scholarly journals Plasma γ-Globin Gene Expression Suggests that Fetal Hematopoietic Cells Contribute to the Pool of Circulating Cell-Free Fetal Nucleic Acids during Pregnancy

2004 ◽  
Vol 50 (4) ◽  
pp. 689-693 ◽  
Author(s):  
Tuangsit Wataganara ◽  
Erik S LeShane ◽  
Angela Y Chen ◽  
Lynn Borgatta ◽  
Inga Peter ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Pregnant Women ◽  
Globin Gene ◽  
Hematopoietic Cells ◽  
First Trimester ◽  
Maternal Plasma ◽  
Plasma Samples ◽  
Globin Mrna ◽  
Reverse Transcription Pcr ◽  
Gapdh Mrna

Abstract Background: Reports of placental mRNA sequences in the plasma of pregnant women suggest that the placenta is the predominant source of cell-free fetal nucleic acids in maternal plasma during pregnancy. We developed an assay for γ-globin mRNA concentrations to determine whether hematopoietic cells also contribute to the pool of fetal mRNA in maternal plasma. Methods: Frozen paired plasma samples obtained from 40 women before and within 20 min after elective first-trimester termination of pregnancy (TOP) were analyzed. Fresh plasma samples from eight nonpregnant individuals were included as controls. Plasma γ-globin mRNA was measured by use of real-time reverse transcription-PCR and analyzed with gestational age. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used to confirm the presence of cell-free RNA in each sample. Results: γ-Globin and GAPDH mRNA sequences were detected in every plasma sample. The concentrations of both messages were significantly increased in pregnancy (P <0.01). The concentrations of γ-globin mRNA were decreased in most women after TOP, but γ-globin mRNA was increased in some patients when TOP was performed later than 9 weeks of gestation. Conclusions: γ-Globin mRNA sequences can be detected and measured in fresh and frozen plasma samples. Plasma γ-globin and GAPDH mRNA concentrations are affected by pregnancy. The increased posttermination γ-globin mRNA concentrations seen in some patients suggest that the source of this message is fetal hematopoietic cells. Further study in pregnant women after 9 weeks of gestation is necessary to evaluate the potential of γ-globin mRNA as a marker for fetomaternal hemorrhage.

scholarly journals Radioimmunoassay of plasma ouabain in healthy and pregnant individuals

2000 ◽  
Vol 165 (3) ◽  
pp. 669-677 ◽  
Author(s):  
O Vakkuri ◽  
SS Arnason ◽  
A Pouta ◽  
O Vuolteenaho ◽  
J Leppaluoto
Keyword(s):  
Pregnant Women ◽  
Human Plasma ◽  
High Performance ◽  
Chemical Nature ◽  
Solid Phase ◽  
Plasma Concentrations ◽  
First Trimester ◽  
Plasma Samples ◽  
Third Trimester ◽  
Endogenous Substances

Ouabain was recently isolated from human plasma, bovine hypothalamus and bovine adrenal in attempts to identify endogenous substances inhibiting the cell membrane sodium pump. A number of radioimmunoassays have been developed in order to study the clinical significance of ouabain. The results have been controversial with regard to the presence and chemical nature of plasma ouabain-like immunoreactivity. We have now measured ouabain in healthy and pregnant individuals using solid-phase extraction of plasma samples followed by a new radioimmunoassay with the extraordinary sensitivity of at least 2 fmol/tube (5 pmol/l). Plasma extracts, a previously isolated human plasma ouabain-like compound and bovine hypothalamic inhibitory factor displaced the tracer in parallel and eluted identically with ouabain in high-performance liquid chromatography. Plasma ouabain immunoreactivity was found to be much lower than reported previously: 12.6+/-1.3 pmol/l in healthy men (mean+/-s.e., n=20) and 9.4+/-0.7 pmol/l in women (n=14). In pregnant women (n=28) plasma ouabain concentration was 16.3+/-4.0 pmol/l during the first trimester, 18.8+/-4.3 pmol/l during the second trimester and 24.3+/-4.0 pmol/l during the third trimester (all P<0.01 compared with non-pregnant women). Plasma ouabain 3-5 days after the delivery was 13.6+/-1.1 pmol/l (n=10, P<0.05-0.01 compared with second and third trimesters). The pregnancy-related changes in the plasma concentrations of ouabain resembled those of cortisol. Therefore cortisol was measured from the same plasma samples and a significant positive correlation was found (r=0.512, P=0.006). The similar profiles of plasma ouabain and cortisol during pregnancy and their rapid decreases postpartum are consistent with the adrenal cortical origin of ouabain and also show that the secretions of these hormones are possibly under the control of same factors.

