Pregnancy-associated plasma protein A in human ovarian follicular fluid.

Human ovarian follicular fluid contains pregnancy-associated plasma protein A (PAPP-A) immunoreactivity as detected by RIA. This PAPP-A was found to be stable to repeated freeze-thaw cycles and not removed by dialysis. Further characterization showed that ovarian follicular PAPP-A bound reversibly to heparin-Sepharose. On gel chromatography follicular PAPP-A coeluted with radioiodinated PAPP-A and pregnancy serum PAPP-A which was determined to have an apparent mol wt of 820,000. In the RIA, serial dilutions of high molecular weight heparin-Sepharose binding proteins gave parallel displacement curves to pooled late pregnancy serum and to the International Reference Preparation, WHO 78/610. All the human ovarian follicular fluids tested had PAPP-A concentrations between 0.317-1.595 IU/liter. The relationship between follicular content of PAPP-A and follicular volume was best expressed by the exponential relationship, y = 164.3 e0.117x. Therefore, ovarian follicular fluid PAPP-A has many physico-chemical similarities to and shares immunological identity with pregnancy-derived PAPP-A. As this pregnancy-associated glycoprotein cannot be detected in the circulation of normal non-pregnant adults, its presence within the Graafian follicle is believed to be of intrafollicular origin for the maintenance of proteolytic homeostasis.

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.

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.

Regulation of insulin-like growth factor-binding protein-3 ternary complex formation in pregnancy.

The IGFs are believed to be important in pregnancy and are implicated in the pathophysiology of pre-eclampsia. In adults the IGFs circulate primarily with IGF-binding protein-3 (IGFBP-3) and an acid-labile glycoprotein (ALS) in a 140 kDa complex which limits IGF bioavailability. Less than 10% of IGFBP-3 is in lower molecular weight forms. We have investigated the developmental regulation of the IGF/IGFBP system in normal and pre-eclamptic pregnancies with particular emphasis on the IGFBP-3 ternary complex. Circulating levels of IGF-I, IGFBP-3 and ALS, and their degree of association in the ternary complex in the fetus increased with gestational age. In neonatal serum from deliveries <35 weeks' gestation IGFBP-3 was predominantly in 30-50 kDa form(s) and ALS was a limiting factor for ternary complex formation. In serum from deliveries >35 weeks both ALS and IGFs were limiting but approximately 25% of IGFBP-3 was unable to form the ternary complex even in the presence of excess ALS and IGF-I. Serum IGFBP-1, -2 and -6 concentrations tended to decrease with increasing gestational age. In pre-eclamptic pregnancies, amniotic fluid IGFBP-2, -3 and -6 levels decreased with gestational age while IGFBP-1 levels did not show the normal decline. We speculate that the endocrine IGF system develops in the fetus during the third trimester of pregnancy when ALS levels increase.

RU 486 induced suppression of placental neutrophil elastase inhibitor levels.

After administration of RU 486 to pregnant cynomolgus monkeys, placental morphology varied from normal to pathological. In all cases (n = 5) circulating and placental PAPP-A levels were markedly suppressed by 76.0 per cent and 55.5 per cent, respectively. When fetal demise occurred within 24 h prior to caesarian section, morphological changes consistent with an active inflammatory (polymorphonuclear leukocytosis) reaction was readily observed at the utero-placental interface, degrading the chorionic villi. Whereas heparin-Sepharose fractionated aqueous extracts of placentae inhibited human neutrophil (or granulocyte) elastase (HGE) activity, extracts of placenta being degraded by host phagocytic-proteolytic defense system were rich in HGE activity. This study establishes: (1) the cynomolgus monkey as a model for PAPP-A studies, (2) that haemochorially implanted placentae produce PAPP-A, a heparin-binding inhibitor of HGE, (3) that administration of progesterone receptor antagonist suppressed placental PAPP-A synthesis, and (4) disrupted protease-protease inhibitor equilibrium at the feto-maternal interface. Thus supporting a role for progesterone in placental PAPP-A production and maintenance of a placental barrier against maternal phagocytic-proteolytic defenses.

Modulation of prostacyclin and thromboxane secretion by cytotrophoblasts from normal and pre-eclamptic human pregnancies.

