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Vascular endothelial growth factor and insulin-like growth factor-1 in polycystic ovary syndrome and their relation to ovarian blood flow
European Journal of Obstetrics & Gynecology and Reproductive Biology 118 (2005) 219–224 www.elsevier.com/locate/ejogrb
Vascular endothelial growth factor and insulin-like growth factor-1 in polycystic ovary syndrome and their relation to ovarian blood flow Diaa Eldeen M. Abd El Aala,*, Safwat A. Mohameda, Ahmed F. Aminea, Abdel-Raheim M.A. Mekib a
Department of Obstetrics and Gynecology, Faculty of Medicine, Assiut University, Egypt b Department of Biochemistry, Faculty of Medicine, Assiut University, Egypt
Received 10 December 2003; received in revised form 8 May 2004; accepted 13 July 2004
Abstract Objectives: (1) To determine the serum levels of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) in women with polycystic ovary syndrome (PCOS). (2) To study Doppler blood flow changes within the ovarian stroma of women with PCOS. (3) To evaluate the relationship between VEGF and IGF-1 and Doppler indices as well as hormonal profile. Setting: Department of Obstetrics and Gynecology, and Department of Biochemistry, Faculty of Medicine, Assiut University, Egypt. Design: Cross-sectional study. Patients and methods: Fifty infertile women with PCOS diagnosed by ultrasound examination and a history of oligomenorrhea, hirsutism and obesity were studied. Serum levels of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and hormonal profile were measured. Doppler blood flow velocity waveforms analysis in both right and left intraovarian arteries was done. Twenty healthy and fertile women with regular menstrual cycles served as a comparison group were similarly studied at the third day of the cycle. Results: The serum levels of VEGF, IGF-1 (4.79 0.91, 253.15 70.07 versus 2.39 0.42, 186.65 42.7) were significantly elevated (P < 0.001 and P < 0.01, respectively) in women with PCOS compared with control. Doppler indices, PI (2.01 0.77, 2.66 1.00 versus 2.98 0.77, 3.75 0.98) and RI (0.77 0.12, 0.82 0.09 versus 0.87 0.09, 0.89 0.09) in both right and left intraovarian vessels were significantly lower in the patients than controls. The VEGF and IGF-1 levels were negatively correlated with RI and PI in the uterine and intraovarian arteries. VEGF level was positively correlated with IGF-1 (r = 0.41, P < 0.05) in women with PCOS. Conclusions: Higher serum levels of VEGF and IGF-1 in PCOS women may be related to the increased vascularity that underlies the increased blood flow demonstrated by Doppler blood flow measurements in these women. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Vascular endothelial growth factor; Insulin-like growth factor; Polycystic ovary syndrome
1. Introduction The polycystic ovary syndrome (PCOS) is a common endocrinopathy and is characterized by infertility, oligomenorrhea and hyperandrogenism. Biochemical abnormalities include hyperandrogenism, acyclic estrogen production, increased levels of lutinizing hormone (LH), decreased levels of sex hormone-binding globulin (SHBG) and hyperinsulinemia. The heterogeneity of the clinical and laboratory
* Corresponding author. Tel.: +105212137; fax: +20 88 333327. E-mail address: [email protected] (D.E.M. Abd El Aal).
features strongly speaks for the multifactorial etiology of the syndrome [6,21]. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is potent angiogenic factor, which is a mitogen for vascular endothelium. VEGF exists in five isoforms resulting from alternative splicing of the same gene, and acts through a family of three tyrosine kinase receptors [15]. VEGF may be involved in the physiological regulation of ovarian angiogenesis. VEGF is expressed and secreted in the human ovary in a manner that suggests a role for this growth factor in both cyclic angiogenesis and regulation of vascular permeability, both of which are critical for ovarian
0301-2115/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2004.07.024
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folliculogenesis and normal reproductive function [17]. Increased expression of VEGF has been described in the hyperthecotic stroma of polycystic ovaries [22,4]. Increased ovarian stromal blood flow in women with PCO has been demonstrated previously by color Doppler blood flow imaging [5]. VEGF may also be a factor responsible for maintaining perifollicular blood flow and regulation of intrafollicular oxygen levels [25]. Insulin-like growth factor-1 (IGF-1) has received considerable attention because it has been demonstrated in the ovary and may influence both differentiation and proliferation in paracrine or autocrine actions. IGF-1 is a single-chain polypeptide that is structurally homologous to proinsulin. Experimental evidence has shown that IGF-1 appears to play contributory roles in human ovarian physiology including follicular development and steriodogenesis [8]. IGF-1 is increasingly linked to the disturbed follicular development in PCOS. The follicular fluid from ovaries in PCOS women contains significantly higher concentrations of IGF-1 when compared with normal women [8]. The purpose of the present study was: (1) To determine the serum levels of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) in women with polycystic ovary syndrome (PCOS). (2) To study Doppler blood flow changes within the ovarian stroma of women with PCOS. (3) To evaluate the relationship between VEGF and IGF-1 and hormonal profile as well as Doppler indices. 