Comparison of serum human pregnancy-specific beta-1-glycoprotein 1 levels in pregnant women with or without preeclampsia.

The aim of this study was to investigate the relationship between the maternal serum levels of pregnancy-specific beta-1-glycoprotein 1 (PSG1) and preeclampsia, and to compare levels of PSG1 in pregnancies with preeclampsia and uneventful pregnancies. A case-control study was conducted in a research and training hospital. A total of 40 women with preeclampsia and 42 healthy pregnant women who were gestational age-matched were included. Serum PSG1 levels were measured using enzyme-linked immunosorbent assay. The maternal serum PSG1 levels were significantly lower in patients with preeclampsia compared with controls (11.60 ± 8.08 vs. 17.58 ± 9.72 ng/mL, p = .003). Circulating PSG1 levels were negatively correlated with age in the preeclampsia and control groups (r = -0.322, p = .043), (r = -0.430, p = .005). PSG1 levels, age, blood urea nitrogen levels and birth weight were significantly associated with high odds of having preeclampsia. Receiver operating characteristic (ROC) curve analysis confirmed that the area under ROC curve was 0.707 (95% CI: [0.595-0.819], p < .001) for PSG1. The optimal cut-off value of PSG1 for detecting preeclampsia was ≤ 11.80 ng/mL. There may be a decrease in PSG1 production in preeclampsia-complicated pregnancies where there are pathologies related to placenta formation. A decline in PSG1 concentrations may reflect placental dysfunction.Impact StatementWhat is already known on this subject? Previous studies have reported abnormal pregnancy-specific glycoprotein (PSG) levels in complicated pregnancies and demonstrated their importance in maintaining a healthy pregnancy. Human PSG homologues have been identified in species with haemochorial placentation such as non-human primates, rats and mice, where foetal cells are in direct contact with the maternal circulation. There are studies in which there is no clear relationship between PSGs and preeclampsia.What the results of this study add? We have demonstrated that circulating PSG1 levels were significantly lower in women with preeclampsia than in healthy pregnant women. There may be a decrease in PSG1 production in preeclampsia-complicated pregnancies where there are pathologies related to placenta formation and function. The results obtained from this current study could be used to clarify the relationship between PSG1 levels and preeclampsia.What the implications are for clinical practice and/or further research? Evaluation of the role of circulating PSG1 levels in preeclampsia would be helpful in order to design further studies to determine the feasibility of using PSG1 as a serum marker to predict the risk of developing preeclampsia. The screening performance of PSG1 for preeclampsia is not yet clinically relevant, but may become so when evaluated together with other placental proteins. This will give a lead to further researches which could focus on the early detection of preeclampsia with the combination of several serum markers.

Serum biomarkers for detecting ectopic pregnancy.

Unless an ectopic pregnancy is visible by ultrasound, diagnosis can be a challenge. Differentiating ectopic pregnancies from intrauterine pregnancies can be impossible without intervention or follow-up. This poses a clinical dilemma to the practitioner given the inherent danger to the mother of tubal rupture of an ectopic pregnancy versus the fear of intervening in the case of a desired pregnancy without certainty of diagnosis. Early diagnostic modalities are clearly lacking, and serum biomarkers are currently being investigated as a solution to need for a rapid and accurate test for ectopic pregnancy.

Changes in serum pregnancy-associated glycoprotein, pregnancy-specific protein B, and progesterone concentrations before and after induction of pregnancy loss in lactating dairy cows.

