Department of Obstetrics and Gynecology, The National Hospital, University of Oslo, 0027 Oslo, Norway
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Abstract |
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Key words: embryo developmental competence/fetal development/implantation/mice/ovarian stimulation
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Introduction |
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We have reported previously that treatment with gonadotrophins impairs pre- and post-implantation development in mice. Increased pre- and post-implantation mortality, low fetal weight and fetal growth retardation were observed in superovulated mice and in animals treated with ovulation induction alone (Ertzeid and Storeng, 1992). Furthermore, delayed implantation and presumably impaired implantation did occur (Ertzeid et al., 1993
). The objective of this study was to evaluate whether impaired embryo quality and/or changes in uterine milieu are responsible for the previously observed adverse effects of superovulation with gonadotrophins on implantation and fetal development in mice. Taking advantage of the fact that the uterus in mice has two horns, an embryo donation model was used (Storeng and Jonsen, 1984
), in which in-vitro-cultured embryos from superovulated and non-stimulated females were transferred to separate uterine horns within the same superovulated or non-stimulated pseudopregnant recipient.
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Materials and methods |
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Gonadotrophin treatment of donor and recipient females
Pregnant mare's serum gonadotrophin (PMSG) (Folligon; Intervet Norge, Oslo, Norway) and human chorionic gonadotrophin (HCG) (Profasi; Serono, Geneva, Switzerland) were used. Donor and recipient females for the experiments were chosen without regard to their oestrous cycle, and were superovulated with an i.p. injection of 10 IU PMSG in 0.1 ml 0.145 mol/l NaCl at 12:00, followed 48 h later by an i.p. injection of 10 IU HCG in 0.1 ml 0.145 mol/l NaCl to induce ovulation. Controls were injected with the vehicle at the appropriate times. Donor females were mated with fertile males, and recipients were rendered pseudopregnant by mating with vasectomized males. The presence of a vaginal plug on the following day indicated successful mating, and this was designated day 1 of gestation. Since the presence of a coagulation plug does not necessarily indicate a pregnancy, it was assumed in this study that vaginal plug-positive mice may fail to become pregnant after transfer, due not only to the quality of the embryos and/or endometrium but also to failure to become pseudopregnant.
Embryo recovery and in-vitro culture
Pregnant donor mice were killed by cervical dislocation on gestational day 2. The oviducts were excised and flushed with M2 medium (Sigma Aldrich Norway AS, Oslo, Norway). Morphologically normal day 2 embryos (2- to 4-cell) were pooled after collection, washed twice in M2 medium and transferred to a 0.05 ml droplet of M16 Medium (Sigma), overlaid with paraffin oil, and incubated at 37°C in a humidified atmosphere of 5% CO2 in air for 48 h. At day 4, normal embryos were pooled and their number and stage of development (compacted morulae, early to fully expanded blastocysts) were recorded.
Embryo transfer
At day 4, normal embryos from control and superovulated mice were divided randomly and transferred to separate uterine horns within the same control or superovulated recipient (see Figure 1). Available normal embryos (two to six embryos) were transferred to the uterine horns of females on day 3 of pseudopregnancy. Briefly, the animals were anaesthetized with an i.p. injection of 0.2 ml Dormicum:Hypnorm (midazolam/fentanyl/fluanison; National Hospital Pharmacy, Oslo, Norway). A paralumbar incision was made on the recipient female and each uterine horn was consecutively pulled through the excision. The uterus was punctured with a needle, and through this hole embryos were transferred to the lumen, using a finely drawn glass pipette. Thereafter, the uterus was replaced in the abdominal cavity and the skin incision closed by clips. One recipient received control embryos in the right uterine horn, while embryos from superovulated donors were transferred in the left uterine horn. The next recipient received embryos derived from superovulated donors in the right uterine horn and control embryos in the left uterine horn. This pattern was followed throughout the experiments in order to alleviate any technically induced influence in the outcome of the embryo transfer. Seventeen replicate experiments with a total of 182 embryo transfers to 91 pseudopregnant recipient mice were performed. A total of 820 embryos was transferred as follows: 116 control embryos and 340 embryos from superovulated mice to control recipients; 134 control embryos and 230 embryos from superovulated mice to superovulated recipients.
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Statistical analysis
The data were analysed using Student's t-test for continuous data and 2 for categorical data. Differences were considered significant at P < 0.05.
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Results |
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Impact of ovarian stimulation on embryo developmental competence
Morphological examination of normal embryos transferred on day 4 revealed that a significantly higher proportion of embryos from control donors had reached the blastocyst stage (61%) than had those from superovulated donors (41%; P < 0.001; Table I).
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The mean weight of live fetuses which developed after transfer of embryos derived from superovulated donors was significantly lower for those obtained from superovulated recipients (0.51 g) than from control recipients (0.72 g; P = 0.006; Table IV).
There were no macroscopic malformations observed in either group of offspring.
