Human Reproduction, Vol. 16, No. 6, 1237-1243,
June 2001
© 2001 European Society of Human Reproduction and Embryology
Superovulation of female mice delays embryonic and fetal development
Leuven University Fertility Center, University Hospital Gasthuisberg, Catholic University Leuven, Leuven, Belgium
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Abstract |
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Mouse and human embryos, cultured in vitro, undergo a delay in development compared with those grown in vivo. This delay can be caused by suboptimal culture conditions, but possible influences of ovarian stimulation cannot be excluded. The objective of this study was to test the hypothesis that both in vitro and in vivo, preimplantation embryonic development and postimplantation fetal development are impaired in superovulated female mice when compared with naturally cycling controls. A delay in in-vitro blastocyst hatching and in-vivo blastocyst formation (P < 0.03 and P < 0.0001 respectively) and a 40% fetal growth retardation (P < 0.0001) were observed after superovulation in comparison with naturally cycling controls. After transfer to non-stimulated foster mothers, blastocysts from stimulated females had a lower implantation rate (P < 0.005), and developed into fewer living fetuses (P < 0.02), more resorption sites (P < 0.02) and had more pronounced growth retardation (P < 0.0001) when compared with blastocysts from naturally cycling controls. In conclusion, superovulation in the mouse causes a delayed embryonic development in vitro and in vivo, an increased abnormal blastocyst formation, a pronounced fetal growth retardation, and an increased number of resorption sites. If this observation in mice can be extrapolated to humans, it may offer an explanation for the delay in embryonic development and the low birth weight observed after IVF.
Key words: blastocysts/fetal growth retardation/implantation/ovarian stimulation/weight
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Mouse and human embryos, when cultured in vitro, undergo a delay in development compared with those grown in vivo. This delay can be caused by suboptimal culture conditions, but possible influences of ovarian stimulation cannot be excluded. Some 20 years ago, an embryo loss of 44% before implantation in mice that had been superovulated was described (Beaumont and Smith, 1975
). Others (Miller and Armstrong, 1981a,b, 1982) described an adverse effect of hormonal treatment on the fertility of superovulated rats and reported a failed or delayed implantation that could be restored by ovariectomy. These authors stated that embryo loss following superovulation was due to excessive oestrogenic secretion after ovulation. In addition, early embryo loss due to superovulation was described (Walton and Armstrong, 1981) that could be rescued by an injection of goat antiserum against pregnant mare serum gonadotrophins (PMSG). In order to determine if implantation failure was due to abnormalities in the blastocysts or the endometrium, decidualization studies and embryo transfers to pseudopregnant recipients were performed (Walton et al., 1982; Walton and Armstrong, 1983). These authors concluded that the uterus of a large proportion of superovulated animals was unable to undergo decidualization in time, whereas embryo transfers to pseudopregnant females resulted in normally developing fetuses, which indicated that hormonally treated oocytes themselves were not affected.
When compared with blastocysts derived from naturally cycling mice, blastocysts that developed in vivo in superovulated mice have fewer microvilli on their surface (Champlin et al., 1987), a reduced [35S]-methionine uptake (Wetzels et al., 1995), and a lower cell number and mitotic index (Elmazar et al., 1989). A reduced cell number and a two-fold decrease in viability post-transfer of embryos from gonadotrophin-stimulated hamster females was also observed (McKirnan and Bavister, 1998). Furthermore, it has been reported (Ertzeid and Storeng, 1992; Ertzeid et al., 1993) that the proportion of abnormal preimplantation embryos increases after superovulation, and that blastocysts have a smaller trophoblastic outgrowth in vitro. In these studies (Ertzeid and Storeng, 1992; Ertzeid et al., 1993), the postimplantation mortality in superovulated mice was increased, while the live fetuses had a reduced fetal weight and a developmental retardation. The authors concluded that the adverse effects of superovulation are probably related to changes in the maternal milieu of the oviduct and/or uterus. However, this study was biased by differences in number of implantation sites when compared with the naturally cycling controls, which can be explained by the high numbers of oocytes after superovulation. Therefore, nutritional deficiency of the fetuses of superovulated mice (Evans et al., 1981; Romero et al., 1992) might be the cause of the observed fetal growth retardation, while possible negative effects on endometrium receptivity (Walton et al., 1982; Walton and Armstrong, 1983) could have influenced implantation and outgrowth. Furthermore, it cannot be excluded that oocyte and embryo quality are also affected by hormonal stimulation. In a previous study (Van der Auwera et al., 1999) an impaired implantation and a lower number of living fetuses has been described after longer exposure of murine embryos to the stimulated oviductal environment before transfer to pseudopregnant, non-stimulated foster mothers. At that time however, no fetal weights were evaluated, and embryos from stimulated females were not compared with embryos of naturally cycling controls.
