Acta Obstetricia et Gynecologica Scandinavica
ISSN: 0001-6349 (Print) 1600-0412 (Online) Journal homepage: http://www.tandfonline.com/loi/iobs20
Serum vascular endothelial growth factor 165 levels and uterine fibroid volume Da-Chung Chen, Jah-Yao Liu, Gwo-Jang Wu, Chih-Hung Ku, Her-Young Su & Chi-Huang Chen To cite this article: Da-Chung Chen, Jah-Yao Liu, Gwo-Jang Wu, Chih-Hung Ku, Her-Young Su & Chi-Huang Chen (2005) Serum vascular endothelial growth factor 165 levels and uterine fibroid volume, Acta Obstetricia et Gynecologica Scandinavica, 84:4, 317-321 To link to this article: http://dx.doi.org/10.1080/j.0001-6349.2005.00621.x
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Acta Obstet Gynecol Scand 2005: 84: 317--321 Printed in Denmark. All rights reserved
Copyright # Acta Obstet Gynecol Scand 2005
Acta Obstetricia et Gynecologica Scandinavica
ORIGINAL ARTICLE
Serum vascular endothelial growth factor165 levels and uterine fibroid volume DA-CHUNG CHEN1,2, JAH-YAO LIU1, GWO-JANG WU1, CHIH-HUNG KU3, HER-YOUNG SU1
AND
CHI-HUANG CHEN1
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From the 1Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, 2Department of Obstetrics and Gynecology, E-Da Hospital, I-Shou University, Kaohsiung County, Taiwan, and 3School of Public Health, National Defense Medical Center, Taipei, Taiwan
Acta Obstet Gynecol Scand 2005; 84: 317–321.
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Acta Obstet Gynecol Scand 84 2005
Background. Vascular endothelial growth factor165 (VEGF165) demonstrates increased expression in uterine fibroids. This study aimed to investigate the relationship between serum VEGF165 levels and uterine fibroid volume. Methods. On a prospective observational basis, 80 women with symptomatic uterine fibroids underwent hysterectomy. Uterine weight was determined after hysterectomy. Six hours before and 48 h after hysterectomy, serum VEGF165 levels were measured using an enzyme-linked immunosorbent assay. Results. Logistic regression analysis showed no correlation between serum VEGF165 levels and uterine weight (r ¼ 0.0037; P > 0.05). The mean serum VEGF165 level declined significantly from 716.31 457.99 to 581.81 403.32 pg/mL after hysterectomy (P < 0.0001). Controlling for age, body mass index, uterine weight, proliferative or secretory phase, and parity, only parous patients (n ¼ 58) were found to have significantly decreased serum VEGF165 levels after hysterectomy (P ¼ 0.000035), in contrast to nulliparous patients (n ¼ 22; P ¼ 0.15). Conclusions. Serum VEGF165 levels do not correlate with uterine fibroid volume, but demonstrate a significant decrease after hysterectomy. The decrease in serum VEGF165 levels after hysterectomy was significant in parous but not in nulliparous patients with uterine fibroids. Serum VEGF165 levels may not predict uterine fibroid development. Keywords: hysterectomy; nulliparous; parous; uterine fibroids; vascular endothelial growth factor Submitted 11 December, 2003 Accepted 15 April, 2004
Uterine leiomyomata or fibroids are the most common smooth muscle tumors in adult women, occurring in 20–40% of women of childbearing age, accounting for significant morbidity and often resulting in major surgery (1). The etiology of fibroids is still unclear. Estrogen and progesterone are believed to play a role as regulators of fibroid growth (2), and many growth factors, including vascular endothelial growth factor (VEGF), epidermal growth factor, transforming growth factor b (3), insulin-like Abbreviations: VEGF: vascular endothelial growth factor.
