is a complex process regulated by a variety of endocrine and paracrine signals (McGee and Hsueh, 2000). The availability of an adequate vascular supply to provide endocrine and paracrine signals may play a key role in the regulation of follicle growth (Redmer and Reynolds, 1996). It is postulated that increased ovarian stromal vascularity may lead to a greater delivery of gonadotrophins to the granulosa cells of the developing follicles. Ovarian stromal vascularity can be assessed by colour or power Doppler ultrasound. Power Doppler is better suited to the study of the ovarian stromal vascularity, as it is more sensitive to lower velocities and essentially angle-independent (Guerriero et al., 1999). It has been reported that ovarian stromal vascularity measured by two-dimensional (2D) power Doppler ultrasound had no predictive value for the ovarian response (Ng et al., 2005a). In combination with three-dimensional (3D) ultrasound, power Doppler provides a unique tool with which to examine the ovarian stromal blood supply as a whole, as opposed to analysis of small individual stromal vessels in 2D planes. It is postulated that ovarian stromal vascularity measured by 3D power Doppler ultrasound might have a role in predicting ovarian response and pregnancy. The objective of this prospective study was to determine the significance of ovarian stromal vascularity in the prediction of the ovarian response and pregnancy of infertile women undergoing the first IVF cycle using a standard regimen of ovarian stimulation by comparing age of women, body mass index (BMI), basal FSH concentration, antral follicle count (AFC) and ovarian stromal vascularity indices measured by 3D power Doppler ultrasound. Materials and methods Consecutive women attending the Department of Obstetrics and Gynaecology, the University of Hong Kong between February 2003 and June 2004 for their first IVF treatment were recruited for study when the following criteria were met: (i) no history of ovarian surgery and (ii) no hormonal treatment in the 6 months prior to the IVF treatment. Women who were aged >40 years at the time of treatment or whose basal FSH concentration on repeated testing on recruitment was 10 IU/l were advised against the IVF treatment according to the recruitment guideline issued by the Hospital Authority in Hong Kong. Poor visualization of ovaries because of abdominal position, an ovarian cyst of 20 mm in diameter and presence of polycystic ovaries (Balen et al., 2003) were retrospectively excluded. Every patient gave a written informed consent prior to participating in the study, which was approved by the Joint Institutional Review Board of the University of Hong Kong and the Hospital Authority. They did not receive any monetary compensation for participation in the study. The results of ovarian stromal vascularity did not affect their IVF treatment. All authors declared no conflict of interest. Indications for IVF treatment included tubal, male, endometriosis, unexplained and mixed factors. Intracytoplasmic sperm injection (ICSI) was performed for couples with severe semen abnormalities where <100,000 motile spermatozoa were recovered after sperm preparation. In case of obstructive or nonobstructive azoospermia, surgically retrieved spermatozoa from epididymis or testis respectively were used for ICSI. The details of the long protocol of ovarian stimulation regimen, gamete handling, standard insemination and ICSI were as previously described (Ng et al., 2000b). Basal FSH concentration was checked on days 2 4 of the period immediately preceding the treatment cycle. All women were pre-treated with buserelin (Suprecur, Hoechst, Frankfurt, Germany) nasal spray 150 μg 4 times a day from the mid-luteal phase of the cycle preceding the treatment cycle. All 3D ultrasound examinations were performed on day 2 of the treatment cycle (prior to ovarian stimulation) at 8 10 a.m. by EHYN using GE Voluson 730 (GE Kretz, Zipf, Austria), after the bladder had been emptied. The details and reliability of 3D volume acquisition and data analysis were as previously described (Ng et al., 2004). AFC was obtained in the multiplanar view and the intra-observer coefficient of variation for AFC was 7%. Both ovaries were then scanned with the power Doppler mode. The setting condition for this study was as follows: frequency mid, dynamic set 2, balance-g >140, smooth 5/5, ensemble 