Plasma Of Pregnant Women Research Articles

Understanding genetic variability: exploring large-scale copy number variants through non-invasive prenatal testing in European populations.

Large-scale copy number variants (CNVs) are structural alterations in the genome that involve the duplication or deletion of DNA segments, contributing to genetic diversity and playing a crucial role in the evolution and development of various diseases and disorders, as they can lead to the dosage imbalance of one or more genes. Massively parallel sequencing (MPS) has revolutionized the field of genetic analysis and contributed significantly to routine clinical diagnosis and screening. It offers a precise method for detecting CNVs with exceptional accuracy. In this context, a non-invasive prenatal test (NIPT) based on the sequencing of cell-free DNA (cfDNA) from pregnant women's plasma using a low-coverage whole genome MPS (WGS) approach represents a valuable source for population studies. Here, we analyzed genomic data of 12,732 pregnant women from the Slovak (9,230), Czech (1,583), and Hungarian (1,919) populations. We identified 5,062 CNVs ranging from 200 kbp and described their basic characteristics and differences between the subject populations. Our results suggest that re-analysis of sequencing data from routine WGS assays has the potential to obtain large-scale CNV population frequencies, which are not well known and may provide valuable information to support the classification and interpretation of this type of genetic variation. Furthermore, this could contribute to expanding knowledge about the central European genome without investing in additional laboratory work, as NIPTs are a relatively widely used screening method.

HSD3B1 is an oxysterol 3β-hydroxysteroid dehydrogenase in human placenta.

Most biologically active oxysterols have a 3β-hydroxy-5-ene function in the ring system with an additional site of oxidation at C-7 or on the side-chain. In blood plasma oxysterols with a 7α-hydroxy group are also observed with the alternative 3-oxo-4-ene function in the ring system formed by ubiquitously expressed 3β-hydroxy-Δ5-C27-steroid oxidoreductase Δ5-isomerase, HSD3B7. However, oxysterols without a 7α-hydroxy group are not substrates for HSD3B7 and are not usually observed with the 3-oxo-4-ene function. Here we report the unexpected identification of oxysterols in plasma derived from umbilical cord blood and blood from pregnant women taken before delivery at 37+ weeks of gestation, of side-chain oxysterols with a 3-oxo-4-ene function but no 7α-hydroxy group. These 3-oxo-4-ene oxysterols were also identified in placenta, leading to the hypothesis that they may be formed by a previously unrecognized 3β-hydroxy-Δ5-C27-steroid oxidoreductase Δ5-isomerase activity of HSD3B1, an enzyme which is highly expressed in placenta. Proof-of-principle experiments confirmed that HSD3B1 has this activity. We speculate that HSD3B1 in placenta is the source of the unexpected 3-oxo-4-ene oxysterols in cord and pregnant women's plasma and may have a role in controlling the abundance of biologically active oxysterols delivered to the fetus.

A Large Data Set; Hepatitis B and C Infection in Pregnant Females

Background: Hepatitis B and C virus (HBV & HCV) infection is on the surge among pregnant women as a result of unsaved dental and cosmetic procedures, reuse of needles, raising the risk of perinatal transmission and HBV & HCV infection in children. Although few studies on the prevalence of HBV and HCV infection during pregnancy have been conducted but the majority of the studies are on very low sample size. The current study is based on large data set. Objectives: The major goal of this study was to find out the frequency of HBV & HCV in pregnant women. Methods: We conducted a retrospective cohort study in Alkhidmat Raazi Hospital Rawalpindi. A total of 31911 pregnant women who delivered between 2016 and 2021 were analyzed and categorized as HBV & HCV infected or HBV & HCV uninfected by billing codes. The birth certificate and hospital discharge records were used to identify infant records related to HBV and HCV-infected pregnant mothers. Pregnant women's plasma samples were tested for HBV and HCV using chemiluminescence ARCHITECT i1000SR® Abbott Diagnostics. Data were analyzed using packages within SPSS software and P ≤ 0.05 was considered significant. Cross-tabulation chi-square analysis was performed with a significance level of 5%. Results: The pregnant women with age under 25 years were 12699 (39.8%), women with age from 26 to 29 years were 9734 (30.5%), while women with age 30 and older were 9478 (29.7). The patients who were primigravida were 9514 (29.8%), multipara having 1-4 children 18545 (58.1%) and those having more than five children were 3852 (12.1%). The prevalence of HBV and HCV in 31911 pregnant women were 170/31911 (0.5%) and 478/31911 (1.5%), respectively. The mean age was 27.9 years. Conclusions: HBV and HCV infection is present in pregnant females, which may ultimately increase the treatment cost and vertical transmission. Keywords: Hepatitis B, Hepatitis C, Infection, Pregnant females