scholarly journals Detection of Chromosome 21-encoded mRNA of Placental Origin in Maternal Plasma

2003 ◽  
Vol 49 (9) ◽  
pp. 1445-1449 ◽  
Author(s):  
Cees B M Oudejans ◽  
Attie T J J Go ◽  
Allerdien Visser ◽  
Monique A M Mulders ◽  
Bart A Westerman ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Placental Tissue ◽  
Rna Isolation ◽  
First Trimester ◽  
Maternal Plasma ◽  
Chromosome 21 ◽  
Placental Lactogen ◽  
Plasma Samples ◽  
Human Placental Lactogen ◽  
Dnase Treatment

Abstract Background: mRNA of placental origin (i.e., human placental lactogen and β-human chorionic gonadotropin) has been demonstrated to be easily detectable in maternal plasma. We tested whether detection of chromosome 21-encoded mRNA of placental origin is possible in maternal plasma obtained during the first trimester. Methods: Plasma samples were obtained from pregnant women between weeks 9–13 of pregnancy. RNA was isolated from 800 or 1600 μL of plasma by silica-based affinity isolation and, after on-column DNase treatment, was subjected to two-step, one-tube reverse transcription-PCR with gene specific primers. Results: Three chromosome 21-encoded genes located within the Down syndrome critical region with overexpression in trisomy 21 placentas were screened for expression in early placental tissue to select their potential use for RNA based plasma screening. One of the chromosome 21-encoded genes (LOC90625) showed strong expression in first trimester placenta similar to CSH1 (human placental lactogen) and was selected for plasma analysis. The RNA isolation assay was validated with CSH1 mRNA, which could be detected in the plasma of all women tested in weeks 9–13 of pregnancy. RNA from the chromosome 21-encoded, placentally expressed gene, LOC90625, was present in maternal first-trimester plasma and could be detected in 60% of maternal plasma samples when 800 μL of plasma was used and in 100% of samples when 1600 μL of plasma was used. Conclusion: The detection of chromosome 21-encoded mRNA of placental origin in maternal plasma during the first trimester may allow development of plasma-RNA-based strategies for prenatal prediction of Down syndrome. LOC90625 is a candidate gene for this purpose.

scholarly journals The Concentration of Circulating Corticotropin-releasing Hormone mRNA in Maternal Plasma Is Increased in Preeclampsia

2003 ◽  
Vol 49 (5) ◽  
pp. 727-731 ◽  
Author(s):  
Enders K O Ng ◽  
Tse N Leung ◽  
Nancy B Y Tsui ◽  
Tze K Lau ◽  
Nirmal S Panesar ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Gestational Age ◽  
Reverse Transcription ◽  
Peripheral Blood ◽  
Maternal Plasma ◽  
Corticotropin Releasing Hormone ◽  
Pcr Assay ◽  
Blood Samples ◽  
Reverse Transcription Pcr ◽  
Plasma Rna

Abstract Background: Increased fetal DNA in maternal plasma/serum has been reported in pregnancies complicated by preeclampsia. We hypothesize that fetal RNA may also be increased in maternal plasma in preeclampsia. Methods: We developed a real-time quantitative reverse transcription-PCR assay to measure the concentration of the mRNA of the corticotropin-releasing hormone (CRH) locus. Peripheral blood samples were obtained from healthy pregnant women both before and 2 h after delivery. Peripheral blood samples were also obtained from women suffering from preeclampsia and controls matched for gestational age. Plasma was harvested from these samples, and RNA was extracted. Plasma RNA was subjected to analysis by the reverse transcription-PCR assay. Results: CRH mRNA was detected in the plasma of 10 healthy pregnant women in the third trimester. CRH mRNA was found to be cleared very rapidly after cesarean section, with no detectable signal by 2 h postpartum. Plasma CRH mRNA concentrations were 1070 and 102 copies/mL, respectively, in 12 preeclamptic women and 10 healthy pregnant women matched for gestational age (Mann–Whitney test, P &lt;0.001). Conclusion: Plasma CRH mRNA represents a new molecular marker for preeclampsia. Maternal plasma RNA is gender- and polymorphism-independent and may allow noninvasive gene-expression profiling of an unborn fetus.

scholarly journals Failure to up-regulate VEGF165b in maternal plasma is a first trimester predictive marker for pre-eclampsia

2009 ◽  
Vol 116 (3) ◽  
pp. 265-272 ◽  
Author(s):  
Victoria L. Bills ◽  
Julia Varet ◽  
Ann Millar ◽  
Steven J. Harper ◽  
Peter W. Soothill ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Predictive Marker ◽  
First Trimester ◽  
Maternal Plasma ◽  
Related Condition ◽  
Increased Risk ◽  
Median Plasma ◽  
Endothelial Growth Factor ◽  
Plasma Marker ◽  
In Pregnancy