2. Subjects and methods This study was carried out in the Department of Obstetrics and Gynecology, Assiut University, Assiut, Egypt. We recruited 70 women. These women had no concomitant pelvic pathology, such as endometriosis, uterine fibroids or ovarian cysts. They divided into two groups according to the following criteria. The polycystic ovary syndrome group (n = 50) had PCO on ultrasound examination and a history of anovulatory menstrual cycles and/or oligomenorrhea, with or without hirsutism, acne and obesity and/or elevated serum LH and/or elevated serum androgen concentrations, who did not receive any treatment for at least 3 months. The normal ovary group (n = 20) had regular ovulatory menstrual cycles and normal ovaries as demonstrated on base line ultrasound examination. The mean age of PCOS and control women were 33.1 (range, 28–43) and 36.2 (range, 28–41) years, respectively. The means body mass index for PCOS and control women were (mean S.D.) 26.9 2.2 and 25.9 2.8 (kg/m2), respectively. 2.1. Pelvic ultrasonography and Doppler blood flow velocity measurements Ultrasound examination was done using an ultrasound duplex system (Acuson Model 128XP/10 computed
sonography system, Mountain View, California, USA). This duplex system combines B-mode imaging (linear and sector probes) and bidirectional pulsed colored Doppler technique with real time spectral analysis. The high pass filter was set on 125 Hz. All examinations were performed at the beginning of a menstrual cycle (day 2 or 3). Doppler examination was done using 5 MHz transvaginal probe with pulsed and color Doppler facilities. Color Doppler was used to identify vessels of interest and served as a guide for pulsed Doppler velocimetry studies. The patients were examined in the dorsal lithotomy position. The transducer is inserted into a condom with a coupling jelly. The transducer was inserted into the vaginal fornix with the aid of lubricating jelly and directed along the paracervical area. In each case, flow velocities were obtained from both main uterine arteries at the level of internal os. Color flow mapping and pulsed Doppler measurements were performed on ovarian stromal blood vessels once normal pelvic findings were confirmed. Areas of maximum color intensity, representing the greatest Doppler frequency shifts, were selected for pulsed Doppler examinations. Blood flow velocity waveforms were thus detected and recorded. We tried to get angles between the pulsed Doppler beam and the vessel close to zero or as low as possible. Blood flow velocity waveforms were stored on light sensitive papers for off-line analysis, and both peak flow velocity and minimum flow velocity were measured, and the resistance indices were calculated using a special computer program. The RI and PI were used as measures of blood flow impedance distal to the point of sampling. All examinations were performed before midday to reduce the effects of diurnal variations in blood flow [27]. 2.2. Biochemical analysis On the morning of the ultrasound examination, blood samples were taken from patients and controls. All women participated in the study were at early follicular phase. Sera were separated and stored at 20 8C until biochemical analysis. Serum FSH and LH were determined by enzyme linked immunosorbent assay (ELISA) kits (Cat No. KIF 4057 and 4023, respectively/Hedix Biotech Inc., USA). Also, serum T was measured by enzyme immunoassay kit (Cat No. XD003, Orion Diagnostica, Finland). In addition, serum VEGF levels were estimated by using ELISA kit (Cat No. 5587,294 Cyt Immune Sciences Inc., USA). The sensitivity of the assay was 0.195 ng/ml. Solid-phase 125I radioimmunoassay were used to determine serum SHBG (125I kit, Cat No. PK SH I, Diagn. Prod. Corp., USA), estradiol (125I kit, Cat No. TKE2I, Diagn. Prod. Corp., USA), and serum IGF-1 (125I kit, Cat No. 1674, Immunotech., France). The sensitivity of the assays of SHBG and IGF-1 were about 0.04 nmol/l and 12 ng/ml, respectively. An informed consent was obtained from all studied patients and participants.
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2.3. Statistical analysis
3.2. Doppler blood flow velocity waveforms
Comparison between groups was carried out by Student’s t-test. Correlation coefficients from linear regression analysis were also applied. P-value less than 0.05 was considered statistically significant. The results were shown as mean and standard deviation.
In the PCO group the mean RI in right and left uterine arteries were 0.94 (S.D. 0.07); 0.95 (S.D. 0.06). They were significantly lower than the control group 0.97 (S.D. 0.06); 0.97 (S.D. 0.07) (P < 0.001). The same relationship could be shown for the mean PI in right and left uterine arteries were 4.08 (S.D. 1.43); 3.22 (S.D. 1.02) and only 4.99 (S.D. 1.38); 3.89 (S.D. 1.37) in the control group (P < 0.001 and P < 0.0001, respectively). The mean RI in right and left intraovarian arteries in the PCO group were 0.77 (S.D. 0.12); 0.82 (S.D. 0.09). They were lower than the control group 0.87 (S.D. 0.09); 0.89 (S.D. 0.09) (P < 0.0001). The same relationship could be shown for the mean PI in right and left intraovarian arteries were 2.01 (S.D. 0.77); 2.66 (S.D. 1.00) and only 2.98 (S.D. 0.77); 3.75 (S.D. 0.98) in the control group (P < 0.0001).
