1 Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research/Leiden University Medical Center, Gorlaeus Laboratories, P.O.Box 9502, 2300 RA Leiden and 2 Division of Reproductive Medicine, Department of Gynaecology, Leiden University Medical Center, Leiden, The Netherlands
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Abstract |
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Key words: gonadotrophin ovarian stimulation/peri-implantation period/VEGF120 expression
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Introduction |
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The mechanism whereby ovarian stimulation leads to a low birthweight is still poorly understood. In order for the embryo to survive, a vital connection between the mother and the embryo in the form of a richly vascularized placenta is essential. Among several angiogenic factors, the vascular endothelial growth factor (VEGF) is considered to be a potent stimulator of angiogenesis, the formation of blood vessels from pre-existing blood vessels. VEGF is a heparin-binding dimeric glycoprotein which induces angiogenesis by stimulating endothelial cell proliferation and migration (Gospodarowicz et al., 1989; Park et al., 1993
) and enhances vascular permeability (Keck et al., 1989
). In rodents, there are four splice variants of this angiogenic factor: VEGF115, VEGF120, VEGF164 and VEGF188 (Breier et al., 1992
; Sugihara et al., 1998
) and each is shorter by one amino acid than the human VEGF (Ferrara and Davis-Smyth, 1997
). These different VEGF isoforms bind to two tyrosine kinase receptors (flt-1 and KDR/flk-1), which are expressed almost exclusively in endothelial cells (Quinn et al., 1993
; Fong et al., 1996
) and are present during the peri-implantation period in decidual cells (Jakeman et al., 1993
; Chakraborty et al., 1995
).
VEGF mRNA expression has been shown to correlate spatially and temporarily with changes in angiogenesis and vascular reactivity at implantation sites, in decidua and placental tissues (Jakeman et al., 1993; Chakraborty et al., 1995
). So far, no studies have been done on the effects of excessive gonadotrophin treatment on angiogenic factors such as VEGF. Therefore, the aim of our research is to study the effects of gonadotrophin stimulation on embryonic development and VEGF expression during the peri-implantation period.
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Materials and methods |
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Riboprobe
The antisense and sense probes were transcribed from a mouse 451 bp cDNA fragment which encodes for the entire sequence of VEGF120 (courtesy of G.Breier, Max-Planck Institute for Physiological and Clinical Research, Germany). In-vitro transcription for the antisense and sense strands using [35S]UTP (ICN, Irvine, CA, USA) were generated with T3 and T7 RNA polymerase respectively using a standard protocol (Boehringer, Mannheim, Germany).
In-situ hybridization
Cryostat sections (20 µm) of the entire implantation sites (area between two intersites) were cut at 20°C whereby every other section was collected, thaw-mounted on 1% (w/v) poly-L-lysine-coated slides and stored at 80°C until hybridization. Sections were fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS) (w/v, pH 7.2) for 60 min at room temperature. They were then washed twice in PBS (5 min each), permeabilized by 0.01 mol/l HCl for 10 min at room temperature, rinsed briefly with diethyl pyrocarbonate (DEPC)-treated water, acetylated with 0.25% (v/v) acetic anhydride in 0.1 mol/l triethanolamine (pH 8.0), washed with saline sodium citrate solution (SSC: 0.30 mol/l NaCl and 0.03 mol/l sodium citrate, pH 7.0) for 10 min, dehydrated in an increasing graded ethanol series and then air-dried. The hybridization mix consisted of 50% (w/v) formamide, 10% (w/v) dextran sulphate, SSC, Denhardt's solution, 10 mmol/l dithiothreitol, 0.01% yeast tRNA (w/v) and 0.01% hsssDNA (w/v). The riboprobes (2.3x106 d.p.m./ml) were added to the hybridization mix and 100 µl of the mix was pipetted on each slide and covered with a 24x50 mm microscopic coverslip. Subsequently, the slides were stacked and sealed in slide boxes, placed inside a moist chamber and hybridized overnight at 53°C. The following morning the cover slips were removed and the slides were washed three times in SSC at room temperature each time for 10 min and, thereafter, the slides were treated with 0.002% RNase A (w/v, in 0.5 mol/l NaCl, pH 7.5) at 37°C for 30 min, dipped shortly in SSC at 37°C and washed three times at 60°C in SSC/50% formamide (v/v; 15 min each) and finally in SSC (5 min). The slides were then dehydrated in a graded alcohol series, air dried and exposed to X-Omat AR film for 23 days. After the development of the films the slides were dipped in NTB3 photoemulsion (Kodak, Rochester, NY, USA) developed after 29 weeks and counterstained with methyl green pyronin.
