Mesenchymal stem cells therapy protects fetuses from resorption and induces Th2 type cytokines profile in abortion prone mouse model.

The imbalance of Th1/Th2 cytokines is well known in recurrent spontaneous abortion (RSA) mouse model. Mesenchymal stem cells (MSCs) possess potent immunoregulatory properties that could modulate the Th1 cytokine responses in benefit of Th2 types. In this study, we aimed to analyze the local and systemic balance of Th1/Th2 cytokines following MSCs therapy. Syngeneic adipose derived MSCs were administered to abortion prone mice during the implantation window. The abortion rate was determined and IL-4, IL-6, IL-12, IL-2, IFN-γ and GM-CSF gene expression was evaluated by Real-Time-PCR in decidual and placental tissues of pregnant mice at day 13.5 of pregnancy. Splenocytes of pregnant mice were co-cultured with mitomycin C treated paternal splenocytes and IL-2, IL-4, IL-10 and IFN-γ cytokines were measured in co-cultures supernatants by ELISA method. Proliferation response of female splenocytes to paternal antigens was also evaluated using the CFSE method. Our results showed a significant reduction in abortion rate following MSCs administration in abortion prone mice. We also observed a significant down-regulation of IL-2 and IFN-γ as well as up-regulation of IL-4 and IL-10 production from pregnant mouse splenocytes following MSCs therapy along with a significant reduction of splenocytes proliferation against paternal antigens. Our findings revealed that MSCs therapy increased the IL-4, IL-6, IL-10 and GM-CSF and at the same time decreased the IL-12, IL-2 and IFN-γ gene expression at feto-maternal interface. Here, we showed that MSCs therapy could modulate the systemic as well as local Th1/Th2 cytokines production along with protection of fetus from resorption in abortion prone mice. The fine balance of Th1/Th2 cytokine response could be considered as one of the possible mechanisms for fetal protection following MSCs therapy.

Pregnancy losses in cattle: potential for improvement.

For heifers, beef and moderate-yielding dairy cows, it appears that the fertilisation rate generally lies between 90% and 100%. For high-producing dairy cows, there is a less substantive body of literature, but it would appear that the fertilisation rate is somewhat lower and possibly more variable. In cattle, the major component of embryo loss occurs in the first 16 days following breeding (Day 0), with emerging evidence of greater losses before Day 8 in high-producing dairy cows. In cattle, late embryo mortality causes serious economic losses because it is often recognised too late to rebreed females. Systemic concentrations of progesterone during both the cycle preceding and following insemination affect embryo survival, with evidence of either excessive or insufficient concentrations being negatively associated with survival rate. The application of direct progesterone supplementation or treatments to increase endogenous output of progesterone to increase embryo survival cannot be recommended at this time. Energy balance and dry matter intake during the first 4 weeks after calving are critically important in determining pregnancies per AI when cows are inseminated at 70-100 days after calving. Level of concentrate supplementation of cows at pasture during the breeding period has minimal effects on conception rates, although sudden reductions in dietary intake should be avoided. For all systems of milk production, more balanced breeding strategies with greater emphasis on fertility and feed intake and/or energy must be developed. There is genetic variability within the Holstein breed for fertility traits, which can be exploited. Genomic technology will not only provide scientists with an improved understanding of the underlying biological processes involved in fertilisation and the establishment of pregnancy, but also, in the future, could identify genes responsible for improved embryo survival. Such information could be incorporated into breeding objectives in order to increase the rate of genetic progress for embryo survival. In addition, there is a range of easily adoptable management factors, under producer control, that can either directly increase embryo survival or ameliorate the consequences of low embryo survival rates. The correction of minor deficits in several areas can have a substantial cumulative positive effect on herd reproductive performance.

Programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) regulate CD4+ T cells to induce Type 2 helper T cell (Th2) bias at the maternal-fetal interface.

