Murine embryos as a direct target for some human autoantibodies in vitro

B.D. Kaider, C.B. Coulam and R.G. Roussev1

The Center for Human Reproduction, Reproductive Immunology, 750 N. Orleans, Chicago, IL 60610, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The involvement of one or another autoantibody in reproductive failure have long been thought to be through post-implantation thrombosis and/or peri-implantation trophoblast dysfunction and/or maternal hormonal imbalance. It can be postulated that the embryo may be a direct target for some autoantibodies prior to implantation. Mouse embryos have been labelled and cultured with affinity purified immunoglobulin (IgG) and IgA from positive for antiphospholipid antibody sera, as well as IgG from positive for antinuclear antibody sera and positive for antithyroid antibody sera. Intact IgG and IgA from healthy individuals were used as controls. All embryos cultured with purified antiphospholipid IgG or IgA, and anti-nuclear IgG exhibited strong immunofluorescence. No difference in fluorescent intensity was observed whether antiphospholipid or anti-nuclear antibodies were used, but the pattern of antibody distribution seemed to be different. Antiphospholipid IgG was more dominant on the zona pellucida, while antiphospholipid IgA and antinuclear IgG had predominant distribution on the embryonic cells. None of the embryos cultured with antithyroid IgG or with control immunoglobulins showed strong immunofluorescence. Embryos cultured with purified antiphospholipid and antinuclear immunoglobulins experienced significant growth impairment or death compared to those cultured with antithyroid or control immunoglobulins.

Key words: antiphospholipids/autoantibodies/embryotoxicity/human/mouse embryos


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years countless papers have been published discussing the association between autoimmune antibodies and reproductive failure (Gleicher et al., 1992Go; Rote et al., 1992Go; Birdsall et al., 1996Go; Rote, 1996Go; Roussev et al., 1996Go; Tartakovsky et al., 1996Go; Katzuragawa et al., 1997). Early studies focused on anticardiolipin and the lupus anticoagulant (Lockshin et al., 1985Go; Cowchock et al., 1992Go; Birkenfeld et al., 1994Go), while later research showed more interest in the other phospholipids (Kalunian et al., 1988Go; Rote et al., 1992Go; Kaider et al., 1996Go; Roussev et al., 1996Go; Gleicher, 1997Go; Branch, 1998Go), as well as antinuclear antibodies (ANA) (Birkenfeld et al., 1994Go; Roussev et al., 1996Go; Iijima et al., 1997Go) and antithyroid antibodies (ATA) (Pratt et al., 1993Go; Roussev et al., 1996Go; Iijima et al., 1997Go). The importance of testing three isotypes [immunoglobulin (Ig)A, IgG, and IgM] of antiphospholipid antibodies has also been stressed by several investigators (Kalunian et al., 1988Go; Kaider et al., 1996Go; Roussev et al., 1996Go).

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, 1988Go). Previous findings (Gharavi et al., 1998Go; Chamley et al., 1998Go) 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., 1992Go). It has been suggested that antiphosphatidyl serine (aPS) can inhibit intercellular fusion, hormone production, and cellular invasion in the trophoblast (Katsuragawa et al., 1997Go). 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, 1996Go).

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., 1994Go; Sher et al., 1994Go; Kaider et al., 1996Go; Coulam et al., 1997Go). However, controversy has developed regarding the significance of the increased APA and pregnancy outcome of the IVF cycles (Birkenfeld et al., 1994Go; Geva et al., 1994Go; Lynch et al., 1994Go; Sher et al., 1994Go; Birdsall et al., 1996Go; Denis et al., 1997Go; Stern et al., 1998Go). 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, 1999Go), 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., 1998Go). 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., 1987Go). 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?