Pre-eclampsia is a pregnancy-related condition characterized by hypertension, proteinuria and endothelial dysfunction. VEGF165b, formed by alternative splicing of VEGF (vascular endothelial growth factor) pre-mRNA, inhibits VEGF165-mediated vasodilation and angiogenesis, but has not been quantified in pregnancy. ELISAs were used to measure means±S.E.M. plasma VEGF165b, sEng (soluble endoglin) and sFlt-1 (soluble fms-like tyrosine kinase-1). At 12 weeks of gestation, the plasma VEGF165b concentration was significantly up-regulated in plasma from women who maintained normal blood pressure throughout their pregnancy (normotensive group, 4.90±1.6 ng/ml; P&lt;0.01, as determined using a Mann-Whitney U test) compared with non-pregnant women (0.40±0.22 ng/ml). In contrast, in patients who later developed pre-eclampsia, VEGF165b levels were lower than in the normotensive group (0.467±0.209 ng/ml), but were no greater than non-pregnant women. At term, plasma VEGF165b concentrations were greater than normal in both pre-eclamptic (3.75±2.24 ng/ml) and normotensive (10.58 ng/ml±3.74 ng/ml; P&gt;0.1 compared with pre-eclampsia) pregnancies. Patients with a lower than median plasma VEGF165b at 12 weeks had elevated sFlt-1 and sEng pre-delivery. Concentrations of sFlt-1 (1.20±0.07 and 1.27±0.18 ng/ml) and sEng (4.4±0.18 and 4.1±0.5 ng/ml) were similar at 12 weeks of gestation in the normotensive and pre-eclamptic groups respectively. Plasma VEGF165b levels were elevated in pregnancy, but this increase is delayed in women that subsequently develop pre-eclampsia. In conclusion, low VEGF165b may therefore be a clinically useful first trimester plasma marker for increased risk of pre-eclampsia.

scholarly journals Maternal Plasma Pyridoxal 5′-Phosphate Concentration Is Inversely Associated with Plasma Cystathionine Concentration across All Trimesters in Healthy Pregnant Women

2019 ◽  
Vol 149 (8) ◽  
pp. 1354-1362
Author(s):  
Maria F Mujica-Coopman ◽  
Dayana R Farias ◽  
Ana B Franco-Sena ◽  
Juliana S Vaz ◽  
Gilberto Kac ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Carbon Metabolism ◽  
Plasma Concentrations ◽  
First Trimester ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Third Trimester ◽  
Metabolite Concentrations ◽  
Mixed Regression ◽  
One Carbon Metabolism

ABSTRACTBackgroundVitamin B-6 (B-6), in the form of pyridoxal 5′phosphate (PLP), is critical for one-carbon metabolism reactions and cellular function. Plasma PLP concentration decreases throughout pregnancy, but the functional consequences of this have not been studied. Plasma cystathionine is a sensitive indicator of suboptimal B-6 status in healthy adults.ObjectivesThe aim of this study was to determine the relation between plasma PLP and cystathionine concentrations, and to assess longitudinal changes in plasma concentrations of metabolites of one-carbon metabolism, including total homocysteine (tHcy), cysteine, methionine, glycine, serine, and glutathione, over the course of pregnancy.DesignThis was a prospective cohort study of 186 healthy Brazilian pregnant women (20–40 y). Plasma PLP and metabolite concentrations were quantified in fasting maternal blood samples collected between 5–13, 20–26, and 30–36 weeks of gestation. Linear mixed regression models were used to determine the association of 1) first-trimester PLP tertiles, and 2) the variation of PLP concentration throughout pregnancy, with related metabolite concentrations across weeks of gestation.ResultsMedian (IQR) PLP concentration decreased from 36.2 (29.2–44.5) to 21.0 (15.9–26.0) to 16.8 (12.9–21.4) nmol/L in the first, second, and third trimester, respectively, whereas cystathionine concentration increased from 63.2 (49.7–78.9) to 122 (98.0–167) to 143 (114–193) nmol/L, respectively (both P < 0.001). The variation of PLP throughout pregnancy was inversely associated with cystathionine concentration across weeks of gestation, after adjusting for confounding factors; β (95% CI) = −0.387 (−0.752, −0.219), P = 0.04. This association significantly differed by trimester and was strongest in the third trimester. Plasma concentrations of glycine, serine, methionine, cysteine, and tHcy decreased, and that of glutathione increased, between the first and second trimesters (all P < 0.05).ConclusionsThe variation of PLP concentration predicted cystathionine concentration throughout pregnancy. Increases in plasma cystathionine across trimesters may reflect maternal intracellular B-6 deficiency.