STUDY QUESTION: Are the immune regulatory molecules programmed cell death-1 (PD-1) and T-cell immunoglobulin mucin-3 (Tim-3) involved in regulating CD4+ T cell function during pregnancy? SUMMARY ANSWER: PD-1 and Tim-3 promote Type 2 helper T cell (Th2) bias and pregnancy maintenance by regulating CD4+ T cell function at the maternal-fetal interface. WHAT IS KNOWN ALREADY: The maternal CD4+ T cell response to fetal antigens is thought to be an important component of maternal-fetal tolerance during pregnancy. PD-1 and Tim-3 are important for limiting immunopathology. The co-expression of PD-1 and Tim-3 on T cells identifies a T cell subset with impaired proliferation and cytokine production. Combined blockade of Tim-3 and PD-1 could restore T cell function to the greatest degree. STUDY DESIGN, SIZE, DURATION: The expression of PD-1 and Tim-3 on CD4+ T cells was analyzed by flow cytometry, and in vitro and in vivo analyses were used to investigate the role of PD-1/Tim-3 signal in the regulation of CD4+ T cells function and pregnancy outcome. PARTICIPANTS/ MATERIALS, SETTING, METHODS: A total of 88 normal pregnant women, 37 women with recurrent spontaneous abortion, 36 normal pregnant mice and 45 abortion-prone mice were included. We measure the expression of PD-1 and Tim-3 on CD4+ T cells and their relationship to the function of CD4+ T cells and pregnancy outcome, as well as the effects of blocking PD-1 and Tim-3 pathways on decidual CD4+ T (dCD4+ T) cells during early pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: PD-1 and Tim-3, by virtue of their up-regulation on dCD4+ T cells during pregnancy, define a specific effector/memory subset of CD4+ T cells and promote Th2 bias at the maternal-fetal interface. Using in vitro and in vivo experiments, we also found that combined targeting of PD-1 and Tim-3 pathways results in decreased production of Th2-type cytokines by dCD4+ T cells and increased fetal resorption of normal pregnant murine models. Moreover, decreased PD-1 and Tim-3 on dCD4+ T cells may be associated with miscarriage. LIMITATIONS AND LIMITS OF CAUTION: Further study is required to examine the mechanism of PD-1 and Tim-3 effects on Th2 cytokine production by CD4+ T cells during pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: These results have important implications for understanding the physiological mechanisms that promote maternal-fetal tolerance. Our study also indicates that targeting Tim-3 and PD-1 pathways may represent novel therapeutic strategies to prevent pregnancy loss. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Basic Research Program of China (2015CB943300); National Nature Science Foundation of China (81490744, 91542116, 31570920, 81070537, 31171437, 81370770, 31270969, 31570920, 91542116); the Key Project of Shanghai Municipal Education Commission (14ZZ013) and the Key Project of Shanghai Basic Research from Shanghai Municipal Science and Technology Commission (12JC1401600). None of the authors have any conflict of interest to declare.

Role of Paternal Antigen-Specific Treg Cells in Successful Implantation.

Maternal lymphocytes recognize fetal antigens, so tolerance is necessary to prevent rejection. Seminal plasma is important for induction of paternal antigen-specific Treg cells in the uterine draining lymph nodes and the pregnant uterus. Elimination of Treg cells during implantation or early pregnancy induces implantation failure or fetal resorption in mice. Immunosuppressive therapy with an anti-TNF antibody or the immunosuppressive agent tacrolimus improves the pregnancy rate in women with repeated implantation failure and recurrent pregnancy loss of unknown etiology, suggesting that Treg cells play an essential role in successful implantation and pregnancy in humans.

Maternal N-Carbamylglutamate Supplementation during Early Pregnancy Enhances Embryonic Survival and Development through Modulation of the Endometrial Proteome in Gilts.

