Aptamers based sensing of pregnancy associated glycoproteins (PAG) of bovine for early pregnancy detection.

Tosyl activated magnetic beads were used for aptamer selection against PAG- 7 and 18 proteins of bovine origin. PAG proteins were immobilized on beads with further addition of biotin tagged aptamer library. The recognition of aptamers with PAG was identified by ST-HRP based approach which was colorimetric in nature. The selected aptamers were sequenced and at the same time several new aptamers were identified. Later M-fold structure and G-quadruplex score of aptamers were analyzed for their selection. Those aptamers having high G value and complex structure were chosen. In dot blot assay, aptamers recognized PAG protein in an animal after 42 days of artificial insemination which later given birth to a healthy calf. Further the cross reactivity with serum of 0th day animal (post AI) or with non pregnant animal serum was minimal. Aptamers have also shown interaction with PAG protein of buffalo origin. These selected aptamers have commercial application especially in development of biosensors for early detection of pregnancy in bovine.

Characterization by Quantitative Serum Proteomics of Immune-Related Prognostic Biomarkers for COVID-19 Symptomatology.

The COVID-19 pandemic caused by SARS-CoV-2 challenges the understanding of factors affecting disease progression and severity. The identification of prognostic biomarkers and physiological processes associated with disease symptoms is relevant for the development of new diagnostic and therapeutic interventions to contribute to the control of this pandemic. To address this challenge, in this study, we used a quantitative proteomics together with multiple data analysis algorithms to characterize serum protein profiles in five cohorts from healthy to SARS-CoV-2-infected recovered (hospital discharge), nonsevere (hospitalized), and severe [at the intensive care unit (ICU)] cases with increasing systemic inflammation in comparison with healthy individuals sampled prior to the COVID-19 pandemic. The results showed significantly dysregulated proteins and associated biological processes and disorders associated to COVID-19. These results corroborated previous findings in COVID-19 studies and highlighted how the representation of dysregulated serum proteins and associated BPs increases with COVID-19 disease symptomatology from asymptomatic to severe cases. The analysis was then focused on novel disease processes and biomarkers that were correlated with disease symptomatology. To contribute to translational medicine, results corroborated the predictive value of selected immune-related biomarkers for disease recovery [Selenoprotein P (SELENOP) and Serum paraoxonase/arylesterase 1 (PON1)], severity [Carboxypeptidase B2 (CBP2)], and symptomatology [Pregnancy zone protein (PZP)] using protein-specific ELISA tests. Our results contributed to the characterization of SARS-CoV-2-host molecular interactions with potential contributions to the monitoring and control of this pandemic by using immune-related biomarkers associated with disease symptomatology.

Hepatokine Pregnancy Zone Protein Governs the Diet-Induced Thermogenesis Through Activating Brown Adipose Tissue.

Intermittent fasting (IF), as a dietary intervention for weight loss, takes effects primarily through increasing energy expenditure. However, whether inter-organ systems play a key role in IF remains unclear. Here, a novel hepatokine, pregnancy zone protein (PZP) is identified, which has significant induction during the refeeding stage of IF. Further, loss of function studies and protein therapeutic experiment in mice revealed that PZP promotes diet-induced thermogenesis through activating brown adipose tissue (BAT). Mechanistically, circulating PZP can bind to cell surface glucose-regulated protein of 78 kDa (GRP78) to promote uncoupling protein 1 (UCP1) expression via a p38 MAPK-ATF2 signaling pathway in BAT. These studies illuminate a systemic regulation in which the IF promotes BAT thermogenesis through the endocrinal system and provide a novel potential target for treating obesity and related disorders.

