J.G. Grudzinskas

Pregnancy-associated plasma protein A: Circulating levels during normal pregnancy

The development of specific and sensitive electroimmunoassays for a recently identified high molecular weight alpha-2 mobile pregnancy-specific protein (pregnancy-associated plasma protein A, PAPP-A or SP4) is described. These assays have permitted the detection of circulating levels of PAPP-A (10 microgram/L) as early as the fifth week of pregnancy. In all 18 subjects studied, the levels of PAPP-A rose from first detection in the first trimester until delivery at term. The development of these assays now permit the evaluation of PAPP-A measurement as a diagnostic test of early pregnancy and as an index of fetal well-being throughout gestation.

Circulating levels of pregnancy zone protein: normal range and the influence of age and gender.

Serum levels of pregnancy zone protein (PZP) were measured in 506 apparently normal males and 329 normal non-pregnant females, the age range being 18 to 70 years. The estimations of PZP were performed by sensitive radio-rocket-line immunoelectrophoresis. The distribution of the data had a marked positive skew in both sexes which was reduced following logarithmic transformation. Serum concentrations in both men and women were found to increase significantly with advancing age. This increase and the mean concentrations were significantly higher in females.

The prediction of pregnancy failure by measurement of pregnancy-associated plasma protein A (PAPP-A) following in vitro fertilization and embryo transfer.

Circulating PAPP-A was measured serially in five patients following successful IVF-ET. PAPP-A concentrations were consistently normal in all three patients in whom pregnancies progressed normally to term. Depressed levels of PAPP-A were observed in one patient who miscarried spontaneously at 17 weeks gestation despite ultrasound evidence of normal fetal development. Circulating PAPP-A was not detected in the fifth patient, whose tubal pregnancy ruptured at 8 weeks. These data are discussed in relation to surveillance of pregnancies following IVF-ET.

Purification of pregnancy-associated plasma protein-A by a two step affinity chromatographic procedure

A high molecular weight pregnancy specific protein (PAPP-A) was purified by immunospecific affinity chromatography. The purification procedure was based on a new method for affinity chromatography using direct coupling of precipitable immune complexes to the gel matrix. The procedure was performed in two steps: a positive immunospecific affinity chromatography followed by a negative affinity chromatography in which balanced amounts of anti-contaminant antibodies were used as ligant. The purified material at a concentration of 300 microgram/ml was tested by immunoelectrophoretic methods and no contaminants were detectable. WHO beta-1 SP1 reference material 78610 contained 45 microgram PAPP-A/ml.

Circulating C3, C4, and C3 Split Products (C3c and C3d) During Normal Pregnancy*

ABSTRACT: The plasma concentrations of the complement components C3 and C4, as well as the split products C3c and C3d, were measured before, during, and after normal pregnancy. Significantly increased values were observed in the C3 and C3d levels in the second and third trimesters of pregnancy. The level of C4 was not significantly affected by pregnancy and C3c could not be detected using electroimmunoassays. These results suggest that the increased C3 split‐product levels observed reflected an increased turnover of native C3 rather than activation of the complement cascade.

Influence of time, temperature and coagulation on the measurement of C3, C3 split products and C4

Quantitative and qualitative immunoelectrophoretic analyses of circulating C3, C3 split products and C4 were performed in matched EDTA plasma and serum obtained from 5 normal subjects and stored for up to 48 h at room temperature (18 degrees C-22 degrees C) and 4 degrees C. Fluctuations in apparent levels of C3 were greater in serum than plasma stored at room temperature, a fall in levels seen by 24 h being followed by a significant increase. By contrast, levels of C3 did not alter if stored at 4 degrees C. C4 levels in both EDTA plasma and serum remained unchanged for 24 h, a slight decrease being seen at 48 h. Levels of C4 remained constant if samples were stored at 4 degrees C. Crossed immunoelectrophoresis revealed a significant progressive decrease in C3 levels and a simultaneous increase in C3c occurring after 4 h in serum and 8 h in EDTA plasma, stored at room temperature. In studies conducted at 4 degrees C, similar but delayed fluctuations were seen. A progressive and significant increase in C3d levels was seen in both plasma and serum samples stored at room temperature, levels rising to 276% (plasma) and 308% (serum) of levels seen at zero time. At 4 degrees C marginal increases in C3d levels only were observed. These results suggest that in vitro degradation of C3 and C4 are readily facilitated by temperature, time and coagulation, and that conditions of collection and storage of samples must be optimized for the accurate definition of activation of the complement cascade.

