Reduced placental growth and hCG secretion in vitro induced by antiphospholipid antibodies but not by anti-Ro or anti-La: studies on sera from women with SLE/PAPS.

Systemic lupus erythematosus (SLE) and primary anti-phospholipid syndrome (PAPS) are autoimmune diseases causing recurrent pregnancy loss. We hypothesized that anti-phospholipid antibodies (aPL), but not anti-Ro and anti-La antibodies, might have a role through direct placental damage. We cultured human placental explants in sera from women with SLE/PAPS with different antibodies. These sera were found to reduce placental growth and increase trophoblastic apoptosis. No effect was found on estradiol or progesterone secretion, but inhibition in betahCG secretion was detected. BetahCG was reduced in women with a history of recurrent pregnancy loss or thromboembolic events, and was also the most sensitive marker when examining the effects of specific antibodies. High titers of aPL were found to cause the largest reduction in betahCG. Anti-Ro and anti-La did not induce placental damage. A strong correlation was found between the rise in the number of different antibodies in the sera and the incidence of recurrent pregnancy loss, which was also accompanied by a decline in the betahCG levels. In conclusion, aPL, but not anti-Ro or anti-La, may cause placental damage in vitro. Thus betahCG levels might constitute a predictive marker for the risk of placental damage and pregnancy loss in women with SLE/PAPS.

A simple method of culture of 11.5-day-old rat embryos in DMEM/F12 and 20% fetal bovine serum.

Post-implantation mouse and rat embryos are usually cultured in sera obtained from various rodents and from humans. We describe here a simple and inexpensive culture medium--Dulbecco's minimal essential medium/Ham's F12 (DMEM/F12) supplemented with 20% fetal bovine serum (FBS)--in which E11.5 rat embryos of the Hebrew University Sabra strain grow for 28 h slightly better than in heat-inactivated 90% rat serum with 10% distilled water and addition of 1 mg mL(-1) glucose. They have good growth and development and a low rate of anomalies, similar to that observed in embryos cultured on rat serum. Their size, total score and olfactory system development is slightly higher than in rat embryos cultured in rat serum. About 7.2% of these embryos exhibit subcutaneous haemorrhages in various areas, but these do not seem to interfere with their growth and development. This method is not appropriate for younger embryos; in E10.5 embryos cultured for 28 h we found increased embryonic death and anomalies.

The effects of antiphospholipid antibodies obtained from women with SLE/APS and associated pregnancy loss on rat embryos and placental explants in culture.

Recurrent fetal loss occurs in approximately 1% of women. Autoimmune causes have been suggested as a factor in some of these cases. High rates of intrauterine fetal growth retardation and increased incidence of prematurity is associated with systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS). We found in previous studies that sera from SLE/APS patients when used as a culture medium for rat embryos were found to reduce embryonic growth and development, induce a high rate of embryonic anomalies and death and damage the yolk sac morphologically and functionally. In order to investigate the direct effect of IgG purified from women with SLE/APS on the growth and viability of embryos, we cultured 11.5-day-old rat embryos in their yolk sacs in the presence of IgG purified from SLE/APS patients with recurrent pregnancy loss (RPL). The IgG affected directly the embryo and yolk sac, reducing their growth. The purified IgG positive for anticardiolipin/anti-DNA antibodies reduced yolk sac and embryonic growth more than sera negative for these antibodies but positive for antiphosphatydilserine and for antilaminin. Monoclonal antiphosphatydilserine reduced yolk sac growth but the embryos remained intact. Following the observed damage to the yolk sac we cultured human placental explants at 5.5-8 weeks of pregnancy in sera from SLE/APS patients for 96 hours and found that these sera reduced placental trophoblastic cell growth, reduced their proliferation rate and increased their rate of apoptosis. Successful treatment of the women resulted in a correction of the damage induced in the cultured rat embryos and in the cultured placental explants.

The effects of IgG purified from women with SLE and associated pregnancy loss on rat embryos in culture.

PROBLEM: Recurrent fetal loss occurs in approximately 1% of women. Autoimmune causes have been suggested as a factor in some of these cases. High rates of intrauterine fetal growth retardation and increased incidence of prematurity is associated with systemic lupus erythematosus (SLE) and the anti-phospholipid syndrome (APS). Autoantibodies from sera of SLE/APS patients affect reproductive outcome in pregnant mice, as was studied in vivo, where injection of immunoglobulin (Ig)G purified from patients with APS to mice caused fetal resorptions and growth retardation. METHODS: In order to investigate the direct effect of IgG purified from women with SLE or APS on the growth and viability of embryos, we cultured 11.5-day old-rat embryos in their yolk sacs in the presence of IgG purified from SLE and APS patients. RESULTS: IgG purified from SLE and recurrent pregnancy loss (RPL) patients affected directly the embryo and yolk sac reducing their growth. The purified IgG positive for anti-cardiolipin/anti-DNA antibodies reduced yolk sac and embryonic growth more than sera negative for these antibodies. CONCLUSION: Various antiphospholipid antibodies affect differently the growth and development of the embryo and the placenta.

The effect of sera from women with systemic lupus erythematosus and/or antiphospholipid syndrome on rat embryos in culture.

Women with systemic lupus erythematosus (SLE) with or without antiphospholipid antibodies (APLA) suffer from a high rate of recurrent abortions perhaps as a result of specific antibodies that may damage the conceptus. We studied the effects of sera from women with SLE--with or without--APLA and recurrent abortions on 10.5-d-old rat embryos in culture. This was compared to the results of culture on sera from control women and on rat sera. In addition, we studied sera from women with SLE with or without APLA after treatment with low doses of aspirin and glucocorticosteroids. Seventy-three percent of embryos cultured in sera from women with SLE with or without APLA were malformed in comparison to only 10.2% in embryos cultured on control sera and 5.4% in embryos cultured on rat sera. The rate of anomalies was reduced to 37.5% in embryos cultured on sera from women with SLE with or without APLA after treatment, as in 6 of 13 sera, the treatment reduced or prevented the occurrence of embryonic anomalies. When sera were divided in to low- and high-risk sera, the effect of treatment was even more significant, as the average percentage of embryonic anomalies per serum was reduced from 81.7 to 44.7%. Specific ultrastructural changes were found in the yolk sacs of the embryos cultured on the sera from women with SLE with or without APLA by transmission electron microscopy and by scanning electron microscopy. It seems that the rat embryo culture system may be an important clinical diagnostic tool to identify women with recurrent abortions in whom the etiology may be immunologic rejection of the embryo and to assess the efficacy of various treatment modalities.