BACKGROUND: Early pregnancy loss is a major concern in humans and animals. N-carbamylglutamate (NCG) has been found to enhance embryonic survival during early pregnancy in rats. However, little is known about the key factors in the endometrium involved in the improvement of embryonic implantation and development induced by maternal NCG supplementation. OBJECTIVES: Our objectives were to investigate whether NCG supplementation during early gestation enhanced embryonic survival and development in gilts and to uncover the related factors using the approach of endometrium proteome analysis with isobaric tags for relative and absolute quantification (iTRAQ). METHODS: Uteruses and embryos/fetuses were obtained on days 14 and 28 of gestation from gilts fed a basal diet that was or was not supplemented with 0.05% NCG. The iTRAQ-based quantitative proteomics approach was performed to explore the endometrium proteome altered by NCG supplementation. RESULTS: Maternal NCG supplementation significantly increased the number of total fetuses and live fetuses on day 28 of gestation by 1.32 and 1.29, respectively (P < 0.05), with a significant decrease in embryonic mortality (P < 0.05). iTRAQ results indicated that a total of 59 proteins showed at least 2-fold differences (P < 0.05), including 52 proteins that were present at higher abundance and 7 proteins present at lower abundance in NCG-supplemented gilts. The differentially expressed proteins primarily are involved in cell adhesion, energy metabolism, lipid metabolism, protein metabolism, antioxidative stress, and immune response. On day 14 of gestation, several proteins closely related to embryonic implantation and development, such as integrin-αv, integrin-ß3, talin, and endothelial nitric oxide synthase, were upregulated (3.7-, 4.1-, 2.4-, and 5.4-fold increases, respectively) by NCG supplementation. CONCLUSION: To our knowledge, our results provide the first evidence that altered abundance of the endometrial proteome induced by NCG supplementation is highly associated with the improvement of embryonic survival and development in gilts.

CXCR3 blockade protects against Listeria monocytogenes infection-induced fetal wastage.

Mammalian pregnancy requires protection against immunological rejection of the developing fetus bearing discordant paternal antigens. Immune evasion in this developmental context entails silenced expression of chemoattractant proteins (chemokines), thereby preventing harmful immune cells from penetrating the maternal-fetal interface. Here, we demonstrate that fetal wastage triggered by prenatal Listeria monocytogenes infection is driven by placental recruitment of CXCL9-producing inflammatory neutrophils and macrophages that promote infiltration of fetal-specific T cells into the decidua. Maternal CD8+ T cells with fetal specificity upregulated expression of the chemokine receptor CXCR3 and, together with neutrophils and macrophages, were essential for L. monocytogenes-induced fetal resorption. Conversely, decidual accumulation of maternal T cells with fetal specificity and fetal wastage were extinguished by CXCR3 blockade or in CXCR3-deficient mice. Remarkably, protection against fetal wastage and in utero L. monocytogenes invasion was maintained even when CXCR3 neutralization was initiated after infection, and this protective effect extended to fetal resorption triggered by partial ablation of immune-suppressive maternal Tregs, which expand during pregnancy to sustain fetal tolerance. Together, our results indicate that functionally overriding chemokine silencing at the maternal-fetal interface promotes the pathogenesis of prenatal infection and suggest that therapeutically reinforcing this pathway represents a universal approach for mitigating immune-mediated pregnancy complications.

Oocyte maturation and embryo survival in nulliparous female pigs (gilts) is improved by feeding a lupin-based high-fibre diet.

Inclusion of high levels of the high-fibre ingredient sugar-beet pulp in pre-mating diets has been shown to increase gonadotrophin concentrations and improve oocyte quality in nulliparous pigs (gilts). This study evaluated the effects of two alternative fibre sources on reproductive performance in gilts. Gilts received one of three diets from 3 weeks before puberty stimulation until Day 19 of the first oestrous cycle: control (39 g kg⁻¹ fibre), bran (500 g kg⁻¹ wheat bran, 65 g kg⁻¹ fibre) or lupin (350 g kg⁻¹ lupin, 118 g kg⁻¹ crude fibre). Diet did not affect circulating LH concentrations or ovarian follicle size. However, a higher percentage of oocytes collected from lupin-supplemented gilts reached metaphase II in vitro compared with those collected from bran-fed or control gilts (89±5% versus 72±5% and 66±5%, respectively; P<0.05). Furthermore, in a second experiment, gilts fed the same lupin-based diet before mating had improved embryo survival (92±5%) on Day 28 after mating compared with control gilts (76±4%; P<0.05). Therefore, feeding a high-fibre diet before mating can improve oocyte quality in gilts without changes in circulating LH, but this effect is dependent on the fibre source.

Adoptive transfer of CD4+CD25+ regulatory T cells for prevention and treatment of spontaneous abortion.