Since embryonic and fetal growth retardation have been described after superovulation in mice, the aim of this study was to determine whether this negative effect is induced by impaired oocyte and/or embryo quality, or by abnormal blastocyst formation in the stimulated oviductal and/or uterine environment. First, in-vitro and in-vivo preimplantation embryonic development after superovulation was observed in comparison with embryos derived from naturally cycling females. Second, in addition to previously reported studies (Ertzeid and Storeng, 1992; Ertzeid et al., 1993), postimplantation fetal development was evaluated after transfers of blastocysts from natural and stimulated donor mice into non-stimulated pseudopregnant foster mothers, to eliminate bias from the study. Possible negative effects of the hormonal stimulation on embryonic and fetal development were evaluated.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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F1 hybrid CBAxC57Bl mice were bred in controlled conditions of 14 h light:10 h darkness rhythm (light on at 06:00) at 22°C. The animals' food consisted of a special mix for mice (Muracon-G, Trouw, Gent, Belgium), and they received water [supplemented with HCl (pH 2.8) in order to prevent algal growth], ad libitum. Adult and 4- to 5-week old prepubertal females were stimulated with 5 IU PMSG (Folligon; Intervet, Brussels, Belgium) and 5 IU human chorionic gonadotrophin, i.p. (HCG; Pregnyl; Organon, Oss, The Netherlands) at 17:00, 48 h apart, in order to induce superovulation. The control group consisted of naturally cycling females of the same strain (without stimulation) that were caged with CBAxC57Bl males, at the same moment as the experimental groups, i.e. at the time of the HCG injection. Only females with a copulation plug were included in the study.
Preimplantation embryonic development
First, in-vitro blastocyst development and hatching of pronucleate ova from stimulated adult (n = 100) and prepubertal mice (n = 100) and naturally cycling females (n = 50) (Table I) were evaluated. The pronucleate ova were collected 20 h after caging males and females, and cultured in human tubal fluid (HTF) medium (Quinn et al., 1985) supplemented with 0.5% bovine serum albumin (BSA). The percentage blastocyst development was evaluated after 96 h of culture, and the number of blastocysts that started hatching was counted by microscopic visualization. After 120 h of culture, the number of totally hatched blastocysts was counted.
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Second, in-vivo preimplantation development of embryos collected from stimulated adult and prepubertal mice and naturally cycling females was followed from early cleavage stages (8-cell, morula on day 3) until blastocyst development before implantation (day 5). Therefore, in-vivo preimplantation development was checked at 65, 69, 73, 89, 93, 97 and 113 h after caging males and females. The female mice were killed by cervical dislocation and their oviducts and/or uteri removed and placed into one drop of HEPES-buffered Earle's Balanced Salt solution (EBSS) supplemented with 0.5% BSA. The oviducts and/or uteri were flushed with medium with a 33-gauge needle to obtain the embryos. The developmental stage and morphology of the embryos found were registered. The numbers of female mice and embryos used at each time interval are presented in Table II
.
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Postimplantation fetal development
In a pilot study, postimplantation fetal development and body weights were checked at day 14 of gestation for stimulated and naturally cycling adult females (numbers of mice used are shown in Table III
). Significant differences in litter size and fetal weight were observed between stimulated and naturally cycling pregnant females. Since nutritional deficiency of the fetuses of superovulated mice (Evans et al., 1981; Romero et al., 1992) might be the cause of the observed fetal growth retardation, a new study was started to eliminate this bias.
