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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Key words: developmental rate/embryo transfer/immunodepression/implantation rate/methylprednisolone
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Introduction |
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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 IVFembyo 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 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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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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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 (MannWhitney 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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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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Discussion |
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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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Notes |
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References |
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Submitted on July 1, 1998; accepted on October 15, 1998.