12, line density 7, and power Doppler map 5. The setting conditions for the subpower Doppler mode were as follows: gain, 6.0; balance, 140; quality, normal; wall motion filter, low 1; and velocity range, 0.9 khz. The 3D ultrasound images were stored for later analysis by EHYN. The results of the ultrasound assessment did not affect subsequent clinical management procedures. The built-in VOCAL (Virtual Organ Computer-Aided Analysis) Imaging Program for the 3D power Doppler histogram analysis was used in the analysis, along with computer algorithms, to measure the ovarian volume and indices of vascularization and blood flow. Vascularization index (VI) measures the number of colour voxels representing the blood vessels in the ovary and is expressed as a percentage of the ovarian volume. Flow index (FI) is the mean colour value in the colour voxels, and represents the average intensity of flow inside the ovary. Vascularization flow index (VFI) is a combination of vascularity and flow (Pairleitner r et al., 1999). During analysis and calculation, the manual mode of the VOCAL Contour Editor was used to cover the whole 3D volume of the ovary with a 15 rotation step. Hence, 12 contour planes were analysed for each ovary to cover 180. When the ultrasound scanning showed no ovarian cyst and serum oestradiol concentrations were below 200 pmol/l, human menopausal gonadotrophin (HMG, Pergonal, Serono, Geneva, Switzerland) injections were started at 300 IU daily for the first 2 days followed by 150 IU daily afterwards. The ovarian response was monitored by serial transvaginal scanning and the HMG dosage was increased if there was no follicle 10 mm after 7 days of stimulation. Human chorionic gonadotrophin (HCG; Profasi, Serono) was given intramuscularly when the leading follicle reached 18 mm in diameter and there were at least three follicles of 16 mm in diameter. Serum oestradiol concentration was measured on the day of HCG administration. Cycles were cancelled when the follicles remained <10 mm after 14 days of stimulation. Oocyte retrieval was performed even when there was only one dominant follicle and was

3 scheduled 36 h after the HCG injection and any visible follicles were aspirated during the procedure. Patients were advised to have two embryos replaced into the uterine cavity 48 h after the retrieval, but replacing three embryos was allowed. Excess good quality embryos were frozen. All fresh embryos were cryopreserved if serum oestradiol on the day of HCG injection was >20,000 pmol/l in order to reduce the risks of OHSS. Luteal phase was supported by two doses of HCG or vaginal progesterone suppositories (Cyclogest vaginal pessaries; Cox Pharmaceuticals, Barnstaple, UK). A urine pregnancy test was done 16 days after embryo transfer. If it was positive, ultrasound examination was performed days later to confirm intrauterine pregnancy and to determine the number of gestational sacs present. Only clinical pregnancies defined by the presence of one or more gestational sacs or the histological confirmation of gestational product in miscarriages were considered. Serum FSH and oestradiol concentrations were measured by a two-site sandwich immunoassay (Automated Chemiluminescence ACS-180 System; Bayer Corporation, New York, NY, USA). The sensitivity of the FSH assay was 0.3 IU/l and the intra-assay and inter-assay coefficients of variation were 1.7 and 2.8% respectively. The sensitivity of the oestradiol assay was 36.7 pmol/l and the intra- and inter-assay coefficients of variation were 8.1 and 8.7% respectively. The mean intra-class correlation coefficient with 95% confidence interval (CI, which reflects the inter-observer reliability) for 3D scanning of ovarian volume, VI, FI and VFI was (95% CI: , ), (95% CI: , ), (95% CI: , ) and (95% CI: , ) respectively. The mean intraclass correlation coefficient for data acquisition of ovarian volume, VI, FI and VFI were (95% CI: , ), (95% CI: , ), (95% CI: , ) and (95% CI: , ) respectively. Statistical analysis The correlation coefficient between AFC and the number of oocytes obtained in the previous study (Ng et al., 2000b) was Assuming that AFC and ovarian 3D power Doppler flow indices had similar correlation coefficients, the sample size required would be 107 to give a test of significance of 0.01 and a power of 0.9 (Sigmastat; Jandel Scientific, San Rafael, CA, USA). As there were no differences