Serum exosomal pregnancy zone protein as a promising biomarker in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a kind of intestinal immune dysfunction disease, and its occurrence and prevalence are on the rise worldwide. As a chronic gastrointestinal disease, its pathogenesis is still unknown. Exosomes are vesicles in various body fluids that carry a variety of substances. They can mediate intercellular communication and long-distance transport of multiple media. In this study, we investigated the protein profile of serum exosomes from healthy people and IBD patients to explore a new serological biomarker for IBD. METHODS: Initially, exosomes were extracted from serum samples, and the proteins within the exosomes were identified by label-free liquid chromatography/mass spectrometry (LC-MS/MS). Western blot and ELISA were used to assess the identified protein. To further analyze the target protein, an acute colitis mouse model was established, and exosomes in colonic tissue and serum were extracted to investigate the protein in them. RESULTS: Firstly, serum exosomes were extracted from samples, and proteins in exosomes were identified by LC-MS/MS. Through statistical analysis, we identified 633 proteins. Among these proteins, pregnancy zone protein (PZP) showed a marked difference between patients with IBD and healthy people, in that its expression level was much higher in the IBD patients This exosomal protein was associated with immunosuppressive effects. Also, the level of PZP in colon tissue exosomes and serum exosomes of acute colitis mice was significantly higher than that of the control group. CONCLUSIONS: Our findings indicated that serum exosome PZP was present at a high level in the IBD patients. Hence it might be a promising biomarker and enhance auxiliary diagnosis of IBD.

Prothrombotic state associated with preeclampsia.

PURPOSE OF REVIEW: Preeclampsia is a common complication of pregnancy and contributes significantly to maternal and fetal morbidity and mortality. A protective hypercoagulable state is often developed during late pregnancy and can evolve into a prothrombotic state in patients with preeclampsia. The underlying mechanism of this prothrombotic transition remains poorly understood. We discuss recent progress in understanding the pathophysiology of preeclampsia and associated prothrombotic state. RECENT FINDINGS: The hypercoagulable state developed during pregnancy is initiated by placental factors and progresses into the prothrombotic state in preeclampsia when the placenta is subjected ischemic and oxidative injuries. The cause of the preeclampsia-induced prothrombotic state is multifactorial, involving not only placental factors but also maternal conditions, which include genetic predisposition, preexisting medical conditions, and conditions acquired during pregnancy. Endotheliopathy is the primary pathology of preeclampsia and contributes to the prothrombotic state by inducing the dysregulation of coagulation, platelets, and adhesive ligands. SUMMARY: Patients with preeclampsia often develop a severe prothrombotic state that predisposes them to life-threatening thrombosis and thromboembolism during and after pregnancy. Early recognition and treatment of this prothrombotic state can improve maternal and infant outcomes of preeclampsia patients.

Establishment of a Sandwich-ELISA for simultaneous quantification of bovine pregnancy-associated glycoprotein in serum and milk.

Bovine pregnancy-associated glycoproteins (boPAG) are expressed by trophoblast cells in the bovine placenta. The multigene family of boPAG belongs to the group of aspartic proteases. The accumulation and circulation in maternal blood and milk has made boPAG very useful and important for pregnancy diagnosis in cattle. The goal of the present study was to develop and validate a new Sandwich-ELISA which allows the detection of boPAG in maternal serum and whole milk. Therefore, 984 serum and 928 milk samples were collected monthly from 231 Holstein Friesian cows (Bos Taurus) from one week after insemination (p.i.) until six weeks postpartum. The ELISA is able to identify a cow as being pregnant at day 30 p.i. in serum and at day 40 p.i in milk with threshold values of 1.0 ng/ml in serum and 0.0165 ng/ml in milk. The postpartum half-life of boPAG was estimated to be 6.4 days in serum and 7.1 days in milk. The boPAG profile established during pregnancy in serum and milk showed a typical pattern. The amount of boPAG found in milk was 1.5 % of the amount of boPAG present in serum. In conclusion, a Sandwich-ELISA has been developed to quantify boPAG in serum and in whole milk simultaneously with the same test procedure. This is time saving for farmers and more efficient for laboratories.

PREGNANCY DIAGNOSIS IN OKAPI (OKAPIA JOHNSTONI) USING BIOPRYN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF PREGNANCY-SPECIFIC PROTEIN-B (PSPB).

Banked serum samples from seven okapi (Okapia johnstoni) with known pregnancy status were evaluated using the BioPRYN wild enzyme-linked immunosorbent assay to detect pregnancy-specific protein B (PSPB). Thirty-six serum samples, 18 from known pregnant and 18 from nonpregnant okapi, were analyzed. Using optical density cutoffs, the BioPRYN wild assay demonstrated a sensitivity of 88% (95% confidence interval, 65%-98%) and a specificity of 100% (95% confidence interval, 81%-100%). In one sample, this test confirmed pregnancy as early as 21 days of gestation; however, two pregnant okapi were reported to be not pregnant at 23 and 38 days of gestation, suggesting sensitivity may be lower in early gestation. Sensitivity improved to 100% when samples were evaluated in okapi at 116 days or greater of gestation. Analysis of PSPB can be used to augment pregnancy diagnosis in okapi, a species that is of high conservation value and has documented pregnancy-associated morbidity.

