Ultrasound features prior to 11 weeks' gestation and first-trimester maternal factors in prediction of hypertensive disorders of pregnancy.

OBJECTIVES: Maternal hypertensive disorders (MHD), including pregnancy-induced hypertension and pre-eclampsia, are estimated to occur in 7-10% of pregnancies worldwide and have significant short- and long-term implications for both mother and fetus. This study aimed to determine the association of conventional and novel early first-trimester ultrasound measures with MHD and whether these ultrasound measures, combined with maternal characteristics and biochemistry, improve the prediction of MHD. METHODS: This was a prospective cohort study of consecutive women with a singleton pregnancy, attending for an early (5 + 1 to 11 + 0 weeks' gestation) ultrasound examination at a private obstetric ultrasound practice between February 2016 and August 2018. Recorded ultrasound measurements included mean sac diameter, yolk sac diameter, crown-rump length, fetal heart rate (FHR), trophoblast thickness, trophoblast volume (TV) and mean uterine artery pulsatility index. Maternal biochemistry was assessed at 10-14 weeks and included beta-human chorionic gonadotropin, pregnancy-associated plasma protein-A (PAPP-A), placental growth factor (PlGF) and maternal serum alpha-fetoprotein. Regression models were fitted for each ultrasound parameter and multiples of the median (MoM) were calculated. All measures were compared between women who had a normotensive outcome and those who subsequently developed MHD. Logistic regression analysis was used to create a prediction model for MHD based on maternal characteristics, ultrasound measurements at 5 + 1 to 11 + 0 weeks' gestation and maternal biochemistry at 10-14 weeks. RESULTS: In total, 1141 women were included in the analysis, of whom 1086 (95.2%) were normotensive at delivery and 55 (4.8%) developed MHD. Women who developed MHD weighed significantly more than did normotensive women (P < 0.0001). Mean MoM values for TV (P = 0.006), PAPP-A (P = 0.031) and PlGF (P = 0.044) were decreased significantly in pregnancies that subsequently developed MHD. The proposed logistic regression model includes maternal weight and height and MoM values for TV, FHR and PlGF, resulting in an area under the receiver-operating-characteristics curve of 0.80 (95% CI, 0.75-0.86). CONCLUSION: The combination of maternal weight and height, TV and FHR, measured prior to 11 weeks' gestation, and first-trimester PlGF appears to have good predictive value for development of MHD later in pregnancy. Copyright © 2020 ISUOG. Published by John Wiley & Sons Ltd.

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.

A novel in vitro co-culture system for the study of maternal decidual endothelial cell-trophoblast interactions in human pregnancy.

Investigation of the pathophysiology of pre-eclampsia (characterised by insufficient invasion of the intrauterine vasculature by cytotrophoblasts) has been hampered by the absence of a suitable animal model, and ethical constraints in clinical studies. We have developed a novel in vitro human cell co-culture system allowing direct assessment of cytotrophoblast invasion of a decidual endothelial cell monolayer from the abluminal side, as occurs in vivo. This model will facilitate detection, at the cellular level, of abnormal endothelial cell-trophoblast functional interactions in pre-eclampsia and other pregnancy disorders with abnormal placentation.

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.

Prenatal screening in the first trimester of pregnancy.

Prenatal screening for fetal abnormalities in an accepted part of modern obstetric management. Improvements on current screening procedures need to address increased diagnostic efficacy and earlier diagnosis. This study evaluates diagnostic efficacy of PAPP-A and F beta-hCG in the detection of first trimester pregnancy abnormalities, including Down syndrome (DS). Of 731 pregnant volunteers, obtained from a mature age population undergoing chorionic villus sampling (CVS), 17 DS and 11 compromised (six numerical (excluding sex chromosome) aneuploidies, five spontaneously failed) pregnancies were detected. Application of an algorithm, which combines PAPP-A and F beta-hCG levels with material age, detected 66.6 per cent of DS pregnancies for a five per cent false positive rate. Similarly, for a 1-2 per cent recall rate, 72.2 per cent of compromised pregnancies were detected. This report supports the notion that prenatal screening at 9-12 weeks of pregnancy is achievable with PAPP-A and F beta hCG quantitation. Whereas mid-gestational screening targetted the detection of fetal abnormalities, screening earlier in pregnancy will detect other pregnancy-related abnormalities, in addition to aneuploidy.