in volume, VI, FI and VFI between the left and the right ovaries, averaged ovarian volume, ovarian VI, FI and VFI were given. The primary outcome measures were the number of oocytes and pregnancy. Secondary measures included serum oestradiol concentration on the day of HCG, the duration and dosage of human menopausal gonadotrophin (HMG) used. Continuous variables were not normally distributed and were given as median (25th 75th centiles), unless indicated. Multiple regression analysis with the least-squares regression was applied to evaluate the predictive values of different parameters on the number of oocytes obtained, serum oestradiol concentration on the day of HCG, the duration and dosage of HMG used. Logistic regression analysis was applied to assess the impact of various ovarian reserve markers and ovarian response in the prediction of pregnancy. Statistical analysis was performed using the Statistical Program for Social Sciences (SPSS Inc., Version 12.0, Chicago, IL, USA). Two-tailed P < 0.05 was considered statistically significant. Results A total of 197 women underwent their first IVF cycle during the study period and 14 women were not eligible because of a history of ovarian surgery. All eligible patients agreed to participate in the study and 72 women were excluded after the ultrasound examination: poor visualization of the ovaries in 20 women, an ovarian cyst in 27 women and polycystic ovaries in 25 women. Therefore, 111 women underwent ovarian stimulation and were included in the final analysis: 28 tubal factors; eight endometriosis; 56 male infertility; 11 unexplained and eight mixed causes. Table 1 summarizes the demographic data and ovarian response. Ranges of the parameters were as follows: age years, BMI kg/m 2, basal FSH concentration IU/l, HMG dosage IU and HMG duration 7 9 days. Two cycles did not proceed to oocyte retrieval because of absent follicular development. These patients were considered to have no oocytes obtained. Oocytes were obtained in all planned retrievals. Failed fertilization was encountered in four cycles. Embryo transfer was postponed in eight cycles because of the risk of OHSS. Embryo transfer was performed in 97 cycles, and 24 clinical pregnancies resulted. The pregnancy rate (PR) was 21.6% per cycle initiated and 24.7% per transfer. Age of women, BMI, basal FSH concentration, AFC and mean ovarian volume, mean ovarian VI, FI and VFI were entered in a stepwise fashion in the multiple regression analysis using the number of oocytes obtained as the dependent variable with a constant included in the equation. Receiver operating characteristic curves could not be generated as the number of oocytes obtained was taken as the primary outcome measure rather than the presence of poor response or cycle cancellation as used in other studies. Therefore Tables 2 4 usefully demonstrate the multiple regression analysis model. AFC had the largest R 2 change, which was followed by age of women and BMI (Table 2). When these parameters were entered in a stepwise fashion in the multiple regression analysis using the HMG duration and dosage as the dependent variables, basal FSH concentration was the only predictive factor (Tables 3 and 4). Serum oestradiol concentration on the day of HCG could not be predicted by these factors. No significant differences in demographic characteristics, ovarian reserve markers, ovarian response and number of embryos replaced were detected between the non-pregnant group and the pregnant group (Table 5). Age of women, BMI, basal FSH concentration, AFC, the number of oocytes obtained, serum serum concentration on the day of HCG, number of embryos replaced, mean ovarian VI, FI and VFI were not predictive of pregnancy when these factors were entered in a conditional forward fashion in multiple logistic regression analysis. 45

