Pathomorphologic and immunohistochemical study on the devastation of rat embryos by antiphospholipid antibody positive serum.

PROBLEM: While relying on previous publications, our aim was to examine the morphologic changes, induced in early rat embryos by intra-uterine exposure to the low-molecular weight fraction of boiled human serum containing antiphospholipid antibodies (APLA) that had been obtained from women with antiphospholipid syndrome (APS). METHOD OF STUDY: Human APLA-positive sera were pooled, boiled, centrifuged and separated by ultrafiltration. The molecular weight fraction lower than 30 kDa was used for the experiments. One hundred and fifty microlitres was injected into one uterine horn of 12 pregnant rats, 5 or 6 days after fertilization, while similarly prepared normal human serum or saline were injected into the contralateral horn. The rats were subsequently sacrificed. Serial sections, obtained from all uterine horns, were stained histologically and immunohistochemically. Normal embryos developed in the control uterine horns, while embryos in the experimental horns were destroyed rapidly. RESULTS: Signs of apoptosis appeared 2 hr following the injection, and 4 hr later all the embryonic cells were apoptotically destroyed. There was only partial damage to cytotrophoblasts and intermediate trophoblasts. CONCLUSION: These findings support the existence of a novel factor in the APLA-positive serum, causing a detrimental effect to the conceptus, without any relation to the antiphospholipid antibodies.

Effect of intrauterine injection of casein on fetal survival in rat: a new pharmacological approach for contraception.

BACKGROUND: The incidental finding of casein as a possible new local pharmacological contraceptive prompted us to assess its validity in an experimental rat model. METHODS: The intrauterine injection of 150 microg of bovine alpha-casein dissolved in 150 microL phosphate-buffered saline (PBS) was performed on one uterine rat horn on days L(5)-L(7), whereas the contralateral horn was used for injection of 150 microL PBS as a control. Intraperitoneal injection of alpha-casein (5 mg/mL, 2 mL/rat) was performed on day L(5). The rats were killed by cervical dislocation on the day L(14). RESULTS: The effect of an alpha-casein on fetal resorption rate was assessed following the unilateral injection of 150 microL of alpha-casein (1 mg/mL in PBS) and compared with the effect obtained following the contralateral injection of 150 microL PBS. The unilateral injection of alpha-casein on day L(5) caused a significant increase in fetal resorption rate as compared with the contralateral uterine horn injected with PBS (p<.00001). The decrease in alpha-casein concentration from 1 to 0.3 mg/mL caused a reduced, but still significant, effect on fetal resorption rate (p<.0001). The injection on days L(6)-L(7) caused a local effect of resorption near the injection site. There was no effect on fetal resorption rate following the injection of alpha-casein intraperitoneally. CONCLUSION: Our data suggest a new pharmacological approach for contraception, based on local intrauterine effect of alpha-casein in an experimental rat model. The exact mechanism of action related to casein should be further studied.

Human decidua-associated protein 200 neutralizes the detrimental effect of serum containing antiphospholipid antibodies on fetal survival in the rat.

PROBLEM: We wanted to examine whether the detrimental effect of serum containing antiphospholipid (APL) antibodies on rat pregnancy outcome can be neutralized by addition of human decidua-associated protein (hDP) 200, a kind of rheumatoid factor, extracted from decidual tissue. METHODS: Fifty microliters of pooled serum, obtained from women having anticardiolipin antibody and lupus anticoagulant and presenting with APL antibody syndrome, added with 100 microL of immunoaffinity purified hDP200 was injected into unilateral uterine horn of each rat on day L5 of rat pregnancy. The contralateral uterine horn was used for injection of 50 microL APL positive serum added with physiologic saline as a control. The rats were killed on day L14, and the uterus of each rat was inspected for the presence of live and resorbed fetuses. RESULTS: The addition of hDP200 to APL positive serum before the intrauterine injection neutralized the detrimental effect of APL serum on fetal resorption rate. Although the neutralizing effect was demonstrated following the addition of hDP200 and immediate intrauterine injection (a decrease in fetal resorption rate, P = 0.004), still the effect was more impressive following the addition of hDP200 and incubation period of 24 hr before the injection, thus causing a significant increase in the number of normal embryos and a significant decrease in fetal resorption rate (P < 0.001). CONCLUSION: Human decidua-associated protein 200, extracted from human decidual tissue neutralizes the detrimental effect of serum containing APL antibodies in an experimental rat model. Further studies are needed to prove this finding.

