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Human Chorionic Gonadotropin-Induced Ovarian Hyperstimulation Syndrome Is Associated with Up-Regulation of Vascular Endothelial Growth Factor

Department of Obstetrics & Gynecology, Chang-Gung University Medical School, Chang-Gung Memorial Hospital, Lin-Kou Medical Center, Tao-Yuan 333, Taiwan

Address all correspondence and requests for reprints to: Hsin-Shih Wang, M.D., Ph.D., Department of Obstetrics & Gynecology, Chang-Gung Memorial Hospital, Lin-Kou Medical Center, 5 Fu-Hsing Street, Kwei-Shan, Tao-Yuan 333, Taiwan. E-mail: . hswang86{at}ms17.hinet.net

Abstract

Ovarian hyperstimulation syndrome (OHSS), a life-threatening complication occurring in stimulated ovarian cycles, arises from treatment with gonadotropin for induction of follicular maturation in infertile women. Clinical characteristics of OHSS include ascites and pleural effusion induced by increased vascular permeability, where vascular endothelial growth factor (VEGF) was suspected to be the culprit. To test whether the effects of human CG (hCG) on the pathogenesis of OHSS were mediated through the VEGF produced by luteinized granulosa cells, we measured estradiol, VEGF, IGF-II levels in serum, and follicular fluid and analyzed their mRNA expression in luteinized granulosa cells obtained from 101 women (58 with OHSS and 43 controls) who underwent in vitro fertilization and embryo transfer. This study presents the first evidence that hCG up-regulated VEGF expression of granulosa cells in the OHSS, not the control groups, and that follicular VEGF worked through an autocrine mechanism using its kinase insert domain-containing receptor, not the fms-like tyrosine kinase receptor. We calculated total follicular production of VEGF, by multiplying follicular concentrations by follicular volumes, and verified that an increase in total follicular production of VEGF accounted for elevated serum levels of VEGF, which was associated with the development of OHSS. These findings demonstrate that through up-regulation of VEGF, hCG plays a significant role in the pathogenesis of OHSS.

OVARIAN HYPERSTIMULATION SYNDROME (OHSS) is a severe complication arising from treatment with gonadotropin for induction of follicular growth and maturation in infertile women. Severe ascites and/or pleural effusion, induced by increased vascular permeability, leading to excessive fluid accumulation, are characteristic for OHSS. There are two distinct forms, with different predisposing factors: the early-onset and the late-onset OHSS. The early-onset OHSS occurs 3–7 d after administration of human CG (hCG) and is related to the magnitude of the preceding ovarian response, whereas the late-onset OHSS is identified 12–17 d after ovulatory dose of hCG and frequently is associated with multiple pregnancies.

Vascular endothelial growth factor (VEGF) is a heparin-binding, 40,000- to 46,000-molecular weight, disulfide-linked homodimeric glycoprotein that dissociates upon reduction into two 20,000- to 23,000-molecular weight subunits (1). In vivo, VEGF induces angiogenesis and possesses a potent effect on vascular permeability. Although kallikrein-kinin and renin-angiotensin systems may also be involved in the hyperpermeability seen in OHSS (2, 3), VEGF was proposed to be the major capillary permeability agent in OHSS ascites fluid, based on the observations that treatment with anti-VEGF antibody decreased the vascular permeability activity of OHSS ascites by approximately 70% (4). In the human ovary, VEGF mRNA and protein are expressed by granulosa and theca cells in late follicular development before ovulation (5). In infertile patients who underwent controlled ovarian hyperstimulation, the expression of VEGF mRNA in luteinized granulosa cells was enhanced by hCG in both dose-dependent and time-dependent manners (6). Additionally, VEGF concentrations in serum, peritoneal fluid, and follicular fluid of patients at risk for OHSS are positively related to the development of the syndrome (7, 8). Extending from these observations, we attempted to further test whether the effects of hCG on the pathogenesis of OHSS could be mediated through the VEGF that was produced by luteinized granulosa cells.

In the ovary, aromatase P450 (9) and IGFs/IGF-binding proteins (IGFBPs) (10, 11, 12) regulate steroidogenesis as well as follicular growth and maturation. In patients who later develop OHSS, serum estradiol levels are highly elevated, suggesting the possible overexpression of aromatase P450 and IGFs/IGFBPs by follicles. Therefore, aromatase P450 and IGFs/IGFBPs, in addition to VEGF, may also be related to the development of OHSS. However, the cause-effect relationship among aromatase P450, IGFs/IGFBPs, and OHSS has not been explored.