We and others have previously observed an imbalance in cytotrophoblast secretion of the vasoactive prostanoids prostacyclin and thromboxane A(2) in pre-eclampsia. To examine the effects of potential modulators of this imbalance, cytotrophoblasts isolated from normal and pre-eclamptic pregnancies were incubated in the presence of lipopolysaccharide, the calcium ionophore A23187, tumour necrosis factor alpha, or interleukin 1beta, with or without the cyclo-oxygenase inhibitor, indomethacin. Further incubations included the drugs tranylcypromine, a prostacyclin synthetase inhibitor (0.1, 10 microM ), or the thromboxane synthetase inhibitor, pirmagrel (0.001, 1 microM ). Results showed that cytotrophoblasts from pre-eclamptic pregnancies had increased thromboxane production and significant stimulation of prostacyclin production by lipopolysaccharide and calcium ionophore. Lipopolysaccharide stimulated thromboxane production in normal cytotrophoblasts, while indomethacin almost completely inhibited production of both prostanoids. Tranylcypromine mildly inhibited prostacyclin production in normal cytotrophoblasts only, whereas pirmagrel strongly inhibited thromboxane production in a dose-related manner, with reciprocal increase in prostacyclin production occurring in cytotrophoblasts from pre-eclamptic pregnancies. This study confirmed that cytotrophoblasts from pre-eclamptic women had increased thromboxane production and showed that pirmagrel, at the relatively low dose of 0.001 microM, was able to normalize the imbalance of thromboxane and prostacylin production and may therefore warrant further investigation as a treatment for pre-eclampsia.

In-vitro secretion of prostanoids by placental villous cytotrophoblasts in pre-eclampsia.

Villous trophoblasts isolated from term placentae of normal pregnancies, and pregnancies complicated by chronic hypertension or pre-eclampsia, were examined over 7 days in primary culture. Low levels of prostaglandin E2 and prostacyclin (measured as 6-keto prostaglandin Fl alpha) were secreted by trophoblast cells from all three clinical groups. Secretion was maximal at day 1 and decreased exponentially thereafter. Thromboxane secretion also fell sequentially from day 1. Thromboxane secretion by pre-eclamptic trophoblasts was three to four times that of cells from normal or chronically hypertensive subjects. Prostanoid secretion by isolated cultured cytotrophoblasts was not dependent on aggregation or morphological alteration, nor related to changes in progesterone or human chorionic gonadotrophin production. Because the local maternal circulation is exposed to substances secreted by this cell population, thromboxane could be the trigger for vasoconstriction and coagulation found within the maternal uteroplacental circulation in pre-eclampsia.

Potential role of pregnancy-associated plasma protein-A in human reproduction.

By radioimmunoassay, pregnancy-associated plasma protein-A (PAPP-A) was undetectable in matched follicular and luteal phase serum samples (n = 17) or in the peripheral circulation of normal males (n = 17). However, seminal plasma (91.5%), cervical mucus (100%) and pre-ovulatory follicular fluid (99.6%) were consistently PaPP-A positive. In addition to PAPP-A, four circulating protease inhibitors (PIs) were detected in pooled seminal plasma whereas pooled follicular fluid contained an additional six. Follicular concentrations of serum PIs were inversely related to molecular size. By contrast, PAPP-A formed a positive concentration gradient across the blood-reproductive tract barrier suggesting PAPP-A production within the reproductive tract. A minor proportion (1.7%) of ejaculated spermatozoa were coated with PAPP-A, as demonstrated by direct immunofluorescence. Since PAPP-A specifically inhibits leucocyte elastase, it is suggested that PAPP-A coated spermatozoa were "selected" to overcome localized phagocytic-proteolytic degradation. The physiological significance of these findings are discussed in relation to human reproduction.

Monitoring of postimplantation embryo viability by measurement of PAPP-A, SP1, and hCG.

After successful in vitro fertilization and embryo transfer, chorionic gonadotropin, pregnancy-specific beta 1-glycoprotein, and pregnancy-associated plasma protein-A were measured in serum samples collected serially from 21 patients. While 14 pregnancies, including one twin pregnancy, progressed successfully to term, the remaining seven pregnancies failed during the first half of gestation. This latter group consisted of three tubally implanted, one anembryonic, and three spontaneously aborted pregnancies. Circulating levels of hCG, SP1, and PAPP-A in the patient with an anembryonic pregnancy were within normal limits. Similarly, 90.5% of the serum samples obtained from women with tubal pregnancies showed hCG levels within normal limits. By contrast, only two of these samples had detectable PAPP-A, of which only one was within normal limits. Of the samples obtained from the patients who spontaneously aborted, including one patient with normal ultrasonic findings up to 48 hours prior to the event, 85.7% had PAPP-A concentration below the 10th percentile, whereas only 16.7% of these samples showed depressed hCG levels. These data suggest that PAPP-A measurement has great clinical potential in the management of compromised early pregnancies.

Pregnancy-associated plasma protein-A and placental protein 5 in human ovarian follicular fluid.