3. Results 3.1. Serum VEGF and IGF-1, LH, FSH, testosterone estrogen and SHBG in PCO and in control In the PCO group the mean serum VEGF concentration was 4.79 ng/ml (S.D. 0.91 ng/ml). It was significantly higher than that in the control: 2.39 ng/ml (S.D. 0.42 ng/ ml) (P < 0.0001) (Table 1). The same relationship could be shown for the mean serum IGF-1 concentration, which was 253.15 ng/ml (S.D. 70.07 ng/ml) and only 186.65 ng/ ml (S.D. 42.7 ng/ml) (P < 0.001) in the control (Table 1). The mean serum concentration of LH in PCO was 14.23 IU/l (S.D. 4.01 IU/l) and significantly higher compared to the control: 8.8 IU/l (S.D. 2.16 IU/l) (P < 0.0001) (Table 1). There was however no difference in FSH level between PCO (6.45 IU/l, S.D. 2.11 IU/l) and the control group: 7.84 IU/l (S.D. 2.02 IU/l) (Table 1). Serum testosterone level was however higher: 4.77 nmol/ l (S.D. 1.58 nmol/l) compared to the control group: 1.35 nmol/l (S.D. 0.31 nmol/l) (P < 0.0001) (Table 1). The serum estrogen was not significantly different between the PCO group: 78.25 pg/ml (S.D. 53.39 pg/ml) and the control: 62.25 pg/ml (S.D. 20.88 pg/ml) (Table 1). In the PCO group the mean serum SHBG level was 58.67 nmol/l (S.D. 15.87 nmol/l) and significantly lower than in the control: 96.88 nmol/l (S.D. 30.69 nmol/l) (range 60.1–145.3 nmol/l) (P < 0.0001) (Table 1).
3.3. Correlation between Doppler indices, and VEGF and IGF-1 There was a strong negative correlation between vascular endothelial growth factor and the resistance index in the right uterine artery (r = 0.92, P < 0.001), left uterine artery (r = 0.85, P < 0.001), right intraovarian artery (r = 0.92, P < 0.001) and left intraovarian artery (r = 0.92, P < 0.001) (Fig. 1). The same strong negative correlation was found between vascular endothelial growth factor and the pulsatility index in the right uterine artery (r = 0.90, P < 0.001), left uterine artery (r = 0.87, P < 0.001), right intraovarian artery (r = 0.93, P < 0.001) and left intraovarian artery (r = 0.93, P < 0.001) (Fig. 2).
Table 1 Serum levels of VEGF, IGF-1 and hormones in women with PCOS and normal control ovaries (mean + S.E.) Variables
Control group; N = 20
PCOS group; N = 50
VEGF (ng/ml) IGF-1 (ng/ml) LH (IU/l) FSH (IU/l) Estradiol (pg/ml) T (nmol/l) T/SHBG SHBG (nmol/l) LH/FSH T/SHBG
2.39 0.09 186.65 9.55 8.8 0.48 7.84 0.45 62.25 4.67 1.35 0.07 0.02 0.001 96.88 6.87 1.13 0.04 0.02 0.01
4.79 0.18b 253.15 14.01a 15.23 0.82a 6.45 0.42 78.25 10.68 4.77 0.32b 0.09 0.01b 58.67 3.17b 2.28 0.17b 0.09 0.01b
FSH, follicle stimulating hormone; LH, lutinizing hormone; SHBG, sex hormone-binding globulin; T, testosterone. a P < 0.01. b P < 0.001.
Fig. 1. Correlation between vascular endothelial growth factor and the resistance index in the uterine and intraovarian vessels.
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Fig. 2. Correlation between vascular endothelial growth factor and the pulsatility index in the uterine and intraovarian vessels.
Fig. 4. Correlation between insulin-like growth factor-1 and the pulsatility index in the uterine and intraovarian vessels.
There was a strong negative correlation between insulinlike growth factor-1 and the resistance index in the right uterine artery (r = 0.95, P < 0.001), left uterine artery (r = 0.80, P < 0.001), right intraovarian artery (r = 0.95, P < 0.001) and left intraovarian artery (r = 0.97, P < 0.001) (Fig. 3). Similar strong negative correlation was found between insulin-like growth factor-1 and the pulsatility index in the right uterine artery (r = 0.82, P < 0.001), left uterine artery (r = 0.78, P < 0.001), right intraovarian artery
(r = 0.88, P < 0.001) and left intraovarian artery (r = 0.85, P < 0.001) (Fig. 4). The VEGF level was positively correlated with IGF-1 (r = 0.41, P < 0.05), LH (r = 0.61, P < 0.001) and T (r = 0.71, P < 0.001), while negatively correlated with SHBG (r = 0.41, P < 0.05) in women with PCOS.
Fig. 3. Correlation between insulin-like growth factor-1 and the resistance index in the uterine and intraovarian vessels.