Prediction of adverse perinatal outcome and the mean time until delivery in twin pregnancies with suspected pre-eclampsia using sFlt-1/PIGF ratio.

PURPOSE: An elevated soluble fms-like tyrosine kinase-1 (sFlt-1) / placental growth factor (PlGF) ratio is associated with adverse perinatal outcome (APO) and the mean time until delivery (MTUD) in singleton pregnancies complicated by pre-eclampsia (PE). Data on APO and MTUD prediction in twin pregnancies using sFlt-1/PlGF ratio are scarce. We evaluated the predictive value of the sFlt-1/PIGF ratio regarding APO and MTUD in twin pregnancies with suspected PE and/or HELLP syndrome. METHODS: This is a single center retrospective cohort study. All twin pregnancies with suspected PE/HELLP and determined sFlt-1/PIGF were included. Composite APO (CAPO) was defined as the presence of at least one of the following outcomes: respiratory distress syndrome (RDS), intubation, admission to neonatal intensive care unit (NICU) and arterial umbilical cord pH value < 7.10. Selective fetal growth restriction (s-FGR) was analyzed separately. RESULTS: For final analysis, 49 twin pregnancies were included. Median sFlt-1/PIGF ratio was not significantly different in patients with CAPO compared to those without (89.45 vs. 62.00, p = 0.669). MTUD was significantly negative correlated with sFlt-1/PIGF ratio (r = -0.409, p < 0.001). For the whole study cohort, ROC analysis revealed no predictive value for sFlt-1/PIGF and CAPO (AUC = 0.618, 95% CI: 0.387-0.849, p = 0.254). However, sFlt-1/PIGF ratio showed a predictive value for s-FGR (AUC = 0.755, 95% CI: 0.545-0.965, p = 0.032). CONCLUSION: In twin pregnancies with PE and/or HELLP, sFlt-1/PIGF ratio may be helpful for s-FGR prediction and decision-making regarding close monitoring of high-risk patients. However, further prospective studies are warranted to define the role of sFlt-1/PlGF ratio as outcome predictor in twin pregnancies.

Serum amino acid concentrations are modified by age, insulin resistance, and BCAT2 rs11548193 and BCKDH rs45500792 polymorphisms in subjects with obesity.

BACKGROUND & AIMS: The amino acid profile of young adults is modified by sex, body mass index (BMI) and insulin resistance (IR). However, we do not know if age or the presence of specific polymorphisms in the genes of BCAT2 and BCKDH contribute to changes in the amino acid profile, especially in subjects with obesity. Therefore, we have evaluated the effect of age, the presence of IR and the polymorphisms of BCAT2 rs11548193 and BCKDH rs45500792 on the concentration of amino acids in subjects with obesity. METHODS: This was a cross-sectional study conducted with 487 subjects with obesity. Participants underwent a physical examination in which their clinical history was obtained and a blood sample was taken for biochemical, hormonal, and DNA analysis. RESULTS: Adults <30 years old with obesity had higher levels of alanine, arginine, aspartate, histidine, leucine, lysine, methionine, phenylalanine, proline, serine and valine than adults ≥30 years old. Interestingly, regardless of age, we found that arginine, aspartate, serine decreased, while proline and tyrosine increased in the presence of IR; tyrosine and sum of branched-chain amino acids (∑BCAA) were the amino acids that increased in the presence of BCAT2 rs11548193 and BCKDH rs45500792 polymorphisms. CONCLUSIONS: We found that the amino acid profiles of subjects with obesity are differentially modified by age, the presence of IR, and the presence of the BCAT2 rs11548193 and BCKDH rs45500792 polymorphisms.

EVALUATION OF A VISUAL ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR PREGNANCY DETECTION IN FOUR UNGULATE SPECIES.

