Department of Obstetrics & 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, Queen Mary Hospital, Pokfulam Road, Hong Kong. Email: nghye{at}hkucc.hku.hk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: ovarian stromal blood flow/polycystic ovary syndrome/three-dimensional power Doppler
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Subjective assessment of the intensity and quantity of coloured areas during colour or power Doppler analysis usually appears to be higher in polycystic than normal ovaries. Increased ovarian stromal blood flow has been considered to be a new parameter to assist in the ultrasound diagnosis of polycystic ovaries. Zaidi et al. (1995) first reported a significantly greater mean ovarian stromal peak systolic blood flow velocity (PSV) and time averaged maximum velocity in PCOS women than infertile women with normal ovaries. Higher serum concentrations of vascular endothelial growth factor (VEGF) were found in PCOS women and were related to the increased ovarian stromal velocities, when compared with women with normal ovaries (Agrawal et al., 1998
). Both pulsatility index (PI) and resistance index (RI) of ovarian stromal vessels were significantly lower in polycystic than normal ovaries (Aleem and Predanic, 1996
; Dolz et al., 1999
), although the mean ovarian PSV was not different between them (Aleem and Predanic, 1996
). Battaglia et al. (1995)
also showed that the presence of ovarian stromal vascularization with low RI had a high diagnostic value for PCOS. Using three-dimensional (3D) ultrasound with power Doppler, Pan et al. (2002)
demonstrated significantly higher ovarian stromal blood flow in PCOS women while Järvelä et al. (2002)
found similar ovarian stromal blood flow between PCOS women and women with normal ovaries. Therefore, conflicting information exists in the literature with respect to ovarian stromal blood flow in PCOS women.
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
). 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 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 two-dimensional (2D) planes.
Infertile women with normal ovaries were chosen as normal controls in the above studies on ovarian stromal blood flow in PCOS. The details of the normal controls were not given in the studies of Battaglia et al. (1995) and Dolz et al. (1999)
. It is possible that ovarian stromal blood flow is reduced in infertile women and therefore fertile women with normal ovaries may be a much better choice for comparison. The aims of this prospective study were (i) to compare the ovarian stromal blood flow measured by 3D power Doppler ultrasound and serum VEGF concentration between fertile women with normal ovaries and infertile women with PCOS, and (ii) to compare the ovarian stromal blood flow and hormonal parameters of PCOS women in relation to their body mass index (BMI) and AFC.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
They attended the Day Care Centre for a transvaginal ultrasound examination and blood test in the early follicular phase (Days 24) of a spontaneous menstrual period in women with regular periods or a withdrawal bleeding in women with long irregular cycles. The details and reliability of 3D volume acquisition and data analysis were as previously described (Chan et al., 2003; Ng et al., 2004
). All 3D ultrasound examinations were performed at 810 am by EHYN using Voluson 730 (Kretz, Zipf, Austria) with a 59 MHz transvaginal probe, 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: 6.0; 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.
The built-in VOCAL (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 15° rotation step. Hence, 12 contour planes were analyzed for each ovary to cover 180°.
The mean intraclass correlation coefficient (ICC) with 95% confidence interval (CI) for 3D scanning of ovarian volume, VI, FI and VFI were 0.9923 (95%CI: 0.9686, 0.9982), 0.9636 (95%CI: 0.8505, 0.9916), 0.9707 (95%CI: 0.8800, 0.9933) and 0.9558 (95%CI: 0.8188, 0.9899), respectively. The mean ICC for data acquisition of ovarian volume, VI, FI and VFI were 0.9898 (95%CI: 0.9693, 0.9967), 0.9984 (95%CI: 0.9951, 0.9995), 0.9733 (95%CI: 0.9196, 0.9913) and 0.9981 (95%CI: 0.9942, 0.9994), respectively.