OBJECTIVES: The purpose of this study was to examine whether adoptive transfer with in vitro expanded CD4+CD25+ regulatory T cells (Tregs) could prevent immune response-mediated spontaneous abortion in mice. STUDY DESIGN: Female CBA/J mice were mated with male Balb/c as the control with normal pregnancy or with DBA/2J mice as a model of spontaneous abortion. The CBA/J mice mated with DBA/2J were treated intravenously with freshly isolated or in vitro expanded Tregs on day 1 or 4 of pregnancy, respectively. The numbers of surviving and reabsorbed fetuses in the different groups of mice were counted on day 14 of pregnancy, and the concentrations of cytokines in individual sera and the supernatants of cultured Tregs were measured by ELISA. RESULTS: Adoptive transfer with freshly isolated Tregs only slightly reduced the fetal resorption rate, which was not significantly different from that of the mice without Treg treatment, regardless of treatment at early stage and implementation of pregnancy. In contrast, adoptive transfer with in vitro expanded Tregs significantly reduced the fetal resorption rates, particularly for treatment at early stage of pregnancy (P<0.05). Furthermore, adoptive transfer with in vitro expanded Tregs at early stage of pregnancy significantly increased the levels of serum IL-10, TGF-ß1, and the ratios of IL-10 to IFN-γ. CONCLUSIONS: Our data clearly indicated that adoptive transfer with in vitro expanded Tregs at early stage of pregnancy protected fetuses from spontaneous abortion by re-establishing immune tolerance in mice.

Folic acid and safflower oil supplementation interacts and protects embryos from maternal diabetes-induced damage.

Maternal diabetes increases the risk of embryo malformations. Folic acid and safflower oil supplementations have been shown to reduce embryo malformations in experimental models of diabetes. In this study we here tested whether folic acid and safflower oil supplementations interact to prevent embryo malformations in diabetic rats, and analyzed whether they act through the regulation of matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), and nitric oxide (NO) and reactive oxygen species production. Diabetes was induced by streptozotocin administration prior to mating. From Day 0.5 of pregnancy, rats did or did not receive folic acid (15 mg/kg) and/or a 6% safflower oil-supplemented diet. Embryos and decidua were explanted on Day 10.5 of gestation for further analysis of embryo resorptions and malformations, MMP-2 and MMP-9 activities, TIMP-1 and TIMP-2 levels, NO production and lipid peroxidation. Maternal diabetes induced resorptions and malformations that were prevented by folic acid and safflower oil supplementation. MMP-2 and MMP-9 activities were increased in embryos and decidua from diabetic rats and decreased with safflower oil and folic acid supplementations. In diabetic animals, the embryonic and decidual TIMPs were increased mainly with safflower oil supplementation in decidua and with folic acid in embryos. NO overproduction was decreased in decidua from diabetic rats treated with folic acid alone and in combination with safflower oil. These treatments also prevented increases in embryonic and decidual lipid peroxidation. In conclusion, folic acid and safflower oil supplementations interact and protect the embryos from diabetes-induced damage through several pathways related to a decrease in pro-inflammatory mediators.

Chronic ethanol exposure and folic acid supplementation: fetal growth and folate status in the maternal and fetal guinea pig.

Chronic ethanol exposure (CEE) can produce developmental abnormalities in the CNS of the embryo and developing fetus. Folic acid (FA) is an important nutrient during pregnancy and low folate status exacerbates ethanol-induced teratogenicity. This study tested the hypotheses that (1) CEE depletes folate stores in the mother and fetus; and (2) maternal FA supplementation maintains folate stores. CEE decreased fetal body, brain, hippocampus weights, and brain to body weight ratio but not hippocampus to body weight ratio. These effects of CEE were not mitigated by maternal FA administration. The FA regimen prevented the CEE-induced decrease of term fetal liver folate. However, it did not affect maternal liver folate or fetal RBC folate at term, and did not mitigate the nutritional deficit-induced decrease of term fetal hippocampus folate. This study suggests that maternal FA supplementation may have differential effects on folate status in the mother and the fetus.

Administration of high-dose intact immunoglobulin has an anti-resorption effect in a mouse model of reproductive failure.