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Purification of antibodies
From a pool of more than 8000 archived sera from patients with different reproductive problems (including recurrent pregnancy loss, unexplained infertility and IVF failure) that had been tested for APA, ANA, and ATA, sera with consistency on their reaction against specific antigen were selected for immunoglobulin purification. Three sera with specific IgG antibodies against phosphatidyl serine (PS), three against phosphatidyl glycerol (PG), and three against phosphatidylcholine (PC) respectively, as well as three sera with a variety of specificities (multispecific), were used. Their IgG with antiphospholipid reactivity was affinity-purified by solid-phase heparin-agarose chromatography (Sigma, St Louis, MO, USA). IgA from two sera and plasma with specific IgA antibodies against phosphatidylinositol (PI) and phosphatidylethanolamine (PE) were isolated by immobilized Jacalin® (specifically IgA binding lectin) affinity chromatography (Pierce, Rockford, IL, USA). Immunoglobulin G from three sera with specific IgG against double stranded (ds)DNA, three sera positive against single stranded (ss)DNA, and two sera positive against histones; and four sera with IgG against thyroglobulin and thyroid microsomal antigens were purified by protein-A sepharose affinity chromatography (Sigma). IgG and IgA were purified from two male and three female normal healthy individuals, negative when tested for APA, ANA, and ATA, and used as controls. The antibody concentrations were adjusted to 10 mg/ml and retested to check their specific activity as purified immunoglobulins. Our collection of patient sera included none that reacted specifically only with cardiolipin (CL). Although APA calibrators [i.e. Harris standards, or anticardiolipin standards, provided by Louisville APL Diagnostics Inc. (Louisville, KY, USA) with known amount of IgG, IgM, and IgA antibodies expressed as IgG anticardiolipin (GPL), IgM anticardiolipin (MPL) and IgA anticardiolipin (APL) units respectively] are also not specific only for anticardiolipin (aCL), they are undeniably positive for this antibody, and were included to assess its possible effects. IgA (A4: 120 APL units) and IgG (GM1: 125.5 GPL units and 83.7 MPL units) from APA calibrators were used. Because the APA calibrators are serum-derived and may contain complement, they were heat-inactivated at 56°C for 30 min. Each antibody has been used separately in the experiments.

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., 1995Go) 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 µl–10 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Immunofluorescence (Figure 1Go)
Embryos cultured with purified Ig from APA, ANA, and ATA positive sera
All embryos cultured with purified IgG or IgA from APA positive sera, whether monospecific, Louisville APL controls, or multispecific, exhibited strong immunofluorescence. There appeared to be no difference in fluorescent intensity whether the APA was IgG or IgA, but the pattern of antibody distribution seemed to be different. IgA APA was bound to the embryonic cells, while the IgG APA was more dominant on the zona pellucida, and only attached to embryonic cells when the embryo became atretic. All embryos cultured with purified IgG from ANA positive sera exhibited immunofluorescence, with the predominant distribution of antibodies on the embryonic cells. None of the embryos cultured with purified IgG from ATA positive sera showed immunofluorescence. The embryos cultured with GM1 Louisville APL controls were also labelled with rabbit anti-human IgM FITC antibodies and exhibited very strong immunofluorescence. However, the embryos cultured only with rabbit anti-human IgM FITC control antibodies showed the same amount of immunofluorescence as well. This was attributed to the non-specific high affinity of the IgM molecule.



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Figure 1. Immunofluorescent views of embryos cultured in immunoglobulin (IgG) isolated from single stranded (ss)DNA positive serum (A, original magnification x60) and multispecific antinuclear antibodies (ANA) positive serum (B, original magnification x100). C: embryo cultured in IgG isolated from multispecific antiphospholipid antibody (APA) positive serum (original magnification x100). D: embryos cultured in IgG isolated from antiphosphatidyl serine (aPS) positive serum (left) and anticardiolipin (aCL) positive serum (right)(original magnification x200). E: embryos cultured with IgA isolated from multispecific APA positive serum (original magnification x60). F: embryos cultured in intact IgG control (original magnification x100). G: embryos cultured in intact IgA control (original magnification x100). H: embryo cultured in IgG isolated from antithyroglobulin positive serum (original magnification x200).

 
Embryos cultured with IgG and IgA isolated from controls
From 30 embryos cultured with purified control IgA or IgG from normal healthy individuals only three showed weak immunofluorescence with IgG.

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 5GoGoGoGo).



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Figure 2. Stages of embryo development in embryotoxicity assay performed with isolated IgG from APA positive sera. BL = blastocyst, EB = early blastula, M = morula, 2–8 = embryos arrested at 2- to 8-cell stage, ATR = atretic (mean ± SD; P < 0.001 for all tested specificities), anti-PC = antiphosphatidyl-choline, anti-PS = antiphosphatidyl serine, anti-PG = antiphosphatidyl glycerol.

