1 Department of Obstetrics and Gynecology, Prince of Wales Hospital (PWH) and 2 Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong (CUHK), Hong Kong
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Prince of Wales Hospital, 3032 Ngan Shing Street, Shatin, New Territories, Hong Kong. e-mail: Lampomui{at}cuhk.edu.hk
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Abstract |
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Key words: hydrosalpinx/mRNA/RTPCR/VEGF
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Introduction |
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Oviductal fluid is a complex mixture of plasma-derived constituents and proteins synthesized by the oviduct epithelium (Verhage et al., 1988; Willis et al., 1994
). Analysis of hydrosalpinx fluid reveals electrolyte concentrations similar to those in serum but with lower levels of total protein and albumin (Ng et al., 1997
; Granot et al., 1998
). Thus, serum transudation may be the main mechanism for the formation of hydrosalpinx fluid, with reduced protein synthesis in the damaged oviduct epithelium. Vascular endothelial growth factor (VEGF), a known permeability enhancing factor, has been localized in human oviductal epithelium (Gordon et al., 1996
). We have also shown that VEGF expression in the healthy oviduct is parallel to the regional and temporal modulation of oviductal fluid secretion (Lam et al., 2003
). This finding suggests that VEGF may be a controller of oviductal secretion by promoting vascular and epithelial permeability and thereby serum transudation into the oviduct lumen. Also, recent studies have shown that VEGF expression can be up-regulated by bacterial lipopolysaccharide (van Der Flier et al., 2000
; Morozumi et al., 2001
; Nielsen et al., 2001
). In this way, VEGF may play an important role in pelvic inflammatory disease for the subsequent hydrosalpinx fluid formation.
This study compares the mRNA expression of VEGF and its receptors (VEGF-R), flt-1 and KDR, in the hydrosalpinx with that in the healthy oviduct, using semi-quantitative reversetranscriptase PCR (RTPCR) analysis. The results may help in the understanding of the mechanisms of hydrosalpinx fluid formation.
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Materials and methods |
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Subjects
Ten infertile women with ultrasound-visible hydrosalpinx, who underwent laparoscopic salpingectomy before IVF cycle, were recruited as the study group. Another ten parous women with no tubal pathology, who underwent laparoscopic tubal sterilization, were recruited as the control group. All subjects had regular monthly menstrual cycles and were not on any exogenous hormone in the past three months. The cycle was divided into five stages that were determined by the date of commencement of last menstrual period (LMP) and were confirmed by appropriate serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol (E2) and progesterone. Two women in the study group, one in the early follicular stage and the other in the periovulatory stage, as well as one woman in the control group who was in the early follicular stage, were excluded because their LMP did not correspond with the serum LH, FSH, E2 and progesterone concentrations.
The stages of the ovulatory cycle, at which the oviduct tissue was collected were matched in the study group and the control group. In each group, there were two women in each stage, namely stage 1 (early follicular phase, day 15), stage 2 (mid-follicular phase, day 612), stage 3 (periovulatory phase, day 1317), stage 4 (early or mid-luteal phase, day 1823), and stage 5 (late luteal phase, day 2428). Informed consent was obtained from each participant.
Oviduct tissue dissection and preparation
Oviduct tissue was excised in the operation of laparoscopic salpingectomy for removal of hydrosalpinx or for sterilization. Excised tissue was immediately placed in 20 ml of HEPES-buffered Quinns human tubal fluid (Irvine Scientific, Santa Ana, CA). This medium was used for all tissue manipulation that was carried out at room temperature. The ampullary part of oviduct tissue, which was defined as the parts 46 cm from the proximal ends (Lam et al., 2003), was collected because it is the site of fertilization and early embryo development.
The lumen of the oviduct was exposed by incising along the antimesenteric border, and the tissue was rinsed to minimize the contamination by hydrosalpinx fluid or blood cells. Only the mucosal layer was dissected off macroscopically. The mucosal strips were cut into 1 mm3 pieces and digested by trypsinization (0.05% trypsin and 0.53 mmol/l ethylenediaminetetraacetic acid; Gibco-BRL, Carlsbad, CA). Immunohistochemistry confirmed that >95% of the cells stained positively for the specific marker cytokeratin, supporting the epithelial cell origin. An aliquot of the cell suspension was placed into an Eppendorf tube and stored at 70°C for subsequent mRNA isolation.
Only two of the investigators performed the tissue dissection and preparation, and the technique was closely monitored to ensure accurate replication. The duration and conditions of the isolation procedures of oviduct mucosal cells were the same for the hydrosalpinx and the healthy oviduct tissue. The procedures were performed under room conditions, and the cells were not subjected to any culture or hypoxic conditions.