Serum from women with preeclampsia partially corrects the abnormal in vitro prostacyclin secretion of preeclamptic villous cytotrophoblasts but not that of prostaglandin E2 or endothelin-1.

OBJECTIVE: This study was conducted (1) to determine in vitro placental villous cytotrophoblast secretion of prostacyclin, prostaglandin E2, and endothelin-1, (2) to examine the effect of serum from normal and preeclamptic women on secretion of these vasoactive substances, and (3) to determine whether responses to these sera by cytotrophoblasts from preeclamptic pregnancies are different from those of normal pregnancies. STUDY DESIGN: Cytotrophoblasts isolated from human placentas collected at cesarean section from normal and preeclamptic women were incubated for 20 hours in 20% (vol/vol) sera from preeclamptic or gestational age-matched normal pregnant women. Levels of prostacyclin (measured as 6-keto-prostaglandin F1alpha), prostaglandin E2, and endothelin-1 were measured in cytotrophoblast supernatants. RESULTS: In normal pregnancy sera preeclamptic cytotrophoblasts secreted significantly lower amounts of prostacyclin and prostaglandin E2 but higher amounts of endothelin-1 than did normal cytotrophoblasts. In preeclamptic sera the abnormality of prostacyclin secretion by preeclamptic cytotrophoblasts was partially corrected, but there was no effect on prostaglandin E2 or endothelin-1 secretion. Preeclamptic sera had no effect on secretion by normal cytotrophoblasts. CONCLUSIONS: The differences between normal and preeclamptic cytotrophoblasts in prostacyclin, PGE2, and endothelin-1 secretion and in response to preeclamptic serum suggest altered arachidonic acid metabolism in preeclampsia.

First trimester aneuploidy screening using nuchal translucency, free beta human chorionic gonadotrophin and maternal age.

Screening for aneuploidy using maternal age has a low detection rate and high false positive rate. Second trimester maternal serum screening increases trisomy 21 detection and decreases the false positive rate. First trimester screening would enable definitive diagnosis with chorionic villus sampling, and simple surgical termination of affected pregnancies would still be an option. Nuchal translucency (NT), free beta human chorionic gonadotrophin (f beta HCG) and maternal age were assessed in 302 patients before chorionic villus sampling. NT positively and f beta HCG negatively correlated with gestation, but neither correlated with maternal age nor with each other. Both NT and f beta HCG were increased in trisomy 21. NT was increased and f beta HCG was decreased in trisomy 18. Multivariate discriminant analysis enabled 87.5% detection of trisomy 21 in this high-risk population, for a 14% false positive rate. In a simulated normal population, using a risk cut-off of 1 in 250, 71% detection was achieved for a 7% false positive rate. The combination of NT, f beta HCG and maternal age is a simple, readily available and viable first trimester screening strategy.

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.

Establishment and application of a normal range for nuchal translucency across the first trimester.

Before chorionic villus sampling at 10-13 weeks' gestation, 453 women had the crown-rump length and nuchal translucency (NT) measured with transabdominal ultrasound. There were 19 aneuploid pregnancies (ten cases of trisomy 21, six of trisomy 18, one of 47 + marker, one 47,XXX, and one 45,X mosaic). Average NT was 1.7 mm (range 0-5 mm), correlating with the crown-rump length, but not maternal age. A static cut-off of 2.5 mm gave a false-positive rate of 1.3 per cent for crown-rump length between 30 and 35 mm, rising to 13 per cent in fetuses with a crown-rump length between 50 and 65 mm. This gave an overall false-positive rate of 5.5 per cent for a detection rate of 30 per cent for trisomy 21. Applying a dynamic action limit (95th centile), the false-positive rate remained at 5 per cent irrespective of the crown-rump length, detecting 30 per cent of trisomy 21 and 36.8 per cent of all aneuploidies. Raising the action limit to the 97.5th centile halved the false-positive rate (2.5 per cent), with no change in trisomy 21 detection and only a slight decrease in aneuploidy detection (31.6 per cent). Aneuploid fetuses showed normal first-trimester growth. NT increases with gestational age, making a dynamic action limit necessary to decrease the false-positive rate, while maintaining aneuploidy detection rates. Aneuploidy does not cause significant first-trimester growth retardation, enabling normal ranges for NT with crown-rump length to apply.

