Effect of age on ovarian stromal flow measured by three-dimensional ultrasound with power Doppler in Chinese women with proven fertility

Human Reproduction Vol.19, No.9 pp. 2132 2137, 2004 Advance Access publication July 8, 2004 DOI: 10.1093/humrep/deh387 Effect of age on ovarian stromal flow measured by three-dimensional ultrasound with power Doppler in Chinese women with proven fertility Ernest Hung Yu Ng 1, Carina Chi Wai Chan, William Shu Biu Yeung and Pak Chung Ho Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong Special Administrative Region, People s Republic of China 1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, The University of Hong Kong, 6/F, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong. E-mail: nghye@hkucc.hku.hk BACKGROUND: Increasing age is associated with reduced ovarian stromal blood flow detected by threedimensional (3D) power Doppler ultrasound in infertile patients. However, little information exists in the literature regarding the effect of age on the ovarian stromal blood flow in fertile women. METHODS: On the second to fourth day of their menstrual cycle, fertile Chinese women who had regular monthly cycles and no history of tubal and ovarian surgery underwent a transvaginal scan with 3D power Doppler to determine total antral follicle count (AFC), total ovarian volume, total ovarian vascularization index (VI)/flow index (FI)/vascularization flow index (VFI). Serum FSH and vascular endothelial growth factor concentrations were checked. RESULTS: Out of 177 women scanned, 138 women were included in the final analysis. Total AFC and serum FSH were significantly different among four age groups (#30, 31 35, 36 40 and $41 years). Total ovarian VI and VFI were significantly lower in women aged $41 years. AFC had the best correlation with the age of women, followed by serum FSH concentration and ovarian 3D power Doppler indices. The rate of decline of total ovarian VI was 0.18% per year. CONCLUSION: Ovarian stromal blood flow was significantly reduced only in women aged $41 years. Key words: fertile women/ovary/stromal blood flow/three-dimensional power Doppler Introduction Fertility is remarkably reduced with increasing age of women in both spontaneous conceptions (Tietze, 1957) and assisted reproduction methods (Templeton et al., 1996). The decline in fertility with ageing is likely to be due to a continuous reduction in the number of primordial follicles after birth. Block (1952) observed that. 250 000 primordial follicles were present at menarche but only a few hundreds or thousands remained at the end of reproductive life. As many women in developed countries delay childbearing to fulfil their personal goals, an early and accurate assessment of the ovarian reserve would be of help in counselling these women about their fertility potentials and perhaps in scheduling pregnancy. We have recently demonstrated in a group of Chinese fertile women that antral follicle count (AFC) of both ovaries is best correlated with the age of women and declines linearly at 3.8% per year or 0.35 follicle per year (Ng et al., 2003). Other markers including serum FSH concentration, serum inhibin B concentration, ovarian volume and peak systolic velocity (PSV) of ovarian stromal vessels determined by two-dimensional (2D) colour Doppler ultrasound did not significantly change with increasing age. Folliculogenesis in the human ovary is a complex process regulated by a variety of endocrine and paracrine signals (McGee and Hsueh, 2000). It has been suggested that 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). Ovarian stromal blood flow can be assessed by colour Doppler and power Doppler ultrasound. Power Doppler imaging is more sensitive than colour Doppler imaging at detecting low velocity flow and hence improves the visualization of small vessels (Guerriero et al., 1999). In particular, ovarian stromal blood flow can now be more