In the present study, we searched for the source that could explain the elevated VEGF serum levels in patients who later developed OHSS, and we investigated whether the VEGF production of granulosa cells in OHSS patients was regulated by hCG. In addition, we also analyzed the associations among expression of aromatase P450, IGFs/IGFBPs, and elevated levels of estradiol in OHSS. Here, we report that supernumerary follicles are the main source of elevated VEGF in patients who eventually develop OHSS and that hCG up-regulates the expression of VEGF.

Materials and Methods

Subjects and protocols

During the period from May 1997 to July 1998, 487 women underwent the controlled ovarian hyperstimulation for in vitro fertilization-embryo transfer (IVF-ET); 58 women who developed ovarian hyperstimulation syndrome (OHSS), including 46 moderate cases and 12 severe cases, were enrolled in the present study. All these OHSS cases were the early-onset form, where clinical symptoms and signs developed within 7 d after the ovulatory dose of hCG (13). The severity of OHSS was categorized according to the revised classification proposed by Golan et al. (14). Moderate OHSS was diagnosed by abdominal distension, enlarged ovaries (over 6 cm in diameter), and ultrasonographic evidence of ascites. Severe OHSS was manifested by additional breathing difficulties and/or hydrothorax, hemoconcentration (hematocrit > 45%), and oliguria. Another group of 43 women, who underwent ovulation induction for IVF-ET but did not develop OHSS, were enrolled as controls. The age of patients ranged from 26–39 yr (mean age, 33.2 yr). Body mass index [weight (kilograms) divided by height squared (meters)] was 23.7 (range, 20.1–27.6) kg/m² . All patients gave written informed consent to participate in the study, and the experimental design was approved by the Institutional Review Board of Chang Gung Memorial Hospital.

The protocol for controlled ovarian hyperstimulation included: 1) Pituitary down-regulation with sc injection of GnRH agonist (1 mg/d; Lupron, Abbott Laboratories, North Chicago, IL) was begun on the 21st day of the previous menstrual cycle. 2) On the third day of the menstruation, im administration of FSH (150–450 IU/d; Metrodin, Industria Farmaceutica Serono S.p.A., Rome, Italy) was started, based on the women’s age and past response history when applicable. 3) hCG (10,000 IU; Pregnyl, N.V. Organon, Oss, Netherlands) was administered im 34–36 h before oocyte retrieval, and ovulation induction was monitored with transvaginal sonography.

During oocyte retrieval, follicular fluid and luteinized granulosa cells from each patient were pooled. Peripheral blood samples were withdrawn from patients at the time when follicles were aspirated, and serum was isolated by centrifugation immediately after blood clotted. Both follicular fluid and serum were stored at -70 C until subsequent assays.

Preparation and culture of luteinized granulosa cells

The use of human luteinized granulosa cells for clinical trials and research involving human subjects was approved by the Human Research Committee of Chang-Gung Memorial Hospital. Pooled follicular aspirates from individual patients were centrifuged at 290 x g for 10 min to form a cell pellet that was resuspended in Medium 199 (Sigma, St. Louis, MO) by vigorous pipetting. Luteinized granulosa cells were isolated from contaminated red blood cells by density-gradient centrifugation in 50% Percoll (Sigma) at 800 x g for 10 min. Granulosa cells were recovered from the interface, resuspended in 3 ml Medium 199, and sieved through a nylon filter with micropores of 70 µm in diameter (Becton Dickinson and Co., Franklin Lakes, NJ). Percoll separation, resuspension, and centrifugation were repeated once, and the resultant cell pellet was resuspended in 14 ml Medium 199 containing 10% FBS, penicillin (100 IU/ml), and streptomycin (100 µg/ml). The number and viability of cells were determined by Trypan blue using a hemocytometer. Approximately 8–14 x 10⁶ luteinized granulosa cells were obtained per retrieval, with cell viability varying from 55–85%.