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In a prospective, randomized study, postimplantation fetal development was evaluated after transferring five early day 4 blastocysts of stimulated (prepubertal and adult) and non-stimulated females into the left and right horn of day 3 pseudopregnant non-stimulated foster mothers respectively. The same superovulation protocol was given as in the previous experiment. Pseudopregnancy was obtained by caging adult CBAxC57/Bl females in a natural non-stimulated cycle with vasectomized CBAxC57/Bl males, 24 h later than the embryo donors (Hogan et al., 1986
). Blastocysts were removed from the stimulated adult and prepubertal donor mice, and from the naturally cycling females at 90–91 h after caging with fertile males. The blastocysts were stored in the incubator at 37°C, 5% CO2 and 100% humidity in air, until the transfer was performed in HTF medium containing 0.5% BSA. Transfers were performed under anaesthesia with i.p. sodium pentobarbital (100 mg/kg; Nembutal; Abbott, Brussels, Belgium). At 92–95 h after caging the donors with fertile males (i.e. 68–71 h after caging the foster mothers with vasectomized males), early blastocysts were transferred into the uteri of the pseudopregnant females with a fine glass pipette under microscopic visualization. Each pseudopregnant foster mother received five blastocysts per uterine horn, derived from stimulated adults, stimulated prepubertals or naturally cycling controls (Figure 1). Numbers of foster mothers and embryos used are shown in Table IV. The foster mothers were killed by cervical dislocation on the 13th day of gestation (i.e. 14-day-old fetuses), the uterine horns were opened, and the number of implantation sites, fetal viability, number of resorption sites and fetal weight were registered for the different groups. Care was taken to remove all pieces of chorion and amnion villi and all amnion fluid by drying the fetuses with a paper towel before fetal weight was measured.
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Placebo-controlled experiment
It can be argued that the effect of stress related to the handling and i.p. injection of the superovulated females might induce endocrine release of hormones with potential negative effects on reproductive outcome. Therefore, an additional control experiment was performed to assess the effect of placebo i.p. injections and animal handling on the number of implantation sites, fetal viability, number of resorption sites and fetal weight in naturally cycling female mice. In the study group, nine adult CBAxC57Bl females were injected i.p. twice with 30 µl physiological saline (the same amount as in previous experiments) at 17:00, 48 h apart. The control group consisted of 11 naturally cycling females of the same strain (without injections or handling) that were caged with CBAxC57Bl males, at the same moment as the experimental groups, i.e. at the time of the second injection. Females with copulation plug were killed by cervical dislocation at the 14th day of gestation, the uterine horns were opened, and the number of implantation sites, fetal viability, number of resorption sites and fetal weight were registered for the two groups.
Statistical evaluation
Differences in the in-vitro development to the blastocyst and hatched blastocyst stages and number of implantation and resorption sites were evaluated using a 
2 test. Differences in the distribution of in-vivo embryo development at specific time intervals were evaluated with Biggers contingency tables for evaluating trends. Differences in fetal weights were evaluated using Student's t-test.
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Results |
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Preimplantation embryonic development
In vitro, the rate of blastocyst formation was similar in the stimulated and non-stimulated groups (Table I
). However, the hatching process started significantly earlier (P < 0.03 on day 5; P < 0.001 on day 6) in blastocysts derived from oocytes obtained in naturally cycling females when compared with blastocysts derived from oocytes obtained in stimulated adults and prepubertals.
A significant delay in the in-vivo development of preimplanted embryos was observed after ovarian stimulation of adult females compared with natural cycle development (Figure 2), and at all time intervals observed. This was expressed by a delay in morula formation (P < 0.0001, at 69 and 73 h after caging), a delay in blastocoele formation (P < 0.001, at 89, 93 and 97 h after caging), a delay in zona lysis (P < 0.001, 97 h after caging) and a delay in blastocyst collapse, i.e. a reduction of the volume and an enlargement of the shape of the blastocyst, just before implantation takes place (P < 0.0001, 113 h after caging). The in-vivo development of preimplanted embryos after superovulation of prepubertal females resulted in a faster morula formation at 69 h after caging (P < 0.0001), followed by a delay in development, when compared with naturally cycling controls. This was expressed by a delay in zona lysis (P < 0.0001 at 97 h after caging) and a delay in blastocyst collapse (P < 0.0001, 113 h after caging). Moreover, a higher prevalence of morphologically abnormal blastocysts was found in prepubertal females after ovarian stimulation (7%) when compared with the controls (0%, P < 0.0001). Blastocyst abnormalities included failed cavitation and/or sticky zona pellucidae, including normal or abnormal blastocysts (Figure 3).