Lactating crossbred dairy cows were synchronized to receive a timed artificial insemination (TAI), and blood samples were collected from all cows from TAI until pregnancy diagnosis 39 d after TAI (period 1), and from pregnant cows from onset of treatment until the end of the experiment (period 2). Cows diagnosed pregnant 39 d after TAI were randomly assigned to 1 of 3 treatments to receive (1) an i.m. injection of saline (CON, n=10); (2) an i.m. injection of PGF(2α) (PGF, n=10); or (3) an intrauterine infusion of 120 mL of hypertonic saline (INF, n=9). During period 1, serum pregnancy-associated glycoprotein (PAG) concentrations began to increase in pregnant cows by 25 d after TAI and differed from those in nonpregnant cows by 27 d after TAI, whereas serum pregnancy-specific protein B (PSPB) concentrations in pregnant cows differed from those in nonpregnant cows by 22 d after TAI. During period 2, time from treatment to cessation of the embryonic heartbeat was greater for PGF than for INF cows (36.0±5.7 vs. 0.2±0.1 h, respectively), and time from treatment to conceptus disappearance was greater for INF than for PGF cows (7.1±3.3 vs. 1.9±0.3 d, respectively). Overall, progesterone concentration was greater for CON and INF than for PGF cows (8.7±2.8, 8.2±3.1, and 1.0±2.3 ng/mL, respectively) due to luteal regression for PGF cows and corpus luteum maintenance for CON and INF cows. Serum PAG and PSPB concentrations differed among CON cows and PGF and INF cows beginning 1 and 2.5 d after treatment for PAG and PSPB, respectively. By 9.5 d after treatment, PAG and PSPB concentrations were similar to those of nonpregnant cows. We conclude that although timing of conceptus expulsion occurred 5.2 d later for INF than for PGF cows, serum PAG and PSPB concentrations decreased at a similar rate from the onset of treatment for both models of pregnancy loss evaluated.

PSG7 and 9 (Pregnancy-Specific ß-1 Glycoproteins 7 and 9): Novel Biomarkers for Preeclampsia.

Background Preeclampsia is pregnancy specific, involving significant maternal endothelial dysfunction. Predictive biomarkers are lacking. We evaluated the biomarker potential, expression, and function of PSG7 (pregnancy-specific ß-1 glycoprotein 7) and PSG9 (pregnancy-specific ß-1 glycoprotein 9) in preeclampsia. Methods and Results At 36 weeks gestation preceding term preeclampsia diagnosis, PSG7 and PSG9 (in Australian cohorts of n=918 and n=979, respectively) were significantly increased before the onset of term preeclampsia (PSG7, P=0.013; PSG9, P=0.0011). In samples collected at 28 to 32 weeks from those with preexisting cardiovascular disease and at high risk of preeclampsia (Manchester Antenatal Vascular Service, UK cohort, n=235), both PSG7 and PSG9 were also significantly increased preceding preeclampsia onset (PSG7, P<0.0001; PSG9, P=0.0003) relative to controls. These changes were validated in the plasma and placentas of patients with established preeclampsia who delivered at <34 weeks gestation (PSG7, P=0.0008; PSG9, P<0.0001). To examine whether PSG7 and PSG9 are associated with increasing disease severity, we measured them in a cohort from South Africa stratified for this outcome, the PROVE (Preeclampsia Obstetric Adverse Events) cohort (n=72). PSG7 (P=0.0027) and PSG9 (P=0.0028) were elevated among patients who were preeclamptic with severe features (PROVE cohort), but not significantly changed in those without severe features or with eclampsia. In syncytialized first trimester cytotrophoblast stem cells, exposure to TNFα (tumor necrosis factor α) or IL-6 (interleukin 6) significantly increased the expression and secretion of PSG7 and PSG9. In contrast, when we treated primary endothelial cells with recombinant PSG7 and PSG9, we only observed modest changes in Flt-1 (FMS-like tyrosine kinase-1) expression and Plgf (placental growth factor) expression, and no other effects on proangiogenic/antiangiogenic or endothelial dysfunction markers were observed. Conclusions Circulating PSG7 and PSG9 are increased before preeclampsia onset and among those with established disease with their production and release potentially driven by placental inflammation.

[Vaccination against influenza A (H1N1) in pregnancy].

AIM: Evaluation of alterations of immune response regulation and possible risk of antenatal development of fetus in postvaccination period in pregnant women immunized against influenza A (H1N1). MATERIALS AND METHODS: Women were vaccinated with MonoGrippol plus vaccine in the II trimester of physiological pregnancy. At certain intervals ofthe vaccination period (before the vaccination, 7 and 30 days after the vaccination) major biochemical markers in blood sera (alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, creatinine, urea) and levels of key cytokines in spontaneous and stimulated test (IL-1alpha, IL-1RA, IL-2, IL-4, IL-10, IFNgamma, TNFalpha) were evaluated. Vaccination safety for the fetus and trophoblast development was evaluated by using human chorionic gonadotropin (HCG), alpha-fetoprotein (AFP) and trophoblasitc beta-1-glycoprotein (TBG) levels. RESULTS: During vaccination in 13% of cases mild local reactions were noted, in 26.1%--general systemic reactions in the form of weakness, dizziness and headaches. Levels of major biochemical markers at days 7 and 30 after the vaccination did not have any significant difference from the initial values (p > 0.05). Cytokine levels in spontaneous and stimulated tests also did not change significantly. Markers of the course of pregnancy and fetus development (HCG, AFP and TBG) in the two groups observed had comparable values. CONCLUSION: Vaccination of pregnant women against influenza A (H1N1) by Russian subunit formulation (MonoGrippol plus) showed reactogenicity comparable to control group by the level of influence on general metabolic and immunologic homeostasis and on the course of pregnancy, which is an evidence of its safety.

