Gonadotrophin stimulation reduces VEGF120 expression in the mouse uterus during the peri-implantation period

R.M. Sibug1,3, F.M. Helmerhorst2, A.M.I. Tijssen1, E.R. de Kloet1 and J. de Koning1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Ovarian stimulation by gonadotrophin treatment exerts negative effects on implantation and embryonic development. We investigated whether gonadotrophin treatment affects VEGF120 mRNA expression during the peri-implantation period. METHODS: Two groups of adult female CD1 mice were used: the hormone-treated group was injected i.p. with urinary human FSH (5 IU in 0.1 ml saline) and urinary HCG (5 IU in 0.1 ml saline). Spontaneously ovulating mice served as controls and received saline injections. The pregnant mice were killed on embryonic development (ED) days 0, 3, 4, 5 and 6 (day of vaginal plug detection is considered as ED0). The uteri with the implanted embryos were processed for in-situ hybridization for VEGF120. A separate group of control and hormone-treated pregnant mice were allowed to give birth. Litter size, birthweight and length of gestational period were noted. RESULTS: Gonadotrophin treatment decreased VEGF120 mRNA levels, delayed implantation, reduced the size of the embryo implantation site on ED5 and ED6 and prolonged the gestational period. CONCLUSIONS: Gonadotrophin treatment reduces VEGF120 expression which may have serious consequences for normal embryonic development. The present data cannot establish whether this effect is a cause or consequence of delayed implantation.

Key words: gonadotrophin ovarian stimulation/peri-implantation period/VEGF120 expression


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Ovarian stimulation with gonadotrophins is routinely used for ovulation induction in human IVF programmes and in the generation of transgenic animals. Numerous studies have shown that exogenous gonadotrophin treatment arrested meiotic development in rat oocytes (Tain et al., 2000Go), decreased the rate of fertilization and increased the incidence of oocyte and embryo degeneration in rodents (Walton et al., 1983Go; Edgar et al., 1987Go) and humans (Lopata, 1983Go). Furthermore, in rodents, increases in the frequency of chromosomal abnormalities (Maudlin and Fraser, 1977Go; Elbling and Colot, 1985Go; Luckett and Mukherjee, 1986Go) and increased pre- and post-implantation mortality (Beaumont and Smith, 1975Go; Ertzeid and Storeng, 1992Go) have been reported. In addition, delayed and impaired implantation (Miller and Armstrong, 1981aGo; Fossum et al,. 1989Go; Ertzeid et al., 1993Go; Ertzeid and Storeng, 2001)Go, retarded fetal growth (Walton et al., 1983Go; Van der Auwera and D'Hooghe, 2001Go) and prolonged gestation periods (Ertzeid et al., 1993Go) have been reported. Additionally, in humans (Doyle et al., 1992Go; Koudstaal et al.2000aGo,bGo) and rodents (Evans et al., 1981Go; Ertzeid and Storeng, 1992Go; Ertzeid et al., 1993Go), ovarian stimulation has been associated with a low fetal/birthweight. Epidemiological studies have shown an association between children with a low birthweight and an increased risk to develop several metabolic, cardiovascular and behavioural pathologies in later life (Barker, 1995Go).

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., 1989Go; Park et al., 1993Go) and enhances vascular permeability (Keck et al., 1989Go). In rodents, there are four splice variants of this angiogenic factor: VEGF115, VEGF120, VEGF164 and VEGF188 (Breier et al., 1992Go; Sugihara et al., 1998Go) and each is shorter by one amino acid than the human VEGF (Ferrara and Davis-Smyth, 1997Go). 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., 1993Go; Fong et al., 1996Go) and are present during the peri-implantation period in decidual cells (Jakeman et al., 1993Go; Chakraborty et al., 1995Go).

