Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP. Two coding substitutions were found in comparison with the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429]PLAP, into wild-type Chinese hamster ovary cells. We determined the k(cat) and K(m), of the PLAP S, F, and D allozymes using the non-physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples.     

Stress and immune mediators in miscarriage.

BACKGROUND: Stress is thought to be abortogenic and psycho-neuro-immunological pathways have been suggested to be involved in triggering miscarriages. From experiments in pregnant mice exposed to stress some insights into the underlying mechanisms have been gained, delineating immunological imbalances as a cause of pregnancy failure. In order to test the validity of the conclusions drawn from murine experiments and the role of stress in human pregnancy loss, the following study was performed. METHODS: We used an established perceived stress questionnaire and measured the stress score of women with a confirmed diagnosis of first trimester spontaneous abortion (n = 94). Decidual tissue was investigated by immunohistochemistry and in-situ hybridization to detect the presence and distribution of immunocompetent decidual cells [CD56(+) natural killer (NK) cells, CD8(+)and CD3(+) T cells, tryptase(+) mast cells (MCT(+)) and tumour necrosis factor (TNF)-alpha(+) cells]. The patient cohort was divided into women experiencing low or high levels of stress. RESULTS: In the decidua of women with high stress scores we observed significantly higher numbers of MCT(+), CD8(+) T cells and TNF-alpha(+) cells per mm(2) tissue (P < or = 0.05). No significant differences between individuals with lower or higher stress scores could be observed with respect to decidual CD56(+) NK and CD3(+) T cells. CONCLUSIONS: Using a questionnaire to score perceived stress in humans may be a valid approach to assess non-biased stress scores. Stress-triggered abortion in humans, identified by a questionnaire, can be linked to immunological imbalances.     

Possible susceptibility of the HLA-DPB1*0402 and HLA-DPB1*04 alleles to unexplained recurrent abortion: analysis by means of polymerase chain reaction-restricted fragment length polymorphism method

PROBLEM: To clarify whether HLA-DP antigens are associated with patient population of unexplained recurrent abortion. METHOD OF STUDY: The frequency of HLA-DPB1 alleles in patients with unexplained recurrent abortion, and the compatibility of HLA-DPB1 alleles between patient couples, were studied using a polymerase chain reaction (PCR)-restricted fragment length polymorphism (RFLP) method. Thirty patients who had a history of unexplained primary recurrent abortion, and their husbands, were typed for HLA-DPB1 genotype. Two hundred and ninety-nine base pair fragments from the second exon of HLA-DPB1 genes were selectively amplified using the PCR-primers. After amplification, the DNAs were digested with restriction endonucleases, and subjected to electrophoresis in a 12% polyacrilamide gel to determine HLA-DPB1 genotype. RESULTS: The frequency of HLA-DPB1*0402 and DPB1*04 alleles in the patient group (n = 30) was significantly increased, as compared to that in the normal fertile women (n = 30). The frequency of HLA-DPB1*04 allele in the patient group was significantly increased, as compared to that in the general population (n = 112). No significant compatibility of HLA-DPB1 alleles could be observed between patient couples and normal fertile couples. CONCLUSION: These findings suggest a possible new class II association with patient population of unexplained recurrent abortion.     

Adenosine Deaminase and Human Reproduction: A Comparative Study of Fertile Women and Women with Recurrent Spontaneous Abortion

PROBLEM: We have investigated the possible role of adenosine deaminase (ADA) genetic polymorphism in human fertility through a comparative study of couples with recurrent spontaneous abortion (RSA) and healthy puerperae. METHOD OF STUDY: Adenosine deaminase phenotype has been determined in 209 women with repeated episodes of unexplained spontaneous abortion (RSA) and their husbands, as well as in 115 healthy pregnant women from the population of Rome. An independent sample of 286 puerperae along with their newborn infants in the population of Penne was also studied. RESULTS: The proportion of carriers of ADA*2 allele, which is associated with the lowest enzymatic activity, is lower among women with RSA than among healthy pregnant women from the same population of Rome. Preliminary observations suggest a protective effect of ADA*2 against the development of autoantibodies in RSA. Such an effect seems to be mediated by an interaction with ABO blood groups. In the population of Penne the proportion of women carrying ADA*2 allele is higher among those who have had two or more previously born children than among women with only one or no children. CONCLUSIONS: The data suggest that women carrying the ADA*2 allele are better protected against the spontaneous loss of embryos and have a higher fertility rate.     