objectively determined by three-dimensional (3D) ultrasound with power Doppler and is well correlated with ovarian response and subsequent IVF outcome (Kupesic and Kurjak, 2002). Vascular endothelial growth factor (VEGF) is one of the key factors regulating angiogenesis in the ovary during the cyclic growth of ovarian follicles and corpus luteum development and maintenance (Abulafia and Sherer, 2000; Geva and Jaffe, 2000). VEGF is a diffusible endothelial cell mitogen with potent angiogenic properties (Senger et al., 1983; Ferrara and Henzel, 1989; Gospodarowicz et al., 1989). Within the ovary, it is expressed in the theca cells (Gordon 2132 Human Reproduction vol. 19 no. 9 q European Society of Human Reproduction and Embryology 2004; all rights reserved

Ovarian stromal blood flow in fertile Chinese women et al., 1996), granulosa and lutein cells (Phillips et al., 1990; Shweiki et al., 1993; Koos, 1995; Neulen et al., 1995) and the interstitial tissue (Kamat et al., 1995). Five VEGF forms have been identified in mammals, resulting from alternative splicing of the single VEGF gene: VEGF 121, VEGF 145, VEGF 165, VEGF 189 and VEGF 206. RT PCR studies demonstrated that mrna encoding VEGF 165 and VEGF 121 were dominant in normal human ovaries (Fujimoto et al., 1998; Otani et al., 1999). Increasing age was associated with reduced ovarian stromal blood flow detected by 3D power Doppler in infertile patients (Kupesic et al., 2003). Information on fertile women regarding the ovarian stromal blood flow measured by 3D power Doppler ultrasound and its relationship to AFC and serum VEGF concentration is scarce in the literature. The aims of this prospective study were to evaluate the effect of age on the ovarian stromal blood flow measured by 3D power Doppler ultrasound and to correlate the 3D power Doppler indices with AFC and serum VEGF concentration in Chinese women with proven fertility. Materials and methods Chinese women attending the Department of Obstetrics and Gynaecology at the University of Hong Kong between August 2002 and August 2003 for normal antenatal care or termination of pregnancy were recruited when the following criteria were met: (i) history of spontaneous conception(s); (ii) regular menstrual cycles with cycle length of 25 35 days with #4 days difference between cycles; and (iii) presence of both ovaries. Exclusion criteria were: (i) history of infertility investigation or treatment; (ii) presence of gynaecological disorders such as menorrhagia or dysfunctional uterine bleeding; (iii) history of tubal or ovarian surgery; (iv) smoking; and (v) history of taking sex hormonal preparation within the last 3 months. Poor visualization of ovaries because of abdominal position, presence of an ovarian cyst of $20 mm in diameter and polycystic ovaries (Adams et al., 1986) were retrospectively excluded. Every patient gave a written informed consent prior to participating in the study, which was approved by the Ethics Committee, Faculty of Medicine, the University of Hong Kong. They did not receive any monetary compensation for participation in the study. Two to three months after delivery or termination of pregnancy, they attended the Day Care Centre for a transvaginal ultrasound examination and blood test at around 08:00 10:00 h in the early follicular phase (day 2 4) of the menstrual cycle. All 3D ultrasound examinations were performed by E.H.Y.N. using Voluson 730 w (Kretz, Austria), after the patients had emptied the bladder. AFC was obtained in the multi-planar 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: Frequency: Mid; Dynamic set: 2; Balance: G. 140; Smooth: 5/5; Ensemble: 12; Line Density: 7; Power Doppler Map: 5; and the setting condition for the sub-power Doppler mode was: Gain: 26.0; Balance: 140; Quality: normal; Wall Motion Filter: low1; Velocity range: 0.9 khz. The 3D ultrasound images were stored for later analysis by E.H.Y.N., who was blinded