Comparison of pregnancy diagnosis in dairy cattle by use of a commercial ELISA and palpation per rectum.

OBJECTIVE: To compare agreement between 2 pregnancy tests in dairy cattle. DESIGN: Evaluation study. ANIMALS: 976 and 507 cattle for phases 1 and 2, respectively. PROCEDURES: Blood samples were collected, and palpation per rectum (PPR) was performed on cattle. Blood samples for the pregnancy-specific protein B (PSPB) ELISA were sent by courier to a commercial laboratory with results returned later. Results of PPR were extracted from herd records. Statistical comparison of results was performed by use of a mixed linear model and N analysis. RESULTS: Of 571 cattle classified as pregnant by the PSPB ELISA in phase 1, 30 (5%) were nonpregnant by PPR. Mean +/- SE adjusted optical density (OD) of cattle classified pregnant by both tests was significantly higher (0.31 +/- 0.01), compared with the adjusted OD of cattle classified pregnant by the PSPB ELISA and nonpregnant by PPR (0.22 +/- 0.02). Of 255 cows classified pregnant by the PSPB ELISA in phase 2, 31 (12%) were nonpregnant by PPR. Mean +/- SE adjusted OD of cattle classified pregnant by both tests was significantly higher (0.26 +/- 0.01), compared with the adjusted OD of cattle classified pregnant by the PSPB ELISA and nonpregnant by PPR (0.21 +/- 0.01). The N value was 0.82 and 0.81 for phases 1 and 2, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Good agreement existed between the 2 tests, especially at longer intervals after insemination. Discrepant results appeared to be attributable to a nonviable fetus, embryonic loss, or fetal loss.

Assessment of ovulation synchronization protocols in goats and use of pregnancy specific protein B (PSPB) enzyme linked immunsorbent assay (ELISA) to determine kid number at birth.

The efficacy of several protocols for ovulation synchronization and timed artificial insemination (TAI) in goats was examined. In addition, the relationship between levels of pregnancy specific protein B (PSPB) during gestation assessed with a commercially available ELISA and the number of offspring at birth was determined. In Experiment 1, 70 does were randomized into four treatments: (1) breed by estrus [BBE], (2) 6-d treatment with a new [C6N], (3) once-used [C61], or (4) twice-used Controled Internal Drug Release (CIDR) device [C62)]. BBE does received two 15 mg doses of prostaglandin-F2α (PGF) at a 10-d interval and were bred 12 h after estrus onset. CIDR groups received a CIDR for 6 d with 15 mg PGF given at CIDR removal. TAI was performed 48 h after CIDR removal and does were given 50 µg GnRH. All does were inseminated with a single dose of frozen semen using a non-surgical, transcervical technique. Pregnancy rates for the BBE, C6N, C61 and C62 treatment groups were 39% ± 12%, 64% ± 12%, 77% ± 12% and 57% ± 12%, respectively, and did not differ. Reuse of CIDRs, even with reuse extending for a total of 21 d, was as effective as new CIDRs for synchronization of ovulation. In Experiment 2, 68 does were randomized into four treatments: (1) BBE, (2) C6N, (3) NC.Synch [NCS], (4) modified NCS [NCSM]. The BBE and C6N groups were as described for Experiment 1. The NCS and NCSM groups received 15 mg PGF on Day 1, 50 µg GnRH on Day 8 and 15 mg PGF on Day 15 (NCS) or Day 15.5 (NCSM). Does were bred by TAI at 72 h (NCS) or 60 h (NCSM) after the second PGF injection. All does in the NCS and NCSM groups received 50 µg GnRH at TAI. Pregnancy rates were 53% ± 12%, 30% ± 11%, 50% ± 11% and 41% ± 12% for does in the BBE, C6N, NCS and NCSM group, respectively, and did not differ. In Experiment 3, 62 does pregnant to TAI were bled at Days 48 and 85 post-insemination for PSPB. Data on kid numbers and birth weights were subsequently recorded. At Day 48 of gestation, PSPB levels for does birthing singletons were lower than for does birthing twins or triplets (25.0 ± 0.1a, 28.8 ± 0.1b and 30.7 ± 0b ng/mL, respectively, abP<0.05). At Day 85 of gestation, PSPB levels were progressively greater for does birthing singletons versus twins versus triplets (27.0 ± 0.1a, 28.5 ± 0.1b and 31.6 ± 0c ng/mL, abcP<0.05). In conclusion, PSPB concentrations detected using a commercially available ELISA at Day 48 or 85 of gestation could distinguish does carrying single versus multiple fetuses.