By sensitive and specific radioimmunoassays PAPP-A and PP5 were detected in follicular aspirates obtained from women undergoing ovarian hyperstimulation for oocyte harvest prior to in vitro fertilization and embryo transfer. Follicular and pregnancy-derived PAPP-A were immunologically and physicochemically indistinguishable. Similarly, pregnancy- and nonpregnancy-derived PP5 were immunologically indistinguishable. However, in addition to the 18- and 36-K species, a larger species having a molecular size greater than 140K was found in the follicular fluid. Mean follicular PAPP-A and PP5 concentrations were 727 mIU/L and 1376 mAU/L, respectively, with no significant correlation between follicular PAPP-A, PP5, and steroid concentrations. There was, however, a significant but negative relationship with follicular volume. Preliminary in vitro studies indicated that both proteins were synthesized by granulosa cells in preparation for follicular rupture. Follicular PP5, like antithrombin III, interacted reversibly with heparin and thrombin affinity matrices, suggesting a potential biological role as a follicular anticoagulant, whereas PAPP-A, a specific and potent inhibitor of leukocyte elastase, contributes to the maintenance of proteolytic homeostasis and the protection of spermatozoa and embryo against proteolytic attack originating from the maternal leukocytes.

Pregnancy-associated plasma protein-A and placental protein 5 in human seminal plasma.

Human seminal plasma contains two glycoproteins which are physiochemically and immunologically indistinguishable from pregnancy-associated plasma protein-A and placental protein 5. Seminal concentrations of both glycoproteins did not correlate with clinical assessment of semen quality. Furthermore, analysis of split ejaculates indicated a nontesticular origin for both proteins, which are possibly secreted into the distal portions of the tract by the accessory glands (prostate gland and seminal vesicles). The physiological significance of these findings has yet to be determined. However, it is suggested that pregnancy-associated plasma protein-A, a known potent inhibitor of leukocyte elastase, protects the deposited sperm against proteolytic attack originating from the localized leukocyte reaction within the female reproductive tract, thus contributing towards sperm survival within this immunologically hostile environment and enabling fertilization to occur.

Effects of clomiphene administration on ovarian function as measured by estradiol and ultrasound.

The growth and rupture of the graafian follicle were studied in 23 women during 40 cycles (20 spontaneous, 20 clomiphene-induced) by the estimation of plasma estradiol levels and ultrasound scan. The mean preovulatory estradiol peak level was 415 pg/ml in spontaneous cycles and 626 pg/ml after clomiphene administration when one follicle was present. Ultrasonic examination revealed the presence of more than one developing follicle in one of 20 spontaneous cycles, and in 11 of 20 cycles after clomiphene treatment. The determination of follicle size and number by ultrasound scan during ovulation induction by the routinely used starting dosage of clomiphene has revealed a previously unrecognized incidence of ovarian overstimulation, and may allow the rationalization of ovulation induction regimens.

Monitoring of postimplantation embryo viability following successful in vitro fertilization and embryo transfer by measurement of placental proteins.

Serum levels of pregnancy-associated plasma protein A (PAPP-A), human chorionic gonadotropin (hCG), and pregnancy-specific beta 1-glycoprotein (SP1) were measured in 21 women after successful in vitro fertilization and embryo transfer. Of the 21 pregnancies, 14, including 1 twin gestation, progressed successfully to term. The remaining seven, composed of tubal (n = 3), anembryonic (n = 1), and spontaneously aborted (n = 3) pregnancies, failed during the first half of pregnancy. Placental protein measurement was of no diagnostic value in the detection of anembryonic pregnancy. Similarly, measurement of hCG and SP1 could not readily distinguish tubal ectopic from normal intrauterine pregnancies. By contrast, the predictive value (38.9%) of a depressed PAPP-A level in conjunction with superior diagnostic sensitivity (70%) and relative risk factor (23.6) proved to be of greater diagnostic value in this potentially lethal condition. In the absence of ultrasonography, the biochemical diagnostic indices were comparable in the prediction of spontaneous abortion. However, in the presence of a live fetus, PAPP-A levels were consistently depressed (sensitivity, 91.7%) many weeks before pregnancy demise. The relative risk factor of depressed PAPP-A levels was 29 times greater than the risk associated with a depressed hCG level. These findings further demonstrate the potential diagnostic value of PAPP-A measurement for monitoring postimplantation embryo viability.

Demonstration of pregnancy-associated plasma protein-A (PAPP-A)-like material in the fallopian tube.

The distribution and concentration of pregnancy-associated plasma protein-A (PAPP-A) in the human fallopian tube were examined by the immunoperoxidase staining technique and radioimmunoassay as part of a detailed study of PAPP-A in the nonpregnant state. PAPP-A-like material was identified in the epithelial cells of the mucosa in all fallopian tube specimens examined (n = 21). The intensity of the staining for PAPP-A was unrelated to the phase of the menstrual cycle. PAPP-A-like material was detected in saline extracts from all tubal tissues (n = 14) but not in any of the sera obtained from the same patients. The tissue concentration (mean +/- standard error of the mean) of immunoreactive PAPP-A varied from 15.2 +/- 1.1 to 30.1 +/- 4.2 micrograms/g protein in different parts of the tubes. No difference in the PAPP-A concentration was found between isthmic, ampullar, and fimbrial part of the tube, but proliferative phase tube seems to contain more PAPP-A than secretory phase tube. The PAPP-A measured in the fallopian tube appears to be similar in molecular size and antigenicity to that of pregnancy.