4. Discussion In the current study, we found that serum VEGF concentrations in women with PCOS were significantly higher than in women with normal ovaries. Doppler blood flow velocities within the ovarian stromal vessels rose in parallel with the rising serum VEGF levels. This is consistent with a previous study [4]. These findings may explain the increased ovarian stromal vascularity [1,3]. In the present study, the serum levels of IGF-1 in PCOS were significantly increased in comparison with controls. Similarly, IGF-1 has been reported to be increased in sera of PCOS women, as compared with healthy ovulatory controls [9,12,20]. IGF-1 seems to have an overall negative effect on normal folliculogenesis and ovulation [11,12] suggested that the pathogenesis of PCOS may involve interrelated abnormalities of the IGF-1 and ovarian steroidogenesis systems. The T levels were significantly correlated with LH and estradiol in PCOS [12]. It has been proposed that the elevated LH often found in this disorder, may increase IGF-1 production by theca cells within the polycystic ovary and enhance androgen production [7,14,18]. In this respect, IGF-1 receptors have been
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detected in thecal cells. In addition, IGF-1 has been shown to stimulate granulosa cell for estrogen production [13]. It has been demonstrated that IGF-1 stimulates aromatase activity in the human ovary [13]. Furthermore, Bergh et al. [7] suggested that lowering IGF-1 levels could potentially lower androgen and estrogen. Our result revealed that VEGF was positively correlated with IGF-1, LH and T but negatively correlated with SHBG in PCOS. It seems that VEGF levels in PCOS are hormone dependent. In this respect, IGF-1 induces VEGF-mRNA and protein production by both an increase in the transcriptional rate of the VEGF gene and the stability of mRNA [26]. Conversely, Agrawal et al. [4] did not observe any correlation between VEGF concentrations and serum levels of LH and T in PCOS patients. It is well established that growth factors are involved in intraovarian regulatory mechanisms. IGF-1 has been shown to induce LH receptors, and LH-mediated angiogenesis has been described previously [16]. Perhaps increased intraovarian concentrations of VEGF are related to increased secretion and pulsatility of LH, an important pathophysiological features of PCOS. There are, however, large fluctuations of LH concentration in women with PCOS over time [2]. Unlike other growth factors responsible for angiogenesis, e.g. basic fibroblast growth factor (bFGF), which is largely intracellular and non-diffusible, VEGF is a soluble, diffusible growth factor. There is also evidence to suggest that angiogenic factors, like bFGF, transforming growth factor-beta, platelet-derived growth factor and nitric oxide, act as agonists to the action of VEGF [10]. Neulen et al. [24] suggested that LH modulates expression of VEGF. Geva et al. [17] showed that the dynamic pattern of VEGF mRNA expression was paralleled with gonadotropin stimulation (LH) and perhaps steroid production. Based on the preferential accumulation of gonadotropins within the dominant follicle, it has been suggested that differential vascular permeability is involved in, and may mediate, follicular secretion [29]. VEGF in the thecal cell layer likely modulates vascular permeability. This modulation may be one mechanism by which differential gonadotropin accumulation is achieved. Thus, the richly vascularized thecal cell layer is probably critical for gonadotropin-dependent growth of the follicle and for access of estrogen-produced in the avascular granulosa cell layer by aromatization of thecal androgen-to the systemic circulation [28]. Our results revealed that ovarian stromal blood flow velocities as confirmed by Doppler blood flow (PI and RI) were significantly higher in PCOS compared with normal ovaries. These findings are consistent with the studies of Agrawal et al. and Loverro et al. [4,23] who found that women with PCOS have elevated serum VEGF concentrations and higher ovarian stromal blood flow velocities than women with normal ovaries. Also they found positive correlations between VEGF and blood flow velocities. Previously, Van Blerkom et al. [25] confirmed VEGF to be responsible for the maintenance of perifollicular blood flow.
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VEGF not only mediates angiogenesis but also induces connective stromal growth by increasing microvascular permeability, which leads to extravasation of plasma proteins. The extravascular matrix thus formed favors in the growth of new blood vessels and fibroblasts, which in turn organize the avascular provisional fibrin matrix into a mature, vascularized connective tissue stroma [22]. The elevated serum VEGF concentrations in women with PCOS may explain in part the dense hyperechgenic and highly vascularized stroma of PCOS as demonstrated by Doppler blood flow [4]. The increased vascularity may result from overexpression of ovarian VEGF in women with PCOS. This hypothesis is supported by demonstration of a strong immunohistochemical staining of VEGF in the ovarian stroma of three patients with PCOS [22]. There were some methodological limitations concerning the measuring of VEGF in peripheral blood concerning the quantification of the contribution from platelets during blood sampling [27]. Unfortunately, this type of measure is quite difficult to judge. This limitation should be considered during revision of the results. The other point is the use of peripheral blood samples as indicators of increased production in the ovaries, the alternative of taking direct measures from the ovaries seems more accurate and precise. In conclusion, in PCOS, the increased vascularity that underlies the increased blood flow demonstrated by Doppler blood flow velocity measurements may be related to the higher serum levels of VEGF and IGF-1. With increasing understanding of the roles of IGF-1 and VEGF in PCOS, future therapeutic modalities will be developed to treat such disturbances in ovarian physiology. VEGF may be fundamental to the aeitiopathogenesis of PCOS. The increased serum VEGF concentrations may be reflected the increased expression of ovarian VEGF, which increases vascular permeability. Thus, it may contribute to the formation of the increased stroma in PCOS. Direct measure of VEGF and IGF-1 in the ovarian tissues of PCOS patients in recommended in future research.