Post-surgical resection prognostic value of combined OPN, MMP7, and PSG9 plasma biomarkers in hepatocellular carcinoma.

Biomarkers for hepatocellular carcinoma (HCC) following curative resection are not currently sufficient for prognostic indication of overall survival (OS) and disease-free survival (DFS). The aim of this study was to investigate the prognostic performance of osteopontin (OPN), matrix metalloproteinase 7 (MMP7), and pregnancy specific glycoprotein 9 (PSG9) in patients with HCC. A total of 179 prospective patients with HCC provided plasma before hepatectomy. Plasma OPN, MMP7, and PSG9 levels were determined by enzyme-linked immunosorbent assay. Correlations between plasma levels, clinical parameters, and outcomes (OS and DFS) were overall analyzed. High OPN ( ⩾ 149.97 ng/mL), MMP7 ( ⩾ 2.28 ng/mL), and PSG9 ( ⩾ 45.59 ng/mL) were prognostic indicators of reduced OS (P < 0.001, P < 0.001, and P = 0.007, respectively). Plasma PSG9 protein level was an independent factor in predicting OS (P = 0.008) and DFS (P = 0.038). Plasma OPN + MMP7 + PSG9 elevation in combination was a prognostic factor for OS (P < 0.001). OPN was demonstrated to be a risk factorassociated OS in stage I patients with HCC and patients with low α-fetoprotein levels ( < 20 ng/mL). These findings suggested that OPN, MMP7, PSG9 and their combined panels may be useful for aiding in tumor recurrence and mortality risk prediction of patients with HCC, particularly in the early stage of HCC carcinogenesis.

Interaction of Pregnancy-Specific Glycoprotein 1 With Integrin Α5ß1 Is a Modulator of Extravillous Trophoblast Functions.

Human pregnancy-specific glycoproteins (PSGs) serve immunomodulatory and pro-angiogenic functions during pregnancy and are mainly expressed by syncytiotrophoblast cells. While PSG mRNA expression in extravillous trophoblasts (EVTs) was reported, the proteins were not previously detected. By immunohistochemistry and immunoblotting, we show that PSGs are expressed by invasive EVTs and co-localize with integrin 5. In addition, we determined that native and recombinant PSG1, the most highly expressed member of the family, binds to 51 and induces the formation of focal adhesion structures resulting in adhesion of primary EVTs and EVT-like cell lines under 21% oxygen and 1% oxygen conditions. Furthermore, we found that PSG1 can simultaneously bind to heparan sulfate in the extracellular matrix and to 51 on the cell membrane. Wound healing assays and single-cell movement tracking showed that immobilized PSG1 enhances EVT migration. Although PSG1 did not affect EVT invasion in the in vitro assays employed, we found that the serum PSG1 concentration is lower in African-American women diagnosed with early-onset and late-onset preeclampsia, a pregnancy pathology characterized by shallow trophoblast invasion, than in their respective healthy controls only when the fetus was a male; therefore, the reduced expression of this molecule should be considered in the context of preeclampsia as a potential therapy.

Antiphospholipid syndrome characteristics and adverse pregnancy outcomes after 20 weeks of pregnancy.