Influence of 17beta-estradiol on the synthesis of reduced neurosteroids in the brain (in vivo) and in glioma cells (in vitro): possible relevance to mental disorders in women.

Brain neurosteroids modulate gamma-aminobutyric acid type A (GABAA) receptor activity, thereby playing a role in mood disorders. Alterations in 17beta-estradiol (E2) and progesterone (P) are also known to play a significant role in psychopathology in women. The aim of the present study was to evaluate the synthesis of dihydroprogesterone (DHP), tetrahydroprogesterone (THP), and the activity of 5alpha-reductase (5alphaR) which regulates the reduction of P to DHP on exposure to supraphysiological levels of E2 in vitro (C6 glioma cells) and in vivo (mouse brain). The results showed that supraphysiological levels of E2 induced a decrease in the accumulation of both neurosteroids, probably by decreasing the activity of 5alphaR. We hypothesize that the high levels of E2 in pregnancy attenuate the increase in the conversion of P to THP in the brain and that the ratio of E2/P modulates the sedative effect of THP. This process may be relevant to psychopathological disorders that are ascribed to drastic alterations in estrogen levels, such as premenstrual syndrome, pregnancy-related mental disorders, and postpartum "blues".

The influence of parturition on the level and synthesis of sulfated and free neurosteroids in rats.

Alterations in neurosteroid levels may play a role in affective disorders including those related to changes in the levels of ovarian steroids. The effects of pregnancy and delivery on circulatory and brain levels of dehydroepiandrosterone (DHEA), pregnenolone (PN), their sulfate esters and the enzymatic activities of sulfatase and sulfotransferase were examined in rats. Our findings indicate an increase, not reflected in the brain cortex, in serum DHEA levels, at the end of pregnancy with a partial decrease following delivery. DHEA sulfate levels in the cortex and PN levels in both serum and cortex decreased following delivery with no changes in its sulfated form. Sulfatase levels were high both before and after delivery with no changes noted in sulfotransferase levels, compared to controls. We speculate that changes in the level or ratio of sulfated and free neurosteroids may play a role in postpartum behavioral disorders due to their antagonistic GABA(A) modulatory effect.

Fetal survival in the rat as affected by intrauterine injection of serum containing antiphospholipid antibodies.

PROBLEM: We wanted to study and examine whether the detrimental effect of antiphospholipid antibodies on gestation is locally derived. Therefore, we established an experimental rat model, enabling to study the intrauterine effect of serum, containing antiphospholipid antibodies, in vivo. METHODS: Sera were obtained from five women, having anticardiolipin antibody and lupus anticoagulant, and presenting with antiphospholipid antibody syndrome. Pooled serum of 150 microL was injected into unilateral uterine horn of each rat on days L2-L7 of rat pregnancy. The contralateral uterine horn was used for injection of 150 microL normal serum or physiologic saline as a control. The rats were sacrificed on day L14, and the uterus of each rat was inspected for the presence of live and resorbed fetuses. RESULTS: A significant effect on fetal resorption and viability was observed following the intrauterine injection of antiphospholipid positive serum (APL positive serum), as compared with the intrauterine injection of normal serum or saline. A significant increase in fetal resorption rate and decrease in fetal viability was noted following the injection of APL positive serum on day L6 (P = 0.005 and 0.004, respectively) and day L7 (P = 0.003 and 0.003, respectively) as compared with normal serum. A comparison with the contralateral injection of saline demonstrated a significant effect of the APL positive serum also following the intrauterine injection on days L3 and L5. CONCLUSIONS: The current study provides an experimental rat model, which can be employed to study the local intrauterine effect of APL positive serum on pregnancy outcome and to further assess the efficacy of various treatment modalities.