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Vascular endothelial growth factor and insulin-like growth factor-1 in polycystic ovary syndrome and their relation to ovarian blood flow Diaa Eldeen M. Abd El Aala,*, Safwat A. Mohameda, Ahmed F. Aminea, Abdel-Raheim M.A. Mekib a
Department of Obstetrics and Gynecology, Faculty of Medicine, Assiut University, Egypt b Department of Biochemistry, Faculty of Medicine, Assiut University, Egypt
Received 10 December 2003; received in revised form 8 May 2004; accepted 13 July 2004
Abstract Objectives: (1) To determine the serum levels of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) in women with polycystic ovary syndrome (PCOS). (2) To study Doppler blood flow changes within the ovarian stroma of women with PCOS. (3) To evaluate the relationship between VEGF and IGF-1 and Doppler indices as well as hormonal profile. Setting: Department of Obstetrics and Gynecology, and Department of Biochemistry, Faculty of Medicine, Assiut University, Egypt. Design: Cross-sectional study. Patients and methods: Fifty infertile women with PCOS diagnosed by ultrasound examination and a history of oligomenorrhea, hirsutism and obesity were studied. Serum levels of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and hormonal profile were measured. Doppler blood flow velocity waveforms analysis in both right and left intraovarian arteries was done. Twenty healthy and fertile women with regular menstrual cycles served as a comparison group were similarly studied at the third day of the cycle. Results: The serum levels of VEGF, IGF-1 (4.79 0.91, 253.15 70.07 versus 2.39 0.42, 186.65 42.7) were significantly elevated (P < 0.001 and P < 0.01, respectively) in women with PCOS compared with control. Doppler indices, PI (2.01 0.77, 2.66 1.00 versus 2.98 0.77, 3.75 0.98) and RI (0.77 0.12, 0.82 0.09 versus 0.87 0.09, 0.89 0.09) in both right and left intraovarian vessels were significantly lower in the patients than controls. The VEGF and IGF-1 levels were negatively correlated with RI and PI in the uterine and intraovarian arteries. VEGF level was positively correlated with IGF-1 (r = 0.41, P < 0.05) in women with PCOS. Conclusions: Higher serum levels of VEGF and IGF-1 in PCOS women may be related to the increased vascularity that underlies the increased blood flow demonstrated by Doppler blood flow measurements in these women. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Vascular endothelial growth factor; Insulin-like growth factor; Polycystic ovary syndrome
1. Introduction The polycystic ovary syndrome (PCOS) is a common endocrinopathy and is characterized by infertility, oligomenorrhea and hyperandrogenism. Biochemical abnormalities include hyperandrogenism, acyclic estrogen production, increased levels of lutinizing hormone (LH), decreased levels of sex hormone-binding globulin (SHBG) and hyperinsulinemia. The heterogeneity of the clinical and laboratory
* Corresponding author. Tel.: +105212137; fax: +20 88 333327. E-mail address: [email protected] (D.E.M. Abd El Aal).
features strongly speaks for the multifactorial etiology of the syndrome [6,21]. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is potent angiogenic factor, which is a mitogen for vascular endothelium. VEGF exists in five isoforms resulting from alternative splicing of the same gene, and acts through a family of three tyrosine kinase receptors [15]. VEGF may be involved in the physiological regulation of ovarian angiogenesis. VEGF is expressed and secreted in the human ovary in a manner that suggests a role for this growth factor in both cyclic angiogenesis and regulation of vascular permeability, both of which are critical for ovarian
0301-2115/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2004.07.024
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folliculogenesis and normal reproductive function [17]. Increased expression of VEGF has been described in the hyperthecotic stroma of polycystic ovaries [22,4]. Increased ovarian stromal blood flow in women with PCO has been demonstrated previously by color Doppler blood flow imaging [5]. VEGF may also be a factor responsible for maintaining perifollicular blood flow and regulation of intrafollicular oxygen levels [25]. Insulin-like growth factor-1 (IGF-1) has received considerable attention because it has been demonstrated in the ovary and may influence both differentiation and proliferation in paracrine or autocrine actions. IGF-1 is a single-chain polypeptide that is structurally homologous to proinsulin. Experimental evidence has shown that IGF-1 appears to play contributory roles in human ovarian physiology including follicular development and steriodogenesis [8]. IGF-1 is increasingly linked to the disturbed follicular development in PCOS. The follicular fluid from ovaries in PCOS women contains significantly higher concentrations of IGF-1 when compared with normal women [8]. The purpose of the present study was: (1) To determine the serum levels of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) in women with polycystic ovary syndrome (PCOS). (2) To study Doppler blood flow changes within the ovarian stroma of women with PCOS. (3) To evaluate the relationship between VEGF and IGF-1 and hormonal profile as well as Doppler indices. 