OBJECTIVE: To assess outcomes after 20 weeks of pregnancy according to autoantibody profile and clinical presentation of maternal antiphospholipid syndrome (APS). METHODS: The present retrospective cohort analysis included women diagnosed with APS at a tertiary medical center in Israel between January 1, 2012, and December 31, 2016. Anticardiolipin antibodies, anti-ß2-glycoprotein antibodies, and lupus anticoagulant were assessed. Participants were stratified by type of APS (obstetric vs thrombotic), antibody profile, and antibody titer (low vs high). Primary composite outcomes were rated as severe (stillbirth, fetal growth restriction at <34 weeks, severe pre-eclampsia, or delivery at <32 weeks) and mild (stillbirth, any fetal growth restriction, any pre-eclampsia, or delivery at <34 weeks). RESULTS: A total of 99 women were included in the analysis. The primary composite outcomes were similar regardless of stratification. Lupus anticoagulant positivity was associated with delivery before 37 weeks. When compared with low antibody titer, high antibody titer was associated delivery at or before 32 weeks (P=0.045) and 34 weeks (P=0.029). CONCLUSION: High antibody titer might be associated with an increased risk of severe prematurity among pregnant women with APS.

Use of circulating pregnancy-specific beta 1 glycoprotein as a marker in carcinoma of the breast in women.

Pregnancy-specific beta 1 glycoprotein (SP1) was determined by radioimmunoassay in sera from 27 normal women, 33 women with benign breast disease, and 191 women with carcinoma of the breast, staged for extent of the disease. All diagnostic groups exhibited substantial overlap in SP1 values. Those with benign breast diseases tended to have values at least as high as those with cancer. Normal patients tended to have slightly lower values, but this difference may well have been due to the younger ages of the normal patients in our sample, because SP1 values tended to increase with age. Immunochemical dilutions of SP1 in the serum with the highest value (10.2 ng/ml) did not differ significantly from standard placental SP1.

Monoclonal antibodies in analysis of oncoplacental protein SP1 in vivo and in vitro.

Three monoclonal antibodies were developed to the placenta-specific glycoprotein SP1. The antibodies were used in the characterization of SP1 in placental tissue, pregnancy serum and urine, cancer serum, and cell culture. The three antibodies reacted similarly with purified SP1 of placental origin in radio- and enzyme immunoassay, and all three decorated the syncytiotrophoblast layer of placental villi in immunoperoxidase staining applied to fixed placental tissue. However, the three antibodies were found to have different and unique specificities and affinities. One antibody of relatively low affinity was used to isolate SP1 from placenta and fibroblast culture medium. The other two antibodies were used to develop a new and simple immunoassay for SP1. A panel of samples including cancer sera, pregnancy sera and urine, as well as cell culture fluids gave similar results in the monoclonal assay as in a conventional radioimmunoassay. Our work with monoclonal antibodies to SP1 provides further evidence for the production of authentic SP1 by fibroblastic cells in vitro and offers improved reagents for the isolation, characterization, and quantitation of an important marker of cancer and fetal development.

Newly established uterine cervical cancer cell line (SKG-III) with Regan isoenzyme, human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein phenotypes.

The production of Regan isoenzyme (heat-stable, L-phenylalanine-sensitive term-placental alkaline phosphatase), human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein by newly characterized human uterine cervical cancer cell lines, SKG-IIIa and SKG-IIIb, is reported. These cell lines were derived from a moderately differentiated epidermoid cancer partially mixed with epidermoid clear-cell components. At the end of the first 4 months in culture 2 sublines with different morphologies were identified. In nude mice, SKG-IIIa produce clear-cell epidermoid cancer with much glycogen, while SKG-IIIb grew as a moderately differentiated epidermoid cancer rich in tonofilaments. The presence of Regan isoenzyme was established by biochemistry, enzyme cytochemistry, immunocytochemistry, and immunoelectrophoresis. However, the copresence of small amounts of early placental alkaline phosphatase was also demonstrated. The alkaline phosphatase specific activities of SKG-IIIa cells and SKG-IIIb cells were 3.7 and 1.4 nmol per mg protein per min, respectively. The existence was proven by radioimmunoassay of human chorionic gonadotropin beta-subunit (SKG-IIIa, 5.0 mlU/mg protein; SKG-IIIb, 4.4 mlU/mg protein), pregnancy-specific beta 1-glycoprotein (SKG-IIIa, 0.7 ng/mg protein) in the culture media as a tumor cell product. The described cell lines may serve as a more representative model system for studies of regulation of oncodevelopmental genes in gynecological tumors in general and in epidermoid cervical cancer in particular.

