Human Reproduction, Vol. 14, No. 1, 207-210,
January 1999
© 1999 European Society of Human Reproduction and Embryology
Effect of 6ß-methylprednisolone on mouse pregnancy rate
Departament de Biologia Cel·lular i Fisiologia, Edifici C, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
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Abstract |
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The aim of this study was to evaluate the effect of methylprednisolone on the pregnancy rate in mice. For this reason, zona pellucida-intact and zona pellucida-free embryos at the blastocyst stage were transferred to recipient mice at day 2.5 of pseudopregnancy. Embryo transfer was performed into non-immunodepressed and immunodepressed groups of recipient mice using 0.3 or 0.6 µg/g of 6ß-methylprednisolone. A higher implantation and developmental rate of zona-free embryos transferred to the immunodepressed group of recipients was observed after using the higher dose of methylprednisolone.
Key words: developmental rate/embryo transfer/immunodepression/implantation rate/methylprednisolone
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Some assisted human reproduction techniques such as intracytoplasmic sperm injection (ICSI), assisted hatching, preimplantation genetic diagnosis or other manipulations of animal embryos (transgenic animal production, splitting or even cloning) usually need partial opening or damaging of the zona pellucida (ZP). Precompacted human and animal embryos with small holes or incisions in their zona are currently replaced into the female genital tract with a lower physical protection., It is still not clear whether the benefits of this procedure could be partially or totally cancelled out by the damage caused by immune cell penetration through the incision, thus decreasing their implantation rate (Willadsen, 1979).
It is well known that there is a rise in uterine lymphocyte concentrations and immune system cell numbers during implantation. Moreover, manipulations for embryo transfer induce an endometrial inflammatory response which normally implies the migration of macrophages and immunocompetent cells to the inflammatory focus (MacMaster et al., 1992; Sandford et al., 1992
). Despite this, it is still not clear whether or not corticosteroid therapy facilitates embryo implantation.
In order to increase the pregnancy rate in human IVF programmes, corticosteroid therapies acting as immunodepresors have been used with variable results.
Previously reported data (Cohen et al., 1990a) suggested that doses of 0.3 µg/g of 6ß-methylprednisolone applied to in-vitro fertilization (IVF)–embyo transfer human patients improved only the implantation rate of micromanipulated embryos. They suggested that immunodepression could diminish the presence of uterine lymphocytes, allowing the embryo to develop normally. Corticosteroid therapy could decrease the number of peripheral immune cells (i.e. segmented neutrophils), which are capable of changing in size and shape and penetrate through the narrow incision of the zona pellucida to the perivitelline space to damage the embryo.
Lee et al. (1994) concluded that a short-term immunodepression using corticosteroid has no effect on pregnancy rates in IVF–embyo transfer patients who received non-micromanipulated, zona-intact embryos. They argued that if corticosteroid therapy acts merely as an anti-inflammatory, as suggested by Cohen et al. (1990b), it is logical that zona-intact embryos do not obtain any advantages from corticosteroid therapy. In contrast, Polak de Fried et al. (1993) reported that immunodepressive doses of 1.0 µg/g of 6ß-methylprednisolone significantly increased implantation rates of non-micromanipulated embryos.
Therefore, to evaluate the effect of corticosteroid therapy on the pregnancy rate, zona-intact and zona-free mouse blastocysts were transferred into recipient control or immunodepressed mice at day 2.5 of pseudopregnancy. Zona-free embryos were used instead of embryos with damaged zonae to potentiate the damaging effect of the maternal immune system. This was performed in order to observe more clearly the effect of immunodepression exerted by the corticosteroid treatment.
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Materials and methods |
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Embryo collection
B6CBAF1 female mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotrophin (PMSG; Laboratories Intervet SA, Salamanca, Spain) followed by 5 IU of human chorionic gonadotrophin (HCG; Farma Leporis SA, Barcelona, Spain) after a 48 h interval, to induce superovulation. Females were housed with male mice of the same strain overnight for mating (Biggers et al., 1971
). At 44–45 h post-HCG the animals were killed by cervical dislocation and the oviducts were flushed to collect 2-cell embryos. Embryos were incubated in medium M16 (Whittingham, 1971) standard conditions (37°C, 5% CO2) until the blastocyst stage was reached.
Embryo manipulation
Embryos at the blastocyst stage were randomly separated into two groups before their transfer to recipient pseudopregnant mice: control group: zona-intact embryos (+ZP); manipulated embryos: zona-free (–ZP) embryos were obtained by removing the zona with an acid Tyrode's solution (pH 2.5) as previously described (Hogan et al., 1994).