growth factor (4), fibroblast growth factor (5), and platelet-derived growth factor, (6) have been implicated in the pathogenesis of fibroids. It has been hypothesized that fibroids originate from somatic mutations in myometrial cells, resulting in progressive loss of growth regulation (7,8). The tumor is a growth of genetically abnormal clones of cells derived from a single progenitor cell, in which the original mutation took place in the myometrium (9). Because the establishment of nutrient blood vessels is fundamental to the growth of all tumors, it has been suggested that angiogenesis may play a role in fibroid development and increased vasculature has been found in #
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perifibroid myometrium (10). A potent angiogenic growth factor, VEGF, has been identified that regulates growth of blood vessels from pre-existing vessels and helps maintain vascular endothelial integrity and is thought to potentially play a role in the pathogenesis of uterine fibroids (11). Using monoclonal antibody immunohistochemistry, Gentry et al. (12) demonstrated the increased expression of VEGF-A antigen in fibroids relative to adjacent myometrium, indicating a possible role for VEGF-A in the pathobiology of fibroids. Agrwal et al. (13) and Torry et al. (14) found that the uterus is a source of VEGF, and hypothesized that hysterectomy should result in a reduction of serum VEGF. The effect of hormone replacement therapy on serum VEGF level has also been investigated in patients with or without a uterus (13,15). Notwithstanding this research, the temporal relationship between hysterectomy and serum VEGF changes remains unknown. Of the five isoforms of VEGF-A (VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206), immunohistochemistry demonstrates higher expression of VEGF165 in fibroids than that in the adjacent myometrium (12). In this study, the serum VEGF165 levels, which are the predominantly secreted VEGF-A isoform, were investigated in an attempt to correlate fibroid volume and hysterectomy. Materials and methods The prospective observational study was approved by the Ethics Committee (Tri-Service General Hospital, Taipei, Taiwan) and was performed in a university medical center after signed informed consent was obtained from patients. Patients with symptomatic fibroids, who underwent diagnostic laparoscopy to exclude pelvic lesions followed by vaginal total hysterectomy that spared the adnexa, were recruited. All were premenopausal non-smokers and had not taken any hormonal medication for at least 3 months prior to surgery. Accurate menstrual histories were recorded. After each hysterectomy, pathologists histologically examined fibroids and performed endometrial dating according to standard criteria (16). Any patients with operative or pathological findings that included endometrial lesions, adenomyosis, degenerative or necrotic fibroids, endometriosis, endometrioma, ovarian cysts, or tumors were excluded from the study. In order to reduce the possible effects of massive fluid replacement, oral food and medication intake, hemodynamic unstable, analgesic, and anesthesia on serum VEGF levels, we set the timing of the blood aspiration at 6 h before and 48 h after hysterectomy. Blood samples, after being allowed to clot for 30 min, were gently subjected to centrifugation at 1500 g for 15 min, separated and aliquoted, and stored without loss of activity at 80 C until assayed. Serum samples were thawed to room temperature and were assayed for VEGF165 with a commercially available enzyme-linked immunosorbent assay kit (Human VEGF QuantikineTM ELISA, R&D Systems, Catalogue No. DVE00, Abingdon, Oxfordshire, UK) (13). The monoclonal antibody contained in this kit was raised against recombinant human VEGF165. #
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The assay utilized a quantitative sandwich enzyme immunoassay technique. The intra-assay precision of the kit, as expressed by the coefficient of variability, was 5.4% (range 4.5–6.7%). The average recovery of VEGF from serum samples was 100% (range 92–115%). Statistical analysis to investigate the correlation between serum VEGF165 alteration and age, body mass index (BMI), parity, proliferative or secretory phase, and uterine weight was performed using the Generalized Estimating Equation (17,18) function in SAS version 8.2 (SAS, Cary, NC, USA). Paired t-test and Student’s t-test were used to compare serum VEGF165 levels before and after hysterectomy as individual indication. P-values of 0.05) (Fig. 1). The mean age, parity, BMI, and uterine weight are listed in Table I. The serum VEGF165 level decreased in 63 (78.75%) and increased in 17 (21.25%) patients after hysterectomy. The mean serum VEGF165 level declined in a statistically significant manner from 716.31 457.99 to 581.81 403.32 pg/mL after surgery (P < 0.0001) (Table I). Comparing proliferative (n ¼ 38) and secretory phase (n ¼ 42) patients, there were no significant differences in patients’ age, parity, BMI, uterine weight, or pre- and posthysterectomy VEGF165 levels (Table II). Controlling for age, BMI, uterine weight, and parity, no difference was found in pre-operative serum VEGF165 levels, when nulliparous patients with fibroids (para ¼ 0) (n ¼ 22) were compared to parous patients (para >0) (n ¼ 58). After hysterectomy, nulliparous patients with fibroids had significantly higher VEGF165 levels than parous patients (P ¼ 0.0065) (Table III). The decrease in serum VEGF165 levels after hysterectomy was significant in parous patients (P ¼ 0.000035), but not in nulliparous patients (P ¼ 0.15) (Table III). Uterine weight (gm)
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2000
R = 0.0037; P > 0.05
1500 1000 500 0
0
500
1000
1500
2000
2500
Pre-hysterectomy serum VEGF165 level (pg/mL)
Fig. 1. Before hysterectomy, logistic regression analysis (r ¼ 0.0037; P > 0.05) found no statistically significant correlation between serum vascular endothelial growth factor165 (VEGF165) levels and uterine weight.