Densitometric and area size analysis
The autoradiograms were quantified using an Olympus image analysis system with the appropriate software (Paes Nederland B.V., Zoeterwoude, The Netherlands). Four to six comparable sections (with a distance of 20 µm for every section) for each implantation site were assessed and compared. Only sections containing the embryos were measured. The VEGF expression in the whole uterus cross-sections including that of the embryo was measured and expressed as integrated optical density. Area size was measured by outlining the periphery of the entire cross-section of the embryo implantation site. Only qualitative analyses were done with the autoradiograms on ED4 since not all of the stimulated mice showed implantation sites.
Statistics
The integrated optical density values and area size in mm2 of four to six sections for each embryo were pooled and were analysed for the different treatment groups. VEGF expression integrated optical density, area size, birthweight and gestational length of the two groups were compared using the independent Student's t-test while the litter size was compared using the Wilcoxon signed rank test. Significance was accepted at P < 0.05.
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Results |
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Expression of VEGF120
On ED0 and ED3 VEGF120 mRNA expression was observed prominently at the epithelial lining of the lumen. There was no significant difference in the VEGF120 mRNA levels between the stimulated and spontaneously ovulating group at these time points (ED0: control 1.1 ± 0.1 versus stimulated 1.7 ± 0.4; ED3: control 1.2 ± 0.3 versus stimulated 0.9 ± 0.2). ED4 control group showed distinct labelling in the primary decidual area of the uterus while a lower signal was observed in the gonadotrophin-treated group. However, the topographical distribution expression pattern of VEGF120 was the same as in the controls. No specific signal was detected using the sense probe.
There was a significant difference in the VEGF120 expression in the embryo implantation sites on ED5 and ED6 (P < 0.05; Figure 1). The gonadotrophin-treated group showed lower VEGF120 mRNA levels than the control group. Labelling was present in both the mesometrial and the antimesometrial areas. There was a low expression in the embryo but no hormone treatment effect was observed (Figure 2
).
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Area size of the implantation site
Macroscopically, the implantation sites in the uterus of the hormone-treated group appeared to be smaller than those in the control group during ED5 and ED6. Measurement of the area of the sections containing the embryos showed that, indeed, gonadotrophin treatment exerted a significant reduction in the area size during these periods (P < 0.05; Figure 3).
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Discussion |
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The present data raise the question whether the reduced VEGF120 expression and delay in implantation are interrelated or separate independent events. VEGF120 expression is probably associated with the implantation process since blastocyst apposition to the uterine epithelium at the initial stage of implantation coincides with increased vascular permeability (Enders and Schlafke, 1967) and a dramatic growth of blood vessels in the maternal endometrium (Moore, 1988
). The fact that VEGF120 expression on ED4 is not increased in the stimulated uterus with no implantation sites indicates that the attachment of the blastocyst to the endometrium is necessary to trigger expression of VEGF120. This, in turn, stimulates vascular permeability as attested by the penetration of the blue dye at the implantation sites. It could therefore be inferred that the low VEGF120 expression in the stimulated implantation sites is a consequence of delayed implantation. On the other hand, it cannot be excluded that the delay in implantation is a result of the decreased VEGF120 expression since it has been reported that the number of implantation sites in rats at day 5 of pregnancy was significantly reduced after VEGF antibody injection (Rabbani and Rogers, 2001
).