To minimize variability in pregnancy capacity among females, the development of treated and control embryos within the same foster mother were compared: so, seven non-micromanipulated (+ZP) blastocysts were transferred to one uterine horn and seven manipulated (–ZP) to the opposite horn. The horn to which each group of embryos was to be transferred was also randomly chosen to diminish the effect of technical skill of the investigator during the transfer procedure (Gardner et al., 1988).
Embryo transfer
Recipient mice were of the same strain as donors. These mice were made pseudopregnant by mating in pro-oestrus with vasoligated OF1 males. The moment a vaginal plug was confirmed was defined as day 1 of pseudopregnancy. Blastocysts were transferred into two groups of pseudopregnant mice on day 2.5 to non-immunodepressed females or immunodepressed females. Mice were killed on day 16 of pregnancy to analyse the number of fetal resorptions and the number of viable fetuses. Animals in which no pregnancy was established, either in controls or test embryo groups, were rejected for analysis.
Mice immunodepression
6ß-Methylprednisolone (Urbason
Soluble; Hoechst Iberica SA, Barcelona, Spain) was used in daily intraperitoneal injections of 0.3 µg/g of animal weight in one group, and 0.6 µg/g of animal weight in the other group during 5 days starting on day 1 of pseudopregnancy. To evaluate the immunodepression of mice, the concentration of immunoglobulin G (IgG) was measured by radioimmunoassay (Mouse IgG NL RID KIT, The Binding Site® Ltd, P.O.Box 4073, Birmingham, UK) in blood serum samples.
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Results |
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Immunodepression
To evaluate the effect of 6ß-methylprednisolone treatment on the immune system of the female mice, the IgG concentrations were determined in blood samples. A statistical non-parametric Mann–Whitney U-test was performed (P < 0.05). The results show a significantly lower IgG concentration in the group of females treated with 0.6 µg/g compared with non-treated and treated (0.3 µg/g) females. However, no statistically significant differences were detected when comparing the group treated with 0.3 µg/g and controls (Table I
).
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6ß-Methylprednisolone analysis
Two different rates were defined to analyse the results. Implantation rate (IMR) was defined as the number of fetal resorptions (R) plus the number of viable fetuses (VF) divided by the total number of embryos transferred (ET) [IMR = (R+VF)/ET] while developmental rate (DR) was defined as the number of viable fetuses divided by the total number of embryos transferred in one uterine horn (DR = VF/ET).
Embryo transfer: 30, 25 and 25 pregnant foster mothers were obtained from non-immunodepressed, treated with 0.3 µg/g of 6ß-methylperdnisolone and depressed with 0.6 µg/g of 6ß-methylprednisolone females respectively (Table II).
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Table II
shows the comparison of gestation rates between +ZP and –ZP embryos (Wilcoxon; P < 0.05). In non-immunodepressed females, no statistical differences in the implantation rate between +ZP and –ZP embryos were detected. However, in the same group of females, a statistically significant lower developmental rate was observed when –ZP embryos were transferred, thus reducing the mean number of developing fetuses. In contrast, both implantation and developmental rates were statistically equivalent when comparing +ZP and –ZP embryos in females treated with 0.3 µg/g 6ß-methylprednisolone. In the group treated with 0.6 µg/g 6ß-methylprednisolone, a statistically significant higher developmental rate was also observed in –ZP embryos when compared to +ZP controls, while implantation rate and mean numbers of resorbed developing fetuses remained equivalent in both groups.
On the other hand, implantation and developmental rates between the different methylprednisolone doses administered were compared (Mann–Whitney U; P < 0.05) (Table III). In the 0.6 µg/g group, statistically significant higher implantation and developmental rates of the –ZP embryos were observed when comparing with other doses (0 and 0.3 µg/g) of 6ß-methylprednisolone.
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Referring to the mean number of resorptions and developing embryos, a lower proportion of resorptions was obtained with +ZP and –ZP embryos, whereas a higher proportion of developing embryos was observed only in –ZP ones when compared to other doses (0 and 0.3 µg/g) of methylprednisolone (Table III
). No statistically significant differences were observed in any of the parameters studied when comparisons were established between the other doses of 6ß-methylprednisolone (0 and 0.3 µg/g).
Finally a correlation between the different parameters analysed and increasing doses of methylprednisolone was established (Tables IV and V) (P < 0.05). No correlation was found in the pregnancy rate of +ZP control group, while a positive one was detected in –ZP embryos (Table IV).