Serum VEGF165 levels are not linked to uterine fibroid volume
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Table I. Demographic characteristics of patients Characteristics
Values (n ¼ 80)
Range
Age (years) Parity (number) Body mass index (kg/m2) Uterine weight (gm) VEGF (prehysterectomy) (pg/mL) VEGF (posthysterectomy) (pg/mL)
45.15 4.63 1.99 1.55 24.45 3.94 315.13 234.80 716.31 457.99* 581.81 403.32*
36–52 0–6 18.49–36.40 80–1480 20.4–2137.6 18.7–1813.5
Values are indicated as mean SD. *P < 0.0001, statistically significant decrease of serum vascular endothelial growth factor165 (VEGF165) level after hysterectomy (paired t-test).
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Discussion
Various reports have described a relationship between VEGF-A and benign reproductive events, cyclic endometrial change, abnormal endometrial bleeding, endometriosis, placental physiology, the development of pre-eclampsia, peritoneal fluid, corpus luteum physiology, ovarian tumors, and uterine fibroid tumors (14,19,20). VEGF’s potent angiogenic, mitogenic, and vascular permeability-enhancing activities are specific to endothelial cells and play a role in both benign and malignant tumor development. The uterus has also been implicated as an important source of VEGF165 (13–15). The specific role of VEGF165 on uterine fibroids remains elusive, as it may underlie angiogenesis that promotes tumor growth rather than promoting smooth muscle mitogenesis (19,21). It has been shown that serum VEGF level is positively associated with tumor volume, distant or lymph node metastasis, and stage in cervical cancer and colorectal cancer (22–24). Intriguingly, the present study failed to identify a relationship between serum VEGF165 levels and fibroid weight, which was used as a substitute for fibroid volume. Nevertheless, the significant decline in serum VEGF165 levels in patients with uterine fibroids after hysterectomy suggests that VEGF165 plays a role in uterine fibroid development. Serum VEGF levels of less than 50 pg/mL may not be sufficient to induce the various biological activities of quiescent endothe-
lium (25). Moon et al. (23) reported serum VEGF levels in cervical cancer patients to have a mean of 407.9 232.2 pg/mL, which were lower than serum levels of VEGF165 in the presently studied patients with fibroids (mean 716.31 457.99 pg/ mL). This difference is likely to be the result of different pathogenesis rather than different serum VEGF165 levels. The expression of VEGF in the human endometrium has been found to exhibit cyclic change throughout the menstrual cycle, with three- to six-fold increases in the secretory versus the proliferative phase (14). In order to address concerns that the different menstrual phases may have produced a bias in this study, the study group was divided by phase and serum levels of VEGF165 were compared and not found to be affected by being in either the proliferative or the secretory phase. This was in concord with immunohistochemical studies of fibroid tissue that have found that the action of VEGF165 on fibroids does not differ between estrogendominant proliferative and progesteronedominant secretory phases (12,26), and may imply an alternative pathway for fibroid growth other than ovarian steroid hormone alteration, independent of menstrual cycles. Accordingly, the bias potentially caused by menstrual phase differences was considered insignificant enough to ignore. Using multiple logistic regression, parity was not found to be related to serum levels of
Table II. Comparison of serum vascular endothelial growth factor165 (VEGF165) level between groups divided by the proliferative and the secretory phase before and after hysterectomy Characteristics
Proliferative phase (n ¼ 38)
Secretory phase (n ¼ 42)
P-value
Age (years) Parity (number) Body mass index (kg/m2) Uterine weight (gm) VEGF (prehysterectomy) (pg/mL) VEGF (posthysterectomy) (pg/mL)
45.05 4.18 1.95 1.51 23.58 3.11 352.58 296.21 683.35 389.94 595.25 158.74
45.24 5.05 2.02 1.60 25.25 4.46 281.02 156.65 746.13 514.81 569.65 412.25
NS NS NS NS NS NS
Values are indicated as mean SD. NS: not significant. #
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D.-C. Chen et al.