4 Table 1. Summary of demographic data and ovarian responses (n = 111). Parameters Median (interquartile range) Age of women (years) 35.0 (32 37) Duration of infertility (years) 4.0 (3 6) Primary ifertility a 82 (73.9) Body mass index (kg/m 2 ) 21.4 ( ) Basal FSH concentration (IU/l) 6.5 ( ) Total antral follicle count 11.0 (8 15) Mean ovarian volume (cm 3 ) 6.21 ( ) Mean ovarian VI (%) 2.05 ( ) Mean ovarian FI (0 100) ( ) Mean ovarian VFI (0 100) 0.63 ( ) HMG dosage (IU) 1800 ( ) HMG duration (days) 10.0 (9 12) Total number of eggs obtained 10.0 (7 13) Oestradiol on HCG day (pmol/l) 11,131 ( ,074) a Given as number (%); VI = vascularization index; FI = flow index; VFI = vascularization flow index; HMG = human menopausal gonadotrophin; HCG = human chorionic gonadotrophin. Table 2. Multiple regression analysis evaluating the values of different parameters in predicting the number of oocytes obtained. B (95% CI) β R 2 change P-value Constant (18.609, ) AFC 0.421(0.204, 0.638) <0.001 Age of women ( 0.809, 0.224) BMI ( 0.720, 0.057) B is the unstandardized coefficient; β is the standardized coefficient. Basal FSH concentration, mean ovarian volume, mean ovarian vascularization index, flow index and vascularization flow index were excluded from the equation. AFC = antral follicle count; BMI = body mass index. Total number of oocytes = AFC age of women BMI; R = 0.539; adjusted R 2 = Table 3. Multiple regression analysis evaluating the values of different parameters in predicting the duration of human menopausal gonadotrophin (HMG) used. B (95% CI) β R 2 change P-value Constant (16.200, ) Basal FSH (0.494, 1.479) <0.001 B is the unstandardized coefficient; β is the standardized coefficient. Age of women, body mass index, antral follicle count, mean ovarian volume, mean ovarian vascularization index, flow index and vascularization flow index were excluded from the equation. HMG duration = basal FSH; R = 0.356; adjusted R 2 =

5 Table 4. Multiple regression analysis evaluating the values of different parameters in predicting the dosage of human menopausal gonadotrophin (HMG) used. B (95% CI) β R 2 change P-value Constant (7.396, ) Basal FSH (0.159, 0.535) <0.001 B is the unstandardized coefficient; β is the standardized coefficient. Age of women, body mass index, antral follicle count, mean ovarian volume, mean ovarian vascularization index, flow index and vascularization flow index were excluded from the equation. HMG dosage = basal FSH; R = 0.330; adjusted R 2 = Table 5. Comparison of demographic data, ovarian reserve markers and ovarian responses between non-pregnant and pregnant patients. Values are given as median (interquartile range). Non-pregnant (n = 73) Pregnant (n = 24) Age of women (years) 35.0 ( ) 35.0 ( ) Primary infertility a 53 (72.6) 18 (75.0) Infertility duration (years) 4.0 ( ) 4.0 ( ) Causes of infertility 1 Tubal 18 (24.7) 7 (29.2) Male 38 (52.1) 12 (50.0) Endometriosis 6 (8.2) 2 (8.3) Unexplained 7 (9.6) 3 (12.5) Mixed 4 (5.5) 0 (0.0) Body mass index (kg/m 2 ) 21.5 ( ) 21.6 ( ) Basal FSH concentration (IU/l) 6.3 ( ) 6.4 ( ) Total antral follicle count 11.0 (8 16) 11.0 (9 13.8) Mean ovarian volume (cm 3 ) 6.20 ( ) 6.70 ( ) Mean ovarian VI (%) 2.02 ( ) 2.08 ( ) Mean ovarian FI (0 100) ( ) ( ) Mean ovarian VFI (0 100) 0.64 ( ) 0.59 ( ) HMG dosage (IU) 1800 ( ) 1800 ( ) HMG duration (days) 11.0 ( ) 10.0 ( ) Serum oestradiol b (pmol/l) 10,618 ( ,097) 11,572 ( ,083) No. of oocytes obtained 10.0 ( ) 10.0 ( ) No. of embryos transferred a One 7 (9.6) 1 (4.2) Two 64 (87.7) 23 (95.8) Three 2 (2.7) 0 (0.0) There were no statistically significant differences between the pregnant and non-pregnant women. See Table 1 for definition of abbreviations. a Given as number (%). b On the day of human chorionic gonadotrophin injection. 47

6 48 Discussion It is important to highlight that the infertile women examined in this prospective study had relatively normal ovarian reserve as suggested by age <40 years at the time of treatment and basal FSH concentration <10 IU/L. In order to avoid confounding factors, women were recruited who were undergoing their first IVF cycle and receiving the same starting dose of HMG after a long protocol of pituitary down-regulation for ovarian stimulation. All patients receiving ovarian stimulation were offered egg collection, unless there was absent follicular development after 14 days of stimulation. Multiple regression analysis was applied to compare the significance of various factors in the prediction of ovarian response. Ovarian response was assessed by the number of oocytes aspirated, serum oestradiol on the day of HCG, the dosage and duration of HMG. It has been demonstrated that AFC achieved the best predictive value in relation to the number of oocytes obtained, followed by age of women and BMI. Basal FSH concentration was the only predictive factor for the HMG duration and dosage. Mean ovarian 3D power Doppler flow indices measured after pituitary down-regulation