2. Subjects and methods This study was carried out in the Department of Obstetrics and Gynecology, Assiut University, Assiut, Egypt. We recruited 70 women. These women had no concomitant pelvic pathology, such as endometriosis, uterine fibroids or ovarian cysts. They divided into two groups according to the following criteria. The polycystic ovary syndrome group (n = 50) had PCO on ultrasound examination and a history of anovulatory menstrual cycles and/or oligomenorrhea, with or without hirsutism, acne and obesity and/or elevated serum LH and/or elevated serum androgen concentrations, who did not receive any treatment for at least 3 months. The normal ovary group (n = 20) had regular ovulatory menstrual cycles and normal ovaries as demonstrated on base line ultrasound examination. The mean age of PCOS and control women were 33.1 (range, 28–43) and 36.2 (range, 28–41) years, respectively. The means body mass index for PCOS and control women were (mean S.D.) 26.9 2.2 and 25.9 2.8 (kg/m2), respectively. 2.1. Pelvic ultrasonography and Doppler blood flow velocity measurements Ultrasound examination was done using an ultrasound duplex system (Acuson Model 128XP/10 computed
sonography system, Mountain View, California, USA). This duplex system combines B-mode imaging (linear and sector probes) and bidirectional pulsed colored Doppler technique with real time spectral analysis. The high pass filter was set on 125 Hz. All examinations were performed at the beginning of a menstrual cycle (day 2 or 3). Doppler examination was done using 5 MHz transvaginal probe with pulsed and color Doppler facilities. Color Doppler was used to identify vessels of interest and served as a guide for pulsed Doppler velocimetry studies. The patients were examined in the dorsal lithotomy position. The transducer is inserted into a condom with a coupling jelly. The transducer was inserted into the vaginal fornix with the aid of lubricating jelly and directed along the paracervical area. In each case, flow velocities were obtained from both main uterine arteries at the level of internal os. Color flow mapping and pulsed Doppler measurements were performed on ovarian stromal blood vessels once normal pelvic findings were confirmed. Areas of maximum color intensity, representing the greatest Doppler frequency shifts, were selected for pulsed Doppler examinations. Blood flow velocity waveforms were thus detected and recorded. We tried to get angles between the pulsed Doppler beam and the vessel close to zero or as low as possible. Blood flow velocity waveforms were stored on light sensitive papers for off-line analysis, and both peak flow velocity and minimum flow velocity were measured, and the resistance indices were calculated using a special computer program. The RI and PI were used as measures of blood flow impedance distal to the point of sampling. All examinations were performed before midday to reduce the effects of diurnal variations in blood flow [27]. 2.2. Biochemical analysis On the morning of the ultrasound examination, blood samples were taken from patients and controls. All women participated in the study were at early follicular phase. Sera were separated and stored at 20 8C until biochemical analysis. Serum FSH and LH were determined by enzyme linked immunosorbent assay (ELISA) kits (Cat No. KIF 4057 and 4023, respectively/Hedix Biotech Inc., USA). Also, serum T was measured by enzyme immunoassay kit (Cat No. XD003, Orion Diagnostica, Finland). In addition, serum VEGF levels were estimated by using ELISA kit (Cat No. 5587,294 Cyt Immune Sciences Inc., USA). The sensitivity of the assay was 0.195 ng/ml. Solid-phase 125I radioimmunoassay were used to determine serum SHBG (125I kit, Cat No. PK SH I, Diagn. Prod. Corp., USA), estradiol (125I kit, Cat No. TKE2I, Diagn. Prod. Corp., USA), and serum IGF-1 (125I kit, Cat No. 1674, Immunotech., France). The sensitivity of the assays of SHBG and IGF-1 were about 0.04 nmol/l and 12 ng/ml, respectively. An informed consent was obtained from all studied patients and participants.
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2.3. Statistical analysis
3.2. Doppler blood flow velocity waveforms
Comparison between groups was carried out by Student’s t-test. Correlation coefficients from linear regression analysis were also applied. P-value less than 0.05 was considered statistically significant. The results were shown as mean and standard deviation.
In the PCO group the mean RI in right and left uterine arteries were 0.94 (S.D. 0.07); 0.95 (S.D. 0.06). They were significantly lower than the control group 0.97 (S.D. 0.06); 0.97 (S.D. 0.07) (P < 0.001). The same relationship could be shown for the mean PI in right and left uterine arteries were 4.08 (S.D. 1.43); 3.22 (S.D. 1.02) and only 4.99 (S.D. 1.38); 3.89 (S.D. 1.37) in the control group (P < 0.001 and P < 0.0001, respectively). The mean RI in right and left intraovarian arteries in the PCO group were 0.77 (S.D. 0.12); 0.82 (S.D. 0.09). They were lower than the control group 0.87 (S.D. 0.09); 0.89 (S.D. 0.09) (P < 0.0001). The same relationship could be shown for the mean PI in right and left intraovarian arteries were 2.01 (S.D. 0.77); 2.66 (S.D. 1.00) and only 2.98 (S.D. 0.77); 3.75 (S.D. 0.98) in the control group (P < 0.0001).