Blood was then taken for the measurement of FSH, LH and VEGF concentrations. The blood samples were processed by centrifuge within 2 h of collection and the supernatant was stored at 20 °C for subsequent analysis. Serum FSH and LH concentrations were measured using commercially available kits (Automated Chemiluminescence System, Bay Corporation, NY). The sensitivity of the FSH assay was 0.3 IU/l and the intra- and inter-assay coefficients of variation were 1.7% and 2.8%, respectively. The sensitivity of the LH assay was 0.07 IU/l and the intra- and inter-assay coefficients of variation were 4.5% and 5.2%, respectively. Serum VEGF was measured by a quantitative sandwich enzyme immunoassay technique (Quantikine, R & D Systems, Oxon, UK), which was designed to measure VEGF165 levels. The minimum detectable VEGF concentration by the assay was 9.0 pg/ml. The inter-assay coefficients of variation were 8.8%, 7.0% and 6.2% at the concentrations of 65, 250 and 1003 pg/ml, respectively, whereas the intra-assay coefficients of variation were 6.7%, 4.5% and 5.1% at the concentrations of 54, 235 and 910 pg/ml, respectively.
Concentrations of serum free testosterone, androstenedione, dehydroepiandrosterone sulphate (DHEAS), sex hormone binding globulin (SHBG) and fasting leptin were checked in PCOS women. Serum free testosterone, androstenedione, DHEAS and leptin were measured by immunoassay (Diagnostic Systems Laboratories, Texas) and serum SHBG concentration was measured by immunoassay (Human Gesellschaft für Biochemica und Diagnostica, Wiesbaden, Germany).
Statistical analysis
The primary outcome measures included VI, FI and VFI 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 MannWhitney U tests, where appropriate. Two way ANOVA was then used for comparison in order to control the difference in the age of women between normal fertile women and PCOS women. Correlation was assessed by the Spearman rank method. P-value (two-tailed) of <0.05 was taken as significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
PCOS women
Total ovarian VI / FI / VFI was negatively correlated with BMI, which was positively correlated with age of women (Table II). Total AFC was positively correlated with serum LH concentration only.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We found significantly higher ovarian stromal blood flow in normal weight PCOS women than their overweight counterparts. No significant difference in RI of ovarian stromal vessels between lean (BMI < 23 kg/m2) and obese (BMI > 25 kg/m2) PCOS women was shown by Battaglia et al. (1996). Similarly, Dolz et al. (1999)
could not find any difference in intraovarian PI and RI between non-obese (BMI < 25 kg/m2) and obese (BMI > 25 kg/m2) PCOS women. We could not give any explanation for an increased ovarian stromal blood flow observed in normal weight PCOS women as both normal weight and overweight PCOS women were of similar age and had comparable hormonal profiles, except the difference in fasting leptin concentration.
Our result differed from that of Pan et al. (2002) but different control groups were used in these two studies. We selected fertile women with normal ovaries while Pan et al. (2002)
chose infertile women with normal ovaries. Moreover, Pan et al. (2002)
found significant differences in age of women and BMI between infertile women with normal ovaries and PCOS but did not control these when comparing ovarian 3D power Doppler flow indices. Our Doppler ultrasound findings also appeared to be contradictory to the histological documentation of increased vascularity in the SteinLeventhal ovaries syndrome (Hughesdon, 1982
) but different methods were employed to determine vacularity and ovarian stromal blood flow.
Our previous study (Ng et al., 2004) addressing the effect of age on ovarian stromal blood flow in normal fertile women revealed that total ovarian VI/FI/VFI was negatively correlated with age of women and was positively correlated with total AFC. In PCOS women, the relationship among the age of women, total AFC and total ovarian VI/FI/VFI seemed to be different. The total ovarian VI/FI/VFI in PCOS women was positively correlated with BMI only but was not correlated with age of women and total AFC. The vascular impedance to blood flow was not influenced by total AFC and no correlation was found between serum hormone concentration and PI and RI of ovarian stromal vessels in PCOS women (Aleem and Predanic, 1996
). In contrast, Dolz et al. (1999)
showed that the total AFC was directly related to the degree of intraovarian vascular flow pattern during colour Doppler analysis. Only 2D ultrasound was used in these two studies (Aleem and Predanic, 1996
; Dolz et al., 1999
).