This study evaluated the use of a commercially available, visual enzyme-linked immunosorbent assay (ELISA) for diagnosis of pregnancy in okapi (Okapia johnstoni), gerenuk (Litocranius walleri), eastern giant eland (Tragelaphus derbianus spp. gigas), and dama gazelle (Nanger dama). This assay has been validated for use in domestic cattle, sheep, goats, and water buffalo. Unlike other blood-based pregnancy associated glycoprotein (PAG) detection methods, this assay does not require sophisticated laboratory equipment for detection or interpretation and can therefore be utilized in many settings. Banked serum samples from gerenuk (n = 11), giant eland (n = 4), dama gazelle (n = 33) and okapi (n = 3) were tested, and a pregnant and nonpregnant sample from each individual were included. The ELISA showed 100% sensitivity and specificity in gerenuk and giant eland samples, and 0% sensitivity in dama gazelle and okapi samples. Using this assay, pregnancy was detected by 7-8 wk gestation in gerenuk and 6 wk in giant eland. These results are consistent with previous studies that were able to accurately detect pregnancy in other members of the family Bovidae, but it is possible that PAGs present in okapi and dama gazelle are structurally dissimilar relative to the intended test target, and are therefore unrecognizable using this test. The faint positivity in the dama gazelle assays may be due to cross-reactivity with other proteins in the sample, or due to inconsistent binding with the dama gazelle PAG. This ELISA appears to be an accurate, rapid, and inexpensive method of point-of-care pregnancy diagnosis in gerenuk and giant eland, but not okapi and dama gazelle. Additional studies should be pursued to further characterize the limits of pregnancy detection using this assay in gerenuk and giant eland, and to investigate the validity of this test in other nondomestic ruminant species.

Induced prostaglandin release alters steroid concentrations but not pregnancy survival in cows.

Embryonic mortality (EM) is a major factor limiting reproductive efficiency in cattle, and despite negative connotations related to reproductive performance, prostaglandin F2α (PGF2α) is capable of being released by the uterus by Day 30 of gestation. Therefore, the objective was to evaluate differences in PGF2α release after an oxytocin challenge between cows with high circulating concentrations of pregnancy-associated glycoproteins (PAGs) vs low PAG because of the association of increased PAG concentrations with pregnancy success. At Day 30 of gestation, pregnant cows were divided into oxytocin treatment (OT; n = 13) and control (CON; n = 12) groups. Treatment cows were further subdivided by circulating PAG concentration (high PAG, n = 7; and low PAG, n = 6). Blood samples were collected every 30 min beginning 1 h before oxytocin administration and continuing for 4 h. Prostaglandin F2α metabolite (PGFM), progesterone, estradiol-17ß (E2), and PAG concentrations were quantified. The peak concentration of PGFM occurred 2 h after oxytocin injection in treatment animals and returned to baseline levels by 4 h. No correlations were observed between PAG and PGFM, progesterone, or E2 concentrations (P > 0.05). There was no difference in initial or final PGFM concentrations between groups (P > 0.05). Progesterone and E2 concentrations decreased in cows after treatment of oxytocin (P < 0.05); however, only progesterone returned to basal concentrations by the end of the sampling period. In summary, cows with high vs low PAG concentrations at Day 30 of gestation have a similar PGFM response to oxytocin challenge.

Soluble fms-like tyrosine kinase to placental growth factor ratio in different stages of early-onset fetal growth restriction and small for gestational age.

INTRODUCTION: Increased soluble fms-like tyrosine kinase to placental growth factor ratio (sFlt-1/PlGF) has been demonstrated in early-onset fetal growth restriction (FGR) and small for gestational age (SGA). sFlt-1/PlGF cut-offs have been described to assess preeclampsia severity; however, sFlt-1/PlGF values present in early-onset SGA and different FGR severity stages remain unknown. Hence, the objective of this study was to describe and compare the sFlt-1/PlGF values and pregnancy outcomes among early-onset SGA/FGR stages. MATERIAL AND METHODS: This is a prospective case-control study conducted at Vall d'Hebron University Hospital. Singleton pregnancies with estimated fetal weight <10th centile and a control group of uncomplicated pregnancies between 20+0 and 31+6  weeks of gestation were enrolled. Study women were classified at diagnosis into different stages, according to estimated fetal weight centile and Doppler ultrasound. sFlt-1/PlGF serum concentrations were measured at diagnosis and, together with pregnancy outcomes, were compared among FGR severity stages, SGA, and controls. Finally, correlations between sFlt-1/PlGF values and time to delivery, gestational age at delivery, days of neonatal admission, and birthweight z-scores were investigated. RESULTS: Among the 207 women enrolled, 32 (15.4%) had uncomplicated pregnancies, 49 (23.7%) pregnancies showed SGA, and 126 (60.9%) involved FGR (92 being stage I, 17 stage II, and 17 stage III). SGA and controls had similar median sFlt-1/PlGF values (25.7 vs 27.1, P > .05) and pregnancy outcomes. However, all FGR stages had significantly poorer outcomes and greater sFlt-1/PlGF values than those of SGA and controls. Furthermore, median values differed significantly among all FGR severity stages (9.76 for stage I; 284.3 for stage II, and 625.02 for stage III, P < .05) increasing with FGR severity as well as the frequency of adverse pregnancy outcomes. Additionally, a significant correlation was found between greater sFlt-1/PlGF ratio values and gestational age at delivery, time from diagnosis to delivery, birthweight z-scores, and time in neonatal intensive care unit (r = -.637, r = -.576, r = -.161, and r = .311, respectively). CONCLUSIONS: Values of sFlt-1/PlGF at diagnosis permit early-onset FGR/SGA severity classification with good correlation with Doppler ultrasound findings and the occurrence of adverse outcomes. Thus, sFlt-1/PlGF could aid in early-onset FGR/SGA severity classification and clinical management when Doppler assessment is not feasible.