Administration of high-dose intact human immunoglobulin (IH-Ig) has been applied to treat a variety of inflammatory and autoimmune diseases, and is expected to have beneficial effects on human fecundity. In the present study, we investigated whether Ig had anti-resorption effects using polyinosinic-polycytidylic acid sodium salt [poly (I:C)]-induced enhancement of fetal resorption in the mating of CBA/J x DBA/2J resorption-prone mouse model. Furthermore, we investigated the mechanism of the effect by examining the mRNA expression of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-10, IL-4 and TGF-beta(1) in spleens and placentas from the resorption-prone model treated with IH-Ig, by reverse transcription (RT)-polymerase chain reaction (PCR). Administration of high-dose IH-Ig significantly reduced the fetal resorption rate from 55% to 10%. This anti-resorption effect, however, was not detected in mice administered with Fab fragments of human Ig. We then performed adoptive transfer experiments to examine whether cellular components could transfer the effect. A remarkable anti-resorption effect was seen in poly (I:C)-injected pregnant recipients transferred with spleen cells from IH-Ig-treated donor mice. The RT-PCR study showed that IH-Ig reduced the expression of IFN-gamma and TNF-alpha mRNA in placentas of poly (I:C)-injected pregnant mice. The present findings demonstrate that intact Ig, particularly its Fc portion, possesses anti-resorption activity. The effect might be attributed to the suppressed production of pro-inflammatory cytokines at the maternofetal interface.

Beneficial effect of supplemental lipoic acid on diabetes-induced pregnancy loss in the mouse.

Uncontrolled diabetes mellitus (DM) is an etiological factor for recurrent pregnancy loss, fetal growth disorders, and major congenital malformations in the offspring. Antioxidant therapy has been advocated to overcome the oxidant-antioxidant disequilibrium inherent in diabetes. The objective of this article was to evaluate the beneficial effects of alpha-lipoic acid (LA) on fetal outcome in a mouse model of streptozotocin (STZ)-induced DM. Timed pregnant mice were made diabetic by intraperitoneal (IP) injection of a single dose of STZ (200 mg/kg) on gestation day (GD) 2. Diabetic animals were supplemented daily with an IP injection of 15 mg/kg of LA starting on GD 4 and continued through GD 12. Fetuses were examined on GD 18 for malformations and growth restriction. Some diabetic mice injected with Evans blue were examined on GD 3.5 and GD 6.5 to evaluate frequency of implantations. STZ-treated mice had all cardinal signs of DM. LA treatment did not normalize blood glucose levels of DM mice. Rates of pregnancy in saline control, DM, and DM + LA groups were 90%, 28%, and 64%, respectively, indicating that LA promotes pregnancy in DM animals. However, postimplantation resorption showed a threefold increase in the DM + LA group. Rates of intrauterine growth restriction and major congenital malformations were also augmented thus indicating that the interaction between DM and LA has deleterious effects on postimplantation embryos.

Combined supplementation of folic acid and vitamin E diminishes diabetes-induced embryotoxicity in rats.

BACKGROUND: Oxidative stress and enhanced apoptosis may be involved in the induction of embryonic dysmorphogenesis in diabetic pregnancy. Administration of folic acid or vitamin E diminishes embryonic dysmorphogenesis. We aimed to evaluate the effect of combined treatment with folic acid and vitamin E on the disturbed development in embryos of diabetic rats. METHODS: Pregnant nondiabetic and diabetic rats were treated with daily injections of 15 mg/kg folic acid or with 5% vitamin E in the diet. A third group received combined treatment. Day 10 and day 11 embryos were evaluated for development and apoptotic profile. RESULTS: We found increased malformations, resorptions, and profound growth retardation in embryos of diabetic rats compared to control embryos. Vitamin E or folic acid alone, or the 2 compounds combined, normalized embryonic demise. Maternal diabetes caused decreased nuclear factor-kappaB (NF-kappaB) activity and B-cell lymphoma 2 (Bcl-2) protein level, and increased Bcl-2-associated x proteins (Bax) in embryos. Supplementation of vitamin E alone normalized the Bax protein level in a diabetic environment. Administration of folic acid to diabetic rats increased NF-kappaB activity and Bcl-2 protein level. Combined treatment normalized Bcl-2 and Bax protein level in a diabetic environment. CONCLUSIONS: Combined supplementation of folic acid and vitamin E to pregnant diabetic rats diminished diabetes-induced malformations and resorptions, concomitant with normalization of apoptotic protein levels. No treatment completely abolished the embryonic demise; therefore, other mechanisms than oxidative stress and apoptosis are likely to be involved in diabetic embryopathy.