Culture dishes (35 mm x 10 mm, Becton Dickinson and Co.) were coated with 10 µg/ml fibronectin (Sigma) in Medium 199 at 37 C for 1 h and washed three times with medium before cells were plated. Cells from each patient were equally divided into eight aliquots. One was harvested immediately (d 0), and others were cultured in seven dishes, each containing 2 ml Medium 199 with 10% FBS and appropriate antibiotics, at 37 C in a humidified 5% CO2–95% air environment. Culture medium (containing FBS and antibiotics) was replaced 24 h later (d 1). Thereafter, culture medium with and without hCG, at a concentration of 100 ng/ml, as suggested by Neulen et al. (6), was replaced every 48 h (d 3, 5, and 7) for the study and the control groups, respectively. Cells were harvested on d 1, 3, 5, and 7 for the extraction of RNA.

Extraction of RNA

Total RNA in luteinized granulosa cells collected from each culture dish was isolated using a guanidium thiocyanate-phenol-chloroform procedure as previously described (15). The luteinized granulosa cells were first lysed in 0.5 ml denaturing solution [4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% (wt/vol) sarcosyl, and 0.1 M 2-mercaptoethanol], followed by an addition of 0.05 ml 2 M sodium acetate, 0.5 ml of phenol, and 0.1 ml of chloroform/isoamyl alcohol (49:1). After being vigorously shaken, the tubes were incubated on ice for 15 min and microcentrifuged at 10,000 x g, at 4 C, for 20 min. The aqueous layer was transferred to another tube and was added with an equal volume of cold isopropanol to precipitate RNA. The pelleted RNA was resuspended in 0.3 ml denaturing solution and reprecipitated with isopropanol. The RNA pellet was then washed twice with 75% ethanol and dissolved in diethyl pyrocarbonate-treated water. The amount of RNA was quantified spectrophotometrically and stored at -70 C until the time of analysis.

Semiquantitative RT-PCR with multiple pairs of primers

Semiquantitative RT-PCR was performed as previously described (15). Briefly, two sets of primers were used simultaneously in each tube (one for target mRNAs and another for ß-actin mRNA used as an internal control). Sequences of primers for human mRNA used in RT-PCR are as follows: ß-actin (size of PCR product, 838 bp): 5' ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG 3' (downstream), 5' CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC 3' (upstream); IGFBP-1 (size of PCR product, 417 bp): 5' TA CAT CTG GCA GTT GGG GTC TCC 3' (downstream), 5' TGC AGA GGC AGG GAG CCC TGA AA 3' (upstream); IGF-I (size of PCR product, 514 bp): 5' ACA TCT CCC ATC TCT CTG GAT TTC CTT TTG C 3' (downstream), 5' CCC TCT ACT TGC GTT CTT CAA ATG TAC TTC C 3' (upstream); IGF-II (size of PCR product, 538 bp): 5' AGT CGA TGC TGG TGC TTC TCA CCT TCT TGG C 3' (downstream), 5' TGC GGC AGT TTT GCT CAC TTC CGA TTG CTG G 3' (upstream); IGF type I receptor (size of PCR product, 540 bp): 5' GAA TGG AGT GCT GTA TGC CTC TGT GAA CC 3' (downstream), 5' GTG AAA TCT TCG GCT ACC ATG CAA TTC CG 3' (upstream); estrogen receptor (size of PCR product, 532 bp): 5' TGC CAA GGA GAC TCG CTA 3' (downstream), 5' TCA ACA TTC TCC CTC CTC 3' (upstream); aromatase P450 (size of PCR product, 272 bp): 5' TTG TTG TTA AAT ATG ATG CC 3' (downstream), 5' ATA CCA GGT CCT GGC TAC TG 3' (upstream); VEGF (size of PCR product, 363 bp): 5' GTG CTG TAG GAA GCT CAT CTC 3' (downstream), 5' CTG TCT TGG GTG CAT TGG AGC C 3' (upstream); fms-like tyrosine kinase-1 (flt-1) (size of PCR product, 239 bp): 5' GAC GTC TAG AGT TTG ACA CGA AGC 3' (downstream), 5' GCA TGC AAC ACT GAG TAA CAT GAC 3' (upstream); and kinase insert domain-containing receptor (KDR) (size of PCR product, 204 bp): 5' TAA GCT TAC CAA AGG GGC ACG ATT CCG T 3' (downstream), 5' TGG ATC CCT GTA ACA GAT GAG ATG CTC C 3' (upstream).

