The Center for Human Reproduction, Reproductive Immunology, 750 N. Orleans, Chicago, IL 60610, USA
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Abstract |
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Key words: antiphospholipids/autoantibodies/embryotoxicity/human/mouse embryos
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Introduction |
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It has long been thought that the mechanism of antiphospholipid antibodies (APA) in recurrent pregnancy loss is through the induction of thrombosis causing infarction of the placenta and decreased blood flow to the fetus (Alarcon-Segovia, 1988). Previous findings (Gharavi et al., 1998
; Chamley et al., 1998
) suggested that the binding of antiphospholipid antibodies to ß2-glycoprotein-I (GPI) phospholipid complex may result in abnormal physiological function of ß2-GPI as a coagulation regulator resulting in impaired trophoblast proliferation and fetal death. A role of APA in the interception of signal transduction processes has been suggested (Gleicher et al., 1992
). It has been suggested that antiphosphatidyl serine (aPS) can inhibit intercellular fusion, hormone production, and cellular invasion in the trophoblast (Katsuragawa et al., 1997
). These may occur by direct interference with intertrophoblastic adherence and intermembrane fusion, alteration of necessary PS-dependent signal transduction pathways, and interference with the endotrophoblastic control of maternal coagulation in the spiral arteries (Rote, 1996
).
In addition to an association between APA and recurrent pregnancy loss, there is a number of studies showing an increased frequency of APA among patients undergoing in-vitro fertilization (IVF) and embryo transfer for the treatment of infertility (Geva et al., 1994; Sher et al., 1994
; Kaider et al., 1996
; Coulam et al., 1997
). However, controversy has developed regarding the significance of the increased APA and pregnancy outcome of the IVF cycles (Birkenfeld et al., 1994
; Geva et al., 1994
; Lynch et al., 1994
; Sher et al., 1994
; Birdsall et al., 1996
; Denis et al., 1997
; Stern et al., 1998
). While explanations for the controversial results, including differences in methodology of performing and interpreting APA assays, are being addressed by the American Society of Reproductive Immunology in the antiphospholipid antibody workshop (Coulam, 1999
), there remains the possibility that autoantibodies utilize a separate mechanism to impair implantation of an otherwise normal embryo that is bypassed by the IVF and embryo transfer procedure (Chamley et al., 1998
). That IgG and IgA are present in the uterus and Fallopian tubes as a transudate from serum and that IVF takes the pre-embryo out of this environment makes this possibility feasible (El-Roeiy et al., 1987
). The present study was designed to answer two questions: firstly, do autoantibodies selectively bind to embryos and, secondly, what effect do these antibodies have on the embryos?
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Materials and methods |
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Mouse embryos
Superovulation was induced in CB6F1/J mice by pregnant mare's serum gonadotrophin (PMSG) (10 IU i.p.) and human chorionic gonadotrophin (HCG) (10 IU i.p. after 48 h) stimulation, and mated with CB6F1/J males. The female mice were killed 48 h after mating. Two-cell embryos were collected by sharp dissection of the Fallopian tubes, and used in the experiments.
Labelling of embryos
The labelling experiments were performed as 10 embryos per dish were cultured for each isolated immunoglobulin. The experiments were performed three times for each of the tested antibodies. In the dishes that were supplemented with purified APA antibodies, 20 µl of adult bovine serum (ABS; Sigma) was added as a source of cofactor. Embryos were cultured with three different amounts of APA calibrator's (Harris controls) antibodies, 15 µl/ml, 35 µl/ml, and 100 µl/ml. On 3 consecutive days, three embryos from each dish were carefully washed in a series of dishes containing 2 ml human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA) each, and incubated for 1 h with monoclonal F(ab')2 anti-human IgG fluorescein isothiocyanate (FITC) or IgA FITC conjugate (Jackson ImmunoResearch, West Grove, PA, USA). The non-specific binding of the conjugated second antibody was controlled by culturing embryos only in the presence of the second antibody. After washing to remove excess conjugate, the embryos were examined for the presence of immunofluorescence using a fluorescent microscope.