Examination of placental proteins in charge-shift immunoelectrophoresis

Three pregnancy-associated proteins, pregnancy-specific beta1-glycoprotein (SP-I), pregnancy-associated plasma protein A(PAPP-A), and placental lactogen (hPL) were examined by charge-shift immunoelectrophoresis. PAPP-A and hPL exhibited hydrophilic properties whereas SP-I was amphiphilic. These observations suggest that SP-I is a cell membrane protein.

Investigations into the molecular heterogeneity of pregnancy-specific β1-glycoprotein (SP1)

The molecular heterogeneity of pregnancy-specific beta 1-glycoprotein (SP1) was examined by analytical immunoelectrophoresis and a radioimmunostaining technique of immunoelectrophoretic plates. Analytical crossed immunoelectrophoretic analysis and radioimmunostaining of these plates demonstrated the presence of normal human serum components in the alpha-mobile precipitate previously considered to be exclusively the high molecular pregnancy-specific protein, SP1 alpha. This observation suggested that two molecular populations were contributing to the alpha-mobile precipitate. Following fractionation of late-pregnancy serum by size chromatography, the radioimmunostaining technique further demonstrated the presence of normal serum components in the intermediate fraction but not in authentic SP1 (i.e., SP1 beta) or the high molecular weight form (SP1 alpha). We suggest that SP1 antigenic determinants are distributed in three different fractions of pregnancy serum, one of which (intermediate fraction) is a complex of authentic SP1 (SP1 beta) and a normal serum protein, whereas non-pregnancy serum components were not demonstrable in the remaining two (i.e., SP1 beta or SP1 alpha).

Demonstration of antigenic determinants specific for the split products of the third complement factor, C3

Crossed anti-C3c immunoelectrophoresis of a plasma sample with in vivo complement activation revealed a pronounced spur formation towards the cathodic region of immunochemical interaction between native C3 and C3c. In situ absorption of the antibody preparation against C determinants of the 3rd complement factor using fresh normal EDTA plasma as antigen demonstrated the presence of C3 split product specific determinants.

Hyperstimulated human preovulatory follicular fluid, luteinized cells of unruptured follicles, and corpus luteum contain pregnancy-associated plasma protein A (PAPP-A)**Supported by grants from the Academy of Finland (02/027), the Cancer Society of Finland, and the Sigrid Juselius Foundation.

Radioimmunoassay, gel filtration, and immunoperoxidase methods were used to study the occurrence, properties, and concentration of pregnancy-associated plasma protein A (PAPP-A) in the human ovary and in the follicular fluid from 97 hyperstimulated follicles from 29 infertile women participating in an in vitro fertilization program. At the detection level of 15 micrograms/l, PAPP-A was found in 83 of 97 follicular fluids, the levels ranging from undetectable to 483 micrograms/l (median, 130 micrograms/l). In gel filtration, PAPP-A immunoreactivity of follicular fluid eluted in the same volume as placental PAPP-A, and the dose-response curves of follicular fluid PAPP-A and purified PAPP-A were parallel. The PAPP-A concentration was not affected by prior treatment with a protease inhibitor. Follicular fluid aspirates containing the ovum had a higher PAPP-A concentration than those in which no ovum was detected (P less than 0.01), whereas no difference was found in the PAPP-A concentrations between follicles yielding an ovum which was fertilized and cleaved and those yielding an unfertilizable oocyte. There was a correlation between PAPP-A and estradiol or progesterone concentrations, and between the PAPP-A concentration and the volume of follicular fluid aspirate. In hyperstimulated unruptured follicles, PAPP-A was localized in the luteinized granulosa and theca interna cells, but not if luteinization was not observed. Corpus luteum cells were also PAPP-A positive, whereas unstimulated Graafian follicles were negative. Our results indicate that PAPP-A appears in the hyperstimulated follicle shortly before ovulation and may thus play a part in the early events of human reproduction.