Angiogenesis plays an important role in both the follicular and luteal phases of an ovarian cycle and VEGF is one of the key factors regulating angiogenesis in the ovary (Abulafia and Sherer, 2000; Geva and Jaffle, 2000
). VEGF is a diffusible endothelia 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 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: VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206. Reverse transcription polymerase chain reaction studies demonstrated that mRNA encoding VEGF165 and VEGF121 were dominant in normal human ovaries (Fujimoto et al., 1998
; Otani et al., 1999
). We did not observe any difference in serum VEGF concentration between normal fertile controls and PCOS women and between normal weight and overweight PCOS women in the present study. These data differed from those of Agrawal et al. (1998)
, who reported that serum VEGF concentration was significantly higher in PCOS women, and were related to the increased ovarian stromal vascularity shown by Doppler blood flow velocity measurement in these women, when compared with women with normal 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 Jaffle, 2000). Elevated serum LH concentration may be a cause of increased ovarian stromal blood flow in PCOS women through the action of prostaglandins (Aleem and Predanic, 1996
). A linear increase in the capillary cross-sectional area of the theca interna was observed after the LH surge in a spontaneous cycle and the increase in capillary area was attributed to vasodilatation rather than increase in the number of vessels (Cavander and Murdoch, 1990
). Prostaglandins E2 and I2 are potent vasodilators, which markedly increase local blood flow (Raud, 1990
). Serum LH concentration was significantly increased in PCOS women than normal fertile controls. When compared with overweight PCOS women, there was also a trend of higher serum LH concentration in normal weight PCOS but the difference did not reach statistical significance. A positive relationship existed between serum LH concentration and increased PSV of ovarian vessels (Aleem and Predanic, 1996
).
Ovarian stromal blood flow has been examined in assisted reproductive methods to predict the ovarian response to gonadotrophins (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
). PCOS patients are more sensitive to the stimulation of gonadotrophins and are at higher risk of ovarian hyperstimulation syndrome (Aboulghar and Mansour, 2003
). Increased ovarian stromal blood flow in PCOS patient, especially in normal weight PCOS women shown in our study, may lead to a greater delivery of gonadotrophins to the granulosa cells of the developing follicles. Therefore, we should incorporate the assessment of the ovarian stromal blood flow in the management of PCOS women undergoing ovulation induction or ovarian stimulation in order to reduce the associated risk of ovarian hyperstimulation syndrome. Further studies are definitely needed in this area.
In summary, normal fertile controls and PCOS women had similar total ovarian 3D power Doppler flow indices and serum VEGF concentration after adjusting the difference in age of women. Normal weight PCOS patients had significantly higher total ovarian 3D power Doppler flow indices than their overweight counterparts.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abulafia O and Sherer DM (2000) Angiogenesis of the ovary. Am J Obstet Gynecol 182, 240246.[ISI][Medline]
Agrawal R, Sladkevicius P, Engmann L, Conway GS, Payne NN, Bekis J, Tan SL, Campbell S and Jacobs HS (1998) Serum vascular endothelial growth factor concentrations and ovarian stromal blood flow are increased in women with polycystic ovaries. Hum Reprod 13, 651655.[Abstract]
Aleem FA and Predanic M (1996) Transvaginal color Doppler determination of the ovarian and uterine blood flow characteristics in polycystic ovary disease. Fertil Steril 65, 510516.[ISI][Medline]
Balen AH, Laven JSE, Tan SL and Dewailly D (2003) Ultrasound assessment of the polycystic ovary: international consensus definitions. Hum Reprod Update 9, 505514.