3. Results 3.1. Serum VEGF and IGF-1, LH, FSH, testosterone estrogen and SHBG in PCO and in control In the PCO group the mean serum VEGF concentration was 4.79 ng/ml (S.D. 0.91 ng/ml). It was significantly higher than that in the control: 2.39 ng/ml (S.D. 0.42 ng/ ml) (P < 0.0001) (Table 1). The same relationship could be shown for the mean serum IGF-1 concentration, which was 253.15 ng/ml (S.D. 70.07 ng/ml) and only 186.65 ng/ ml (S.D. 42.7 ng/ml) (P < 0.001) in the control (Table 1). The mean serum concentration of LH in PCO was 14.23 IU/l (S.D. 4.01 IU/l) and significantly higher compared to the control: 8.8 IU/l (S.D. 2.16 IU/l) (P < 0.0001) (Table 1). There was however no difference in FSH level between PCO (6.45 IU/l, S.D. 2.11 IU/l) and the control group: 7.84 IU/l (S.D. 2.02 IU/l) (Table 1). Serum testosterone level was however higher: 4.77 nmol/ l (S.D. 1.58 nmol/l) compared to the control group: 1.35 nmol/l (S.D. 0.31 nmol/l) (P < 0.0001) (Table 1). The serum estrogen was not significantly different between the PCO group: 78.25 pg/ml (S.D. 53.39 pg/ml) and the control: 62.25 pg/ml (S.D. 20.88 pg/ml) (Table 1). In the PCO group the mean serum SHBG level was 58.67 nmol/l (S.D. 15.87 nmol/l) and significantly lower than in the control: 96.88 nmol/l (S.D. 30.69 nmol/l) (range 60.1–145.3 nmol/l) (P < 0.0001) (Table 1).
3.3. Correlation between Doppler indices, and VEGF and IGF-1 There was a strong negative correlation between vascular endothelial growth factor and the resistance index in the right uterine artery (r = 0.92, P < 0.001), left uterine artery (r = 0.85, P < 0.001), right intraovarian artery (r = 0.92, P < 0.001) and left intraovarian artery (r = 0.92, P < 0.001) (Fig. 1). The same strong negative correlation was found between vascular endothelial growth factor and the pulsatility index in the right uterine artery (r = 0.90, P < 0.001), left uterine artery (r = 0.87, P < 0.001), right intraovarian artery (r = 0.93, P < 0.001) and left intraovarian artery (r = 0.93, P < 0.001) (Fig. 2).
Table 1 Serum levels of VEGF, IGF-1 and hormones in women with PCOS and normal control ovaries (mean + S.E.) Variables
Control group; N = 20
PCOS group; N = 50
VEGF (ng/ml) IGF-1 (ng/ml) LH (IU/l) FSH (IU/l) Estradiol (pg/ml) T (nmol/l) T/SHBG SHBG (nmol/l) LH/FSH T/SHBG
2.39 0.09 186.65 9.55 8.8 0.48 7.84 0.45 62.25 4.67 1.35 0.07 0.02 0.001 96.88 6.87 1.13 0.04 0.02 0.01
4.79 0.18b 253.15 14.01a 15.23 0.82a 6.45 0.42 78.25 10.68 4.77 0.32b 0.09 0.01b 58.67 3.17b 2.28 0.17b 0.09 0.01b
FSH, follicle stimulating hormone; LH, lutinizing hormone; SHBG, sex hormone-binding globulin; T, testosterone. a P < 0.01. b P < 0.001.
Fig. 1. Correlation between vascular endothelial growth factor and the resistance index in the uterine and intraovarian vessels.
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Fig. 2. Correlation between vascular endothelial growth factor and the pulsatility index in the uterine and intraovarian vessels.
Fig. 4. Correlation between insulin-like growth factor-1 and the pulsatility index in the uterine and intraovarian vessels.
There was a strong negative correlation between insulinlike growth factor-1 and the resistance index in the right uterine artery (r = 0.95, P < 0.001), left uterine artery (r = 0.80, P < 0.001), right intraovarian artery (r = 0.95, P < 0.001) and left intraovarian artery (r = 0.97, P < 0.001) (Fig. 3). Similar strong negative correlation was found between insulin-like growth factor-1 and the pulsatility index in the right uterine artery (r = 0.82, P < 0.001), left uterine artery (r = 0.78, P < 0.001), right intraovarian artery
(r = 0.88, P < 0.001) and left intraovarian artery (r = 0.85, P < 0.001) (Fig. 4). The VEGF level was positively correlated with IGF-1 (r = 0.41, P < 0.05), LH (r = 0.61, P < 0.001) and T (r = 0.71, P < 0.001), while negatively correlated with SHBG (r = 0.41, P < 0.05) in women with PCOS.
Fig. 3. Correlation between insulin-like growth factor-1 and the resistance index in the uterine and intraovarian vessels.