Suppressed Cell-Mediated Immunity and Monocyte and Natural Killer Cell Activity Following Allogeneic Immunization of Women with Spontaneous Recurrent Abortion

Spontaneous recurrent abortion (SRA) has been treated by means of immunization with paternal or third-party white blood cells, yet the immunological basis for SRA and for the role of immunization protocols in pregnancy outcome remains controversial. To elucidate this question, nine women with SRA were immunized with paternal mononuclear cells and studied before and 2 weeks after immunization. Seven women who became pregnant gave birth to live newborns. Secretion of the T helper 1 cytokines IL-2 and interferon- by patients' mononuclear cells decreased, while production of IL-10 increased. The levels of natural killer and lymphokine-activated killer cell-mediated cytotoxicity were markedly decreased. Monocyte functions such as secretion of IL-l, tumor necrosis factor a, IL-6, and cytotoxic activity decreased concurrently with elevations in IL-10 and transforming growth factor secretion. Production of IL-12, a pivotal regulatory cytokine, decreased. Furthermore, B7/1 expression on patients' mononuclear cells was downregulated. This resulted in a decrease in monocyte costimulatory activity of purified T cells with soluble anti-CD3, paralleled by a decline in allogeneic proliferative responses. These results suggest that the improved pregnancy success rate in women with SRA following immunization may be partly related to suppression of cell-mediated immunity and monocyte and natural killer cell activity.     

Psychosocial factors regulating natural-killer cell activity in recurrent spontaneous abortions

PROBLEM: The preconceptional natural-killer cell (NK) activity predicts subsequent miscarriage among women with unexplained recurrent spontaneous abortion (RSA). Psycho-neuro-immuno-endocrine network has recently been proposed as a mechanism for abortions. We therefore examined which psychosocial factors influenced the NK activity among women with RSA. METHOD OF STUDY: We measured the preconceptional NK activity of 61 women with a history two consecutive unexplained first-trimester miscarriages and no live births. We also administered semi-structured interviews and a battery of self-report questionnaires to assess their social support, personality, self-esteem and psychiatric symptoms. RESULTS: The preconceptional NK activity was negatively correlated with the women's neuroticism personality trait (r= -0.32, P = 0.01) and current depressive symptoms (r = -0.26, P= 0.05), and positively correlated with their self-esteem (r = 0.34, P = 0.01). CONCLUSIONS: In addition to several substances such as transforming-growth-factor beta and granulocyte-macrophase colony-stimulating factor, we found that low neuroticism, low depression scale score and high self-esteem contributed to high NK activity among women with RSA.     

Partial duplication 16q: report of two affected siblings resulting from a maternal translocation and literature review

Two siblings with a partial duplication 16q, born to a woman with a balanced translocation (6;16), are described. The first infant died at 8 weeks of age; the second died at 4 months. Fifteen other cases of duplications involving 16q have been reported, all of them derived from a balanced parental translocation. The most frequent physical findings have included dysmorphic facies characterized by high forehead, prominent nose, antimongoloid slant, malformed ears, and micrognathia, as well as flexion contractures of the joints, deformity of the feet, and genital hypoplasia in the male. Anorectal, intestinal and cardiac malformations were less frequent findings. Most of the affected infants died at ages ranging from 8 days to 6 months. The few with longer survival (up to 6 years) had a shorter, more distal segment duplication of chromosome 16. Although intrauterine growth retardation and microcephaly were not always present at birth, most of the infants had postnatal growth failure. The phenotypic and clinical findings of the two infants in this report are compared with those of previously reported cases, from which there appears to be correlation of the length of the 16q duplication with clinical phenotype and survivals.     

Endocrinology: Randomized trial of misoprostol and cervagem in combination with a reduced dose of mifepristone for induction of abortion

Mifepristone (600 mg) in combination with a prostaglandin hasbeen demonstrated to be a safe, acceptable alternative to vacuumaspiration for induction of abortion in the first 9 weeks ofpregnancy. However, the efficacy and side-effects of differentprostaglandins used in combination with mifepristone have notbeen assessed in a randomized trial. In this study, 800 womenseeking an abortion at gestational age 63 days amenorrhoea wererandomized to receive either 0.5 mg gemeprost by vaginal pessary(group I) or 600 µg misoprostol (group II) by mouth –48h after taking 200 mg mifepristone by mouth. The side-effectsand number of complete abortions were used as measures of efficacy.There was no significant difference in the rate of completeabortion between group I [96.7%; 95% confidence interval (CI)94.9–98.5%, n = 391] and group II (94.6%; 95% CI 92.3–96.9,n = 386). It was not possible to assess the outcome with certaintyin the remaining 23 women. However, there were significantlymore ongoing pregnancies in the women who received misoprostolthan in those who received gemeprost (nine versus one, P <0.01) and in eight of these 10 women the gestation was >49days. Fewer women in group II required analgesia than in groupI (48 versus 60%, P < 0.001) although the number requestingopiate was similar in each group (6.9 versus 5.2%, P > 0.4).The incidence of nausea and vomiting after misoprostol (47.8and 21.9% respectively) was higher (P < 0.001) than aftergemeprost (33.9 and 12% respectively). The incidence of infectionand heavy bleeding was low in both groups (<2%) and onlyone woman required blood transfusion. We conclude that the recommendeddose of mifepristone and gemeprost can be reduced without impairingclinical efficacy in pregnancies up to 63 days amenorrhoea.Misoprostol is a safe alternative prostaglandin but has a higherincidence of ongoing pregnancies especially at gestation after49 days amenorrhoea.     