Culturing procedure (embryotoxicity assay)
The collection and culturing of the embryos for this test, as previously described and validated (Roussev et al., 1995) were the same as described above except that the embryos remained in the dishes for the entire 3 day period. At least 15 embryos, from three different mice, were cultured in 2 ml HTF medium (Irvine Scientific) supplemented with 1% vol/vol adult bovine albumin, and 100 µl10 mg/ml of one of the purified antibodies. The tests were performed with each of the isolated immunoglobulins three times on 3 different days. On the third day, embryos were examined to determine their stage of development. The following developmental stages were recorded: blastocyst, early blastocyst, morula, 2- to 8-cell stage, and atretic. The labelling experiments and embryotoxicity assay were performed in parallel.
Statistical analysis
The results from embryotoxicity test between the groups were analysed by Fisher's exact test and/or Student's t-test on Statistical Package for Social Sciences (SSPS) software. The data are presented as percentage mean ± SD of three different experiments.
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Results |
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General observations
Embryos that were removed from culture and labelled during the 2- to 4-cell stage showed stronger immunofluorescence on the zona pellucida, with the embryonic cells being weakly immunofluorescent. Embryos that were removed from culture and labelled in the morula or early blastula stage showed little immunofluorescence on the zona, and significant labelling on the embryonic cells. No differences in fluorescence intensity in earlier or later stages of embryo development were observed.
Embryotoxicity assay
Embryos cultured with controls
An average of 70% of the embryos cultured with purified IgG control antibodies reached the blastula stage, 10% early blastula, and 20% morula. None of the embryos became atretic, or arrested in the 2- to 8-cell stage. Of those cultured with purified IgA control antibodies, on average 60% reached the blastula stage, 10% early blastula, and 30% morula (Figures 2, 3, 4 and 5).
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Embryos cultured with IgG isolated from ANA positive sera
All embryos cultured in the presence of purified IgG from ANA positive sera experienced significant growth impairment or death when compared to those cultured in intact control IgG. Of those embryos cultured with the IgG from multispecific ANA positive sera, on average 10% reached the blastula stage, 20% early blastula, 10% morula, 30% arrested at the 2- to 8-cell stage, and 30% became atretic. Of the IgG purified from monospecific positive ANA sera, the anti-ssDNA was associated with the most damage to embryos with none reaching the blastula stage, 20% early blastula, 20% morula, 30% arresting at the 2- to 8-cell stage, and 30% becoming atretic (Figure 4).
Embryos cultured with IgG isolated from ATA positive sera
All embryos cultured in the presence of purified IgG from ATA positive sera experienced growth success similar to those cultured in intact control IgG antibodies; on average 60% reached the blastula stage, 30% early blastula, and 10% morula. Only 10% arrested at the 2- to 8-cell stage, and none became atretic (Figure 5).
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Discussion |
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There is, however, mounting evidence to suggest that these autoantibodies may play a role in implantation failure as well. Several studies have indicated higher prevalence rates of autoantibodies in patients with IVF failure than those who succeed on IVF (Kalunian et al., 1988; Birkenfeld et al., 1994
; Sher et al., 1994
; Coulam et al., 1997
; Geva et al., 1997
; Stern et al., 1998
). It was demonstrated that autoantibodies found in the serum were also present in the follicular fluid of the same women (El-Roeiy et al., 1987
). Consequently, it is reasonable to suspect that these antibodies may have an effect on implantation. The question then becomes whether the antibodies affect the uterus or the embryo, or both?