Semi-quantitative RTPCR
mRNA was extracted from the epithelial cells of ampullary regions of oviduct using the Oligotex Direct mRNA kit according to the manufacturers instructions (Qiagen, Hilden, Germany). Integrity of RNA was checked by electrophoresis of samples in a 2% agarose gel stained with ethidium bromide. mRNA (1.6 µg) was treated with 1 U of RNase-free DNase (Boehringer Mannheim/Hoffman-La Roche, Basel, Switzerland) before reverse transcription to remove contaminating genomic DNA. Then, the DNase was inactivated by incubating for 5 min at 90°C, and 100 ng of mRNA was used for cDNA synthesis with Multiscribe reverse transcriptase (PE Biosystems, Foster City, CA). All the resultant cDNA was then used for PCR with Amplitaq Gold DNA polymerase (PE Biosystems). Each PCR cycle consisted of denaturation at 95°C for 45 s, annealing at 55°C for 45 s and extension at 72°C for 1 min. Thirty cycles were performed, followed by a final extension at 72°C for 5 min.
Forward and reverse primers specific to VEGF were derived from the published primer sequences (Krussel et al., 2000). The sequences of the oligonucleotide primers were forward GGGCAGAATCAT CACGA, and reverse TGGTCTGCATTCACATTTG (Mwgag Biotech, Ebersberg, Germany). To ensure that the detected product resulted from the amplification of cDNA rather than contaminating genomic DNA, primers have been designed to cross exonexon boundaries. It was shown that VEGF165 and, to a lesser extent, VEGF121 are the predominant isoforms of VEGF in bovine oviduct; however, each of the VEGF isoforms showed the same pattern of expression in oviduct (Gabler et al., 1999
). Therefore, primers have been specifically designed to detect all VEGF isoforms for increased sensitivity. Primers were tested with cDNA obtained from luteal phase human endometrium, a known source of VEGF mRNA, and the identity of the PCR products was confirmed by sequence analysis (Krussel et al., 2000
).
Forward and reverse primers specific to the VEGF-R, KDR and flt-1, were also derived from the published cDNA sequences (de Vries et al., 1992; Charnock-Jones et al., 1994
). The sequences of the oligonucleotide primers for KDR were forward ACGCTGACA TGTACGGTCTAT, and reverse GCCAAGCTTGTACCATGTGAG (Mwgag Biotech). The sequences of the oligonucleotide primers for flt-1 were forward GTCACAGAAGAGGATGAAGGTGTCTA, and reverse CACAGTCCGGCACGTAGGTGATT (Mwgag Biotech). Primers have been tested with cDNA obtained from ovarian microvascular endothelium, a known source of VEGF-R mRNA, and the identity of the PCR products has been confirmed by sequence analysis (Ratcliffe et al., 1999
; Krussel et al., 2000
).
-actin was co-amplified with VEGF to provide a semi-quantitative internal control for RNA quantity and PCR efficiency.
-actin is commonly used as a standard when comparing samples under different hormonal conditions (LaPolt et al., 1990
; Tilly et al., 1992
; Mattioli et al., 2001
) as it is constitutively expressed (Soutar et al., 1997
). It has also been specifically used for human oviduct tissue and its expression is not affected by gonadotropins or sex hormones (Briton-Jones et al., 2001
). Forward and reverse primers specific to
-actin were also derived from the published primer sequences (Soutar et al., 1997
). The sequences of the
-actin primers were forward ATCGTGGGGCGCCCCAGGCAC, and reverse CTCCTTAATGTCACGCACGATTTC (Mwgag Biotech).
The RTPCR assay was validated. The amount of co-amplified product for VEGF or VEGF-R (experimental) and -actin (internal standard) primer sets was linear and parallel with increasing amounts of cDNA. The cycle number and primers concentrations were optimized in the exponentially increasing phase of detectable product. Thirty cycles of PCR were performed for each cDNA sample with 100 pmol/l VEGF or VEGF-R primer and 5 pmol/l
-actin primer added. In order to estimate within-assay variability, RNA from five oviduct tissues collected from women in various phases of an ovulatory cycle was combined and reverse transcribed to form a pool that was amplified as four replicates during the PCR. Within-assay variability typically ranged from <1 to 12%.
Twenty percent of each PCR was separated by gel electrophoresis on 2% agarose gel with 0.5 µg/ml ethidium bromide in Trisborate ethelenediaminetetraacetic acid (TBE) buffer. The separated PCR products were visualized under ultraviolet light. A video camera sent the UV-illuminated gel image to a computer, where the software package Gel Doc (Model GS-1000; Bio-Rad Laboratories, Hercules, CA) enabled an image of the gel to be recorded. The integrated optical density (IOD) was determined for each PCR product by the Molecular Analyst Software (Bio-Rad Laboratories). The IOD ratio between the PCR-amplified VEGF or VEGF-R product (297 base pairs for VEGF, 421 base pairs for KDR and 414 base pairs for flt-1) and its simultaneously amplified control -actin (543 base pairs) was obtained for each sample. The ratio, VEGF/
-actin ratio or VEGF-R/
-actin ratio, was normalized to the lowest value for subsequent analysis.