A half microgram of total RNA from each sample was used for RT using a Gene Amp RNA PCR kit (Perkin-Elmer Corp., Cetus, MA). In each tube containing two downstream primers, one for ß-actin mRNA and another for a target sequence, total RNA was reversely transcribed with murine leukemia virus reverse transcriptase (50 IU) in a final vol of 40 µl. Inside a DNA thermal cycler (Perkin-Elmer Corp.), RT took place at 42 C for 15 min, then reagents were heated to 99 C for 3 min to denature the murine leukemia virus reverse transcriptase, rapidly cooled to 4 C and stored at 4 C. After RT, the resultant cDNA was subjected to 25 cycles PCR containing 2 upstream primers, one for ß-actin and another for a target mRNA. Each cycle consisted of 94 C for 1 min (denature), 60 C (annealing temperature for IGF-I, IGF-II, VEGF, estrogen receptor, flt-1, and KDR) or 55 C (annealing temperature for IGFBP-1, type 1-IGF receptor, and aromatase P450) for 1 min, and extension at 72 C for 2 min. After 25 cycles, the mixtures were maintained at 72 C for 7 min, then cooled rapidly and stored at 4 C. For determining the size of the PCR products, 15 µl of the reaction products mixed with 3 µl loading dye [0.25% (wt/vol) bromophenol blue, 0.25% (wt/vol) xylene cyanol, and 30% (vol/vol) glycerol in water] was separated by 2% agarose (Promega Corp., Madison, WI) gel electrophoresis, containing a minimal amount of ethidium bromide. After electrophoresis, the intensity of RT-PCR products was visualized under UV light and analyzed with an image analyzer (Gel Doc 1000; Bio-Rad Laboratories, Inc., Hercules, CA).

Immunoassays for measurement of VEGF, IGF-II, and estradiol

Concentrations of VEGF in serum and follicular fluid were determined with an ELISA kit (Quantikine; R & D Systems, Minneapolis, MN) that employed the quantitative sandwich enzyme immunoassay technique. The minimum detection limit of the assay was 15.6 pg/ml. The intraassay coefficients of variation were 6.7% at 55.5 pg/ml, 4.5% at 235 pg/ml, and 5.4% at 925 pg/ml (n = 12). The interassay coefficients of variation were 8.8% at 65.3 pg/ml, 7.2% at 252 pg/ml, and 6.3% at 975 pg/ml (n = 8).

Serum and follicular fluid IGF-II levels were measured using an ELISA kit (Active IGF-II; Diagnostic Systems Laboratories, Inc., Webster, TX). The minimum detection limit of the assay was 50 ng/ml. The intraassay coefficients of variation were 7.6% at 286 ng/ml, 4.5% at 416 ng/ml, and 3.5% at 675 ng/ml (n = 10). The interassay coefficients of variation were 10.4% at 295 ng/ml, 8.5% at 452 ng/ml, and 7.8% at 722 ng/ml (n = 6).

Estradiol levels in both serum and follicular fluid were determined by immunofluorometric assays (Pharmacia, Turku, Finland). The detection limit of the estradiol assay was 13.6 pg/ml. The intraassay coefficients of variation were 6.7% at 142 pg/ml and 4.9% at 423 pg/ml (n = 16). The interassay coefficients of variation were 8.6% at 155 pg/ml and 6.8% at 510 pg/ml (n = 12). Manufacturers’ recommendations were followed exactly for all immunoassays.

Statistical analysis

Differences in concentrations of VEGF, IGF-II, and estradiol in both serum and follicular fluid were scrutinized by Student’s t test. Expression of mRNA for target genes among different groups was also analyzed by Student’s t test. To explore the difference in pregnancy rates affected by the severity of OHSS, z test for instances (zI test) was employed. In all cases, a P value less than 0.05 was considered statistically significant.

Results

The incidence of OHSS in the patient population at our institute, during 1997–1998, was 11.9% (58/487), including 9.4% (46/487) moderate and 2.5% (12/487) severe cases. The numbers of oocytes retrieved from patients who later developed moderate or severe OHSS were significantly greater, compared with controls (P < 0.0001 and P < 0.0001, respectively) (Table 1). Likewise, the pregnancy rates were significantly higher in patients with severe and moderate OHSS than in controls (P < 0.02 and P < 0.05, respectively) (Table 1). However, between patients who developed moderate OHSS and who developed severe OHSS, the difference in numbers of retrieved oocytes and in pregnancy rates did not reach a statistical significance. Neither were the abortion rates significantly different among the three groups (Table 1).