Metformin prevents embryonic resorption induced by hyperandrogenisation with dehydroepiandrosterone in mice.

The present study examined the mechanism by which metformin prevents dehydroepiandrosterone (DHEA)-induced embryonic resorption in mice. Treatment with DHEA (6 mg/100 g bodyweight, 24 and 48 h post implantation) induced 88 +/- 1 % embryonic resorption and the diminution of both serum oestradiol (E) and progesterone (P) levels. However, when metformin (50 mg/kg bodyweight) was given together with DHEA, embryo resorption (43 +/- 3% v. 35 +/- 5% in controls) and both serum E and P levels were not significantly different from controls. Glucose and insulin levels were increased in the DHEA-treated mice but when metformin was administered together with DHEA these parameters were similar to control values. Treatment with DHEA increased ovarian oxidative stress and diminished uterine nitric oxide synthase (NOS) activity; however, when metformin was administered together with DHEA, both ovarian oxidative stress and uterine NOS activity were not different from controls. Metformin treatment did not modify the percentage of CD4(+) and CD8(+) T cells from both axillar and retroperitoneal lymph nodes but prevented the increase of serum tumour necrosis factor +/- produced in DHEA-treated mice. These results show that metformin acts in DHEA-induced embryonic resorption in mice by modulating endocrine parameters, ovarian oxidative stress and uterine NOS activity.

Protection from ethanol-induced limb malformations by the superoxide dismutase/catalase mimetic, EUK-134.

Based on previous in vitro studies that have illustrated prevention of ethanol-induced cell death by antioxidants, using an in vivo model, we have tested the anti-teratogenic potential of a potent synthetic superoxide dismutase plus catalase mimetic, EUK-134. The developing limb of C57BL/6J mice, which is sensitive to ethanol-induced reduction defects, served as the model system. On their ninth day of pregnancy, C57BL/6J mice were administered ethanol (two intraperitoneal doses of 2.9 g/kg given 4 h apart) alone or in combination with EUK-134 (two doses of 10 mg/kg). Pregnant control mice were similarly treated with either vehicle or EUK-134, alone. Within 15 h of the initial ethanol exposure, excessive apoptotic cell death was observed in the apical ectodermal ridge (AER) of the newly forming forelimb buds. Forelimb defects, including postaxial ectrodactyly, metacarpal, and ulnar deficiencies, occurred in 67.3% of the ethanol-exposed fetuses that were examined at 18 days of gestation. The right forelimbs were preferentially affected. No limb malformations were observed in control fetuses. Cell death in the AER of embryos concurrently exposed to ethanol and EUK-134 was notably reduced compared with that in embryos from ethanol-treated dams. Additionally, the antioxidant treatment reduced the incidence of forelimb malformations to 35.9%. This work illustrates that antioxidants can significantly improve the adverse developmental outcome that results from ethanol exposure in utero, diminishing the incidence and severity of major malformations that result from exposure to this important human teratogen.

Serum hormone characterization and exogeneous hormone rescue of bromodichloromethane-induced pregnancy loss in the F344 rat.

Previously, we demonstrated that bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss in F344 rats when given on gestational days (GD) 6-10, encompassing the luteinizing hormone (LH)-dependent period of pregnancy (GD 7-10). Pregnancy loss, i.e., full-litter resorption, was associated with reduced serum progesterone levels; however, we were unable to identify an effect on serum LH. Here, we reevaluated serum LH levels using the more sensitive technique, DELFIA(R). We further sought to better define the temporal pattern of endocrine disruption caused by BDCM during pregnancy with more frequent sampling. Lastly, we attempted to prevent BDCM-induced pregnancy loss using exogenous progesterone or human chorionic gonadotropin (hCG), an LH-agonist. BDCM, in 10% Alkamuls(R), was dosed at 75 mg/kg/day by gavage to F344 rats on GD 6-10 (plug day = GD 0). BDCM-induced pregnancy loss was associated with marked reductions in serum progesterone and LH on GD 10. The decrease in serum LH consistently preceded the decrease in progesterone. In the hormone replacement studies, BDCM and progesterone were administered on GD 6-10, hCG on GD 8-10. BDCM was delivered at 100 mg/kg/day, progesterone at 10 mg/kg twice daily, and hCG at 0.5 IU/0.2 ml/rat. Both progesterone and hCG prevented BDCM-induced pregnancy loss. Thus, BDCM-induced pregnancy loss was associated with marked GD-10 reductions in serum LH and corresponding decreases in progesterone. Furthermore, coadministration of an LH agonist prevented pregnancy loss, supporting the hypothesis that BDCM-induced pregnancy loss in the rat occurs via an LH-mediated mode of action.