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Discussion |
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A negative effect of ovarian stimulation on oocyte/embryo developmental competence was observed as transfer of embryos from superovulated donors resulted in a significantly lower implantation rate in control recipients compared with that of embryos from control donors. No difference in post-implantation mortality was observed, indicating an early loss of non-viable embryos recovered from superovulated donors. The reduced weight of live fetuses after transfer to control recipients of embryos from superovulated donors was not statistically significant, but may suggest some negative effect of superovulation even on viable embryos.
In previous studies, increased pre-implantation mortality after superovulation has been reported in mice (Beaumont and Smith, 1975; Ertzeid and Storeng, 1992
) and rats (Miller and Armstrong, 1981
). Furthermore, in hamsters, embryos produced by superovulated females have been observed to be less viable upon transfer than embryos recovered from controls (McKiernan and Bavister, 1998
).
The causes of the loss of developmental competence of embryos from superovulated donors remain unknown. Chromosomal abnormalities may account for reduced viability of embryos from superovulated females because superovulation has been found to increase the proportion of chromosomal abnormalities in murine embryos (Elbling and Colot, 1985, 1987
; Luckett and Mukherjee, 1986
) as well as in oocytes in rats (Tain et al., 2000
). As ovarian stimulation produces a cascade of hormonal and physiological events, oocytes mature in an environment different from that of naturally matured oocytes (Foote and Ellington, 1988
), and variation in the timing of ovulation may also occur (Allen and McLaren, 1971
). New evidence from molecular biology studies of mammalian oogenesis has implicated a role for gonadotrophins in the control of meiosis in mammalian oocytes (Picton et al., 1998
). In mice as well as in humans, there is evidence for steroids being regulators of gene expression, and that embryo morphology and rate of developmentboth of which reflect embryo qualityhave a genetic basis (Warner et al., 1998
). In the present study, within the group of normal day 4 embryos from superovulated donors, the proportion of morulae was higher than from control donors, indicating developmental retardation which may reflect reduced embryo quality.
A negative effect of ovarian stimulation on uterine receptivity was also observed. The implantation rate was significantly reduced and the mortality greatly increased in superovulated recipients compared with controls. The mean weight of live fetuses in stimulated recipients was reduced, indicating impaired implantation and gestation.
Successful implantation depends on embryo quality, uterine receptivity and synchronization of embryo development and endometrial maturation. The relative contribution of the endometrium to the success rate is not known, and there are no accepted criteria for evaluating endometrial receptivity. However, preparation of the endometrium is primarily under the control of ovarian steroid hormones, and increasing evidence suggests that their effects are mediated by locally produced cytokines which then exert their action in an autocrine or paracrine manner (Klentzeris, 1997; Stewart and Cullinan, 1997
; Beier and Beier-Hellwig, 1998
; Simón et al., 1998a
; Giudice, 1999
). In the present study, exogenous administration of gonadotrophins, affecting the concentrations of circulating ovarian steroids (Ertzeid and Storeng, 1992
), may have changed the local expression of cytokines in the endometrium in superovulated recipients, and hence its receptivity. Female mice were chosen without regard to their oestrous cycle. However, the adverse effects of gonadotrophins presumably cannot be attributed to asynchrony, as an earlier study has shown that superovulation after synchronization also increased embryonic loss (Beaumont and Smith, 1975
).
Previous studies in immature rats, superovulated with PMSG, but without HCG as ovulation induction, reported pregnancy failure due to changes in the uterine milieu (Walton et al., 1982; Walton and Armstrong, 1983
). In agreement with the results presented here, a higher implantation rate on gestational day 5 was observed when embryos from superovulated donor mice were transferred to non-stimulated than to stimulated recipient mice (Fossum et al., 1989
). Compared with this study, the implantation rate in the current study in superovulated as well as control recipients was lower, possibly due to a period of embryonic in-vitro culture in this study, as well as the difference in the strain of mice used. Furthermore, a stimulated oviductal environment has also been shown to have a negative influence on the implantation capacity of mouse embryos (Van der Auwera et al., 1999
). In humans, synthetic oestrogen has been used as an effective emergency contraceptive agent to prevent implantation (Haspels, 1976
). In IVF, ovarian stimulation with high oestradiol concentrations has been reported to be detrimental to implantation and pregnancy rates (Pellicer et al., 1996
; Simón et al., 1998b
; Valbueña et al., 1999
; Ng et al., 2000
). While high serum oestradiol concentrations in fresh IVF cycles significantly reduced the implantation rate, the implantation and pregnancy rates in frozenthawed cycles for surplus embryos were similar. Hence, the impairment in implantation was attributed to hostile environment in the endometrium (Ng et al., 2000
).
In conclusion, ovarian stimulation impairs implantation and fetal development in mice. These experiments, using the mouse embryo donation model, indicate that reduced embryo quality or developmental competence as well as changes in the uterine milieu are responsible for the adverse effects observed.
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Notes |
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References |
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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, 437448.[Abstract]
Beier, H.M. and Beier-Hellwig, K. (1998) Molecular and cellular aspects of endometrial receptivity. Hum. Reprod. Update, 4, 448458.
Brinster, R.L. (1975) Teratogen testing using preimplantation mammalian embryos. In Shepard, T.H., Miller, J.R., Marrois, M. (eds), Methods for Detection of Environmental Agents that Produce Congenital Defects. North Holland Publishing Co., Amsterdam/Oxford American Elsevier Publishing Co. Inc., New York, pp. 113123.