Detection of pregnancy-associated glycoproteins in milk and blood as a test for early pregnancy in dairy cows.

The objective of our study was to evaluate 2 pregnancy-associated glycoprotein (PAG)-based enzyme-linked immunosorbent assays (ELISAs) for use with either blood or milk. From 12 dairy farms, 116 Montbéliarde or Holstein cows were selected that had either undergone artificial insemination (AI; n = 102) or had calved (n = 14) 2-3 months earlier and had not undergone any further AI. Serum, plasma, and milk were obtained from all cows; serum and plasma were analyzed using the blood pregnancy test and milk using the milk pregnancy test. No false-positive results were observed when samples of the 14 noninseminated cows were tested. Cows undergoing AI were sampled at ~16, 30, and 41 days post-AI. An additional milk sample was taken at ~53 days post-AI. To establish whether the inseminated cows were pregnant, the cows were subjected to transrectal ultrasonography (TU) on or around day 41. Of the 102 inseminated cows, 63 were confirmed pregnant by TU. By day 30, the serum, plasma, and milk ELISAs demonstrated 100%, 100%, and 98.1% sensitivity and 88.6%, 88.9%, and 90.3% specificity, respectively, with potential pregnancy losses 30-41 days post-AI. Accuracy obtained on serum, plasma, and milk at ~41 days post-AI and on milk at ~53 days post-AI ranged from 97.4% to 100%. There were no differences of practical significance in performance between the blood and milk ELISAs for the sampling dates chosen. This new diagnostic capability with milk samples offers a major improvement in bovine reproductive management.

Pregnancy-specific glycoprotein 9 (PSG9), a driver for colorectal cancer, enhances angiogenesis via activation of SMAD4.

PSG9 is a member of the pregnancy-specific glycoprotein (PSG) family and has been shown to contribute to the progression of colorectal cancer (CRC) and cancer-related angiogenesis. Here, we aim to investigate abnormal PSG9 levels in patients with CRC and to emphasize the role of PSG9 in driving tumorigenesis. Serum from 140 patients with CRC and 125 healthy controls as well as 74 paired tumors and adjacent normal tissue were used to determine PSG9 levels. We discovered that PSG9 was significantly increased in serum (P<0.001) and in tumor tissues (P<0.001) from patients with CRC. Interestingly, the increased PSG9 levels correlated with poor survival (P=0.009) and microvessel density (MVD) (P=0.034). The overexpression of PSG9 strongly promoted the proliferation and migration of HCT-116 and HT-29 cells. However, PSG9 depletion inhibited the proliferation of SW-480 cells. Using a human umbilical vein endothelial cell tube-forming assay, we found that PSG9 promoted angiogenesis. The overexpression of PSG9 also increased the growth of tumor xenografts in nude mice. Co-immunoprecipitation experiments revealed that PSG9 was bound to SMAD4. The PSG9/SMAD4 complex recruited cytoplasmic SMAD2/3 to form a complex, which enhanced SMAD4 nuclear retention. The PSG9 and SMAD4 complex activated the expression of multiple angiogenesis-related genes (included IGFBP-3, PDGF-AA, GM-CSF, and VEGFA). Together, our findings illustrate the innovative mechanism by which PSG9 drives the progression of CRC and tumor angiogenesis. This occurs via nuclear translocation of PSG9/SMAD4, which activates angiogenic cytokines. Therefore, our study may provide evidence for novel treatment strategies by targeting PSG9 in antiangiogenic cancer therapy.

Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle.