Correlations of the expression of γδ T cells and their co-stimulatory molecules TIGIT, PD-1, ICOS and BTLA with PR and PIBF in the peripheral blood and decidual tissues of women with unexplained recurrent spontaneous abortion.

Semi-allogeneic embryos are not rejected by the maternal immune system due to maternal-fetal immune tolerance. Progesterone (P) receptor (PR)-expressing γδ T cells are present in healthy pregnant women. In the presence of P, these cells secrete an immunomodulatory protein called progesterone-induced blocking factor (PIBF), which can facilitate immune escape and is important in preventing embryonic rejection. This work investigated the correlations of the expression of γδ T cells and their co-stimulatory molecules T cell immunoglobulin and ITIM domain (TIGIT), programmed cell death 1 (PD-1), inducible co-stimulator (ICOS) and B and T lymphocyte attenuator (BTLA) with progesterone receptor (PR) and progesterone-induced blocking factor (PIBF) in peripheral blood and decidual tissue in women with unexplained recurrent spontaneous abortion (URSA) and normal pregnant (NP) women. We confirmed that γδ T cell proportions and PIBF expression in the peripheral blood and decidua of URSA women decreased significantly, while PR expression in decidua decreased. However, TIGIT, PD-1, ICOS and BTLA expression in γδ T cells in peripheral blood did not change, while TIGIT and PD-1 expression in γδ T cells in decidua increased significantly. Under the action of PHA-P (10 µg/ml), co-blocking of TIGIT (15 µg/ml) and PD-1 (10 µg/ml) antibodies further induced γδ T cell proliferation, but PIBF levels in the culture medium supernatant did not change. At 10-10 M P, γδ T cells proliferated significantly, and PIBF concentrations in the culture medium supernatant increased. γδ T cells co-cultured with P, TIGIT and PD-1 blocking antibodies showed the most significant proliferation, and PIBF concentrations in the culture medium supernatant were the highest. These results confirm that P is necessary for PIBF production. The TIGIT and PD-1 pathways participate in γδ T cell proliferation and activation and PIBF expression and play important roles in maintaining pregnancy.

The Association between Plasma ERVWE1 Concentrations and Affective Symptoms during Pregnancy: Is This a Friendly Alien?

Human endogenous retroviruses (HERVs) comprise 8% of the human genome, and HERV DNA was reported to be essential in human embryonic development. Specifically, HERV-W encodes a protein, syncytin-1, alternatively known as ERVWE1 (Human Endogenous Retrovirus W EnvC7-1 Envelope Protein), participating in human placental morphogenesis and having a role in immune system regulation. Syncytin-1 activity is increased in neuropsychiatric disorders, autoimmune diseases, and cancer. In our study, forty-four women in the third trimester of pregnancy were tested for ERVWE1 plasma levels. In concomitance with blood samples the following rating scales were administered to women: the Edinburgh Postnatal Depression Scale (EPDS), State Anxiety Inventory (STAI-S), Trait Anxiety Inventory (STAI-T), and Prenatal Attachment Inventory (PAI). We found that higher ERVWE1 protein plasma levels were significantly associated with higher PAI scores (p = 0.02), an earlier gestational age at the time of blood collection (p = 0.01), a longer duration of symptoms (p = 0.03), and fewer lifetime attempted suicides (p = 0.02). Our results seem to support the role of ERVWE1 in maintaining clinical psychiatric symptoms as a result of potential prolonged inflammation. At the same time, this protein may have a protective role in pregnant women by a reduction of suicidal behavior and a better mother-fetus relationship.