Blood fatty acid analysis reveals similar n-3 fatty acid composition in non-pregnant and pregnant women and their neonates in an Israeli pilot study

Maternal docosahexaenoic acid (DHA) is required during pregnancy to supply for normal fetal growth and development. This pilot study aimed to assess the unknown fatty acid (FA) composition in a cohort of non-pregnant and pregnant Israeli women at term and their offspring on a normal diet without n-3 FA supplementation. The fatty acid profile, analyzed using gas chromatography, showed significantly higher plasma monounsaturated (MUFA) and lower n-6 FA percent distribution with similar n-3 index, in pregnant compared to non-pregnant women. RBC exhibited significantly higher MUFA with similar n-3 index, in pregnant compared to non-pregnant women. N-3 FA significantly correlated between neonates’ plasma, with higher n-3 index, and pregnant women's DHA. Conclusion: DHA levels in non-pregnant and pregnant Israeli women at term were comparable and the DHA in pregnant women's plasma positively correlated with their neonate's level, suggesting an efficient mother-fetus FA transfer and/or fetal fatty acid metabolism to longer FA products.

Association of vitamin D and gene variants in the vitamin D metabolic pathway with preterm birth

ObjectivesThe aim of this study was to explore the association of vitamin D (VitD) levels during pregnancy and its metabolic pathway genes with the risk for preterm birth (PTB) among pregnant women in southeast China. MethodsThis study was conducted in Zhoushan Maternal and Child Health Hospital, Zhejiang, from August 2011 to May 2018. Plasma 25-hydroxyvitamin vitamin D [25(OH)D] levels in three trimesters and single-nucleotide morphisms in the VitD metabolic pathway were measured. Relevant information was collected using questionnaires and an electronic medical recorder system. Multiple statistical methods including linear regression, logistic regression, and crossover analysis were applied. ResultsThe prospective cohort study included 3465 pregnant women, of which 202 were PTB (week of gestation at delivery: 33.38 ± 4.05), accounting for 5.8%. After adjusting for potential confounders, VitD sufficiency (≥30 ng/mL) in the second and third trimesters was associated with longer gestational age at delivery compared with VitD deficiency (<20 ng/mL). However, no significant association was found between VitD with the risk for PTB. rs7041, rs10210408, and rs2228171 were associated with gestational week and the risk for PTB. Significant associations were found of rs10210408, rs2209314, rs1155563, rs2544381 and the status of VitD in the second and third trimester with the gestational week. We also found that rs7041 and VitD in the second trimester might exert interaction on gestational week and the risk for PTB (Pinter = 0.038; Pinter = 0.019); rs16846876 and VitD in the second trimester might exert interaction on gestational week (Pinter = 0.024); rs4334089 and VitD in the third trimester might exert interaction on gestational week (Pinter = 0.024). Similar results were found when we tested pregnant women's plasma 25(OH)D in the first and second trimesters. ConclusionsWomen with VitD deficiency were associated with shorter gestational weeks. Single-nucleotide morphisms in VitD metabolic pathway genes were significantly associated with gestation week and the risk for PTB, mainly in vitamin D–binding protein (GC) and low-density lipoprotein-related protein 2 (LRP2)genes. Additionally, maternal VitD with GC gene and maternal VitD with vitamin D receptor (VDR) gene might exert interactions on the risk for PTB.