Using a mouse model, it was demonstrated (Tartakovsky et al., 1996) that both the maternal (uterine) and embryonic compartment are defective as a result of their exposure to aCL. Embryos derived from aCL immunized mice were absorbed and never implanted even after their removal from the aCL environment by transfer into a normal uterus. The authors concluded that the harmful effect of aCL on implantation might be due to binding of aCL to embryos following APA secretion into the uterine lumen. Similar conclusions were drawn from previous studies related to different models of pregnancy loss (Tartakovsky and Ben-Yair, 1991
). Anticardiolipin but not control antibodies were shown to bind specifically to the trophectoderm cell lineage of embryonic growth (Sthoeger et al., 1993
). The findings in the present study support those of Sthoeger et al. (1993) in that APA and ANA bind directly to embryos in vitro, while ATA do not. The precise epitopes that these antibodies are binding to are not clear because there are no nuclear antigens or phospholipids on the surface of the zona. Perhaps they are recognizing some glycerol moiety or protein cofactor. The binding, however, is specific as ATA and control antibodies show no evidence of binding. Recent evidence indicates that APA may not bind directly to phospholipid but rather to phospholipid-binding proteins or to a complex of both (Chamley, 1997
; Chamley et al., 1998
). We excluded the possibility of the ß2-GPI role in the observed phenomena because of the reactions with positively charged phospholipids and PE (which antibodies required different co-factor than the others and from controls). The albumin was added to support the embryo's growth, and the intact (control) IgG, IgA did not react in the presence of ABS. For phospholipid antibodies without ABS the bindings were still positive, but stronger when ABS was in the culture media.
Evidence exists that APA have a direct negative effect on the embryos. Anticardiolipin was found in 50% of women undergoing IVF and embryo transfer who had abnormal embryo morphology compared with 13% with normal embryo morphology (P = 0.001) (Azem et al., 1998). A set of nuclear proteins is transiently synthesized in mice at the 2-cell stage as well as changes in embryonic chromatin composition suggesting that early embryos possess epitopes both for ANA and APA (Clarke, 1992
). Immunofluorescence staining of living cells after incubation with ANA is still one of the useful methods for identification of these antibodies. The data in this study suggest that certain APA and ANA may have a greater effect on embryos than others. Specifically 70% of the embryos cultured in the presence of aPC, and 60% of those cultured with anti-ssDNA, were killed or had their development arrested at the 2- to 8-cell stage. Interestingly, while both IgA and IgG from CL APL calibrator controls (Louisville APL Laboratories) bound to embryos and exhibited immunofluorescence, the IgA antibodies had no negative impact on embryo development, while those cultured with IgG antibodies all arrested at two cells or became atretic. This observation may be attributable to the specificities present in each of the respective controls. Thus it appears that a number of APA and ANA can have detrimental effects on pre-embryos. Our results from embryotoxicity tests when using different concentrations of immunoglobulins from APL calibrators suggested the importance of the APA titres in vivo. With this in mind the value of screening for single APA in individuals with implantation failure is brought into question. We therefore recommend a complete evaluation of ANA (Purvis et al., 1996
) and APA panels consisting of seven phospholipids and three isotypes (Coulam et al., 1997
) when investigating women experiencing reproductive failure. Recent research has significantly advanced understanding of the association between autoantibodies with reproductive failure. A key advance has been the elucidation of the antigenic specificities of APA, i.e. different autoantibodies have different effects (Roubey, 1996
). Thus, there may be several different mechanisms, affecting several different tissue types, by which autoantibodies exert their effect on reproductive success and different autoantibody specificities may play roles in each of these pathways. Yet some studies report results of aCL and aPS as indicative of APA in general (Birdsall et al., 1996
). In the present study, aPS antibodies showed dramatically less activity than aPC or aPE, for example. It has been previously reported that in IVF failure patients, there was a 21.4% prevalence of aPC (Kaider et al., 1996
). In fact, among those patients who had at least one positive APA, 81.8% had aPC. There was one patient in this group who had aPC in all three isotypes and no other detectable APA. Only 2.4% of IVF failure patients had elevated aPS, and 7.1% had elevated aCL. It is possible that aPS plays a significant role in first trimester loss through altered trophoblast function and aCL is primarily involved in thrombosis, while aPC affects implantation. Further investigation into the role of this antibody is necessary, and warranted by preliminary findings.
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Notes |
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References |
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Submitted on February 15, 1999; accepted on June 21, 1999.