Successful implantation and pregnancy is dependent on a synchronized interaction between the preimplantation embryo and maternal endometrium. In accordance with other studies (Miller and Armstrong, 1981a; Fossum et al., 1989
; Ertzeid et al., 1993
; Ertzeid and Storeng, 2001
), our results show that this synchronization process appeared to have been altered by the exogenous gonadotrophins injected since embryo implantation was delayed in the stimulated animals. However, it is not known which factors are specifically involved. There are data which show that uterine receptivity factors and/or signals coming from the developing oocyte/embryos (Ertzeid et al., 1993
; Van der Auwera and D'Hooghe, 2001
) and oviductal factors (Van der Auwera et al., 1999
) are involved. Additionally, it may be deduced that the delayed implantation was probably the cause of a longer gestation period in the hormone-treated mice.
The gonadotrophins may have exerted their effects directly and/or indirectly. A direct effect of HCG may be plausible (Fanchin et al., 2001; Rao, 2001
). On the other hand, indirect effects of the gonadotrophins via estrogen and progesterone cannot be ruled out. The establishment of a receptive uterus for supporting embryo implantation and development is primarily dependent on the coordinated functions of these two ovarian hormones (Psychoyos, 1973
). It is also known that pharmacological doses of estrogen change endometrial histology and inhibit implantation (Pellicer et al., 1996
; Simon et al., 1998
; Valbuena et al., 1999
) and as such may have contributed to delayed implantation. Moreover, in-vitro studies have shown that high estradiol levels exert direct toxic effects on the embryo (Valbuena et al., 2001
), which may contribute to a higher embryo blockade and a slower embryo growth. While a report showed no change on estrogen levels (Ertzeid and Storeng, 1992
), the majority of studies on ovulation induction by gonadotrophins reported high serum levels of estradiol (Miller and Armstrong, 1981b
; Yun et al., 1987
) and progesterone (Yun et al., 1987
). Estrogen stimulates VEGF expression in the uterus (Cullinan-Bove and Koos, 1993
; Shifren et al., 1996
; Hyder et al., 2000
), and therefore a higher expression is expected after gonadotrophin treatment. This was not the case here: VEGF120 expression was not altered on ED0 and ED3 and was reduced on ED5 and ED6. The precise role of progesterone remains unclear although an increase in progesterone levels (Ertzeid and Storeng, 1992
) and a high luteal estradiol:progesterone ratio (Safro et al., 1990
) have been shown to exert adverse effects on the implantation embryo.
The observed time-dependent difference in the induction of VEGF120 expression might have occurred as a result of processes taking place somewhere between the time of gonadotrophin injections and implantation. VEGF120 expression and secretion by the uterus can be induced by both FSH and LH/HCG receptor-activated pathways (Koos, 1995). VEGF120 expression was not affected by stimulation a few hours after fertilization and 1 day before the onset of implantation but gonadotrophin injection may have already had an effect on the uterus and/or oocyte before fertilization. This view fits well with the important role of a well-timed and characteristic pattern of the pre-ovulatory LH surge necessary to initiate timely processes for the production of healthy oocytes (de Koning et al., 2001
). In view of this, it could be deduced that gonadotrophin treatment may have exerted an impairment effect as early as oocyte development.
We cannot offer a definitive explanation as to why the embryo implantation sites in gonadotrophin-treated mice are smaller than those in the controls. One probable explanation is a delayed growth as a consequence of the delayed implantation. In addition, the increased litter size may partly account for this effect since our present data and those of others (Romero et al., 1992) have shown an inverse correlation between birthweight and litter size. An increased litter size causes overcrowding effects leading to nutritional defects which, in turn, can hamper the growth of the embryo (Evans et al., 1981
).
In summary, the presence of intense VEGF expression in the endometrium and decidual cells during the peri-implantation period, as seen here and in other studies (Jakeman et al., 1993; Shweiki et al., 1993
; Chakraborty et al., 1995
), and the activation of the gene as early as the blastula stage (Krussel et al., 2001
) indicates an important role for VEGF in normal development of the embryo and supporting tissues. Furthermore, our data show negative effects of exogenous gonadotrophins used in ovulation induction procedures and necessitate in-depth research to unravel the complexities surrounding their adverse effects.
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Acknowledgements |
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Notes |
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References |
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Submitted on November 23, 2001; accepted on January 24, 2002.