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., 1993Go; Chakraborty et al., 1995Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals and experimental procedure
Adult female CD1 mice (8–10 weeks; Charles River, Sulzfeld, Germany) were housed five per cage upon arrival and allowed to acclimatize for 1 week. They had free access to food and water with a 12:12 dark:light cycle (lights on at 08:00). Mice were used irrespective of the day of the cycle and injected i.p. with urinary human (h)FSH [Metrodin; 5 IU in 0.1 ml saline; Serono, Coinsins, Switzerland] at 13:00 and 48 h later with urinary HCG (Pregnyl; 5 IU in 0.1 ml saline; Organon, Oss, The Netherlands). Control animals received saline injections. For fertilization, males (one per two females) were present throughout the entire hormone treatment period. The females were examined every morning until the day after the HCG injection and successful mating was confirmed by detection of the vaginal plug. The mice that displayed a vaginal plug before the injection of HCG were discarded from the experiment. The day of detection of the plug was considered as embryonic development day 0 (ED0). At the time of termination (ED0, ED3 to ED6), the pregnant mice were injected with Chicago blue dye (Sigma–Aldrich, Steinheim, Germany) through the jugular vein under isofluorane anaesthesia to visualize the implantation sites (Psychoyos, 1973Go). Five to 10 min after injection of the dye, the whole uterus with the embryos was dissected out and frozen in dry-ice ethanol-cooled isopentane. The frozen materials were stored at –80°C till processing and one or two embryo implantation sites per pregnant mouse were used. A separate group of control and hormone-treated pregnant mice were left behind and allowed to give birth. Litter size, birthweight measured as nest weight/litter size, and length of gestation were noted at the time of birth. All animal experiments were in accordance with the governmental guidelines for care and use of laboratory animals and approved by the Animal Care Committee of the University of Leiden.

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 2–3 days. After the development of the films the slides were dipped in NTB3 photoemulsion (Kodak, Rochester, NY, USA) developed after 2–9 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Implantation sites
Implantation of the embryos was delayed in stimulated mice. The implantation sites, as indicated by the blue bands, were distinct and always present along the length of the uterus of all the control mice starting ED4. In the hormone-treated mice these blue bands were not clearly detectable on ED4 but from ED5 onwards distinct implantation sites were clearly seen. All examined implantation sites with blue bands from ED4 to ED6 contained implanted embryos.

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 1Go). 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 2Go).



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Figure 1. Expression of VEGF120 in the implantation site on embryonic day 5 (ED5) and embryonic day 6 (ED6). Mice treated with urinary human FSH and urinary HCG (stim; n = 12 (ED5); 8 (ED6)) showed lower levels of VEGF120 mRNA than the spontaneously ovulating mice [(control; n = 9 (ED5); 7 (ED6)].*P < 0.05, significantly different from the control.

 


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Figure 2. Autoradiograms of the embryo implantation on embryonic day 5. The control group (A) showed a higher intensity of labellingat the mesometrial pole than the gonadotrophin-treated group (B). Note also that uterine epithelium along the antimesometrial chamber isstill intact in the hormone-treated uterus while it is already degraded in the control uterus. Scale bar = 20 µm. Exposure time: 15 days.e = embryo; u = uterine epithelium; mc = mesometrial area.

 
Time course of VEGF mRNA expression
There were more cells expressing VEGF120 with increasing intensity as development progresses. On ED4, the first cells which showed VEGF120 expression were the decidual cells immediately surrounding the mesometrial and antimesometrial chamber. The distribution pattern of expression spread out extensively in the outlying decidual areas at later stages of development.

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 3Go).



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Figure 3. Area size of the implantation site on embryonic day 5 (ED5) and embryonic day 6 (ED6). Mice treated with urinary FSH and urinary HCG [stim; n = 12 (ED5); 8 (ED6) showed a smaller area size than the spontaneously ovulating mice (control; n = 9 (ED5); 7 (ED6)]. *P < 0.05, significantly different from the control.

 
Litter size, birthweight and length of gestational period
The length of gestation in gonadotrophin-treated mice was significantly longer by 1 day in comparison with the control mice (P < 0.05; Table IGo). There was no significant difference in the birthweight and litter size between the two groups. However, in the latter parameter it should be noted that the distribution range between the two groups diverged greatly. In the control group one litter had <10 pups and the other eight litters had 10–20 pups while in the gonadotrophin-treated group two litters had <10 pups, two litters with 10–20 pups and three litters with >20 pups. Regardless of treatment, there is a negative correlation between litter size and birthweight (P < 0.001; Figure 4Go).


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Table I. Effects of urinary human FSH and urinary HCG on litter size, birthweight and gestational length
 


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Figure 4. Negative correlation between litter size and birthweight in spontaneously ovulated (control) and urinary human FSHand urinary HCG-treated (stim) mice. Y = 2.038 – 0.033*X;R > 2 = 0.799; P < 0.001.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Our results show for the first time that gonadotrophins for ovarian stimulation and ovulation induction, as applied in human fertilization and transgenic animal programmes, decreased VEGF120 expression during the peri-implantation period. Furthermore, embryo implantation was delayed, the area size of the embryo implantation site was reduced and the gestational period was prolonged. These adverse effects of stimulation may have occurred as early as oocyte development.

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, 1967Go) and a dramatic growth of blood vessels in the maternal endometrium (Moore, 1988Go). 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, 2001Go).