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For the mean number of resorptions, a negative correlation was observed in +ZP and –ZP embryos when increasing the methylprednisolone dose, while for developing fetuses a positive correlation was established only in the –ZP embryos group (Table V
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Several factors may influence the processes of implantation and embryonic development. First, the use of acid Tyrode during embryo manipulation may decrease the viability of the blastomeres (Gordon et al., 1988
; Nichols and Gardner, 1989; Cohen et al., 1990a; Van Golde et al., 1996). Furthermore, during implantation there is an increase in the number of uterine lymphocytes and of immune cells, because the transfer methodology used in mice induces a local inflammatory response, and the focus of this response is surrounded by macrophages and immunocompetent cells. Uterine cells secrete cytokines such as interleukins 1 and 6 and tumour necrosis factor 
; these substances may produce a negative environment for the embryo, decreasing its chances of early development and reducing the growth of the trophoblast. Finally, the ZP protects the embryo during its journey through the genital tract, and prevents the penetration of immunocompetent cells in the perivitelline space (due to recognition of the major histocompatibility complex or to inherent embryo transfer damage).
Taking into account all these factors, one would expect to find decreased pregnancy rates in embryos deprived of the ZP when compared to normal, +ZP embryos. However, using low doses of 6-ß-methylprednisolone (0.3 µg/g) a significant decrease in the DR when comparing +ZP and –ZP embryos (Table II) was not found, as already shown by Cohen et al. (1990b), indicating a certain positive effect of glucocorticoids on the development of –ZP embryos. The high variability in the results of this group may explain the fact that at this low concentration, the decrease in the concentrations of IgG was not significant (Table I), thus indicating that differences must be small, if any.
The positive effect of glucocorticoids on the IMR and DR of –ZP embryos became more evident when using a higher dose (0.6 µg/g) (Tables II and III). This suggests that an immunodepressive treatment could decrease the presence of lymphocytes and of the other cells of the immune system in the uterus, and allow better development and implantation of the embryos.
It is known that glucocorticoids have an anti-inflammatory effect, and inhibit the latter stages of the immune response, decreasing the blastogenic reaction of T-cells and the activity of natural killer cells, and modulating macrophage activity. This would prevent their penetration of the perivitelline space, and would also facilitate the development of the embryo, expressed as an increase of the DR. However, since the embryo is protected by the ZP, the use of glucocorticoids should not visibly improve the DR of +ZP embryos.
Looking at the females immunodepressed with the higher dose of glucocorticoids (0.6 µg/g) it may seem contradictory to find a higher DR for –ZP embryos than for controls +ZP (Table II). However, it must be considered that, in vitro, the ZP often thickens and hardens, making embryo hatching all the more difficult. On the other hand, if the ZP is fully dissolved, hatching becomes automatic. Furthermore, with high doses of glucocorticoids, the IgG concentrations decrease significantly (Table I), and the recognition of embryonic antigens capable of inducing an immune response [macrophages, immunocompetent cells, NK and lymphokine activated killer (LAK) cells] will also decrease. This would facilitate the process of embryo development and implantation (Tables IV and V).
In contrast, one must also take into account the possibility that immunodepression may facilitate opportunistic bacterial or viral infection. The transfer technique used in this work can induce lesions that may become locally infected. The transvaginal embryo transfer used in humans (Cohen et al., 1990b) can also facilitate penetration of bacteria or viruses capable of producing a local intrauterine infection. Thus, immunodepressive agents must be carefully used, to keep a balance between the increase in DR and IMR and the risk of exogenous infections facilitated by embryo transfer. The results presented here suggest that the doses used may keep this balance in the mouse. The use of a higher dose of 6ß-methylprednisolone would have to be adapted to the human situation, to determine if immunodepression could increase the DR and IMR of embryos with a damaged ZP.
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Acknowledgments |
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This work has been partially supported by Fondo de Investigación Sanitaria project 95/1773. We wish to thank Laboratoris Fornells for the IgG analysis and Dr Barceló and Dr Saez for their support in the statistical analysis.
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Notes |
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1 To whom correspondence should be addressed
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Biggers, J.D., Whitten, W.K. and Whittingham, D.G. (1971) The culture of mouse embryos in vitro. In Daniel, J.C, Jr (ed.), Methods in Mammalian Embryology. Freeman, San Francisco, p. 86–116.
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Gardner, R.L. (1988) Embryo transfer and manipulation. In Beynen, A.C. and Solleveld, H.A. (eds), New Developments in Biosciences: Their Implication for Laboratory Animal Science. Proceedings of the First Symposium of the Federation of European Animal Science Associations. Martinus Nijholf, Dordrecht, p. 147–162.
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Submitted on July 1, 1998; accepted on October 15, 1998.
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