Table III. Comparison of serum vascular endothelial growth factor165 (VEGF165) level between parous and nulliparous patients before and after hysterectomy Characteristics
Parous patients (n ¼ 58)
Nulliparous patients (n ¼ 22)
P-value
Age (years) Body mass index (kg/m2) Uterine weight (gm) VEGF (pg/mL) Pre- versus posthysterectomy
45.24 4.59 24.34 3.79 289.22 189.17
44.91 4.85 24.75 4.39 383.41 321.75
NS NS NS
668.74 477.21† versus 507.33 385.51*†
841.71 335.01z versus 778.17 390.94*z
–
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*P ¼ 0.0065. †P ¼ 0.000035. zP ¼ 0.15. Values are indicated as mean SD. NS: not significant. After hysterectomy, nulliparous patients with fibroids had significantly higher VEGF165 levels than parous patients (*P ¼ 0.0065). The decrease in serum VEGF165 levels after hysterectomy was significant in parous patients (†P ¼ 0.000035), but not in nulliparous patients (zP ¼ 0.15) (Student’s t-test).
VEGF165 before hysterectomy. Interestingly, parous patients with fibroids showed a significant decline in serum levels of VEGF165 after hysterectomy, but not in nulliparous patients. Marshall et al. (27,28) reported that in 95 061 premenopausal women at risk of fibroids, the risk of fibroids was negatively associated with parity and lastterm birth age. Women with at least two fullterm pregnancies had half the risk of developing fibroids relative to nulliparous women. They proposed a regulatory effect on fibroids mediated via steroid hormones involved in pregnancy. Why the significant decline in VEGF165 serum levels only occurred in parous patients with uterine fibroids after hysterectomy remains unclear. During pregnancy, the expression of VEGF-A increases in gestational products and in the maternal circulation as gestation progresses (29–32). Antenatal physiological adaptations that include hypertrophy of the uterine muscles and vasculature and postpartum involution may underlie the more significant VEGF changes seen in parous patients that underwent hysterectomy. The change seen in parous patients may be related to this previous physiological accommodation. A failing of this study was the lack of a normal control group, which may have helped to more accurately define serum VEGF165 levels for women with or without fibroids. Unfortunately, it is not always possible to obtain a true ‘normal control’ without histological assessment or laparoscopy to exclude pelvic lesions. Another failing was that tissue expression of VEGF and its receptors were not investigated, which may help explain the inconsistencies observed between serum VEGF165 levels and uterine fibroid volume. With the advent of anti-VEGF antibodies and anti-angiogenic agents, medical rather than surgical treatment for uterine fibroids may become possible (33). A better understanding of #
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serum and tissue expression of VEGF may allow such clinical applications to be developed. In brief, this study failed to identify any statistically significant correlation between serum VEGF165 levels and uterine fibroid volume. Serum VEGF165 levels do not appear to predict uterine fibroid development, but demonstrate significant decreases after hysterectomy. The decrease in serum VEGF165 levels after hysterectomy was only found to be significant in parous, as opposed to nulliparous patients with uterine fibroids.
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Address for correspondence: Chi-Huang Chen Department of Obstetrics and Gynecology Tri-Service General Hospital National Defense Medical Center 325, Section 2 Cheng-Kung Road Neihu Taipei 114 Taiwan e-mail:
[email protected] #
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