to the age of women during both scanning and analysis. The built-in VOCAL w (virtual organ computer-aided analysis) Imaging Program for the 3D power Doppler histogram analysis was used to determine 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) made by multiplying VI and FI is a combination of vascularity and flow (Pairleitner et al., 1999). During the analysis and calculation, the manual mode of the VOCAL Contour Editor was used to cover the whole 3D volume of the ovary with a 158 rotation step. Hence, 12 contour planes were analysed for each ovary to cover 1808. To assess the reliability of 3D scanning and data acquisition, the ovaries of 15 patients were scanned twice and each 3D dataset was analysed twice using VOCAL. The mean intraclass correlation coefficient (ICC) with 95% confidence interval (CI) was calculated by the one-way random effects model (Järvelä et al., 2003; Raine- Fenning et al., 2003). The mean (95% CI) ICC for 3D scanning of ovarian volume, VI, FI and VFI were 0.9923 (0.9686, 0.9982), 0.9636 (0.8505, 0.9916), 0.9707 (0.8800, 0.9933) and 0.9558 (0.8188, 0.9899) respectively. The mean (95% CI) ICC for data acquisition of ovarian volume, VI, FI and VFI were 0.9898 (0.9693, 0.9967), 0.9984 (0.9951, 0.9995), 0.9733 (0.9196, 0.9913) and 0.9981 (0.9942, 0.9994) respectively. Blood was then taken for the measurement of FSH and VEGF concentrations. The blood samples were processed by centrifuge within 2 h of collection and the supernatant was stored at 2208C for subsequent analysis. Serum FSH was measured by an immunoassay (Bayer Corporation, USA) and the inter-assay and intra-assay coefficients of variation were 2.8 and 1.7% respectively. Serum VEGF was measured by a quantitative sandwich enzyme immunoassay technique (Quantikine w ; R&D Systems, UK), which was designed to measure VEGF 165 levels. The minimum detectable VEGF concentration by the assay was 9.0 pg/ml. The inter-assay coefficients of variation (CV) were 8.8, 7.0 and 6.2% at concentrations of 65, 250 and 1003 pg/ml respectively, whereas the intra-assay CV were 6.7, 4.5 and 5.1% at concentrations of 54, 235 and 910 pg/ml respectively. Statistical analysis Women aged #30, 31 35, 36 40 and $41 years were classified as groups I, II, III and IV respectively. The primary outcome measures included VI/FI/VFI of both ovaries, AFC of both ovaries, and serum VEGF concentration. Continuous variables were not normally distributed and were given as median (range), unless indicated. Statistical tests were carried out by Kruskal Wallis and Mann Whitney U-tests, where appropriate. Correlation was assessed by the Spearman rank method. The effect of age on markers of ovarian reserve was examined by a linear regression model. Two-tailed P, 0.05 was considered significant. Table I. Summary of demographic data and ovarian reserve markers Parameters Median (range) Age (years) 38.0 (25 45) Total antral follicle count 10.0 (2 23) Total ovarian volume (cm 3 ) 10.62 (4.24 38.10) Total ovarian VI (%) 1.93 (0.03 36.97) Total ovarian FI (0 200) 51.06 (19.18 95.64) Total ovarian VFI (0 200) 0.54 (0.01 26.44) Serum FSH (IU/l) 6.30 (1.0 16.9) Serum VEGF (pg/ml) 276.35 (52.3 1288.8) VI ¼ total ovarian vascularization index; FI ¼ flow index; VFI ¼ vascularization flow index; VEGF ¼ vascular endothelial growth factor. 2133

E.H.Y.Ng et al. Table II. Comparison of markers of ovarian reserve in different age groups Median (range) Group I Group II Group III Group IV (n ¼ 15) (n ¼ 32) (n ¼ 61) (n ¼ 30) Age (years) 29.0 (25 30) 33.0 (31 35) 39.0 (36 40) 42.0 (41 45) Total antral follicle count 12.0 a (10 19) 15.0 a (5 26) 11.0 a (2 20) 6.0 a (2 16) Total ovarian volume (cm 3 ) 9.83 (5.61 27.08) 12.39 (4.24 24.78) 10.23 (4.66 38.10) 11.10 (5.83 23.71) Total ovarian VI (%) 2.73 (0.09 12.47) 2.15 (0.12 36.97) 2.44 b (0.08 21.14) 1.30 b (0.03 10.51) Total ovarian FI (0 200) 54.77 (37.78 67.25) 51.39 (27.80 95.64) 51.65 (19.18 86.50) 45.62 (27.24 66.21) Total ovarian VFI (0 200) 0.68 (0.02 4.09) 0.54 (0.03 17.62) 0.62 c (0.01 7.95) 0.32 c (0.01 26.44) Serum FSH (IU/l) 5.50 d (1.0 7.0) 5.75 d (1.4 13.7) 6.70 d (2.5 12.0) 8.05 d (1.9 16.9) Serum VEGF (pg/ml) 270.93 (135.3 730.5) 257.61 (113.0 844.6) 295.04 (52.3 1288.8) 262.20 (61.3 862.4) a P, 0.001 among four groups and between groups III and IV; P ¼ 0.04 between groups I and III; P ¼ 0.007 between groups II and III. b P ¼ 0.022 between groups III and IV. c P ¼ 0.027 between groups III and IV. d P, 0.001 among four groups; P ¼ 0.005 between groups I and III; P ¼ 0.027 between groups II and III; P ¼ 0.015 between groups III and IV. VI ¼ total ovarian vascularization index; FI ¼ flow index; VFI ¼ vascularization flow index; VEGF ¼ vascular endothelial growth factor; NS ¼ not significant. Table III. Correlation coefficients of age of women and total antral follicle count with ultrasound and hormonal markers of ovarian reserve Age Age (years) 20.501 Total antral follicle count 20.501 a Total ovarian volume (cm 3 ) 20.069 0.198 b Total ovarian VI (%) 20.203 b 0.211 b Total ovarian FI (0 200) 20.182 c 0.176 c Total ovarian VFI (0 200) 20.192 d 0.209 b Serum FSH 0.407 a 20.363 a Serum VEGF 20.005 0.173 a P, 0.001. b P ¼ 0.02. c P ¼ 0.04. d P ¼ 0.03. Total antral follicle count Results A total of 229 eligible women were approached and agreed to participate in the study. Only 177 women attended the clinic for ultrasound examination and others defaulted the appointment because of personal reasons. After ultrasound examination, 39 women were excluded from the study: poor visualization of ovaries in seven women, an ovarian cyst in 17 women and polycystic ovaries in 15 women. Therefore, 138 women were included in the final analysis: 15 in group I; 32 in group II; 61 in group III and 30 in group IV. Their demographic data and markers of ovarian reserve are summarized in Table I. Significant differences were found in total AFC and serum FSH concentration among the four groups (Table II). Total ovarian VI and VFI were significantly lower in group IV than in group III. Age of women was negatively correlated with total AFC, total ovarian VI/FI/VFI and was positively correlated with serum FSH concentration (Table III). Total AFC was positively correlated with total ovarian volume and total ovarian VI/FI/VFI and was negatively correlated with age of women and serum FSH concentration (Table III). Serum VEGF concentration was significantly correlated with total ovarian volume only (r ¼ 0.223; P ¼ 0.019). In the linear regression model, the rate of decline of total ovarian VI was 0.18% per year (95% CI ¼ 0.003 0.37, 2134 Figure 1. Effect of the age of women on total ovarian vascularization index (VI). P ¼ 0.047) (Figure 1). The rate of decrease of the total AFC was 0.52 follicle per year (95% CI ¼ 0.36 0.67, P, 0.001) and serum FSH concentration increased by 0.23 IU/l per year (95% CI ¼ 0.14 0.32, P, 0.001). On the other hand, there was no significant decline of total ovarian FI or VFI with respect to age. Discussion Different hormonal and ultrasound markers for ovarian reserve have been examined in assisted reproduction methods to predict the ovarian response to gonadotrophins, including early follicular serum FSH (Scott and Hofmann et al., 1995; Sharara et al., 1998), serum inhibin B (Seifer et al., 1997, 1999; Tinkanen et al., 1999; Dzik et al., 2000), serum anti- Müllerian hormone (Seifer et al., 2002; Van Rooij et al., 2002; Fanchin et al., 2003), ovarian volume (Syrop et al., 1995, 1999; Lass et al., 1997), AFC (Tomás et al., 1997; Chang et al., 1998a,b; Frattarelli et al., 2000; Ng et al., 2000; Hsieh et al., 2001; Nahum et al., 2001; Kupesic and Kurjak, 2002; Popovic-Todorovic et al., 2003) and ovarian stromal blood flow (Zaidi et al., 1996; Engmann et al., 1999; Kupesic and Kurjak, 2002; Kupesic et al., 2003; Popovic- Todorovic et al., 2003).