Effects of Sample Handling and Analytical Procedures on Thyroid Hormone Concentrations in Pregnant Women's Plasma.

Maternal thyroid function is a critical mediator of fetal brain development. Pregnancy-related physiologic changes and handling conditions of blood samples may influence thyroid hormone biomarkers. We investigated the reliability of thyroid hormone biomarkers in plasma of pregnant women under various handling conditions. We enrolled 17 pregnant women; collected serum and plasma were immediately frozen. Additional plasma aliquots were subjected to different handling conditions before the analysis of thyroid biomarkers: storage at room temperature for 24 or 48 hours before freezing and an extra freeze-thaw cycle. We estimated free thyroid hormone indices in plasma based on T3 uptake. High correlations between plasma and serum (>0.94) and intraclass correlation coefficients for plasma handling conditions (0.96 to 1.00) indicated excellent reliability for all thyroid hormone biomarkers. Delayed freezing and freeze-thaw cycles did not affect reliability of biomarkers of thyroid function in plasma during pregnancy. See video abstract at, http://links.lww.com/EDE/B180.

Pregnancy favors the expansion of circulating functional follicular helper T Cells

Pregnancy favors antibody production, and some evidence has suggested a direct effect of estrogen on B cells. The impact of pregnancy on circulating follicular helper T (TFH) cells, typically identified by the expression of CD45RO and CXCR5, has not been previously investigated. Here, the percentage of TFH cells, co-expressing or not PD-1, ICOS, or CXCR3 markers was significantly higher in pregnant women (PW) as compared with non-pregnant ones (nPW). Furthermore, the percentage of CXCR3+ TFH cells able to produce IL-6, IL-21, and IL-10 was significantly higher in PW than nPW. Interestingly, anti-CMV and anti-HBs antibody titers were significantly higher in the plasma of PW and were directly correlated with IL-21-producing CXCR3+ TFH cells. Finally, peripheral estrogen levels, but not progesterone, were positively related to either PD-1+ CXCR3+ TFH cells or plasma anti-CMV and anti-HBs IgG antibodies. In summary, our data suggests a positive effect of pregnancy on the proportion of CD4+ T cell subset specialized in helping B cells. This phenomenon, which could be related to the high estrogen levels produced during pregnancy, may help to explain why pregnancy favor humoral immunity.

Fructose Levels Are Markedly Elevated in Cerebrospinal Fluid Compared to Plasma in Pregnant Women

BackgroundFructose, unlike glucose, promotes feeding behavior in rodents and its ingestion exerts differential effects in the human brain. However, plasma fructose is typically 1/1000th of glucose levels and it is unclear to what extent fructose crosses the blood-brain barrier. We investigated whether local endogenous central nervous system (CNS) fructose production from glucose via the polyol pathway (glucose→sorbitol→fructose) contributes to brain exposure to fructose.MethodsIn this observational study, fasting glucose, sorbitol and fructose concentrations were measured using gas-chromatography-liquid mass spectroscopy in cerebrospinal fluid (CSF), maternal plasma, and venous cord blood collected from 25 pregnant women (6 lean, 10 overweight/obese, and 9 T2DM/gestational DM) undergoing spinal anesthesia and elective cesarean section.ResultsAs expected, CSF glucose was ~60% of plasma glucose levels. In contrast, fructose was nearly 20-fold higher in CSF than in plasma (p < 0.001), and CSF sorbitol was ~9-times higher than plasma levels (p < 0.001). Moreover, CSF fructose correlated positively with CSF glucose (ρ 0.45, p = 0.02) and sorbitol levels (ρ 0.75, p < 0.001). Cord blood sorbitol was also ~7-fold higher than maternal plasma sorbitol levels (p = 0.001). There were no differences in plasma, CSF, and cord blood glucose, fructose, or sorbitol levels between groups.ConclusionsThese data raise the possibility that fructose may be produced endogenously in the human brain and that the effects of fructose in the human brain and placenta may extend beyond its dietary consumption.