were not predictive of ovarian response and pregnancy in the IVF treatment. Mean peak systolic velocity of ovarian stromal vessels prior to pituitary down-regulation was found to be significantly related to the number of follicles, after controlling for age of women (Zaidi et al., 1996). Similarly, ovarian stromal peak systolic velocity after pituitary down-regulation was the most important single independent predictor of the number of oocytes obtained in patients with normal basal FSH concentration, when compared with age of women, basal FSH concentration, oestradiol concentration or FSH:LH ratio (Engmann et al., 1999). Bassil et al. (1997) reported that women with resistance index of ovarian blood flow >0.56 had a significantly longer stimulation and a significantly lower mean number of oocytes retrieved. BMI and AFC were not included in these three studies. Both Zaidi et al. (1996) and Engmann et al. (1999) recruited patients with polycystic ovaries and used different starting doses of gonadotrophins. Popovic-Todorovic et al. (2003) determined ovarian stromal blood flow by 2D power Doppler ultrasound and a semiquantitative score was allocated to each ovary according to the number and area of the Power Doppler signals. Total Doppler score was the sum of scores for each ovary: score 1 for poor flow; score 2 for moderate flow and score 3 for good flow. The number of oocytes was predicted by AFC, total Doppler score, serum testosterone concentration and smoking status. The scoring for power Doppler signals was subjective. Using 3D ultrasound with power Doppler, Kupesic and Kurjak (2002) demonstrated that AFC achieved the best predictive value for successful IVF outcome, followed by ovarian stromal FI, oestradiol on the day of HCG, total ovarian volume, total ovarian stromal area and age of women. No pregnancy occurred in patients with low mean ovarian FI <11 (Kupesic et al., 2003). In a previous study with a similar design, it was demonstrated that ovarian stromal vascularity indices measured by 2D power Doppler ultrasound had no predictive value for the number of oocytes obtained, serum oestradiol concentration on the day of HCG, the duration and dosage of HMG used (Ng et al., 2005a). As the ovarian stromal vascularity can now be objectively measured by 3D power Doppler ultrasound, it is postulated that total ovarian 3D power Doppler indices might play a role in predicting the ovarian response and pregnancy. In theory, increased ovarian stromal vascularity leads to a greater delivery of gonadotrophins to the ovary and the developing follicles, leading to a higher number of oocytes, a shorter duration of stimulation and lesser amount of gonadotrophins required. This hypothesis could not be confirmed, and the results obtained were different from that of Kupesic and Kurjak (2002) and Kupesic et al. (2003), who used varying starting doses ( IU) of gonadotrophin. AFC remained the best predictive factor for the number of oocytes obtained (Ng et al., 2000b, 2005b). A previous study performed in fertile Chinese women showed that ovarian stromal vascularity was significantly lower after the age of 41 and the rate of decline of total ovarian VI was only 0.18% per year (Ng et al., 2004). Similarly, peak systolic velocity of ovarian stromal vessels determined by 2D colour Doppler ultrasound did not significantly change with increasing age (Ng et al., 2003). The results are contradictory to those of Pan et al. (2002) and Kupesic et al. (2003). Pan et al. (2002) measured ovarian vascularity by 3D power Doppler ultrasound in 100 consecutive normal women without ultrasound features of polycystic ovaries, who were classified into three groups according to the menstrual pattern. Ovarian VI, FI and VFI decreased significantly in the order of premenopause, perimenopause and then postmenopausal. In 56 consecutive infertile patients undergoing their first IVF cycle, Kupesic et al. (2003) found that AFC, total ovarian volume and mean ovarian FI were negatively related to the age of patients and decreased linearly with advancing age. In summary, mean ovarian 3D power Doppler flow indices measured after pituitary down-regulation were not predictive of the ovarian response and pregnancy in the IVF treatment. Acknowledgements This study was funded by the Hong Kong Research Grant Council (HKU 7280/01M). 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