 


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Figure 3. Stages of embryo development in embryotoxicity assay performed with isolated IgA from APA positive sera. BL = blastocyst, EB = early blastula, M = morula, 2–8 = embryos arrrested at 2- to 8-cell stage, ATR = atretic [mean ± SD;P < 0.001 for antiphosphatidylinositol (anti-PI) and phosphatidylethanolamine (PE) tested specificities].

 


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Figure 4. Stages of embryo development in embryotoxicity assay performed with isolated IgG from ANA positive sera. BL = blastocyst, EB = early blastula, M = morula, 2–8 = embryos arrested at 2- to 8-cell stage, ATR = atretic (mean ± SD; P < 0.001 for all tested specificities), dsDNA = double stranded DNA, ssDNA = single stranded DNA, Hist = histone.

 


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Figure 5. Stages of embryo development in embryotoxicity assay performed with isolated IgG from ATA positive sera. BL = blastocyst, EB = early blastula, M = morula, 2–8 = embryos arrested at 2- to 8-cell stage, ATR = atretic, TG = thyroglobulin, mcrs = microsomal (mean ± SD; no significant differences with controls).

 
Embryos cultured with Ig purified from APA positive sera
All embryos cultured in the presence of purified APA IgG experienced significant growth impairment or death when compared to those cultured in control antibodies (Figure 2Go). Of those embryos cultured with the multispecific APA IgG, 20% reached the blastula stage, 10% early blastula, 30% morula, and the remaining 40% arrested at the 2- to 8-cell stage. Of the IgG isolated from positive APA sera with monospecific antibodies that were tested, the antiphosphatidylcholine (aPC) was associated with the most damage to embryos, with none reaching the blastula stage, 20% early blastula, 10% morula, 40% arresting at the 2- to 8-cell stage, and 30% becoming atretic. Anti-PI and anti-PE IgA impaired embryos growth (Figure 3Go) but not as much as IgG. Of the embryos cultured with IgA isolated from APL calibrators (A4), 50% grew to the blastula stage regardless of antibody concentration, while most of those cultured with 15–35 µl/ml of GM1 (IgG and IgM) antibodies arrested at two cells, and all of those cultured with 100 µl/ml became atretic (Figures 2 and 3GoGo).

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 4Go).

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 5Go).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
For years, investigators have studied the role of autoantibodies in recurrent pregnancy loss and intrauterine growth retardation (IUGR) (Gleicher et al., 1992Go; Rote et al., 1992Go; Birdsall et al., 1996Go; Roussev et al., 1996Go; Tartakovsky et al., 1996Go; Gleicher, 1997Go; Katzuragawa et al., 1997). Much evidence has been found which suggests that APA, in particular, can induce thrombosis in the spiral arteries, causing infarction of the placenta and decreased blood supply to the fetus (Alarcon-Segovia, 1988Go; Gharavi et al., 1998Go). Depending on the severity of the infarction, this can result in late first, second or third trimester miscarriage or IUGR. Katsuragawa et al. (1997) have also reported evidence strongly suggesting that APA, particularly aPS but not aCL, can dramatically impair several trophoblast functions including intercellular fusion, hormone production and cellular invasion causing reproductive failure in the first trimester.

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., 1988Go; Birkenfeld et al., 1994Go; Sher et al., 1994Go; Coulam et al., 1997Go; Geva et al., 1997Go; Stern et al., 1998Go). It was demonstrated that autoantibodies found in the serum were also present in the follicular fluid of the same women (El-Roeiy et al., 1987Go). 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., 1996Go) 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, 1991Go). Anticardiolipin but not control antibodies were shown to bind specifically to the trophectoderm cell lineage of embryonic growth (Sthoeger et al., 1993Go). 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, 1997Go; Chamley et al., 1998Go). 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., 1998Go). 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, 1992Go). 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., 1996Go) and APA panels consisting of seven phospholipids and three isotypes (Coulam et al., 1997Go) 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, 1996Go). 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., 1996Go). 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., 1996Go). 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.


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 15, 1999; accepted on June 21, 1999.