Prediction of Fetal Growth Restriction by Analyzing the Messenger RNAs of Angiogenic Factor in the Plasma of Pregnant Women.

To predict the occurrence of fetal growth restriction (FGR) by analyzing messenger RNA (mRNA) expression levels of vascular endothelial growth factor receptor 1 (fms-like tyrosine kinase 1 [Flt-1]) in maternal blood. Eleven women with FGR were matched with 88 controls. Plasma samples were obtained during each trimester. The Flt-1 mRNA expression levels were compared between groups. Predicted probabilities were calculated, and sensitivity-specificity (receiver-operating characteristic [ROC]) curves were assessed based on regression models for each trimester measurement and possible combinations of measurements. The mRNA levels of the FGR group during all trimesters were significantly higher than those of the control group. The ROC curve of combined first and second trimester data yielded a detection rate of 60% at a 10% false-positive rate, with an area under curve of 0.79. The Flt-1 mRNA expression in maternal blood can be used as a marker to predict the development of FGR, long before a clinical diagnosis is made.

Increased Levels of Cell-Free Human Placental Lactogen mRNA at 28–32 Gestational Weeks in Plasma of Pregnant Women With Placenta Previa and Invasive Placenta

We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive placenta (n = 5), and normal pregnancies (n = 92). Median (range) hPL mRNA was significantly higher in women with placenta previa, 782 (10-2301) copies/mL of plasma, and in those with invasive placenta, 615 (522-2102) copies/mL of plasma, when compared to normal pregnancies, 90 (4-4407) copies/mL of plasma, P < .01 and P < .05, respectively. We found a sensitivity of 100% and a specificity of 61.5% for the prediction of invasive placenta among women with placenta previa. In conclusion, expression of hPL mRNA is increased in plasma of women with placenta previa and invasive placenta at 28 to 32 weeks of gestation.

Characterization of Extracellular Vesicles in Plasma of Pregnant Women Using Multicolor Flow Cytometry and Nanoparticle Tracking Analysis

previously been studied using flow cytometry based on different syncytiotrophoblast specific antibodies. In most of these studies, indirect antibody labeling was applied, which, compared to direct labeling, is more susceptible to nonspecific binding. Systemic inflammation, which is a characteristic feature of PE, leads to activa tion of cells circulating in maternal blood including leucocytes, endothelium, platelets, and red blood cells (RBC) with increased release of EVs. Therefore, the authors of this study decided to explore EVs of these cells together with STBMs in samples of platelet‐free plasma (PFP) in nonpregnant (Non‐ P) controls, normal pregnant (Norm‐P), and women with late onset PE. Correct identification of the proper phenotype is crucial for any study on quantification and functional characterization of STBMs and EVs since the STBMs in plasma from pregnant women only account for a minor fraction of all EVs and because of considerable cr oss reactivity with blood‐cell derived EVs. This identification was achieved by multicolor flow cytometry using directly conjugated antibodies targeting multiple markers of EVs. This very unique approach reduces the problem of unspecific binding. For the development of this assay, EVs were prepared in vitro from pure populations of leucocytes, endothelium, platelets, and RBC and were examined for suitable antibodies to analyze the corresponding EVs in vivo. Antibody labeling of EVs was not only compared to parent cells, but each marker was tested for cross reactivity with STBMs. STBMs were prepared in vitro from maternal‐side eluate of dual‐ perfused placenta lobes (pSTBM). It has been shown before that this elegant method is superior to other in vitro techniques, such as mechanical dissection of placental tissue or culture of villous explants, in providing functionally intact STBMs. Based on the in vitro data, a panel of markers was defined for in vivo characterization of STBMs simultaneously with EVs of the different intravascular cell types using flow cytometry. EVs including STBMs were prepared by ultracentrifugation of platelet free plasma samples of three groups of women: nonpregnant controls, normal pregnant, and women with late onset PE. After labeling with two separate antibody panels, the EVs wer e subjected to flow cytometry. The first panel with three color labeled antibodies aimed to identify the placental‐derived EVs; the second panel with five color labeled antibodies was shown to be very effective in identifying EVs derived from platelets, RBC, leucocytes, and endothelial cells.

