Leuven University Fertility Center, University Hospital Gasthuisberg, Catholic University Leuven, Leuven, Belgium
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: blastocysts/fetal growth retardation/implantation/ovarian stimulation/weight
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
|
|
|
|
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.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
).
|
|
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.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 2131% 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 1020% are reached with embryo transfers on days 23. 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 groupsa time difference which is unlikely to explain the 2132% 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.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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]
Bronson, F.H., Dagg, C.P. and Snell, G.D. (1966) Reproduction. In Green, E.L. (ed.), Biology of the Laboratory Mouse. The Blakiston Division, McGraw-Hill, New York, pp. 187204.
Champlin, A.K., Kuzia, S.J., Rice, B.A. et al. (1987) Cell surface characteristics of blastocysts from spontaneously ovulating and gonadotropin-treated mice. Biol. Reprod., 36, 439444.[Abstract]
Check, J.H., O'Shaughnessy, A., Lurie, D. et al. (1995) Evaluation of the mechanism for higher pregnancy rates in donor oocyte recipients by comparison of fresh with frozen embryo transfer pregnancy rates in a shared oocyte programme. Hum. Reprod., 10, 30223027.[Abstract]
Edwards, R.G. and Gates, A.H. (1959) Timing of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs of adult mice treated with gonadotrophins. J. Endocrinol., 18, 292304.[ISI]
Elmazar, M.M.A., Vogel, R. and Spielmann, H. (1989) Maternal factors influencing development of embryos from mice superovulated with gonadotropins. Reprod. Toxicol., 3, 135138.[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. and Storeng, R. (2000) The impact of ovarian hyperstimulation on implantation and fetal development in mice. Hum. Reprod., 16, 221225.
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]
Evans, M.I., Schulman, J.D., Golden, L. et al. (1981) Superovulation-induced intrauterine growth retardation in mice. Am. J. Obstet. Gynecol., 141, 433435.[ISI][Medline]
FIVNAT (1995) Pregnancies and births resulting from in vitro fertilization: French national registry, analysis of data 1986 to 1990. Fertil. Steril., 64, 746756.[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, 714.[ISI][Medline]
Hogan, B., Costantini, F. and Lacy, E. (1986) Manipulating the Mouse Embryo. New York, Cold Spring Harbor, pp. 3039.
McKirnan, 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. (1981a) Superovulatory doses of pregnant mare serum gonadotropin cause delayed implantation and infertility in immature rats. Biol. Reprod., 25, 253260.[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, 261271.[ISI][Medline]
Miller, B.G. and Armstrong, D.T. (1982) Infertility in superovulated immature rats: role of ovarian steroid hypersecretion. Biol. Reprod., 26, 861868.[ISI][Medline]
Paulson, R.J., Sauer, M.V. and Lobo, R.A. (1990) Embryo implantation after human in vitro fertilization: importance of endometrial receptivity. Fertil. Steril., 53, 870874.[ISI][Medline]
Petersen, K., Hornnes, P.J., Ellingsen, S. et al. (1995) Perinatal outcome after in vitro fertilisation. Acta Obstet. Gynecol. Scand., 74, 129131.[ISI][Medline]
Quinn, P., Kerin, J.F. and Warnes, G.M. (1985) Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil. Steril., 44, 493498.[ISI][Medline]
Romero, A., Villamayor, F., Grau, M.T. et al. (1992) Relationship between fetal weight and litter size in rats: application to reproductive toxicology studies. Reprod. Toxicol., 6, 453456.[ISI][Medline]
Snell, G.D., Fekete, E., Hummel, K.P. et al. (1940) The relation of mating, ovulation and the estrous smear in the house mouse to time of day. Anat. Rec., 76, 3954.
Sundström, I., Ildgruben, A. and Högberg, U. (1997) Treatment-related and treatment-independent deliveries among infertile couples, a long-term follow-up. Acta Obstet. Gynecol. Scand., 76, 238243.[ISI][Medline]
Van der Auwera, I., Meuleman, C. and Koninckx, P.R. (1994) Human menopausal gonadotrophin increases pregnancy rate in comparison with clomiphene citrate during replacement cycles of frozen/thawed pronucleate ova. Hum. Reprod., 9, 15561560.[Abstract]
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. Human Reprod., 14, 25702574.
Walton, E.A. and Armstrong, D.T. (1981) Ovarian function and early embryo development in immature rats given a superovulatory dose of PMSG, later neutralised by antiserum. Biol. Reprod., 25, 272280.[ISI][Medline]
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]
Wetzels, A.M.M., Artz, M.T., Goverde, H.J.M. et al. (1995) Gonadotropin hyperstimulation influences the 35S-methionine metabolism of mouse preimplantation embryos. J. Assist. Reprod. Genet., 12, 744746.[ISI][Medline]
Submitted on November 16, 2000; accepted on February 12, 2001.