Bassil S, Wyns C, Toussaint-Demylle D, Nisolle M, Gordts S and Donnez J (1997) The relationship between ovarian vascularity and the duration of stimulation in in-vitro fertilization. Hum Reprod 12, 12401245.[CrossRef][ISI][Medline]
Battaglia C, Artini PG, D'Ambrogio G, Genazzani AD and Genazzani AR (1995) The role of color Doppler imaging in the diagnosis of polycystic ovary syndrome. Am J Obstet Gynecol 172, 108113.[CrossRef][ISI][Medline]
Battaglia C, Artini PG, Genazzani AD, Sgherzi MR, Salvatori M, Giulini S and Volpe A (1996) Color Doppler analysis in lean and obese women with polycystic ovary syndrome. Ultrasound Obstet Gynecol 7, 342346.[CrossRef][ISI][Medline]
Cavander JL and Murdoch WJ (1990) Morphological studies of the microcirculatory system of periovulatory ovine follicles. Biol Reprod 42, 139149.
Chan CCW, Ng EHY, Li CF and Ho PC (2003) Impaired ovarian blood flow and reduced antral follicle count following laparoscopic salpingectomy for ectopic pregnancies. Hum Reprod 18, 21752180.
Dolz M, Osborne NG, Blanes J, Raga F, Abad-Velasco L, Villalobos A, Pellicer A and Bonilla-Musoles F (1999) Polycystic ovarian syndrome: assessment with color Doppler angiography and three-dimensional ultrasonography. J Ultrasound Med 18, 303313.
Engmann L, Sladkevicius P, Agrawal R, Bekir JS, Campbell S and Tan SL (1999) Value of ovarian stromal blood flow velocity measurement after pituitary suppression in the prediction of ovarian responsiveness and outcome of in vitro fertilization treatment. Fertil Steril 71, 2229.[CrossRef][ISI][Medline]
Ferrara N and Henzel WJ (1989) Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 161, 851858.[CrossRef][ISI][Medline]
Franks S (1995) Polycystic ovary syndrome. N Engl J Med 333, 853861.
Fujimoto J, Sakaguchi H, Hirose R, Ichigo S and Tamaya T (1998) Biologic implications of expression of vascular endothelial growth factor subtypes in ovarian carcinoma. Cancer 83, 25282533.[CrossRef][ISI][Medline]
Geva E and Jaffle RB (2000) Role of vascular endothelial growth factor in ovarian physiology and pathology. Fertil Steril 74, 429438.[CrossRef][ISI][Medline]
Gordon JD, Mesiano S, Zaloudek CJ and Jaffe RB (1996) Vascular endothelial growth factor localization in human ovary and fallopian tubes: possible role in reproductive function and ovarian cyst formation. J Clin Endocrinol Metab 81, 353359.[Abstract]
Gospodarowicz D, Abraham JA and Schilling J (1989) Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc Natl Acad Sci USA 86, 73117315.
Guerriero S, Ajossa S, Lai MP, Risalvato A, Paoletti AM and Melis GB (1999) Clinical applications of colour Doppler energy imaging in the female reproductive tract and pregnancy. Hum Reprod Update 5, 515529.
Hughesdon PE (1982) Morphology and morphogenesis of the SteinLeventhal ovary and of so-called hyperthecosis. Obstet Gynecol Surv 37, 5977.[Medline]
Järvelä IY, Mason HD, Sladkevicius P, Kelly S, Ojha K, Campbell S and Nargund G (2002) Characterization of normal and polycystic ovaries using three-dimensional power Doppler ultrasonography. J Assist Reprod Genet 19, 582590.[CrossRef][ISI][Medline]
Kamat BR, Brown LF, Manseau EJ, Senger DR and Dvorak HF (1995) Expression of vascular permeability factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Role in corpus luteum development. Am J Pathol 146, 157165.[Abstract]
Koos RD (1995) Increased expression of vascular endothelial growth/permeability factor in the rat ovary following an ovulatory gonadotropin stimulus: potential roles in follicle rupture. Biol Reprod 52, 14261435.[Abstract]
Kupesic S and Kurjak A (2002) Predictors of IVF outcome by three-dimensional ultrasound. Hum Reprod 17, 950955.