Relationship of Dimethylglycine, Choline, and Betaine with Oxoproline in Plasma of Pregnant Women and Their Newborn Infants ,

Choline and glycine are inter-related through their roles in methyl metabolism. Choline is metabolized to betaine, which donates a methyl group to homocysteine to form methionine, also generating dimethylglycine, which is further metabolized to glycine. Choline is transported across the placenta and is higher in fetal than maternal plasma. Placental glycine transfer, however, is limited and poor glycine status has been suggested in preterm infants. Insufficient glycine for glutathione (GSH) synthesis results in increased metabolism of γ-glutamyl cysteine to 5-oxoproline. We measured plasma 5-oxoproline as a metabolic indicator to address whether choline, via dimethylglycine, contributes physiologically relevant amounts of glycine in pregnancy. Blood was collected from healthy term pregnant women and their newborn infants at delivery (n = 46) and nonpregnant healthy women (n = 19) as a reference group. Plasma choline, betaine, dimethylglycine, homocysteine, methionine, and 5-oxoproline were quantified by HPLC-tandem MS. Plasma choline was 45% higher, but betaine was 63% lower and dimethylglycine was 28% lower in pregnant than nonpregnant women (P < 0.01). Higher white blood cell choline dehydrogenase messenger RNA levels in a random subset of pregnant (n = 8) than nonpregnant women (n = 7) (P < 0.01) suggest increased betaine and dimethylglycine turnover rather than decreased synthesis. Plasma choline, betaine, and dimethylglycine were higher (P < 0.001) in fetal plasma (36.4 ± 13, 29.4 ± 1.0, and 2.44 ± 0.12 μmol/L, respectively) than maternal plasma (15.3 ± 0.42, 14.1 ± 0.6 and 1.81 ± 0.12 μmol/L, respectively). Concentrations of 5-oxoproline and dimethylglycine were inversely (P < 0.05) correlated in maternal (Spearman rho = −0.35) and fetal plasma (Spearman rho = −0.32), suggesting that choline, via dimethylglycine, contributes glycine for GSH synthesis in human development.

Cell-Free mRNA Concentrations of Plasminogen Activator Inhibitor-1 and Tissue-Type Plasminogen Activator Are Increased in the Plasma of Pregnant Women with Preeclampsia

Detection of placental mRNA in maternal plasma has been reported in high-risk pregnancies. We attempted to investigate the concentrations of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (tPA) mRNA in maternal plasma in preeclampsia. Peripheral blood samples were obtained from healthy pregnant women before and after delivery and also from women with or without preeclampsia. Plasma was isolated from these samples, and RNA was extracted. Plasma PAI-1 and tPA mRNA concentrations were then measured by use of reverse transcription PCR assays. The concentrations were converted into multiples of the median (MoM) of the controls adjusted for gestational age. Data were stratified and analyzed according to the clinical severity of preeclampsia and quantitative distribution of blood pressure and proteinuria. The median (minimum-maximum) PAI-1 mRNA MoM values for women with preeclampsia and controls were 2.48 (0.82-8.53) and 1.00 (0.41-2.33), respectively, whereas the median (minimum-maximum) tPA mRNA MoM values were 3.33 (1.01-10.58) and 1.00 (0.95-1.20), respectively. The concentrations of both PAI-1 and tPA mRNA were significantly increased in cases of preeclampsia, compared with controls (P <0.0001). The MoM values of both mRNA species were directly correlated with the severity of preeclampsia and were greatest among a subgroup of hemolysis, increased liver enzymes, and low platelets pregnancies. Maternal plasma PAI-1 and tPA mRNAs are significantly increased in patients with preeclampsia and are positively correlated with the severity of preeclampsia.