View larger version (25K): [in this window] [in a new window]   Figure 1. A, Correlation between number of retrieved oocytes and serum estradiol levels on the day of follicle aspiration; B, correlation between number of retrieved oocytes and serum levels of VEGF on the day of oocyte retrieval; C, correlation between serum levels and estimated TFP of estradiol on the day of ovum retrieval; D, correlation between serum levels and estimated TFP of VEGF on the day of ovum retrieval. The total of 101 cases, consisting of 58 OHSS and 43 controls in this study, are included in these panels. The degree of freedom (d.f.) is 99 when analyzed by lineal regression.

In luteinized granulosa cells from both controls and patients who developed OHSS, expression of IGFBP-1, IGF-I, IGF-II, type 1-IGF receptor, estrogen receptor, aromatase P450, and VEGF was demonstrated at various molecular sizes, depending on the primer designed for each mRNA (Fig. 2). RT-PCR clearly demonstrated that the band at 838 bp represents the expression of ß-actin mRNA and bands at designated sizes corresponding to expression of different target genes (Fig. 2). Expression of mRNA for KDR (one of the two VEGF receptors) in luteinized granulosa cells from both controls and patients (the right panel of Fig. 3) who later developed OHSS was detected as the band at 204 bp (lane 1 of Fig. 3), whereas the band at 239 bp expected for flt-1 (another VEGF receptor) was undetectable (lane 2 of Fig. 3).

View larger version (43K): [in this window] [in a new window]   Figure 2. A representative gel of 2% agarose gel electrophoresis of the semiquantitative RT-PCR products. The band corresponding to the PCR products of ß-actin (838 bp) is shown in each lane, whereas a 363-bp band corresponding to the PCR products of VEGF is shown in lane 7. In addition to the internal control (ß-actin) shown as the upper band in all lanes, PCR products corresponding to target genes are: IGFBP-1 (lane 1), IGF-I (lane 2), IGF-II (lane 3), type 1-IGF receptor (lane 4), estrogen receptor (lane 5), aromatase P450 (lane 6), and VEGF (lane 7). Molecular markers (100-bp ladder) are shown in lane M.

View larger version (69K): [in this window] [in a new window]   Figure 3. A representative gel of 2% agarose gel electrophoresis of RT-PCR products for flt-1 and KDR (VEGF receptors) in luteinized granulosa cells obtained from controls and patients developing OHSS. The band corresponding to the PCR products of ß-actin (838 bp) is shown in each lane. A 204-bp band corresponding to the PCR products of KDR is shown in lane 1. However, a 239-bp band corresponding to the PCR products of flt-1 is undetected (lane 2). Molecular markers (100-bp ladder) are shown in lane M.

View larger version (31K): [in this window] [in a new window]   Figure 4. Densitometric units of PCR products for IGFBP-1, IGF-I, IGF-II, type 1-IGF receptor (IGFR-1), estrogen receptor (ER), aromatase P450, VEGF, and a VEGF receptor (KDR) in luteinized granulosa cells obtained from moderate (n = 46) and severe OHSS (n = 12) and controls (n = 43) on the day of oocyte retrieval. The data were normalized by the intensity of ß-actin, which was set as 100 arbitrary units. Data shown are the means ± SD.

View larger version (34K): [in this window] [in a new window]   Figure 5. The changes in mRNA expression of VEGF (shown in densitometric units) in luteinized granulosa cells obtained from moderate (n = 46) and severe OHSS (n = 12) and controls (n = 43) after being cultured with or without hCG for 2, 4, and 6 d (d 3, 5, and 7). The data were normalized by the intensity of ß-actin, which was set as 100 arbitrary units. Data shown are the means ± SD. Pairs of data with statistically significant difference are indicated, with individual P values shown as follows: a, P = 0.021; b, P = 0.0002; c, P = 0.0008; d, P = 0.038; e, P = 0.0012; f, P = 0.0013.

View larger version (31K): [in this window] [in a new window]   Figure 6. The changes in mRNA expression of IGF-II (shown in densitometric units) in luteinized granulosa cells obtained from moderate (n = 46) and severe OHSS (n = 12) and controls (n = 43) after being cultured with or without hCG for 2, 4, and 6 d (d 3, 5, and 7). The data were normalized by the intensity of ß-actin, which was set as 100 arbitrary units. Data shown are the means ± SD. Pairs of data with statistically significant difference are indicated, with individual P values shown as follows: a, P = 0.048; b, P = 0.002; c, P = 0.001.