Exposure-disease continuum for 2-chloro-2'-deoxyadenosine, a prototype ocular teratogen. 3. Intervention with PK11195.

BACKGROUND: Treatment of pregnant mice with 2-chloro-2'-deoxyadenosine (2CdA) on Day 8 of gestation induces microphthalmia through a mechanism linked to the p53 tumor suppressor pathway. The present study defines the response of Day 8 mouse embryos through time with respect to pharmacologic intervention with PK11195, a ligand of the mitochondrial peripheral benzodiazepine receptor (Bzrp). METHODS: Pregnant CD-1 mice dosed with 2CdA with or without PK11195 on gestation Day 8 provided fetuses for teratologic evaluation on Day 14 and Day 17; HPLC measured pyridine nucleotides (NADH/NAD+) at 1.5 hr, RT-PCR measured mitochondrial 16S rRNA abundance at 3.0 hr, and p53 protein induction was assessed with immunostaining at 4.5 hr postexposure. RESULTS: The mean incidences of malformed fetuses were significantly higher in the 7.5 mg/kg 2CdA treatment group (50.2% malformed) vs. the 2CdA + 4.0 mg/kg PK11195 co-treatment group (4.4% malformed). Malformed fetuses displayed a range of ocular defects that included microphthalmia and keratolenticular dysgenesis (Peters anomaly). No malformations were observed in the control or PK11195 alone groups. PK11195 also protected litters from increased resorption rates and fetal weight reduction. It did not rescue early effects on NADH balance (1.5 hr) or 16S rRNA expression (3.0 hr); however, the p53 response (4.5 hr) was downgraded in 2CdA + PK11195 embryos vs. 2CdA alone. By delaying the administration of PK11195 in 1.5 hr intervals it was determined that the window for protection closed between 4.5 to 6.0 hr after 2CdA. CONCLUSIONS: The capacity of PK11195 to suppress the pathogenesis of microphthalmia implies a critical role for mitochondrial peripheral benzodiazepine receptors in the p53-dependent mode of action of 2CdA on ocular development.

Biological activity of the suppressor cells inducer factor secreted by the Jeg-3 choriocarcinoma cell line.

PROBLEM: We wanted to further study the mechanisms of immune suppression and suppression inducing capacities by choriocarcinoma products, e.g. both crude human choriocarcinoma supernatant (HCS) and especially an active fraction obtained by high performance liquid chromatography (HPLC) from the culture supernatant of the Jeg-3 human choriocarcinoma cell line, since it appeared by weight and charge criteria to be a different molecular species than the low molecular weight fraction previously isolated from mouse and human term placenta. It was important to know whether the purified material was active in vivo as it was in vitro. Therefore, we tested the effects of HCS in vivo in three systems: prevention of fetal demise in the CBA/J x DBA/2 abortion prone murine mating combination, where the effects of the HPLC purified fraction were also monitored as well as by a cell transfer system, where the suppression is revealed by a local GVH/HVG assay, and finally enhancement the survival of a mildly immunogenic tumor allograft. METHODS: An active fraction was isolated from HCS by ion exchange HPLC. Female CBA/J were mated with DBA/2 and the influence of 3 intraperitoneal injections of both crude HCS and the active fraction was evaluated by monitoring the percentage of fetal resorptions. Simultaneously, on the day when resorptions were counted, maternal splenocytes from these females were harvested and were injected by the subcutaneous way in C3H/HEJ hind feet. The lymph node reactivity (HVG + GVH) was assessed by [3H]thymidine intake by cells harvested from the draining popliteal lymph nodes. For assessment of influence of HCS on allograft rejection, BALB/b (H-2b) mice received a subcutaneous injection of allogeneic P815 tumor cells (H-2d). The influence of HCS injections on tumor survival was analyzed by regular measurements of the mean tumor diameter. RESULTS: Intraperitoneal injection of HCS reduced fetal resorptions from 24.7 to 13%. Injection of the in vitro active fraction induced the same rate of reduction. The mean intensity of HvG/GvH reaction was 13400 cpm per lymph node when splenocytes from the control group were injected compared to 2900 cpm when splenocytes from treated mice were used. P815 tumor allografts were completely rejected in all cases after 21 days. Weekly subcutaneous injections of HCS prolonged tumor survival in all cases up to at least 30 days. CONCLUSION: The fraction isolated from HCS increased very efficiently the survival of allografts as well as those of allogeneic fetuses in a resorption prone murine mating. The choriocarcinoma cell line might prove to be a useful source of immunosuppressive materials, which could otherwise be important for the fetal-maternal tolerance and a successful pregnancy.