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, 1981aGo; Fossum et al., 1989Go; Ertzeid et al., 1993Go; Ertzeid and Storeng, 2001Go), 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., 1993Go; Van der Auwera and D'Hooghe, 2001Go) and oviductal factors (Van der Auwera et al., 1999Go) 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., 2001Go; Rao, 2001Go). 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, 1973Go). It is also known that pharmacological doses of estrogen change endometrial histology and inhibit implantation (Pellicer et al., 1996Go; Simon et al., 1998Go; Valbuena et al., 1999Go) 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., 2001Go), 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, 1992Go), the majority of studies on ovulation induction by gonadotrophins reported high serum levels of estradiol (Miller and Armstrong, 1981bGo; Yun et al., 1987Go) and progesterone (Yun et al., 1987Go). Estrogen stimulates VEGF expression in the uterus (Cullinan-Bove and Koos, 1993Go; Shifren et al., 1996Go; Hyder et al., 2000Go), 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, 1992Go) and a high luteal estradiol:progesterone ratio (Safro et al., 1990Go) 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, 1995Go). 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., 2001Go). 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., 1992Go) 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., 1981Go).

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., 1993Go; Shweiki et al., 1993Go; Chakraborty et al., 1995Go), and the activation of the gene as early as the blastula stage (Krussel et al., 2001Go) 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Prof. A.C.Gittenberg and Dr M.van de Ruiter (Department of Anatomy, LUMC, Leiden) for helping us in securing the plasmid containing the mouse VEGF120.


    Notes
 
3 To whom correspondence should be addressed at: Division of Medical Pharmacology, LACDR/LUMC, Wassenaarseweg 72, 2333 AL Leiden University, The Netherlands. E-mail: r.sibug{at}lacdr.leidenuniv.nl Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Barker, D.J. (1995) Intrauterine programming of adult disease. Mol. Med. Today, 1, 418–423.[ISI][Medline]

Beaumont, H.M. and Smith, A.F. (1975) Embryonic mortality during the pre- and post-implantation periods of pregnancy in mature mice after superovulation. J. Reprod. Fertil., 45, 437–448.[Abstract]

Breier, G., Albrecht, U., Sterrer, S. and Risau,W. (1992) Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development, 114, 521–532.[Abstract]

Chakraborty, I., Das, S.K. and Dey, S.K. (1995) Differential expression of vascular endothelial growth factor and its receptor mRNAs in the mouse uterus around the time of implantation. J. Endocrinol., 147, 339–352.[Abstract]

Cullinan-Bove, K. and Koos, R.D. (1993) Vascular endothelial growth factor/vascular permeability factor expression in the rat uterus: rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth. Endocrinology, 133, 829–837.[Abstract]

de Koning, J., Lambalk, C.B., Helmerhorst, F.M. and Helder, M.N. (2001) Is GnRH self-priming an obligatory feature of the reproductive cycle? Hum. Reprod., 16, 209–214.[Abstract/Free Full Text]

Doyle, P., Beral, V. and Maconochie, N. (1992) Preterm delivery, low birthweight and small-for-gestational-age in liveborn singleton babies resulting from in-vitro fertilization. Hum. Reprod., 7, 425–428.[Abstract]

Edgar, D.H., Whalley, K.M. and Mills, J.A. (1987) Effects of high-dose and multiple-dose gonadotropin stimulation on mouse oocyte quality as assessed by preimplantation development following in vitro fertilization. J. In Vitro Fertil. Embryo Transfer, 4, 273–276.[ISI][Medline]

Elbling, L. and Colot, M. (1985) Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice. Mutat. Res., 147, 189–195.[ISI][Medline]

Enders, A.C. and Schlafke, S. (1967) A morphological analysis of the early implantation stages in the rat. Am. J. Anat., 120, 185–226.[ISI]

Ertzeid, G. and Storeng, R. (1992) Adverse effects of gonadotrophin treatment on pre- and postimplantation development in mice. J. Reprod. Fertil., 96, 649–655.[Abstract]

Ertzeid, G. and Storeng, R. (2001) The impact of ovarian stimulation on implantation and fetal development in mice. Hum. Reprod., 16, 221–225.[Abstract/Free Full Text]

Ertzeid, G., Storeng, R. and Lyberg, T. (1993) Treatment with gonadotropins impaired implantation and fetal development in mice. J. Assist. Reprod. Genet., 10, 286–291.[ISI][Medline]