Detection of SNPs in the Plasma of Pregnant Women and in the Urine of Kidney Transplant Recipients by Mass Spectrometry

Recently, it has been discovered that cell-free fetal DNA is smaller than corresponding maternal DNA. Therefore, circulating fetal DNA can be enriched by size-fractionation. Such a selection improves the non-invasive prenatal diagnosis of paternally inherited single gene mutations. Recent studies showed that MALDI-TOF mass spectrometry (MS) can be used to reliably detect fetal-specific single-nucleotide polymorphisms (SNPs) in maternal plasma. In this study, we looked at whether the size-fractionation approach could improve the detection of paternally inherited SNPs by MS assay. Our results indicated that the size-fractionation approach improved the analysis of paternally inherited SNP alleles. Our previous studies showed that donor-derived STR sequences could be detected in the urine of kidney transplant recipients. Here, we also examined whether donor-specific SNPs could be detected in recipient's urine by MS.

Detection of Apoptotic Fetal Cells in Plasma of Pregnant Women

Fetal DNA is present in plasma of pregnant women in amounts almost 1000-fold higher than the DNA present in intact fetal cells circulating in maternal peripheral blood (1)(2). The clinical usefulness of fetal DNA from plasma for noninvasive prenatal diagnosis has been demonstrated recently through analysis of the fetal RhD status by PCR-based approaches (3)(4)(5). We explored the possibility that not all of this fetal DNA is soluble and cell-free, but that part of it is still cell-associated. Heparin blood samples of 25–30 mL were obtained after informed consent from 38 pregnant women attending the Prenatal Diagnostic Center at week 7–16 of gestation. Several weeks after blood withdrawal, chorionic villus sampling or amniocentesis was performed as planned at each patient’s first visit. The protocol was approved by the Committee of Medical Ethics of the University Hospital ‘Vrije Universiteit’. Blood samples were processed on the day of withdrawal and were centrifuged on a discontinuous Percoll gradient as described previously (6)(7). In brief, after dilution of the heparin blood in Hanks’ balanced salt solution; 1:2 dilution (Life Technologies), blood was layered on a 5-step Percoll density gradient consisting of 15 mL of 600 mL/L and 5 mL each of 550, 500, 450, and 400 mL/L Percoll in Hanks’ balanced salt solution. After centrifugation for 25 min at 1000 g at room temperature, plasma samples were removed from the upper part …

Beta-endrophin immunoreactivity during human pregnancy.

The human placenta has been implicated as a source of numerous peptide hormones during pregnancy. Since the immunoassay detection of the proopiomelanocortin derived peptide beta-endorphin (beta E) in placental extracts in 1978, it has remained uncertain whether placental beta E immunoreactivity (IR) is 1) secreted into the maternal circulation and 2) opiate receptor active during pregnancy. To elucidate the nature of beta E IR in the placenta, both beta E IR and N-alpha-acetylated beta E (Ac beta E) IR were simultaneously measured in extracts of human pituitaries, placentas, and plasma by two homologous RIAs. Pituitary extracts (n = 6) contained 38 +/- 7 nmol beta E IR per g wet wt tissue (mean +/- SEM), of which only 20 +/- 4 pmol/g were Ac beta E IR. Term placental extracts (n = 19) had 201 +/- 30 fmol/g wet wt total beta E IR and 30 +/- 3 fmol/g wet wt total Ac beta E IR, which comprised 15% of total beta E IR in placental extracts. Total plasma beta E IR rose from 28 weeks gestation (8.5 +/- 0.3 fmol/mL, n = 159) to peak at labor (50 +/- 4 fmol/mL, n = 98; P < 0.01) but total Ac beta E IR was found in only four 28-week (1.7 +/- 0.9 fmol/mL) and 42 labor plasma samples (0.9 +/- 0.1 fmol/mL). Gel filtration chromatography of placental and pituitary extracts showed that while less than 1% of the beta E31-size material was acetylated in the pituitary, up to 60% of the beta E31-size material in placental extracts was acetylated. In pooled third trimester plasma extracts, however, only 4% of the beta E31-size material was acetylated. Furthermore, the ratio of beta E31:beta-lipotropin in pituitary extracts (n = 3) was 0.5; pooled plasma-0.5, and placental extracts (n = 5)-1.2. These data indicate that 1) the placenta extensively N-alpha-acetylates beta E31 destroying its opiate bioactivity while the pituitary does not; 2) beta E IR in pregnant women's plasma is similar to pituitary beta E IR, being mostly nonacetylated and similar in size to beta-lipotropin. These findings are consistent with a pituitary source for the elevated plasma beta E IR found during late pregnancy which may, in turn, be a consequence of elevated plasma concentrations of placentally secreted plasma corticotropin-releasing factor IR present during the third trimester.