Hormone assays
Both serum LH and FSH were quantified by using a two-site sandwich immunoassay and direct chemiluminescence technology (Bayer, Tarrytown, NY) (Letellier et al., 1996). Serum E2 and progesterone were both quantified using a competitive immunoassay and direct chemiluminescence technology (Bayer) (Levesque et al., 1997
). The principles of the hormone assays as well as their interassay coefficients of variation were as previously described (Lam et al., 2003
).
Statistics
The results were analysed using the SPSS software. One-way analysis of variance was used to evaluate the differences in serum hormone concentrations in various menstrual stages. A paired sample t-test was used to examine the differences in serum hormone concentrations between the study group and the control group. The differences in the mRNA expression of VEGF and its receptors between the two groups were evaluated by the independent samples t-test. Statistical significance was accepted at P < 0.05.
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Results |
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Discussion |
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In our study, the observation of increased mRNA expression of VEGF and its receptor flt-1 in the hydrosalpinx supports the notion that VEGF may play an important role in hydrosalpinx fluid formation, possibly mediated via the flt-1 pathway by promoting permeability. The mechanisms of increased VEGF expression in the hydrosalpinx remain unclear. We speculated that up-regulation of VEGF expression by bacterial lipopolysaccharide (van Der Flier et al., 2000; Morozumi et al., 2001
; Nielsen et al., 2001
) or by paracrine control of other inflammatory cytokines such as interleukin-6 (Cohen et al., 1996
; Nakahara et al., 2003
) may play a role. However, the significance of hydrostatic pressure in regulating epithelial VEGF expression in the hydrosalpinx is unclear, as hydrostatic pressure will reduce the VEGF expression in the endothelial cells instead (Gan et al., 2000
; Conklin et al., 2002
).
VEGF binds to two tyrosine kinase receptors, flt-1 and KDR (Shibuya et al., 1990; Terman et al., 1991
). There is evidence that flt-1 and KDR may mediate different functions of VEGF. Our results suggest that flt-1 may be the main receptor responsible for the actions of VEGF in the hydrosalpinx. This postulation is supported by the previous finding that flt-1 instead of KDR was implicated in increased expression of serine proteases such as matrix metalloproteinases (Wang and Keiser, 1998
), a known effect of hydrosalpinx fluid (Jastrow et al., 2002
).
It is well established that the female reproductive system undergoes a number of programmed cyclical processes along the course of an ovulatory cycle. The cyclical modulation of mRNA expression of VEGF and its receptor flt-1, with the strongest expression in the periovulatory phase, has been demonstrated in healthy oviduct in our previous study (Lam et al., 2003). Because of the small sample size (n = 2 in each menstrual stage for each group), our current study was not powered to evaluate this effect of the ovulatory cycle. The boxplots showing the variation of the mRNA expression of VEGF and its receptors in different menstrual stages supported the similar pattern of cyclical modulation in healthy oviduct and possibly in hydrosalpinx as well. However, the effect of the ovulatory cycle on the expression of VEGF and its receptors in hydrosalpinx requires further evaluation with a larger sample size.
It is well established that VEGF, an important angiogenic factor, is up-regulated by hypoxia (Minchenko et al., 1994). Therefore, for the study on VEGF, it is very important to control the experimental conditions to minimize the confounding effects by hypoxia. In our study, fresh rather than cultured oviduct mucosal cells were collected, and the isolation procedures of all the samples were performed under the same conditions by only two investigators. Nevertheless, we recognize the limitation that our study has only examined the expression of VEGF at the gene transcriptional level in isolated oviduct mucosal cells. Future studies on VEGF expression at the protein level in intact epithelium of the hydrosalpinx, for example by western blot analysis, will be helpful to confirm the findings in-vivo.
Although VEGF may be an important mediator for hydrosalpinx fluid formation, inhibition of VEGF activities cannot prevent epithelial damage and occlusion of the oviduct after pelvic inflammatory disease. Therefore, the clinical applicability of VEGF inhibitors in preserving fertility potential after pelvic inflammatory disease is very limited. Nevertheless, whether VEGF inhibitors can reduce serum transudation and therefore hydrosalpinx fluid formation, especially in women with mild pelvic infection without tubal occlusion, requires further elucidation. If this potential treatment modality is found to be effective, it may improve the IVF outcomes for infertile women with hydrosalpinx.
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Submitted on May 27, 2003; resubmitted on June 23, 2003; accepted on July 22, 2003.