Evans, M.I., Schulman, D., Golden, L. and Mukherjee, A.B. (1981) Superovulation-induced intrauterine growth retardation in mice. Am. J. Obstet. Gynecol., 141, 433–435.[ISI][Medline]

Fanchin, R., Peltier, E., Frydman, R. and de Ziegler, D. (2001) Human chorionic gonadotropin: does it affect human endometrial morphology in vivo? Semin. Reprod. Med., 19, 31–35.[ISI][Medline]

Ferrara, N. and Davis-Smyth, T. (1997) The biology of vascular endothelial growth factor. Endocr. Rev., 18, 4–25.[Abstract/Free Full Text]

Fong, G.H., Klingensmith, J., Wood, C.R., Rossant, J. and Breitman, M.L. (1996) Regulation of flt-1 expression during mouse embryogenesis suggests a role in the establishment of vascular endothelium. Dev. Dyn., 207, 1–10.[ISI][Medline]

Fossum, G.T., Davidson, A. and Paulson, R.J. (1989) Ovarian hyperstimulation inhibits embryo implantation in the mouse. J. In Vitro Fertil. Embryo Transfer, 6, 7–10.[ISI][Medline]

Gospodarowicz, D., Abraham, J.A. and Schilling, J. (1989) Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc. Natl Acad. Sci. USA, 86, 7311–7315.[Abstract]

Hyder, S.M., Huang, J.C., Nawaz, Z., Boettger-Tong, H., Makela, S., Chiappetta, C. and Stancel, G.M. (2000) Regulation of vascular endothelial growth factor expression by estrogens and progestins. Environ. Health. Perspect.108 (Suppl. 5), 785–790.

Jakeman, L.B., Armanini, M., Phillips, H.S. and Ferrara, N. (1993) Developmental expression of binding sites and messenger ribonucleic acid for vascular endothelial growth factor suggests a role for this protein in vasculogenesis and angiogenesis. Endocrinology, 133, 848–859.[Abstract]

Keck, P.J., Hauser, S.D., Krivi, G., Sanzo, K., Warren, T., Feder, J. and Connolly, D.T. (1989) Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science, 246, 1309–1312.[ISI][Medline]

Koos, R.D. (1995) Increased expression of vascular endothelial growth/permeability factor in the rat ovary following an ovulatory gonadotropin stimulus: potential roles in follicle rupture. Biol. Reprod., 52, 1426–1435.[Abstract]

Koudstaal. J., Braat, D.F.D., Bruinse, H.W., Naaktgeboren, N., Vermeiden, J.P. and Visser, G.H. (2000a) Obstetric outcome of singleton pregnancies after IVF: a matched control study in four Dutch university hospitals. Hum. Reprod., 15, 1819–1825.[Abstract/Free Full Text]

Koudstaal, J., Bruinse, H.W., Helmerhorst, F.M., Vermeiden, J.P., Willemsen, W.N. and Visser, G.H. (2000b) Obstetric outcome of twin pregnancies after in-vitro fertilization: a matched control study in four Dutch university hospitals. Hum. Reprod., 15, 935–940.[Abstract/Free Full Text]

Krussel, J.S., Behr, B., Milki, A.A. Hirchenhain, J., Wen, Y., Bielfeld, P. and Polan, M. (2001) Vascular endothelial growth factor (VEGF) mRNA splice variants are differentially expressed in human blastocysts. Mol. Hum. Reprod., 7, 57–63.[Abstract/Free Full Text]

Lopata, A. (1983) Concepts in human in vitro fertilization and embryo transfer. Fertil. Steril., 40, 289–301.[ISI][Medline]

Luckett, D.C. and Mukherjee, A.B. (1986) Embryonic characteristics in superovulated mouse strains. Comparative analyses of the incidence of chromosomal aberrations, morphological malformations, and mortality of embryos from two strains of superovulated mice. J. Hered., 77, 39–42.[ISI][Medline]

Maudlin, I. and Fraser, L.R. (1977) The effect of PMSG dose on the incidence of chromosomal anomalies in mouse embryos fertilized in vitro. J. Reprod. Fertil., 50, 275–280.[Abstract]

Miller, B.G. and Armstrong, D.T. (1981a) Superovulatory doses of pregnant mare serum gonadotropin cause delayed implantation and infertility in immature rats. Biol. Reprod., 25, 253–260.[ISI][Medline]

Miller, B.G. and Armstrong, D.T. (1981b) Effects of a superovulatory dose of pregnant mare serum gonadotropin on ovarian function, serum estradiol, and progesterone levels and early embryo development in immature rats. Biol. Reprod., 25, 261–271.[ISI][Medline]

Moore, M.W. (1988) The Developing Human. W.B.Saunders, Philadelphia.