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Postimplantation fetal development
In the pilot study, five superovulated females and three naturally cycling females were not pregnant. In each group, 97 fetuses were evaluated for weight deriving from six superovulated pregnant females and 11 pregnant controls. A 40% growth retardation of 14-day-old fetuses after superovulation was observed compared with control fetuses (96 ± 29 versus 159 ± 17 mg, P < 0.0001, Table III
). This observation was biased by a significant difference (P < 0.005) in the number of implantation sites per mouse in the superovulated group (16.1 ± 5.1) when compared with the natural cycling controls (8.7 ± 2.3). This may be explained by the high numbers of oocytes after superovulation in the stimulated females. Similarly, significantly more resorption sites (22%) were found in stimulated females, compared with naturally cycling controls (7.6%, P < 0.01).
The prospective randomized transfer experiment showed that the implantation rate of blastocysts from stimulated prepubertals was lower (P < 0.005) compared with the controls (Table IV). Moreover, transfers of blastocysts from stimulated prepubertals resulted in fewer living fetuses (P < 0.005), more resorption sites (P < 0.005) and a growth retardation of 31% when compared with naturally cycling controls (110 ± 21 versus 159 ± 18 mg, P < 0.0001, Table IV). Similarly, transferred blastocysts from stimulated adults showed a trend towards a lower implantation rate (P = 0.08). These blastocysts resulted again in fewer living fetuses (P < 0.02), more resorption sites (P < 0.02) and a growth retardation of 21% when compared with the naturally cycling controls (126 ± 23 versus 159 ± 18 mg, P < 0.0001).
Placebo controls
The additional control experiment showed no differences in the placebo-injected group (nine females, 77 fetuses) compared with controls (11 females, 106 fetuses). The mean number of implantation sites per female were 8.6 ± 2.4 and 9.6 ± 1.6, the mean number of resorption sites per female were 0.6 ± 0.7 and 0.8 ± 0.7, and the mean fetal weights were 281.4 ± 32.1 and 281.1 ± 35.0 mg in the placebo-injected and control groups respectively.
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Discussion |
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This study clearly demonstrates that preimplantation embryonic development in vitro and in vivo is negatively influenced by the ovarian stimulation itself, and results in an impaired blastocyst formation and fetal growth retardation at day 14 of gestation. This study confirms the results of others (Ertzeid and Storeng, 2000
) who showed, in a similar transfer experiment, a decreased implantation rate, a higher postimplantation fetal mortality and a lower mean fetal weight of embryos from superovulated donor mice. This negative effect can be explained by an impaired oocyte quality, by a negative influence of the stimulated oviductal and/or uterine environment on the developing embryos (Van der Auwera et al., 1999), by an impaired receptivity of the endometrium (Walton et al., 1982; Fossum et al., 1989; Paulson et al., 1990; Van der Auwera et al., 1994; Check et al., 1995) or by nutritional deficiency due to overcrowded uteri (Evans et al., 1981). In the in-vitro culture experiment, rates of blastocyst development, hatching and hatched blastocysts were evaluated. This experiment was designed to evaluate the influence of the hormonal stimulation on oocyte quality, since the pronucleate ova were only exposed for a maximum of 10 h to the maternal environment after ovulation. Only about half of the embryos collected from stimulated females started hatching or were totally hatched when compared with embryos collected from naturally cycling controls. These results suggest that aberrant development had already been initiated before the pronuclear stage. The number of inner cell mass cells and trophectoderm were not counted, and probably would have provided more detailed results.
The evaluation of in-vivo preimplantation embryo development showed a delay in blastocyst formation, zona lysis and blastocyst collapse of embryos obtained in stimulated mice when compared with naturally cycling controls. In this experiment however, no distinction could be made between a negative effect of the hormonal stimulation on oocyte quality, or a negative effect of the stimulated oviductal and/or uterine environment on further developmental capacity of these oocytes. Thus, it is speculated that this would be a combined effect on both oocyte quality and embryonic developmental capacity.
