Yoshichika Suzuki

Clinical Applications of Serum Placental Protein 14 (PP14) Measurement in the IVF‐ET Cycle

Objective: Placental protein 14 (PP14) is known to be one of the endometrial proteins that reflect endometrial functioning throughout the menstrual cycle. In this study, we examined PP14 as a marker for human endometrial receptivity in order to predict the outcome of in vitro fertilization and the embryo‐transfer (IVF‐ET) cycle.

Immunohistochemical localization of new placental proteins (PP20, PP25, PP26) in normal pregnancy and gestational trophoblastic disease

Summary Placental Proteins 20, 25, and 26 (PP20, PP25, PP26) were isolated from a soluble fraction of term placenta and characterized by Bohn (1985, 1991). To clarify the localization of these proteins, normal placenta, ectopic pregnancy, endometrium, and trophoblastic disease were studied by indirect immunohistochemical method. Each protein was localized in cytoplasm of syncytiotrophoblast (ST) of early and term placenta. Positive staining for PP25 and PP26 was recognized in the extravillus trophoblast (ET) of basal plate and membrane. Proliferative and secretory phase endometria were not stained for these proteins. However, PP25 and PP26 were localized on decidualized endometrium from ectopic pregnant patients and PP26 showed positive staining on decidua. Although PP20, 25, and 26 were localized on ST of placenta, there was a possibility of extra placental production. PP26 was localized in syncytio and intermediate trophoblastic cells of gestational trophoblastic disease. PP26 is proposed to be a useful new histochemical marker of trophoblastic disease.

Concentration of placental protein 19 in body fluid and placental tissues

SummaryPlacental protein 19 (PP19) is one of the new placental tissue proteins identified in extracts from human term placenta by Bohn and Winkler [1]. We measured the PP19 concentration in body fluids and placental tissue by radioimmunoassay; the minimum detectable dose of standard was 1.5 ng/ml. Although ethylene diamine tetraacetic acid (EDTA-2K) inhibited the immunoreaction between PP19 (225/242) and anti-PP19 antibody (632 ZA), the PP19 concentration did not differ between serum and heparin and sodium citrate plasmas. The serum PP19 concentration was increased by hemolysis. In blood cell fractions separated by the Ficoll-Paque/Macrodex method, polymorphonuclear leukocyte fraction contained the highest PP19 concentration. The circulating serum PP19 concentration was 4.5±1.1 ng/ml (mean ± standard deviation) in the proliferative phase (n=8) and 5.1±1.6 ng/ml in the secretory phase (n=7) for nonpregnant women, and 4.6±2.2 ng/ml from men (n=12). Seminal plasma (n=8) contained 212.2±99.7 ng/ml. The maternal serum PP19 concentration in 291 normal pregnancies increased from 6.2 ng/ml (median) at 6–7 weeks of gestation to 34.1 ng/ml at 38–39 weeks. The mean PP19 concentration was higher in amniotic fluid and retroplacental blood, but lower in umbilical cord blood than that in circulating maternal serum. In hydatidiform mole, vesicular fluid contained high PP19 concentration (1154.6±659.5 ng/ml), although these maternal serum concentration was not statistically higher than normal range. The chorionic villous trophoblast contained more PP19 than decidua, chorion, and amnion. These results suggest that PP19 has an extraplacental source, even though the chorionic villous trophoblast may be the main source throughout pregnancy.