4. Discussion In the current study, we found that serum VEGF concentrations in women with PCOS were significantly higher than in women with normal ovaries. Doppler blood flow velocities within the ovarian stromal vessels rose in parallel with the rising serum VEGF levels. This is consistent with a previous study [4]. These findings may explain the increased ovarian stromal vascularity [1,3]. In the present study, the serum levels of IGF-1 in PCOS were significantly increased in comparison with controls. Similarly, IGF-1 has been reported to be increased in sera of PCOS women, as compared with healthy ovulatory controls [9,12,20]. IGF-1 seems to have an overall negative effect on normal folliculogenesis and ovulation [11,12] suggested that the pathogenesis of PCOS may involve interrelated abnormalities of the IGF-1 and ovarian steroidogenesis systems. The T levels were significantly correlated with LH and estradiol in PCOS [12]. It has been proposed that the elevated LH often found in this disorder, may increase IGF-1 production by theca cells within the polycystic ovary and enhance androgen production [7,14,18]. In this respect, IGF-1 receptors have been
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detected in thecal cells. In addition, IGF-1 has been shown to stimulate granulosa cell for estrogen production [13]. It has been demonstrated that IGF-1 stimulates aromatase activity in the human ovary [13]. Furthermore, Bergh et al. [7] suggested that lowering IGF-1 levels could potentially lower androgen and estrogen. Our result revealed that VEGF was positively correlated with IGF-1, LH and T but negatively correlated with SHBG in PCOS. It seems that VEGF levels in PCOS are hormone dependent. In this respect, IGF-1 induces VEGF-mRNA and protein production by both an increase in the transcriptional rate of the VEGF gene and the stability of mRNA [26]. Conversely, Agrawal et al. [4] did not observe any correlation between VEGF concentrations and serum levels of LH and T in PCOS patients. It is well established that growth factors are involved in intraovarian regulatory mechanisms. IGF-1 has been shown to induce LH receptors, and LH-mediated angiogenesis has been described previously [16]. Perhaps increased intraovarian concentrations of VEGF are related to increased secretion and pulsatility of LH, an important pathophysiological features of PCOS. There are, however, large fluctuations of LH concentration in women with PCOS over time [2]. Unlike other growth factors responsible for angiogenesis, e.g. basic fibroblast growth factor (bFGF), which is largely intracellular and non-diffusible, VEGF is a soluble, diffusible growth factor. There is also evidence to suggest that angiogenic factors, like bFGF, transforming growth factor-beta, platelet-derived growth factor and nitric oxide, act as agonists to the action of VEGF [10]. Neulen et al. [24] suggested that LH modulates expression of VEGF. Geva et al. [17] showed that the dynamic pattern of VEGF mRNA expression was paralleled with gonadotropin stimulation (LH) and perhaps steroid production. Based on the preferential accumulation of gonadotropins within the dominant follicle, it has been suggested that differential vascular permeability is involved in, and may mediate, follicular secretion [29]. VEGF in the thecal cell layer likely modulates vascular permeability. This modulation may be one mechanism by which differential gonadotropin accumulation is achieved. Thus, the richly vascularized thecal cell layer is probably critical for gonadotropin-dependent growth of the follicle and for access of estrogen-produced in the avascular granulosa cell layer by aromatization of thecal androgen-to the systemic circulation [28]. Our results revealed that ovarian stromal blood flow velocities as confirmed by Doppler blood flow (PI and RI) were significantly higher in PCOS compared with normal ovaries. These findings are consistent with the studies of Agrawal et al. and Loverro et al. [4,23] who found that women with PCOS have elevated serum VEGF concentrations and higher ovarian stromal blood flow velocities than women with normal ovaries. Also they found positive correlations between VEGF and blood flow velocities. Previously, Van Blerkom et al. [25] confirmed VEGF to be responsible for the maintenance of perifollicular blood flow.
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VEGF not only mediates angiogenesis but also induces connective stromal growth by increasing microvascular permeability, which leads to extravasation of plasma proteins. The extravascular matrix thus formed favors in the growth of new blood vessels and fibroblasts, which in turn organize the avascular provisional fibrin matrix into a mature, vascularized connective tissue stroma [22]. The elevated serum VEGF concentrations in women with PCOS may explain in part the dense hyperechgenic and highly vascularized stroma of PCOS as demonstrated by Doppler blood flow [4]. The increased vascularity may result from overexpression of ovarian VEGF in women with PCOS. This hypothesis is supported by demonstration of a strong immunohistochemical staining of VEGF in the ovarian stroma of three patients with PCOS [22]. There were some methodological limitations concerning the measuring of VEGF in peripheral blood concerning the quantification of the contribution from platelets during blood sampling [27]. Unfortunately, this type of measure is quite difficult to judge. This limitation should be considered during revision of the results. The other point is the use of peripheral blood samples as indicators of increased production in the ovaries, the alternative of taking direct measures from the ovaries seems more accurate and precise. In conclusion, in PCOS, the increased vascularity that underlies the increased blood flow demonstrated by Doppler blood flow velocity measurements may be related to the higher serum levels of VEGF and IGF-1. With increasing understanding of the roles of IGF-1 and VEGF in PCOS, future therapeutic modalities will be developed to treat such disturbances in ovarian physiology. VEGF may be fundamental to the aeitiopathogenesis of PCOS. The increased serum VEGF concentrations may be reflected the increased expression of ovarian VEGF, which increases vascular permeability. Thus, it may contribute to the formation of the increased stroma in PCOS. Direct measure of VEGF and IGF-1 in the ovarian tissues of PCOS patients in recommended in future research.
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