New predictors of early impaired placentation preceding miscarriage before 10 weeks of gestation in IVF pregnancies: A prospective study.

INTRODUCTION: In a recent study of 10,011 pregnant women, 95% of miscarriages occurred before routine ultrasound scan at 11-14 weeks. Our study aimed to identify early first trimester parameters which may predict miscarriage before 10 weeks of gestation for in vitro fertilization (IVF) pregnancies. METHODS: A cohort of 115 healthy IVF patients with a singleton viable embryo in early first trimester were studied in a tertiary university-affiliated medical center (April 2017-June 2018). Calculations included gestational age (GA); ultrasound evaluation of crown-rump length (CRL), mean gestational sac diameter (GSD) and volume (GSV), mean yolk sac diameter (YSD) and volume (YSV); fetal heart rate (FHR), mean uterine arteries pulsatility index (UtA-PI); and maternal blood placental protein 13 (PP13) levels. Patients were divided into three groups by GA; and early miscarriage versus ongoing pregnancy after GA 10 weeks. RESULTS: Early fetal loss occurred in 14.8% of patients; miscarriage group had higher discrepancy between calculated and measured GA (P < 0.001), lower GSD and GSV (P = 0.005 and P = 0.02, respectively), significantly different YSD and YSV, and lower GSD/YSD and GSV/YSV ratios (P = 0.001 and P = 0.003, respectively). UtA-PI/CRL ratio was higher in patients with miscarriage at GA 46-48 days and GA >48 days (P = 0.034 and P = 0.026, respectively). PP13/CRL ratio was higher in patients with miscarriage at GA >48 days (P = 0.041). DISCUSSION: In IVF pregnancies with live embryo at first ultrasound scan, high UtA-PI/CRL and maternal blood PP13/CRL ratios may indicate impaired placentation preceded early pregnancy loss. A larger cohort is needed to further verify these predictions.

Selection, identification, and application of DNA aptamers against bovine pregnancy-associated glycoproteins 4.

The bovine pregnancy-associated glycoproteins (bPAGs) have been widely used as robust markers for early diagnosis of pregnancy in the cattle. The current immune recognition methods for detecting bPAGs are limited and, to a certain extent, are associated with high costs and poor stability of the antibody. Aptamers that are more stable and easily synthesized than antibodies might serve as suitable candidates for the development of rapid detection methods. This paper describes selection and characterization of bPAG4 aptamers and theirs applicability to detect bPAG4 in the serum. In this work, the recombinant bovine pregnancy-associated glycoproteins 4 (bPAG4) with a relative molecular mass of about 48 kDa was successfully expressed in human embryonic kidney 293 (HEK 293) cells. Subsequently, the ssDNA aptamers were selected by systematic evolution of ligands by exponential enrichment (SELEX) using magnetic beads (MB) coated with bPAG4 as target. After 9 rounds of selection, three aptamers with high affinity to bPAG4 (Kd = 11.7~40.2 nM) were identified. The selected aptamers were successfully used in enzyme-linked aptamer assay (ELAA) to detect bPAG4 at a detection limit of 0.09 ng/mL. Meanwhile, it has been successfully applied for the detection of bPAG4 in serum samples. This work demonstrated that the selected aptamers could be used as promising affinity probes in the development of inexpensive, simple, and sensitive analysis methods for detecting bPAGs. Graphical abstract.

Applied use of interferon-tau stimulated genes expression in polymorphonuclear cells to detect pregnancy compared to other early predictors in beef cattle.