Trophoblast antigen levels in the first trimester of a trisomy 22 pregnancy.

We report trophoblast antigen (pregnancy-associated plasma protein-A, PAPP-A; free beta-human chorionic gonadotrophin, F beta hCG) expression in a trimosy 22 pregnancy. Maternal concentrations of these antigens were depressed prior to detection of abnormalities by ultrasonography. Immunohistochemical findings were consistent with depressed marker expression.

Triple test for prenatal detection of Edwards syndrome (trisomy 18).

OBJECTIVE(S): To demonstrate the limitation of complete reliance on computer generated interpretations and to highlight the need for understanding of pregnancy-related biochemistry when offering prenatal screening. METHODS: Four cases of cytogenetically confirmed trisomy 18 pregnancies are presented. All four cases underwent prenatal screening (Triple Test-AFP, uE3, t beta-hCG) at midgestation and risk assessment by the alpha algorithm. RESULTS: All four cases of trisomy 18 were assessed as being at low risk for DS and/or open NTD. Although marker levels were not consistent with either of these clinical situations, they were indicative of a compromised pregnancy. Circulating levels of trophoblast-derived antigens (uE3, t beta-hCG) were depressed (< or = 0.5 MoM) in all four cases. Further investigations (ultrasonography, amniocentesis) confirmed a trisomy 18 fetus. CONCLUSIONS: Risk assessment by computer based algorithms relies on maternal factors and specific DS/NTD marker profiles. Aberrant marker profiles are not distinguished from normal. Therefore, it is essential that prenatal screening is offered only by those competent in pregnancy biochemistry and able to identify these abnormal situations.

Isolation of ERp72 from guinea pig term placentae using heparin Sepharose affinity chromatography.

The mammalian placenta synthesises many varied antigens, including proteins, such as hormones, enzymes and protease inhibitors. In this report, we isolated and purified the two protein isomerase-related protein precursor ERp72 isoforms from aqueous extracts of guinea pig placenta, by four (4) chromatographic procedures; i) affinity chromatography on immobilised heparin, ii) gel filtration (Ultrogel AcA-54), iii) anion exchange chromatography (Mono-Q), and, iv) negative immunoaffinity chromatography. From 20 term placentae, the final yield of ERp72 isoforms was 2.4mg (Mr 71.5 kDa) and 1.5mg (Mr 75.8 kDa). Identity was confirmed by NH2-terminal amino acid sequencing which demonstrated 85% homology to human ERp72. By indirect immunofluorecence. ER p72 expression was demonstrated in tunicamycin stressed pre-implantation embryos and unfertilised oocytes. These findings demonstrate the potential for immunological monitoring of ERp72 expression, by cultured oocytes and embryos, during manipulation by assisted reproductive technologies.

Influence of fetectomy on serum pregnancy-associated plasma protein-A concentrations in the baboon.

In baboons as in humans, the placenta is a source of various peptides, including pregnancy-associated plasma protein-A (PAPP-A). However, our present understanding of the regulation of PAPP-A production is incomplete. We have demonstrated that after fetectomy, the baboon placenta retains steroidogenic capacity and is maintained in utero until delivered spontaneously close to term. We have suggested, therefore, that fetectomy provides a valuable in vivo approach to elucidating the role(s) of the fetus, and of the hormones (e.g., estrogen and progesterone) dependent upon the presence of the fetus, in the regulation of placental steroidogenesis during primate pregnancy. Therefore in the present study we utilized the fetectomy model to evaluate the respective roles of the fetus, estrogen, and progesterone on placental PAPP-A. Estradiol, progesterone, and PAPP-A concentrations were determined by RIA in maternal blood collected under ketamine anesthesia on Days 78-100 (n = 5), Days 102-144 (n = 4), and Days 146-164 (n = 3) of gestation (term = Day 184) in control baboons (Papio anubis) and on Days 110-164 in baboons fetectomized on Day 100 (n = 9). Studies were also conducted in five animals in which placental estrogen was increased by maternal treatment on Days 70-100 with androstenedione and in three animals treated on Days 140-164 with the antiestrogen, ethamoxytriphetol (MER-25; 25 mg/day/kg BW).(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnosis and management of extrauterine pregnancies.