The design of the postimplantation fetal development experiment, in transferring stimulated and control blastocysts into pseudopregnant normal cycling females, allowed the effects of reduced endometrial receptivity or of nutrient deficiency due to overcrowded uteri to be excluded. In this study, a lower implantation rate, fewer living fetuses and more resorption sites were observed in 14-day-old pregnancies if blastocysts were obtained from stimulated females when compared with naturally cycling controls. These observations confirm the results of our previous study (Van der Auwera et al., 1999), that exposure to the stimulated oviductal environment is detrimental for embryonic implantation and fetal viability. Moreover, a serious fetal growth retardation of 21–31% was observed at day 14 of pregnancy when blastocysts were obtained from stimulated adult and prepubertal females in comparison with naturally cycling controls. Both adult and prepubertal donors were used, since adults were superovulated without regard to their oestrous cycle, and this could interfere with their stimulation. Superovulation of prepubertals, however, does not interfere with their own oestrous cycle (which is probably more comparable with pituitary down-regulation during a human IVF cycle).
Since the naturally cycling donor females in the transfer experiment were not injected with placebo, the results of the transfer experiment could be biased by possible endocrine release of hormones or other factors induced by the stress of handling and injecting the superovulated females. Therefore, the placebo-controlled study was performed. Although this control study was not performed simultaneously, and in these controls no transfers were performed, no evidence for this bias could be detected.
It is difficult to extrapolate these results to the human situation after assisted reproduction. However, a similar negative effect of ovarian stimulation on oocyte and embryo quality seems likely in IVF, since in most centres implantation rates of only 10–20% are reached with embryo transfers on days 2–3. In IVF however, other factors also compromise implantation and viability. Possible negative effects of sub-optimal culture conditions might be involved, as well as negative effects of the stimulated oviductal and uterine environment after transfer on the embryonic competence, and/or an impaired endometrium receptivity, which all could result in a delayed implantation. Nevertheless, the results of the current study may in part offer an explanation for the delay in human embryonic development after IVF, and for the low birth weight often observed after assisted reproductive technologies (FIVNAT, 1995; Petersen et al., 1995; Sundström et al., 1997). We speculate that the delay in human embryonic development after IVF, and the lower birth weight of IVF children, may be related to the negative influence of the ovulation induction on oocyte and embryo developmental competence.
The difference in embryonic development between superovulated mothers and naturally cycling controls may be biased by differences in ovulation time. Although both groups were caged with males at the same moment, others (Allen and McLaren, 1971) reported 5 h differences in ovulation and further development. In the current experiment, naturally cycling females were expected to ovulate between 03:00 and 06:00 as calculated in relation to the day/night rhythm (Snell et al., 1940; Bronson et al., 1966), while the hormone-treated females were expected to ovulate between 11 and 14 h after HCG (Edwards and Gates, 1959; Beaumont and Smith, 1975), i.e. between 04:00 and 07:00. As a result, there was only 1 h difference in ovulation time between both groups—a time difference which is unlikely to explain the 21–32% fetal growth retardation observed in the stimulated prepubertal females.
In conclusion, in a mouse model for IVF, ovarian stimulation itself caused a delay in embryonic development both in vitro and in vivo, an increase in abnormal blastocyst formation, a fetal growth retardation of 22%, and an increase in the number of resorption sites.
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Notes |
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1 To whom correspondence should be addressed at: Leuven University Fertility Center, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: Ingrid.Vanderauwera{at}uz.kuleuven.ac.be
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Submitted on November 16, 2000; accepted on February 12, 2001.