Pregnancy-associated protein-A does not improve predictability of pregnancy success or failure over human chorionic gonadotropin levels in early normal and abnormal pregnancy.

In the present study we sought to compare levels of PAPP-A and hCG produced by different types of pregnancy: normal, ectopic, threatened abortion and molar pregnancy after evacuation. The gestations ranged from 13 to 122 days. Serum levels of both PAPP-A and hCG were measured and compared. Chi squares analysis were predictive only for increasing trends in hCG as well as decreasing trends of both hCG and PAPP-A. Analysis of variance and linear discriminant function used to evaluate results suggested that PAPP-A did not improve predictability of hCG. The values of PAPP-A levels for the postevacuation molar pregnancies barely exceeded the lower limit of detection; thus, no meaningful comparisons could be made.

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.

Failure of implantation in human in vitro fertilization and embryo transfer patients: the effects of altered progesterone/estrogen ratios in humans and mice.

Daily blood samples were taken for progesterone (P) and estradiol (E2) measurements from women who showed a platelet response consistent with the presence of viable embryos after in vitro fertilization and embryo transfer procedures. A comparison of steroid levels between those women who became pregnant and those who did not revealed the following: at and after the time of transfer, women who failed to become pregnant had significantly higher E2 levels and a lower ratio of P/E2 than women who became pregnant. The P/E2 ratio was a better predictor of implantation failure than was the absolute level of either hormone. Experiments were done in mice to test the hypothesis that P could protect implantation of the embryo against the inhibitory effects of high E2. In mice, implantation was inhibited by relatively high levels of E2. This effect was overcome by concomitant administration of P. There was a significant dose-response-related interaction of P with the E2.

Hyperstimulated human preovulatory follicular fluid, luteinized cells of unruptured follicles, and corpus luteum contain pregnancy-associated plasma protein A (PAPP-A).

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.

The value of ultrasound, gonadotropin, and estradiol measurements for precise ovulation prediction.

The exact prediction of ovulation is becoming more important in the management of infertile women. Graafian follicle diameter, measured by ultrasound and plasma follicle-stimulating hormone, luteinizing hormone, and estradiol levels were compared retrospectively as predictors of ovulation in 14 normal women in whom ovulation was dated by conventional ultrasound techniques. Follicle diameter was found to be a better predictor of the anticipated time of ovulation than endocrine estimations for short-term as well as long-term predictions in normal women. The relationship between follicle diameter and plasma estradiol for each day before ovulation was linear but contained a great amount of scatter, suggesting that the assessment of normality of follicular development in infertile women may not be possible with the use of these parameters.

Placental proteins in the diagnosis and evaluation of the "elusive" early pregnancy.

For many many years much time and effort has been invested to identify, purify, and quantify markers of significant reproductive events. The search for such markers has been recently intensified with the widespread acceptance of IVF and ET as a treatment regimen for the management of certain causes of infertility. Now it is possible to monitor ovarian folliculogenesis and time of ovulation, successful fertilization, and subsequent nidation and the ensuing pregnancy. Following successful fertilization and zygote development, EPF, a protein complex which suppresses in vitro lymphocyte rosette formation can be detected within 24 hours. Shortly after implantation hCG can be measured in the maternal circulation. The developing trophoblastic tissue continues to synthesize and secrete into the maternal circulation many varied proteins including SP1, PAPP-A, and PP5. To date hCG is the mainstay of rapid and early pregnancy diagnosis and is an accepted biochemical marker of successful implantation and trophoblastic activity. Combination of hCG and EPF measurement offers the clinician new insight into his infertile patient for now it is possible to monitor successful fertilization and implantation as separate independent events. Such diagnostic assays should also provide new insight into early embryo mortality rate at the pre- and postimplantation stage and hence indicate the selection pressures acting on an embryo. It is the application of these assays which has led to the definition of subclinical abortions and to the description of biochemical pregnancies. The value of these proteins for the management of compromised early pregnancies has to be more fully examined. Although the differentiation between a normal and abnormal pregnancy is more readily ascertained by serial sampling and analysis of the time taken for circulating levels to double (3). Preliminary data suggest the potential of EPF and PAPP-A measurement in such clinical conditions is enormous. Our studies suggest that PAPP-A, at present, is the only biochemical marker which can predict pregnancy abortion, even in the presence of normal ultrasound scans, many weeks prior to the event, thus implying a vital biological role for PAPP-A in normal pregnancy (52). Despite the need for more intensive evaluation of these early pregnancy indicators the search for that "demmed elusive" marker has more than "Those Frenchies" earnestly looking for him here and there.