This study was based on 16 women provisionally diagnosed as having extrauterine pregnancies. Of these, 13 (81.3%) were confirmed as positive at operation. Patients were managed according to 1 of 3 regimens; 1) methotrexate (n = 4), 2) methotrexate followed by surgery (n = 3) and 3) surgery (n = 6). Serial blood samples, collected before and after treatment, were analyzed for ovarian (oestradiol, E2; progesterone, P4) uterine (placental protein 14, PP14) and placental markers (chorionic gonadotrophin, HCG; pregnancy-associated plasma protein-A (PAPP-A). Of the pretreatment samples, only 30.4% and 41.7% were depressed for PP14 and HCG, respectively. By contrast, the diagnostic value of PAPP-A (77.8%) and P4 (87.5%) was greater. Biochemical monitoring of treatment was best achieved with trophoblastic derived antigens (HCG), whereas antigens of maternal origin demonstrated widely varied responses. This study established the effectiveness of chemotherapy for treatment of tubal pregnancies as an alternative to surgery, but if a biochemical marker is required, the marker of choice is HCG.

Progesterone antagonist RU 486 accommodates but does not induce labour and delivery in primates.

The hormonal mechanisms of parturition in primates remain controversial. Even so, the well-known decrease of plasma progesterone concentration near term is considered by many as the 'labour inducer'. The progesterone antagonist RU 486, which blocks progesterone activity at the cellular receptor level, appears to be a useful hormonal tool by which to study this tissue. Here, we tested its capacity to induce labour and delivery. A total of 23 Cynomolgus monkeys (Macaca fascicularis), within 9-17 days of expected term, were assigned to four different protocols to study various doses, routes and regimens of RU 486 administration. Observations included uterine contractile patterns, pharmacokinetics of RU 486 in plasma and passage of RU 486 into breast milk. None of the protocols tested successfully induced labour resulting in vaginal delivery within 24 h. Instead, the data demonstrate that blockade of progesterone activity by the progesterone antagonist was not sufficient by itself to achieve parturition in these primates. Uterine myometrial contractile activity under RU 486 exposure was not sufficient to induce labour and delivery. Moreover, the progesterone antagonist concentration in breast milk was very low, indicating little passage to suckling newborn infants.

Human preovulatory follicular fluid: inhibin and free steroids related to optimal follicular maturation in ovarian stimulation regimes and possible function in ovulation.

Concentrations of inhibin, total and free oestradiol and progesterone were determined in preovulatory follicular fluid from 15 women undergoing in-vitro fertilization and embryo transfer treatment. The women underwent ovarian stimulation using clomiphene citrate and human menopausal gonadotrophin (HMG) (69 follicular fluid samples) in one cycle, and gonadotrophin-releasing hormone agonist (GnRHa) and HMG stimulation in the next treatment cycle (64 follicular fluid samples). The women thereby served as their own control. Inhibin, total oestradiol and progesterone were measured by radioimmunoassay. Concentrations of free steroid were calculated after quantitation of the steroid binding proteins, i.e. sex-hormone binding globulin (SHBG), cortisol binding protein (CBP) and albumin. Levels of inhibin and free and total progesterone were significantly higher in follicular fluids collected after stimulation with the GnRHa compared to the clomiphene regime (P less than 0.05, P less than 0.001, P less than 0.001, respectively). In contrast, levels of total and free oestradiol in follicular fluid were significantly lower after stimulation with GnRHa than after clomiphene stimulation (P less than 0.001). These results indicate that the follicles have achieved a more optimal maturation during the GnRHa regimen than during the clomiphene regime. It is suggested that the concentration of free biologically active steroids in follicular fluid, released into the peritoneal cavity during ovulation, may be physiologically important in stimulating the oviduct and the uterus in connection with ovulation, pre-embryo development and implantation.