Combined treatment with vitamin E and vitamin C decreases oxidative stress and improves fetal outcome in experimental diabetic pregnancy.

The aim was to investigate whether dietary supplementation of a combination of the two antioxidants, vitamin E and vitamin C, would protect the fetus in diabetic rat pregnancy at a lower dose than previously used. Normal and streptozotocin-induced diabetic rats were mated and given standard food or food supplemented with either 0.5% vitamin E + 1% vitamin C or 2% vitamin E + 4% vitamin C. At gestational d 20, gross morphology and weights of fetuses were evaluated. Vitamins E and C and thiobarbituric acid reactive substances were measured in maternal and fetal compartments. In addition, protein carbonylation was estimated in fetal liver. Maternal diabetes increased the rate of malformation and resorption in the offspring. High-dose antioxidant supplementation decreased fetal dysmorphogenesis to near normal levels. The low-dose group showed malformations and resorptions at an intermediate rate between the untreated and the high-dose groups. Thiobarbituric acid reactive substances were increased in fetal livers of diabetic rats and reduced to normal levels already by low-dose antioxidative treatment. Protein carbonylation rate was also increased in fetal liver of diabetic rats; it was normalized by high-dose treatment but only partially reduced by low-dose antioxidants. We conclude that combined antioxidative treatment with vitamins E and C decreases fetal malformation rate and diminishes oxygen radical-related tissue damage. However, no synergistic effect between the two antioxidants was noted, a result that may influence future attempts to design antiteratogenic treatments in diabetic pregnancy. Oxidatively modified proteins may be teratogenically important mediators in diabetic embryopathy.

Abnormal development of the sinuatrial venous valve and posterior hindbrain may contribute to late fetal resorption of vitamin A-deficient rat embryos.

BACKGROUND: Normal embryonic development and survival in utero is dependent on an adequate supply of vitamin A. Embryos from vitamin A-deficient (VAD) pregnant rats fed an inadequate amount of all-trans retinoic acid (atRA; 12 microg per g of diet or approximately 230 microg per rat per day) exhibit severe developmental abnormalities of the anterior cardinal vein and hindbrain by embryonic day (E) 12.5 and die shortly thereafter. METHODS: In the present study, we sought to determine whether supplementation of VAD-RA supported (12 microg per g of diet) pregnant rats with retinol (ROL) at the late-gastrula (presomite or rat E9.5) or early somite stages (E10.5), or provision of higher levels of atRA throughout this period could prevent abnormalities in the developing cardiovascular and nervous systems. RESULTS: A newly described defect in the sinuatrial venus valve along with enlarged anterior cardinal veins and nervous system abnormalities and the later death of embryos are prevented by supplementing pregnant animals with ROL on the morning of E9.5. If ROL supplementation is delayed by 1 day (E10.5), most embryos are abnormal and die by E18.5. Supplementation of VAD rats with atRA (250 microg per g of diet) between E8.5 and E10.5 also prevents the cardiovascular and nervous system abnormalities and a significant number of these embryos survive to parturition. Thus, high levels of atRA can obviate the need for ROL between E9.5 and E10.5. CONCLUSIONS: These results support an essential role for retinoid signaling between the late gastrula and early somite stages in the rat embryo for normal morphogenesis of the primitive heart tube and the posterior hindbrain. Further, these results suggest that embryonic death occurring at midgestation in the VAD rat may be linked to the abnormal development of one or both of these embryonic structures.