View larger version (28K): [in this window] [in a new window]   Figure 7. The changes in mRNA expression of IGF-I (shown in densitometric units) in luteinized granulosa cells obtained from moderate (n = 46) and severe OHSS (n = 12) and controls (n = 43) after being cultured with or without hCG for 2, 4, and 6 d (d 3, 5, and 7). The data were normalized by the intensity of ß-actin, which was set as 100 arbitrary units. Data shown are the means ± SD.

Increased levels of VEGF may promote higher pregnancy rates in patients who later develop OHSS

There are two distinct forms of OHSS, with different predisposing factors (13). The early-onset OHSS is usually identified 3–7 d after administration of hCG and is related to the magnitude of the preceding ovarian response. In contrast, the severer, late-onset OHSS occurs 12–17 d after an ovulatory dose of hCG and only takes place in the presence of pregnancy (13). In this study, only the early-onset OHSS patients were enrolled. Nevertheless, pregnancy rates were significantly higher in patients with both severe and moderate OHSS, compared with controls (Table 1), similar to those previously reported (16). Both higher pregnancy rates and higher serum levels of VEGF were observed in women who later developed OHSS (Tables 1 and 2). Given the role of VEGF in stimulation of endothelial proliferation and angiogenesis (17), VEGF may help the vascularization of secretory endometrium, preparing it for the implantation of embryos. The higher VEGF levels in follicular fluid in patients who later developed OHSS might also contribute to the angiogenesis of the corpus luteum after ovulation and, in turn, promote its function of steroidogenesis (18).

High-affinity receptors for VEGF have been reported to be localized exclusively on vascular endothelial cells (19). Both receptors for VEGF, KDR and flt-1, are homodimeric tyrosine kinases. Because only expression of mRNA for KDR, but not flt-1, was detected in luteinized granulosa cells from both controls and patients who developed OHSS (Fig. 3), these results suggest that VEGF produced by luteinized granulosa cells may play a role in autocrine regulation on the angiogenesis of the corpus luteum through the VEGF receptor KDR. Sufficient blood supply to the corpus luteum may contribute to the higher pregnancy rate observed in patients who later develop OHSS.

In patients with OHSS, increased TFP of VEGF and estradiol accounts for their elevated serum levels that are associated with clinical symptoms of the disease

Preovulatory human follicular fluid, which contains abundant VEGF, a potent vascular permeability factor (17), has been shown to increase capillary permeability (20). Our results also demonstrated that serum levels of VEGF in both moderate and severe OHSS groups were significantly higher than those in the controls (Table 2), in accordance with previous findings (8, 21). Follicular fluid contained much higher concentrations of VEGF, about 5- to 8-fold of serum levels in our study (Table 2). Hence, we hypothesized that ovaries might be the main source for the circulating VEGF. Although the difference in follicular fluid VEGF concentrations between the controls and patients who developed OHSS did not reach a statistical significance, we still could observe the higher follicular VEGF concentrations in the OHSS groups (Table 2). Furthermore, the number of retrieved oocytes was positively correlated with serum VEGF levels (P < 0.01) (Fig. 1B), and a greater number of oocytes were retrieved in patients who later developed OHSS than in the controls (Table 1). Therefore, we also took the total volume of follicular fluid in each patient into consideration. The estimated TFP of VEGF thus could be derived from the multiplication of follicular fluid levels of VEGF by the total volume of follicular fluid. These data indicate that elevated concentrations of serum VEGF in OHSS groups result from the increased total production of VEGF from the supernumerary follicles (Table 2 and Fig. 1D). The same mechanism is likely applicable for the case of estradiol (Table 2 and Fig. 1C). Collectively, elevated serum VEGF levels in patients who will develop OHSS seem to be produced by follicular granulosa cells, resulting in its clinical symptoms and signs, such as ascites, breathing difficulties and/or hydrothorax, hemoconcentration (hematocrit > 45%), and oliguria.

hCG up-regulates VEGF expression of luteinized granulosa cells in patients with OHSS

The gene for human VEGF is organized into eight exons, separated by seven introns. Five isoforms of VEGF arise via alternative splicing of mature mRNA, resulting in two cell-associated (VEGF206 and VEGF189) and three soluble (VEGF165, VEGF145, and VEGF121) proteins (22, 23). All isoforms contain exons 1–5 and 8. However, VEGF165 lacks the residues encoded by exon 6 and VEGF145 lacks the residues encoded by exon 7, whereas VEGF121 lacks the residues encoded by exons 6 and 7 (23, 24). The primers for VEGF RT-PCR used in this study detected regions containing exons 1–4 of VEGF mRNA, which were the common part shared by VEGF206, VEGF189, VEGF165, VEGF145, and VEGF121. Thus, the difference in mRNA expression of these isoforms could not be detected by this set of primers used in our study.