Radioimmunoassay of inhibin based on synthetic human inhibin alpha-chain peptide.

Polyclonal rabbit antisera were produced against cyclic human inhibin [(Cys6, Tyr7) alpha-(6-30)NH2] peptide, covalently conjugated to bovine serum albumin. The tyrosine residue introduced at position 7 facilitated the oxidative incorporation of radiolabel (125I) to yield a tracer with specific activity of 73.9 Ci/g. These reagents were used to develop a homologous equilibrium radioimmunoassay for human inhibin, with polyethylene glycol, 200 g/L, serving as the separation phase. At a detection limit of 2 micrograms/L (n = 7), immunoactive inhibin was detectable in human pre-ovulatory follicular fluid (128 micrograms/L), seminal plasma (2374 micrograms/L), amniotic fluid (66 micrograms/L), and placental extract (347 micrograms/L). We also demonstrated inhibin immunoreactivity in biological fluids from other mammalian species: macaque, chimpanzee, porcine, and bovine, but not rodent (guinea pig). Although the antisera were raised against a nonbioactive inhibin peptide, immunoglobulins fractionated on Protein A-Sepharose neutralized the bioactivity of human ovarian inhibin. Further characterization of inhibin immuno- and bioactivity was undertaken with immobilized heparin, divalent metal cations, and dye ligands. Only heparin-Sepharose distinguished between immuno- and bioactive inhibin.

Immunization of merino ewes with a synthetic inhibin peptide or with preparations obtained from bovine and porcine follicular fluids by immunoaffinity chromatography result in different effects on ovulation rate and on plasma gonadotrophin concentrations.

Ewes were immunized with either a synthetic peptide (peptide 1-32) that has an amino acid sequence identity with the first 32 amino acids at the amino terminal of the alpha-subunit of porcine inhibin, or with bovine or porcine monoclonal antibody purified inhibin (bMPI and pMPI respectively), obtained by immunochromatography from follicular fluids. The peptide 1-32 was conjugated to albumin before use. Peptide 1-32 and bMPI increased ovulation rate and number of follicles (greater than or equal to 5 mm diameter). Although bMPI increased plasma FSH concentration the peptide did not. pMPI had no effect on ovarian activity but markedly elevated both plasma FSH and LH concentrations. The plasma LH concentration was lowered in ewes immunized with peptide 1-32. It appears, therefore, that ovulation rate can be increased following increased plasma FSH concentrations at luteolysis or in the absence of such an increase. Conversely, greatly increased plasma gonadotrophin concentrations at luteolysis (pMPI) were not followed by an increase in ovulation rate. Antibodies in the plasma of ewes immunized with peptide 1-32 and bMPI bound to iodinated synthetic human inhibin alpha-chain 6-30 peptide. The results suggest that ovulation rate is at least partly determined by intraovarian factors.

Pregnancy-associated plasma protein A interaction with heparin: a critical appraisal.

Positive affinity chromatography on heparin-Sepharose has proved a most crucial step in the purification of pregnancy-associated plasma protein A (PAPP-A). In this chromatographic procedure, PAPP-A was purified almost 500-fold from term pregnancy serum. Further purification was achieved by gel filtration and negative immunoaffinity chromatography. Both PAPP-A and free heparin inhibited granulocyte elastase (HGE) activity. Whereas free heparin inhibited only in hypotonic buffers, PAPP-A inhibited HGE in hypertonic buffers also. However, PAPP-A did not inhibit other proteases (trypsin, chymotrypsin, plasmin, fibroblast collagenase) or proteolytic cascades (complement activation). Since heparin was not detected in the purified PAPP-A, the inhibition of HGE was not due to desorbed or leeched heparin ligand.