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, 189195.[ISI][Medline]
Elbling, L. and Colot, M. (1987) Persistence of SCE-inducing damage in mouse embryos and fetuses following superovulation in mice. Mutat. Res., 176, 117122.[ISI][Medline]
Ertzeid, G. and Storeng, R. (1992) Adverse effects of gonadotrophin treatment on pre- and postimplantation development in mice. J. Reprod. Fertil., 96, 649655.[Abstract]
Ertzeid, G., Storeng, R. and Lyberg, T. (1993) Treatment with gonadotropins impaired implantation and fetal development in mice. J. Assist. Reprod. Genet., 10, 286291.[ISI][Medline]
Foote, R.H. and Ellington, J.E. (1988) Is a superovulated oocyte normal? Theriogenology, 29, 111123.[ISI]
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, 710.[ISI][Medline]
Giudice, L.C. (1999) Potential biochemical markers of uterine receptivity. Hum. Reprod., 14 (Suppl. 2), 316.[Medline]
Haspels, A.A. (1976) Interception: post-coital estrogens in 3016 women. Contraception, 14, 375381.[ISI][Medline]
Klentzeris, L.D. (1997) The role of endometrium in implantation. Hum. Reprod., 12 (Natl. Suppl., JBFS 2), 170175.
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, 3942.[ISI][Medline]
Maman, E., Lunenfeld, E., Levy, A. et al. (1998) Obstetric outcome of singleton pregnancies conceived by in vitro fertilization and ovulation induction compared with those conceived spontaneously. Fertil. Steril., 70, 240245.[ISI][Medline]
McKiernan, S.H. and Bavister, B.D. (1998) Gonadotrophin stimulation of donor females decreases post-implantation viability of cultured one-cell hamster embryos. Hum. Reprod., 13, 724729.[Abstract]
Miller, B.G. and Armstrong, D.T. (1981) Effects of a superovulatory dose of pregnant mare serum gonadotrophin on ovarian function; serum estradiol, and progesterone levels and early embryo development in immature rats. Biol. Reprod., 25, 261271.[ISI][Medline]
Ng, E.H.Y., Yeung, W.S.B., Lau, E.Y.L. et al. (2000) High serum oestradiol concentrations in fresh IVF cycles do not impair implantation and pregnancy rates in subsequent frozen-thawed embryo transfer cycles. Hum. Reprod., 15, 250255.
Pellicer, A., Valbuena, D., Cano, F. et al. (1996) Lower implantation rates in high responders: evidence for an altered endocrine milieu during the preimplantation period. Fertil. Steril., 65, 11901195.[ISI][Medline]
Picton, H., Briggs, D. and Gosden, R. (1998) The molecular basis of oocyte growth and development. Mol. Cell. Endocrinol., 145, 2737.[ISI][Medline]
Simón, C., Moreno, C., Remohí, J. et al. (1998a) Molecular interactions between embryo and uterus in the adhesion phase of human implantation. Hum. Reprod., 13 (Suppl. 3), 219236.
Simón, C., Garcia Velasco, J., Valbueña, D. et al. (1998b) Increased uterine receptivity by decreasing estradiol levels during the preimplantation period in high responder patients by using an FSH step-down regimen. Fertil. Steril., 70, 234239.[ISI][Medline]
Stewart, C.L. and Cullinan, E.B. (1997) Preimplantation development of the mammalian embryo and its regulation by growth factors. Dev. Genet., 21, 91101.[ISI][Medline]
Storeng, R. and Jonsen, J. (1984) Recovery of mouse embryos after short-term in vitro exposure to toxic nickel chloride. Toxicol. Lett., 20, 8591.[ISI][Medline]
Tain, C.F., Goh, V.H.H. and Ng, S.C. (2000) Effect of hyperstimulation with gonadotrophins and age of females on oocytes and their metaphase II status in rats. Mol. Reprod. Dev., 55, 104108.[ISI][Medline]
Tanbo, T., Dale, P.O., Lunde, O. et al. (1995) Obstetric outcome in singleton pregnancies after assisted reproduction. Obstet. Gynecol., 86, 188192.
Valbueña, D., Jasper, M., Remohí, J. et al. (1999) Ovarian stimulation and endometrial receptivity. Hum. Reprod., 14 (Suppl. 2), 107111[Medline]
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, 25702574.
Walton, E.A. and Armstrong, D.T. (1983) Oocyte normality after superovulation in immature rats. J. Reprod. Fertil., 67, 309314.[Abstract]
Walton, E.A., Huntley, S., Kennedy, T.G. et al. (1982) Possible causes of implantation failure in superovulated immature rats. Biol. Reprod., 27, 847852.[ISI][Medline]
Warner, C.M., Cao, W., Exley, G.E. et al. (1998) Genetic regulation of egg and embryo survival. Hum. Reprod., 13 (Suppl. 3), 178190.[Abstract]
Submitted on September 1, 2000; accepted on November 2, 2000.