In bovine ovarian follicles, analysis of VEGF transcript by RT-PCR showed that granulosa and theca cells express predominantly the small isoforms (VEGF121 and VEGF165) (25). VEGF121, VEGF145, and VEGF165 are soluble proteins, whereas VEGF189 and VEGF206 are almost completely sequestered in the extracellular matrix and largely insoluble (23, 26). Even so, VEGF189 and VEGF206 can be released in a bioactively diffusible form after being proteolytically cleaved by heparinase, plasminogen activators, and collagenase (27). Therefore, detectable VEGFs in follicular fluid and serum, by ELISA, are most likely the soluble VEGF121, VEGF145, or VEGF165, and the smaller molecules proteolyzed from insoluble VEGF189 or VEGF206.

This study is the first to track the changes in expression of VEGF mRNA induced by hCG in luteinized granulosa cells obtained from patients who will develop OHSS. Previous studies have demonstrated that human luteinized granulosa cells produced VEGF and expressed mRNA for VEGF (5, 6). Short-time-course (1-h interval, for 5 h) experiments have also indicated that the mRNA expression of VEGF in human luteinized granulosa cells (from patients undergoing in vitro fertilization without developing OHSS) was dose- and time-dependently enhanced by hCG (6). In the presence of hCG, we have observed that luteinized granulosa cells from patients who later developed severe OHSS expressed a greater amount of mRNA for VEGF, compared with those from controls (Fig. 5). In luteinized granulosa cells from the severe OHSS group, we also verified that hCG up-regulated the mRNA expression of VEGF in our culture system (Fig. 5). Although we did not measure the protein levels of VEGF in these culture media, mRNA levels of VEGF were recently shown to be positively correlated with its protein levels (28). Collectively, these results indicate that hCG up-regulates VEGF production by granulosa cells, accounting for its increased serum levels in patients who later develop OHSS.

IGFs, IGF receptors, IGFBP-I, estrogen receptors, aromatase, and VEGF receptors

In the present study, low expression of mRNA for KDR (one of the two VEGF receptors) in luteinized granulosa cells from both controls and patients who developed OHSS was detected (Figs. 3 and 4), whereas that for flt-1 (another VEGF receptor) was undetectable (Fig. 3). Our observation of low expression for KDR in the human follicles is similar to that in bovine ovarian follicles, where the mRNA expression of KDR and flt-1 was very weak in granulosa cells, without any regulatory change during follicular growth (25). Given that high-affinity receptors for VEGF were localized exclusively on vascular endothelial cells (19), granulosa cells in ovarian follicles may not be the major target of VEGF. Even so, VEGF may still be important for angiogenesis of corpus luteum after ovulation, assuring the luteal function in steroidogenesis (18).

As with higher pregnancy rates, high serum estradiol levels were also found in patients who developed moderate or severe OHSS (Tables 1 and 2). Elevated serum estradiol levels were hypothesized to be detrimental to endometrial receptivity, leading to lower implantation rates and lower pregnancy rates (29, 30). However, this speculation was not supported by the present study and others (16, 31). Patients with polycystic ovarian syndrome, who have been noted to be particularly sensitive to exogenous gonadotropin therapy, frequently experienced OHSS episodes during IVF-ET cycles (32). An increased aromatase activity in granulosa cells from patients with polycystic ovarian syndrome was suggested to account for the high production of estradiol and, in turn, elevate the incidence of OHSS (33). However, described as follows, our results do not support the hypothesis that the elevated aromatase activity causes the increased estradiol levels in patients with OHSS. First, aromatase P450 mRNA expression in granulosa cells from the OHSS group was similar to that from controls (Fig. 4). Second, although serum levels of estradiol in both moderate and severe OHSS groups were significantly higher than those in the controls, the difference in follicular fluid estradiol concentrations between the controls and patients who developed OHSS did not reach a statistical significance (Table 2). Collectively, these results suggest that higher concentrations of serum estradiol in patients with moderate and severe OHSS most likely reflect the summation of follicular estradiol produced by a greater number of well-developed follicles in these patients (Table 1) rather than a direct result from the increased aromatase P450 activity in granulosa cells.