The present study aimed to compare the accuracy of a commercial PAG-ELISA test (Bovine Preg Test 29) and bovine pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) for diagnosing pregnancy at Day 28 after insemination in dairy cows. Transrectal ultrasonography (TRUS) was performed in 100 Holstein-Friesian cows at Day 28 after artificial insemination (AI; Day 0) to diagnose pregnancy. After TRUS examination, blood sample was collected from the coccygeal vessels of each cow to measure the concentrations of bPAGs by PAG-RIA test and Bovine Preg Test 29. Milk samples were collected at Days 0, 21 and 28 for measurement of progesterone (P4) by ELISA test. The cows were re-examined by TRUS at Day 42 to confirm the pregnancy diagnoses. The actual gold standard was based on TRUS outcomes at Day 28 that agreed with the outcomes of PAG-RIA test or PAG-ELISA test. If the outcomes of TRUS at Day 28 and PAG-RIA test and PAG-ELISA test did not agree, the gold standard was based on the outcome of TRUS at Day 42. Out of 100 inseminated cows, 41 were confirmed pregnant at Day 28 after AI. Based on the actual gold standard, the sensitivity of TRUS, PAG-ELISA and PAG-RIA tests for diagnosing pregnant cows at Day 28 were 92.7%, 90.2% and 100%, while the specificity of the three tests for diagnosing non-pregnant cows were 91.5%, 98.3% and 94.4%, respectively. The overall accuracy of the three tests were 92%, 95% and 97%, respectively. The degree of agreement (Kappa±S.E.) between PAG-RIA and PAG-ELISA test was 0.90 ±0.04. The degrees of agreement between PAG-RIA and PAG-ELISA and TRUS at Day 28 were 0.80±0.05 and 0.76±0.06, respectively. In conclusion, the commercial PAG-ELISA test is a highly accurate method for diagnosing early pregnancy in dairy cows on Day 28 after AI and may be used as an alternative method to the TRUS and the PAG-RIA test.

Estrogen regulation of placental P-450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid levels and activity during baboon pregnancy.

The present study was conducted to determine whether estrogen regulates the P-450 cholesterol side-chain cleavage (P-450scc) enzyme component of the progesterone biosynthetic pathway in the placenta during the second half of baboon pregnancy. Placental estrogen formation was suppressed by removing the fetus, i.e. fetectomy, and thus fetal adrenal C19-steroid estrogen precursors, on day 100 of baboon gestation (term = 184 days). P-450scc activity and messenger ribonucleic acid (mRNA) levels were then determined in placentas obtained on day 160 after fetectomy alone and after fetectomy and s.c. administration of the estrogen precursor and rostenedione or estradiol benzoate. Placentas were maintained in situ after fetectomy, and placental villi were comprised of syncytiotrophoblasts that seemed morphologically normal, based on their histology and immunocytochemical expression of pregnancy-specific-beta 1-glycoprotein. In untreated baboons, peripheral serum estradiol increased with advancing gestation, and mean (+/-SE) concentrations were 1.22 +/- 0.05 ng/ml on days 101-160 of gestation. After fetectomy serum estradiol concentrations decreased to 24% (P < 0.01) of normal. Androstenedione or estradiol administration after fetectomy increased serum estradiol levels to values that were 57% (P < 0.01) of, or 90% (P < 0.001) greater than intact controls, respectively,. Placental P-450scc specific activity, determined on a mitochondrial-enriched fraction of villous tissue, was 281.1 +/- 15.0 pmol pregnenolone plus progesterone formed per mg mitochondrial protein in untreated control baboons. Fetectomy resulted in a 52% decrease (P < 0.001) in placental P-450scc activity. Administration of androstenedione or estradiol after fetectomy increased P-450scc activity to values that were not significantly different from control. P-450scc mRNA levels were quantified by competitive RT-PCR. P-450scc mRNA levels in placental villous tissue of fetectomized baboons was 38% lower (P < 0.01) than that in the intact controls (110.9 +/- 5.9 attomoles/microgram RNA). The administration of androstenedione after fetectomy restored P-450scc mRNA to a level that was not different from the untreated controls. The results of this study show that there was close association between the levels of estrogen and the specific activity of and the mRNA levels for placental P-450scc in the second half of baboon pregnancy. Therefore, we propose that the P-450scc enzyme that catalyzes the conversion of substrate cholesterol to pregnenolone is regulated, for the most part, by estrogen in the primate placenta.

Evidence for the presence of oncoplacental SP1-like protein in normal nonpregnant serum.

To seek the pregnancy-specific beta 1-glycoprotein (SP1) in nonpregnant serum, normal human serum was applied to immunoadsorbent containing monoclonal anti-SP1 antibodies. SP1 eluted with 8 M urea was further analyzed by sodium dodecyl sulfate-gel electrophoresis and immunoblotting. A SP1-positive band with the same electrophoretic mobility as purified placental SP1 was found. The results suggest that serum from normal nonpregnant subjects contains material closely related to the placental protein SP1. The mean serum concentrations of SP1 were similar in men and women, ranging from 1.1-3.4 ng/ml.