We aimed to evaluate the accuracy of Interferon-tau stimulated genes (ISG) abundance in peripheral blood polymorphonuclear cells (PMNs) on D20 after fixed-time artificial insemination (FTAI; D0) as a pregnancy diagnosis method against CL evaluation by Doppler ultrasonography and progesterone (P4) concentrations on D20, as well as Pregnancy Associated Glycoproteins (PAG) concentrations on D25. Additionally, we evaluated the potential of ISG abundance in PMNs as pregnancy loss predictors. Nelore heifers (n = 103) and cows (n = 144) underwent estrous synchronization and were artificially inseminated on D0. Pregnancy was diagnosed by B-mode ultrasonography on D30 and D70, and after the final diagnosis, females were classified in four groups: Pregnant; Non-pregnant; Functional CL on D20 but non-pregnant (CL-NP) and Pregnancy loss between D30 and D70 (PL). After determining cutoff values, the Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accuracy (ACC) were determined for each method. All methods were classified as significant (P < 0.05) predictors of pregnancy. Both ISG expression and PAG concentrations were greater (P < 0.05) in pregnant females than in non-pregnant and CL-NP females but did not differ (P > 0.05) from the PL group. ISG15 expression was greater (P < 0.05) in heifers than in cows, but this difference was not found in OAS1 expression and PAG concentrations. All the methods evaluated were proven to be adequate predictors of pregnancy, but greater accuracies were obtained through PAG concentrations and Doppler-US, due to the decreased number of false positive and false negative results.

Colorimetric enzyme-linked aptamer assay utilizing hybridization chain reaction for determination of bovine pregnancy-associated glycoproteins.

DNA aptamers that bind to bovine pregnancy-associated glycoproteins (bPAGs) were selected by the systematic evolution of ligands by exponential enrichment (SELEX) procedure coupled to surface plasmon resonance (SPR) and high-throughput sequencing (HTS) technology. After seven rounds of selection using carboxylated magnetic beads (MB) coated with bovine pregnancy-associated glycoproteins 9 (bPAG9) and bovine serum albumin (BSA) as target and counter targets, respectively, two aptamers designated as A1 and A24 showed high affinities to bPAG9 (Kd = 1.04 and 2.5 nM). Moreover, the specificity was determined by testing the non-targets bPAG4, bPAG6, bPAG16, BSA, and ovalbumin (OVA). Results showed that two aptamers demonstrated broad group specificity to bPAG family. Subsequently, a colorimetric sandwich enzyme-linked aptamer assay was developed for ultrasensitive detection of bPAG9 based on hybridization chain reaction (HCR) amplification strategy. The method exhibited a broad determination from 0.134 to 134 ng/mL with a detection limit of 0.037 ng/mL. The method has been successfully applied to determine bPAGs in real samples. The results demonstrate that the developed aptamers could be used as promising molecular probes for the development of pregnancy diagnostic tools. Graphical abstract In this study, we first selected aptamers against bovine pregnancy-associated glycoproteins (bPAGs) as molecular recognition elements and then developed a colorimetric enzyme-linked aptamer assay utilizing hybridization chain reaction to detect bPAGs in the serum.The GA can't be deleted, the modified GA can not upload. So themodified GA and figures will be send to CorrAdmin3@spi-global.com.

Detection of twin pregnancies in ewes by pregnancy-associated glycoprotein assay and transabdominal ultrasonography.

The objectives of the present study were to evaluate and compare the accuracy of transabdominal ultrasonography and pregnancy-associated glycoprotein (PAG) assay in the diagnosis of twin pregnancies in ewes and to evaluate the utility of the PAG assay for predicting fetal gender in singleton pregnancies. The animals in the study consisted of 179 pregnant ewes. The number of fetuses in the ewe was determined using transabdominal ultrasonography between days 40 and 60 (on days 40, 45, 50, 55, and 60). Blood samples were collected from all the ewes on the same day to determine the PAG concentrations. The results found were highly sensitive for the detection of twin pregnancies by transabdominal ultrasonography. The accuracy of transabdominal ultrasonography in detecting twin pregnancies was found to be higher on day 60 than on other days (P < 0.05). The sensitivities of PAG assay in detecting twin pregnancies on days 40, 45, 50, 55, and 60 were 91.67%, 66.67%, 81.82%, 88.89%, and 33.33%, respectively. The accuracies of the PAG assay in detecting twin pregnancies on days 40, 45, and 50 were found to be statistically significant higher than other days (P < 0.05). The PAG assay had low sensitivity and specificity for predicting fetal gender. It was concluded that twin pregnancies in ewes can be diagnosed with high accuracy using transabdominal ultrasonography on day 60 of pregnancy and as well as using the PAG assay during the early days of pregnancy (on days 40, 45, and 50). The PAG assay is not useful for predicting fetal gender.