Isolation of pregnancy-associated plasma protein A

Summary Pregnancy-associated plasma protein A (PAPP-A) was purified from normal term maternal serum in this study using a three step chromatographic procedure with molecular sieve chromatography, heparin-Sepharose affinity chromatography and DEAE-Sephacel chromatography. Thirty milliliter of the maternal serum samples were applied to the column for sieve chromatography and eluted according to molecular weight. PAPP-A was detected at the high molecular weight fraction area via radioimmunoassay for PAPP-A, and 91% of the total amount of PAPP-A in the maternal samples was recovered. These PAPP-A containing fractions were applied to heparin-Sepharose column and eluted in a stepwise increase of NaCl 0.15, 0.30 and 0.60 M in 0.15 M Tris-HCl buffer, pH 7.8. Ninety-five percent of PAPP-A in the applied samples was recovered after 0.6 M NaCl. The pooled fractions, which were containing PAPP-A after heparin-Sepharose affinity chromatography, were then applied on a DEAE-Sephacel Chromatography and eluted in a stepwise increase of NaCl 0.15, 0.30 and 0.45 M in 0.01 M acetate buffer, pH 5.5. Ninety-two percent of PAPP-A in applied samples was recovered after 0.30 M NaCl. Finally PAPP-A containing fractions were concentrated 10 times and 3.8 IU (1.7 mg) of PAPP-A was isolated. The purification schedule removed approximately 99% of total protein in the maternal serum while 74% of PAPP-A was recovered. The purification factor (fold), which was calculated as the increase in specific activity (mlU:PAPP-A/mg:protein) in comparison with the starting value in maternal serum, was 526. And the purity(mg:PAPP-A/mg:protein) of the final product was 68.4%. Analysis of the final purified material using SDS-PAGE demonstrated a single band of 200 kDa, and western blot analysis showed that purified main protein was PAPP-A. The immunological identity between purified PAPP-A in this study and PAPP-A donated by Teisner et al. was recognized by crossed immunoelectrophoresis and tandem crossed immunoelectrophoresis. The PAPP-A purified in this study by the three step chromatographic procedure will hopefully be utilized for the development of a sensitive and convenient assay for PAPP-A and its standard material, furthermore for the basic study of clarifying the biological function of PAPP-A in human body.

Placental Protein 19 in Clinical Blood Coagulation Disorder

The human placenta is a highly functional organ able to synthesize a wide variety of biologically active proteins, including hormones, enzymes, proenzymes, activators, inhibitors, immunoregulatory factors, transport and storage proteins, receptors, and structual proteins [1], Moreover, many proteins of unknown function have been discovered and isolated in placental extracts.

Dynamics and synthesis of new placental tissue protein PP19

Summary Placental protein 19 (PP19) is a new placental tissue protein identified in extracts from the human term placenta by Bohn and Winkler (1985) . We measured PP19 concentrations in body fluids and placental tissue culture media by radioimmunoassay. The minimum detectable dose of standard PP19 was 15 ng/ml. HCG and PP19 concentrations in media from placental tissue culture rose markedly at 144 hours, but were not increased by adding 0.1 mM cycloheximide. The circulating serum PP19 concentration was 4.5±1.1 ng/ml (mean±standard deviation) in the proliferative phase (n=8) and 5.1±1.6 ng/ml in the secretory phase (n=7) for nonpregnant women and 4.6±2.2 ng/ml from mean (n=12). Seminal plasma (n=8) contained 212.3±99.7 ng/ml. The maternal serum PP19 concentration in 291 normal pregnancies increased from 6.2 ng/ml (median) at 6–7 weeks of gestation to 34.1 ng/ml at 38–39 weeks. The mean PP19 concentration was higher in amniotic fluid and retroplacental blood but lower in umbilical cord blood than in circulating maternal serum. In hydatidiform mole, vesicular fluid contained high PP19 concentrations (1154.6±659.5 ng/ml), although these were not statistically higher than the normal range. The immunological identity of standard PP19 and PP19-like immunoreactive substances in the maternal serum, retroplacental blood, vesicular fluid from hydatidiform mole, and seminal plasma was indicated by parallelism between their dose-response curves in serial dilutions. An elevated (>10 ng/ml) serum PP19 concentration was observed in endometriosis (2 of 2 samples), squamous cell carcinoma of uterine cervix (2 of 11), ovarian adenocarcinoma (11 of 19), choriocarcinoma (1 of 5), invasive mole (3 of 11) and gastric adenocarcinoma (3 of 4). The level was low in myoma uteri (6 of 6), benign ovarian cyst (3 of 3), and endometrial carcinoma stage I (7 of 7). Ascites (2 of 5) and ovarian tumor fluid content (6 of 8) showed high PP19 concentrations. A metastatic gestational choriocarcinoma case showed elevated serum PP19 level in accordance with occurrence of DIC. In blood cell fractions separated by the Ficoll-Paque/Macrodex method, PMN fraction contained the highest PP19 concentration. These results indicate that PP19 has an extraplacental source even though the placental villous trophoblast may be the main source throughout pregnancy, and that PP19 may be associated with polymorphonuclear leukocyte function although the exact biological function has yet to be clarified.