IGF-I and IGF-II stimulate steroidogenic acute regulatory protein mRNA and protein expression in human granulosa-luteal cells (34). A synergistic interaction of insulin with IGF-II in human granulosa cells has also been demonstrated (35). These observations suggest an important role of IGF-I and IGF-II in human ovarian steroidogenesis. In the present study, significantly lower IGF-II levels in follicular fluid from patients who would develop severe or moderate OHSS were noted, compared with controls (Table 2). Noteworthily, all of these patients were given hCG (10,000 IU) 36 h before aspiration of follicular fluid. When cultured in the presence of hCG for 2, 4, and 6 d, IGF-II expression was much less in luteinized granulosa cells isolated from patients who would develop severe OHSS than in those obtained from controls (d 3, 5, and 7; Fig. 6). In contrast, no such change was detected in the expression of IGF-I (Fig. 7). Despite lower expression of IGF-II in OHSS granulosa cells, significantly higher pregnancy rates that require sufficient steroidogenesis were found in patients with severe OHSS (Table 1). These observations imply that, in patients who will develop OHSS, the IGF/IGFBPs pathways regulate steroidogenesis in luteinized granulosa cells predominantly through IGF-I rather than IGF-II, especially in the presence of hCG.

Although follicular fluid IGF-II levels were significantly lower in patients with OHSS, serum IGF-II levels were similar in both OHSS patients and controls on the day of oocyte retrieval (Table 2). In addition, concentrations of serum IGF-II were 1.6- to 1.8-fold higher than those in follicular fluid in all groups studied on the day of oocyte retrieval (Table 2). Because IGF-I and IGF-II can be synthesized de novo in a variety of tissues (36), the IGF-II production by ovarian follicles during follicular growth and maturation seems unable to affect the serum levels of IGF-II. Therefore, the role of down-regulation of IGF-II in follicular fluid, if any, in the pathophysiology of OHSS has yet to be studied.

To our knowledge, the present study provides the first evidence that hCG enhances in vitro expression of VEGF mRNA and attenuates the expression of IGF-II mRNA by luteinized granulosa cells in patients with severe OHSS but not by those in the controls. In addition to the increased number of oocytes in patients with OHSS, up-regulation of VEGF in luteinized granulosa cells by hCG collaboratively accounts for the increased serum levels of VEGF, resulting in the clinical manifestations of OHSS. The present study demonstrates a significant role of hCG, through up-regulation of VEGF, in the pathogenesis of OHSS, although the coexistence of other mechanisms cannot be eliminated (2, 3). These results strengthen the commonly accepted notion that patients displaying high preovulatory serum levels of estradiol, e.g. more than 4000 pg/ml, after stimulation with gonadotropins, have a high risk for OHSS. Thus, in these patients, we have to use hCG with greater caution to avoid the development of OHSS. Moreover, the critical role of VEGF in the pathophysiology of OHSS warrants the future development of specific agents targeting VEGF for its prevention and treatment.

Acknowledgments

We are indebted to Drs. Chi-Long Lee, Chun-Kai Chen, Chia-Wei Wang, and Hong-Yuan Huang for providing invaluable clinical specimens. We also thank Mei-Li Wang, Chieh-Yu Lin, Ming-Jer Hsu, and Hsuan Jung for excellent technical assistance; Hui-Ting Yang for secretarial work; and Shih-Tien Wang, of Northwestern University, for editing expertise.

Footnotes

This work was supported by funding from the Chang Gung Memorial Hospital (to T.-H.W.; CMRP-890) and the National Science Council, Taiwan [to T.-H.W. (NSC89-2314-B-182A-103, NSC90-2314-B-182A-020, and NSC90-2314-B-182A-150) and to H.-S.W (NSC89-2314-B-182-041, NSC89-2314-B-182-142, NSC89-2314-B-182-077, NSC90-2314-B-182-040, and NSC90-2314-B-182-107)]. This work was presented in part at the 57th Annual Meeting of The American Society for Reproductive Medicine, October 20–25, 2001, Orlando, Florida.

Abbreviations: flt, fms-like tyrosine kinase; hCG, human CG; IGFBP, IGF-binding proteins; IVF-ET, in vitro fertilization-embryo transfer; KDR, kinase insert domain-containing receptor; OHSS, ovarian hyperstimulation syndrome; TFP, total follicular production; VEGF, vascular endothelial growth factor.

Received January 14, 2002.

Accepted March 25, 2002.

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