The emerging role of immunoregulation of fibrinogen-related procoagulant Fgl2 in the success or spontaneous abortion of early pregnancy in mice and humans.

PROBLEM: Abortion of chromosomally normal embryos in the CBA X DBA/2 mating combination is triggered by release of Th1 cytokines (tumor necrosis factor [TNF]-alpha, interferon [IFN]-gamma, and interleukin [IL]-1), which cause abortion via a novel prothrombinase, Fgl2, and polymorphonuclear leukocytes. The site of activation may be maternal vascular endothelium on arteries and veins nourishing the placenta. Activation of coagulation is also prominent in spontaneous abortion of chromosomally normal human embryos. We asked where is Fgl2 up-regulated in the uterus in murine abortions, and if similar Fgl2 expression occurs in human pregnancy failure. METHODS: Control CBA X DBA/2 pregnant mice, or from mice injected with TNF-alpha + IFN-gamma on day 7.5 of gestation, were removed on day 8.5, fixed, sectioned, and subject to in situ hybridization for Fgl2. Sections were also stained for fibrin. Elective first trimester termination samples or biopsies taken early in the course of a recurrent miscarriage were similarly fixed, sectioned, and analyzed by in situ hybridization. Control and cytokine-treated mice were anticoagulated with heparin, an activator of antithrombin III, and/or the direct anti-thrombin inhibitor hirudin. RESULTS: Low level Fgl2 expression localized to basal decidua remote from the embryo was noted in control mice; cytokine treatment, which causes greater than 80% of abortions, produced a striking up-regulation in this area as well as in a band at the junction of decidua and myometrium. Trophoblast also became strikingly positive. Fgl2 expression was associated with increased fibrin staining. Anticoagulation significantly protected against abortions, but doses were limited by the complication of retroplacental hemorrhage. In tissue from normal first trimester pregnancy, minimal Fgl2 positivity was seen in some villous syncytiotrophoblast, in villous stroma, cytotrophoblast, and in some cells in decidua. In spontaneous abortion of normal embryo, striking Fgl2 positivity was seen in syncytiotrophoblast and extravillous cytotrophoblast, in association with areas of thrombus formation. CONCLUSIONS: Fgl2 appears to be physiologically expressed and may protect against the internal danger of maternal and/or fetal bleeding during pregnancy and at parturition; a role in inhibiting transplacental traffic is also possible. External dangers in the form of stress, endotoxin, and antigens eliciting Th1 cytokine responses upregulate Fgl2 prothrombinase in trophoblast as well as in decidua, which results in spontaneous abortion of immunogenetically "weaker" embryos.     

Pharmacokinetics of a novel oral slow-release form of misoprostol

BACKGROUND: The pharmacokinetics of a novel slow-release (SR) misoprostol was studied and compared to conventional misoprostol. METHODS: Thirty-one women, pregnant between 8 and 12 weeks, requesting surgical abortion were randomly allocated to receive orally 400 microg conventional misoprostol, 400 microg SR misoprostol or 800 microg SR misoprostol. Venous blood samples were taken at 0, 30, 60, 120, 240 and 360 min after the administration of misoprostol. Misoprostol acid (MPA) was determined in serum samples using liquid chromatography/tandem mass spectrometry. RESULTS: Serum peak concentration (Cmax) was highest for conventional oral misoprostol. The time to peak concentration (Tmax) was similar for all groups. The area under the curve up to 360 min was similar for conventional and for 800 microg SR misoprostol and significantly greater for these groups compared to 400 microg SR misoprostol (P = 0.013). CONCLUSION: The new SR form of misoprostol demonstrated lower peak levels but longer-lasting elevation in plasma levels compared to conventional oral misoprostol. The AUC for 800 microg SR misoprostol was similar to that of 400 microg of conventional oral misoprostol. SR misoprostol may offer an alternative to repeated administration of oral misoprostol or to vaginal administration.