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 A. J. Watkins, D. Platt, T. Papenbrock, A. Wilkins, J. J. Eckert, W. Y. Kwong, C. Osmond, M. Hanson, and T. P. Fleming From the Cover: Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure PNAS, March 27, 2007; 104(13): 5449 - 5454. [Abstract] [Full Text] [PDF]
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A. Sato, E. Otsu, H. Negishi, T. Utsunomiya, and T. Arima Aberrant DNA methylation of imprinted loci in superovulated oocytes Hum. Reprod., January 1, 2007; 22(1): 26 - 35. [Abstract] [Full Text] [PDF]
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R.M. Sibug, N. Datson, A.M.I. Tijssen, M. Morsink, J. de Koning, E.R. de Kloet, and F.M. Helmerhorst Effects of urinary and recombinant gonadotrophins on gene expression profiles during the murine peri-implantation period Hum. Reprod., January 1, 2007; 22(1): 75 - 82. [Abstract] [Full Text] [PDF]
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H.-C. Ruan, X.-M. Zhu, Q. Luo, A.-X. Liu, Y.-L. Qian, C.-Y. Zhou, F. Jin, H.-F. Huang, and J.-Z. Sheng Ovarian stimulation with GnRH agonist, but not GnRH antagonist, partially restores the expression of endometrial integrin beta3 and leukaemia-inhibitory factor and improves uterine receptivity in mice Hum. Reprod., October 1, 2006; 21(10): 2521 - 2529. [Abstract] [Full Text] [PDF]
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 R. L. Kelley, K. L. Kind, M. Lane, R. L. Robker, J. G. Thompson, and L. J. Edwards Recombinant human follicle-stimulating hormone alters maternal ovarian hormone concentrations and the uterus and perturbs fetal development in mice Am J Physiol Endocrinol Metab, October 1, 2006; 291(4): E761 - E770. [Abstract] [Full Text] [PDF]
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 N. S. Macklon, R. L. Stouffer, L. C. Giudice, and B. C. J. M. Fauser The Science behind 25 Years of Ovarian Stimulation for in Vitro Fertilization Endocr. Rev., April 1, 2006; 27(2): 170 - 207. [Abstract] [Full Text] [PDF]
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C. De Geyter, M. De Geyter, S. Steimann, H. Zhang, and W. Holzgreve Comparative birth weights of singletons born after assisted reproduction and natural conception in previously infertile women Hum. Reprod., March 1, 2006; 21(3): 705 - 712. [Abstract] [Full Text] [PDF]
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L. J. Edwards, K. L. Kind, D. T. Armstrong, and J. G. Thompson Effects of recombinant human follicle-stimulating hormone on embryo development in mice Am J Physiol Endocrinol Metab, May 1, 2005; 288(5): E845 - E851. [Abstract] [Full Text] [PDF]
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R.M. Sibug, J. de Koning, A.M.I. Tijssen, M.C. de Ruiter, E.R. de Kloet, and F.M. Helmerhorst Urinary gonadotrophins but not recombinant gonadotrophins reduce expression of VEGF120 and its receptors flt-1 and flk-1 in the mouse uterus during the peri-implantation period Hum. Reprod., March 1, 2005; 20(3): 649 - 656. [Abstract] [Full Text] [PDF]
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D. Zudova, A. J. Wyrobek, J. Bishop, and F. Marchetti Impaired fertility in T-stock female mice after superovulation Reproduction, November 1, 2004; 128(5): 573 - 581. [Abstract] [Full Text] [PDF]
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 R. Roberts, J. Stark, A. Iatropoulou, D. L. Becker, S. Franks, and K. Hardy Energy Substrate Metabolism of Mouse Cumulus-Oocyte Complexes: Response to Follicle-Stimulating Hormone Is Mediated by the Phosphatidylinositol 3-Kinase Pathway and Is Associated with Oocyte Maturation Biol Reprod, July 1, 2004; 71(1): 199 - 209. [Abstract] [Full Text] [PDF]
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D. Lucifero, J.R. Chaillet, and J. M. Trasler Potential significance of genomic imprinting defects for reproduction and assisted reproductive technology Hum. Reprod. Update, January 1, 2004; 10(1): 3 - 18. [Abstract] [Full Text] [PDF]
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 R. L. Krisher The effect of oocyte quality on development J Anim Sci, January 1, 2004; 82(13_suppl): E14 - 23. [Abstract] [Full Text] [PDF]
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C. M.H. Combelles and D. F. Albertini Assessment of Oocyte Quality Following Repeated Gonadotropin Stimulation in the Mouse Biol Reprod, March 1, 2003; 68(3): 812 - 821. [Abstract] [Full Text] [PDF]
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R.M. Sibug, F.M. Helmerhorst, A.M.I. Tijssen, E.R. de Kloet, and J. de Koning Gonadotrophin stimulation reduces VEGF120 expression in the mouse uterus during the peri-implantation period Hum. Reprod., June 1, 2002; 17(6): 1643 - 1648. [Abstract] [Full Text] [PDF]
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