Ovarian stromal blood flow in fertile Chinese women Zaidi et al. (1996) first demonstrated that PSV of ovarian stromal blood vessels measured by 2D colour Doppler prior to pituitary down-regulation was significantly correlated with the ovarian response, after controlling for patients age. Those with normal ovarian responses had significantly higher velocity than poor responders (10.2 ^ 5.8 versus 5.2 ^ 4.2 cm/s) and other colour Doppler flow indices were not useful. PSV of ovarian stromal blood vessels obtained after pituitary down-regulation was also predictive of the ovarian response and the IVF outcome (Engmann et al., 1999). Bassil et al. (1997) reported that women with resistance index (RI) of ovarian blood vessels.0.56 had a significantly longer stimulation and a significantly lower mean number of oocytes retrieved. Recently, ovarian stromal blood flow assessed by power Doppler ultrasound has also been useful in the prediction of ovarian response, whether using 2D (Popovic-Todorovic et al., 2003) or 3D (Kupesic and Kurjak, 2002) ultrasound. In 56 consecutive infertile patients undergoing their first IVF cycle, Kupesic et al. (2003) found statistically significant differences in AFC, total ovarian volume and mean ovarian FI among four age groups (#30, 31 35, 36 40 and.40 years). AFC, total ovarian volume and mean ovarian FI were negatively correlated with the age of patients and decreased linearly with advancing age. Pan et al. (2002a) measured ovarian blood flow 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. Pre-menopausal women had menstruation within the last 3 months; peri-menopausal women had last menstruated between 3 and 12 months and post-menopausal women had no periods within the last 12 months. Ovarian VI, FI and VFI decreased significantly in the order of pre-menopause, peri-menopause and then postmenopausal. The effect of age on ovarian stromal blood flow of fertile women could not be determined in this study because detailed characteristics of these women regarding age, previous pregnancy and smoking habit were not given. AFC was not documented. This is the first study addressing the effects of age on the ovarian stromal blood flow measured by 3D power Doppler ultrasound and its relationship to AFC and serum VEGF concentration in women with proven fertility. In order to ascertain normal fertility, we recruited study subjects from women who had a recent history of spontaneous pregnancy and excluded those who had undergone tubal sterilization. Patients with tubal surgery were not recruited because both AFC and ovarian stromal 3D power Doppler indices were reduced after laparoscopic salpingectomy for ectopic pregnancy (Chan et al., 2003). There is an adverse effect of smoking on ovarian function which is dose dependent (Shiverick and Salafia, 1999) and therefore smokers were not recruited in this study either. Women with polycystic ovaries were excluded from the analysis to avoid bias due to increased ovarian stromal flow in these women demonstrated by 3D power Doppler ultrasound (Pan et al., 2002b). Our previous study (Ng et al., 2003) could not find any effect of age on mean PSV of ovarian stromal blood vessels determined by 2D colour Doppler ultrasound. Power Doppler is better suited to the study of the ovarian stromal blood flow as it is more sensitive to lower velocities and essentially angle independent (Rubin et al., 1994). In combination with 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. Total ovarian VI and VFI were significantly lower in women aged $41 years. Ovarian 3D power Doppler indices were negatively correlated with age of women but their correlation coefficients were much smaller when compared with those for AFC and serum FSH. In contrast, Kupesic et al. (2003) noted a good correlation between mean ovarian FI and age of infertile women with a correlation coefficient of 20.68. These infertile patients had early follicular serum FSH concentration, 10 IU/l and the 3D ultrasound examinations were performed after confirmation of pituitary down-regulation. Our data suggest that reduction in ovarian stromal blood flow with increasing age is a relatively late phenomenon and ovarian stromal