Kupesic S, Kurjak A, Bjelos D and Vujisic S (2003) Three-dimensional ultrasonographic ovarian measurements and in vitro fertilization outcome are related to age. Fertil Steril 79, 190197.[CrossRef][ISI][Medline]
McGee EA and Hsueh AJ (2000) Initial and cyclic recruitment of ovarian follicles. Endocr Rev 21, 200214.
Neulen J, Yan Z, Raczek S, Weindel K, Keck C, Weich HA, Marme D and Breckwoldt (1995) Human chorionic gonadotropin-dependent expression of vascular endothelial growth factor/vascular permeability factor in human granulosa cells: importance in ovarian hyperstimulation syndrome. J Clin Endocrinol Metab 80, 19671971.[Abstract]
Ng EHY, Chan CCW, Yeung WSB and Ho PC (2004) Effect of age on ovarian stromal flow measured by three-dimensional ultrasound with power Doppler in Chinese women with proven fertility. Hum Reprod 19, 21322137.
Otani N, Minami S, Yamoto M, Shikone T, Otani H, Nishiyama R, Otani T and Nakano R (1999) The vascular endothelial growth factor/fms-like tyrosine kinase system in human ovary during the menstrual cycle and early pregnancy. J Clin Endocrinol Metab 84, 38453851.
Pairleitner H, Steiner H, Hasenoehrl G and Staudach A (1999) Three-dimensional power Doppler sonography: imaging and quantifying blood flow and vascularization. Ultrasound Obstet Gynecol 14, 139143.[CrossRef][ISI][Medline]
Pan HA, Wu MH, Cheng YC, Li CH and Chang FM (2002) Quantification of Doppler signal in polycystic ovary syndrome using three-dimensional power Doppler ultrasonography: a possible new marker for diagnosis. Hum Reprod 17, 201206.
Phillips HS, Hains J, Leung DW and Ferrara N (1990) Vascular endothelial growth factor is expressed in rat corpus luteum. Endocrinology 127, 965977.[Abstract]
Popovic-Todorovic B, Loft A, Lindhard A, Bangsboll S, Andersson AM and Andersen AN (2003) A prospective study of predictive factors of ovarian response in standard IVF/ICSI patients treated with recombinant FSH. A suggestion for a recombinant FSH dosage normogram. Hum Reprod 18, 781787.
Raud J (1990) Vasodilatation and inhibition of mediator release represent two distinct mechanisms of prostaglandin modulation of acute mast cell-dependent inflammation. Br J Pharmacol 99, 449454.[ISI][Medline]
Redmer D and Reynolds L (1996) Angiogenesis in the ovary. Rev Reprod 1, 182192.
Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group (2004) Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Hum Reprod 19, 4147.
Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS and Dvorak HF (1983) Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219, 983985.[ISI][Medline]
Shweiki D, Itin A, Neufeld G, Gitay-Goren H and Keshet E (1993) Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis. J Clin Invest 91, 22352243.[ISI][Medline]
WHO expert consultation (2004) Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet 363, 157163.[CrossRef][ISI][Medline]
Zaidi J, Campbell S, Pittrof R, Kyei-Mensah A, Shaker A, Jacobs HS and Tan SL (1995) Ovarian stromal blood flow in women with polycystic ovariesa possible new marker for diagnosis? Hum Reprod 10, 19921996.[Abstract]
Zaidi J, Barber J, Kyei-mensah A, Bekir J, Campbell S and Tan SL (1996) Relationship of ovarian stromal blood flow at the baseline ultrasound scan to subsequent follicular response in an in vitro fertilization program. Obstet Gynecol 88, 779784.
Submitted on November 2, 2004; resubmitted on December 28, 2004; accepted on February 18, 2005.
|