A sensitive and specific radioimmunoassay for oxytocin (author's transl)

Recently, measurements of oxytocin content in serum have been reported from several institutes using radioimmunoassay (RIA) techniques. But these assay values showed significant differences, and the plasma levels of this hormone related to pregnancy and labor are still unclear. In order to clarify those problems, a sensitive and specific RIA system has been developed, and moreover, in this RIA system, any purification or extraction procedures of plasma samples were unnecessary.Antiserum against oxytocin was produced in New Zealand white rabbits with multiple intradermal and intrasplenal injections of oxytocin-bovine serum albumin (BSA) complex with complete Freund's adjuvant. For the coupling of oxytocin with BSA, water soluble carbodiimide was used. After twelve weeks from the first immunization, antiserum capable of binding with 45-50% of125 I-oxytocin at a final dilution of 1 : 72000 was obtained.Iodination of synthetic oxytocin was done with the chloramine T method with some modifications, and the purification of iodinated oxytocin was achieved by using a Sephadex G-25 fine gel filtration (0.8 X 10 cm) eluted with 0.1M acetic acid. Further purification with a Dowex ion exchange column chromatography gave no benefit on this assay system.In the RIA procedure, 125I-oxytocin was added after a 24 hour-preincubation, and a double antibody method was used for the separation of B/F hormones. All measurements were done in duplicate, and 0.05M phosphate buffer pH 7.5 containing 0.01M EDTA-2Na, 0.015M NaN3, 2.5 X 10-5M o-phenanthlorine and 0.1% human serum albumin was used.A linear dose response curve was obtained at an oxytocin concentration between 250 μU/ml and 16 μU/ml, and the minimal detectable dose was 2 μU/ml.Cross-reactivities with Arg-vasopressin and Lys-vasopressin were less than 0.2%. The values of inter-assay coefficient of variation obtained from five different assays with two pooled plasma were 18.6% and 15.8%. The values of intra-assay coefficient of variation obtained from five different concentrations of pooled plasma were less than 4.9%.The recovery rate was improved with the immobilization of plasma samples, and in this RIA system, anti-oxytocin serum, normal rabbit serum and anti-rabbit gamma globulin goat serum as well as plasma samples were used after immobilization.The serial dilution of pregnant women's plasma gave reasonable assay values. From these results, this RIA system is thought to be satisfactory for measuring oxytocin in plasma.Blood samples were obtained from normal pregnant women at Osaka University hospital at 2-3 pm. Sampling was done from the antecubital vein with a previously cooled and heparinized syringe, and the blood was immediately mixed with an ice cold solution, containing 0.01M EDTA and 2.5 X 10-5M o-phenanthlorine for the inhibition of oxytocinase activity. After immobilization, the plasma samples were kept in a deep freezer (-20°C) and in this condition no change was observed in the assay values at least within one month.The plasma oxytocin levels during normal pregnancy, evaluated by this RIA system, showed a gradual rise towards the estimated delivery date, and the mean value at the 10th month of pregnancy was 12.5 μU/ml.