blood flow is unlikely to be an early marker of ovarian reserve tests. The significance of AFC has been extensively evaluated in infertile patients undergoing assisted reproduction treatment (Tomás et al., 1997; Chang et al., 1998a,b; Ng et al., 2000;Fratarelli et al., 2000; Hsieh et al., 2001; Nahum et al., 2001; Kupesic and Kurjak, 2002; Popovic-Todorovic et al., 2003). We previously demonstrated that AFC achieved the best predictive value, followed by early follicular serum FSH concentration, body mass index and age of women in a prospective study of 128 infertile patients undergoing the first IVF cycle using a standard regimen of ovarian stimulation (Ng et al., 2000). AFC has also been shown to be the best reflection of reproductive age in normal women with proven fertility among other hormonal and ultrasound markers of ovarian reserve (Scheffer et al., 1999, 2003; Ng et al., 2003). The present study demonstrated that ovarian 3D power Doppler indices were positively correlated with AFC. In a linear regression model, the rate of decline of total ovarian VI was 0.18% per year and there was no significant decline of total ovarian FI and VFI with age. Further longitudinal studies exploring the impact of ovarian stromal blood flow on the rate of decline of AFC in normal women are warranted. The rate of decrease of AFC with respect to age was 0.52 follicle per year (95% CI 0.36 0.67) and was comparable to 0.35 follicle per year (95% CI 0.26 0.45) in our previous study (Ng et al., 2003). The rate of decline of AFC in Chinese women (Chang et al., 1998b; Ng et al., 2003) appeared to be much lower than those reported in Caucasians. AFC decreased by 0.95 follicle per year (Reuss et al., 1996) or by 8.2% per year (95% CI 5.2 11.2%) (Scheffer et al., 1999). The reasons for such difference remain unknown. The rate of decrease in ovarian stromal flow in fertile Caucasian women is not currently available and it would be interesting to see if there is any difference in the decrease in ovarian stromal flow between Chinese and Caucasians. Angiogenesis plays an important role in both the follicular and luteal phases of an ovarian cycle and VEGF is one of 2135

E.H.Y.Ng et al. the key factors regulating angiogenesis in the ovary (Abulafia and Sherer, 2000; Geva and Jaffe, 2000). Higher serum concentrations of VEGF were found in women with polycystic ovaries and may be related to the increased ovarian stromal vascularity shown by Doppler blood flow velocity measurement in these women, when compared with women without polycystic ovaries (Agrawal et al., 1998a). Serum VEGF concentration rose in the luteal phase in parallel with changes with ovarian and uterine blood flow velocities (Agrawal et al., 1998b). In the present study, we did not find any difference in serum VEGF concentration among the four age groups. The serum VEGF concentration was significantly correlated with total ovarian volume only and there was no significant correlation with AFC and ovarian stromal 3D power Doppler indices. Our data suggested that serum VEGF concentration did not reflect ovarian stromal blood flow. These data differed from the report by Agrawal et al. (1998a), who demonstrated that serum VEGF concentrations were positively correlated with PSV and time-averaged maximum flow velocity of ovarian stromal vessels measured in both women with and without polycystic ovaries. The regulation of ovarian angiogenesis is a complex process and other vasoactive and angiogenic factors including angiopoietin, angiogenin and fibroblast growth factor are also involved (Geva and Jaffe, 2000). The early follicular phase FSH level taken prior to the treatment cycle is widely used in many IVF programmes. Although there was a positive correlation of age with serum FSH concentration, the median serum FSH concentrations were below the normal range of FSH levels (,10 IU/l) in all four age groups. The age-dependent hormonal changes are a relatively late phenomenon and only occur when the number of follicles is much reduced (te Velde and Pearson, 2002). In summary, total ovarian VI and VFI were significantly lower in women aged $41 years. 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