Park, J.E., Keller, G.A. and Ferrara, N. (1993) The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol. Biol. Cell, 4, 1317–1326.[Abstract]

Pellicer, A., Valbueña, D., Cano, F., Remohí, J. and Simón, C. (1996) Lower implantation rates in high responders: evidence for an altered endocrine milieu during the preimplantation period. Fertil. Steril., 65, 1190–1195.[ISI][Medline]

Psychoyos, A. (1973) Hormonal control of ovoimplantation. Vitam. Horm., 31, 201–256.[Medline]

Quinn, T.P., Peters, K.G., De Vries, C., Ferrara, N. and Williams, L.T. (1993) Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Proc. Natl Acad. Sci. USA, 90, 7533–7537.[Abstract/Free Full Text]

Rabbani, M.M. and Rogers, P.A. (2001) Role of vascular endothelial growth factor in endometrial vascular events before implantation in rats. Reproduction, 122, 85–90.[Abstract/Free Full Text]

Rao, C.V. (2001) An overview of the past, present, and future of nongonadal LH/hCG actions in reproductive biology and medicine. Semin. Reprod. Med., 19, 7–17.[ISI][Medline]

Romero, A., Villamayor, F., Grau, M.T., Sacristan, A. and Ortiz, J.A. (1992) Relationship between fetal weight and litter size in rats: application to reproductive toxicology studies. Reprod. Toxicol., 6, 453–456.[ISI][Medline]

Safro, E., O'Neill, C. and Saunders, D.M. (1990) Elevated luteal phase estradiol:progesterone ratio in mice causes implantation failure by creating a uterine environment that suppresses embryonic metabolism. Fertil. Steril., 56, 1150–1153.

Shifren, J.L., Tseng, J.F., Zaloudek, C.J., Ryan, I.P., Meng, Y.G., Ferrara, N., Jaffe, R.B. and Taylor, R.N. (1996) Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J. Clin. Endocrinol. Metab, 81, 3112–3118.[Abstract]

Shweiki, D., Itin, A., Neufeld, G., Gitay-Goren, H. and Keshet, E. (1993) Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis. J. Clin. Invest., 91, 2235–2243.[ISI][Medline]

Simón, C., Garcia Velasco, J.J., Valbueña, D., Peinado, J.A., Moreno, C., Remohí, J. and Pellicer, A. (1998) Increasing uterine receptivity by decreasing estradiol levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen. Fertil. Steril., 70, 234–239.[ISI][Medline]

Sugihara, T., Wadhwa, R., Kaul, S.C.and Mitsui, Y. (1998) A novel alternatively spliced form of murine vascular endothelial growth factor, VEGF 115. J. Biol. Chem., 273, 3033–3038.[Abstract/Free Full Text]

Tain, C.F., Goh, V.H. and Ng, S.C. (2000) Effects of hyperstimulation with gonadotrophins and age of females on oocytes and their metaphase II status in rats. Mol. Reprod. Dev., 55, 104–108.[ISI][Medline]

Valbueña, D., Jasper, M., Remohí, J. Pellicer, A. and Simón, C. (1999) Ovarian stimulation and endometrial receptivity. Hum. Reprod., 14 (Suppl. 2), 107–111.[Medline]

Valbueña, D., Martin, J., de Pablo, J.L., Remohí, J., Pellicer, A. and Simón, C. (2001) Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo. Fertil. Steril., 76, 962–968.[ISI][Medline]

Van der Auwera, I. and D'Hooghe, T. (2001) Superovulation of female mice delays embryonic and fetal development. Hum. Reprod., 16, 1237–1243.[Abstract/Free Full Text]

Van der Auwera, I., Pijnenborg, R. and Koninckx, P.R. (1999) The influence of in-vitro culture versus stimulated and untreated oviductal environment on mouse embryo development and implantation. Hum. Reprod., 14, 2570–2574.[Abstract/Free Full Text]

Walton, E.A., Evans, G. and Armstrong, D.T. (1983) Ovulation response and fertilization failure in immature rats induced to superovulate. J. Reprod. Fertil., 67, 91–96.[Abstract]

Yun, Y.W., Yuen, B.H. and Moon, Y.S. (1987) Effects of superovulatory doses of pregnant mare serum gonadotropin on oocyte quality and ovulatory and steroid hormone responses in rats. Gamete Res., 16, 109–120.[ISI][Medline]

Submitted on November 23, 2001; accepted on January 24, 2002.