scholarly journals Presence of Filterable and Nonfilterable mRNA in the Plasma of Cancer Patients and Healthy Individuals

2002 ◽  
Vol 48 (8) ◽  
pp. 1212-1217 ◽  
Author(s):  
Enders KO Ng ◽  
Nancy BY Tsui ◽  
Nicole YL Lam ◽  
Rossa WK Chiu ◽  
Simon CH Yu ◽  
...  
Keyword(s):  
Real Time ◽  
Globin Gene ◽  
Interquartile Range ◽  
Plasma Samples ◽  
Healthy Individuals ◽  
Pcr Assay ◽  
Mrna Concentration ◽  
Gapdh Mrna ◽  
Significant Difference ◽  
Plasma Rna

Abstract Background: As RNA is labile, we investigated whether circulating RNA in human plasma may be present in a particle-associated form. Methods: Blood was collected from 27 healthy individuals and 16 hepatocellular carcinoma (HCC) patients. The plasma from each individual was processed by two means: filtration through filters with different pore sizes (from 5 μm to 0.22 μm) and ultracentrifugation. We assessed plasma RNA content by a real-time quantitative reverse transcription-PCR assay for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts and plasma DNA by a real-time quantitative PCR assay for the β-globin gene. Results: The plasma GAPDH mRNA concentrations in the healthy individuals were significantly different in every pair of these filter sizes (P &lt;0.05 for each pair). Overall, the plasma GAPDH mRNA concentration was higher by a median of 15-fold (interquartile range, 10- to 24-fold) in the paired unfiltered sample than in the sample filtered through a 0.22 μm filter. In contrast, no significant difference was seen in β-globin DNA concentrations among different pore-size-filtered plasma samples (P = 0.455). Similarly, a significant difference was observed for RNA, but not DNA, between unfiltered plasma and ultracentrifuged plasma (P &lt;0.05). No significant difference in GAPDH mRNA concentrations was seen between the 0.22-μm-filtered plasma samples and the ultracentrifuged plasma samples (P &gt;0.05). In HCC patients, filtration with a 0.22 μm filter produced a median 9.3-fold (interquartile range, 6.9- to 311-fold) reduction in GAPDH mRNA concentration in plasma. Plasma GAPDH mRNA concentrations in HCC patients were significantly higher than those in healthy individuals, both with or without filtration (P &lt;0.0 5 for filtered plasma samples; P &lt;0.005 for unfiltered plasma samples). Conclusions: A substantial proportion of plasma mRNA species is particle-associated. In HCC patients, both circulating particle- and non-particle-associated plasma RNA are increased.

scholarly journals Non-invasive Fetal RHD and RHCE Genotyping Using Real-time PCR Testing of Maternal Plasma in RhD-negative Pregnancies

2005 ◽  
Vol 53 (3) ◽  
pp. 301-305 ◽  
Author(s):  
Ilona Hromadnikova ◽  
Lenka Vechetova ◽  
Klara Vesela ◽  
Blanka Benesova ◽  
Jindrich Doucha ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Real Time ◽  
Real Time Pcr ◽  
Maternal Plasma ◽  
Plasma Samples ◽  
Maternal Circulation ◽  
Exon 2 ◽  
Non Invasive ◽  
Fetal Dna ◽  
Exon 10

We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation.

scholarly journals Time Profile of Appearance and Disappearance of Circulating Placenta-Derived mRNA in Maternal Plasma

2006 ◽  
Vol 52 (2) ◽  
pp. 313-316 ◽  
Author(s):  
Rossa WK Chiu ◽  
Wing-bong Lui ◽  
Mei-chun Cheung ◽  
Nihal Kumta ◽  
Antonio Farina ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Plasma Concentrations ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Time Profile ◽  
Placental Lactogen ◽  
Human Placental Lactogen ◽  
Blood Samples ◽  
Reverse Transcription Pcr ◽  
Kinetics Of

Abstract Background: Fetal RNA of placental origin has been detected in the plasma of pregnant women, but the timing of the first appearance and the detailed kinetics of postdelivery clearance of such circulating RNA have not been studied. Methods: To address the timing of the first appearance of circulating placental RNA, we collected serial maternal blood samples from 47 women who had conceived by assisted reproductive procedures. To address the postdelivery clearance kinetics, we collected serial postdelivery blood samples from 6 pregnant women who had delivered by cesarean section. Placenta-derived transcripts were sought by real-time quantitative reverse transcription-PCR. Results: The earliest gestational age at which human placental lactogen and human chorionic gonadotropin β-subunit mRNAs were detectable in a proportion of the pregnant women was the 4th week of gestation. The postdelivery study indicated that the median apparent half-life for the clearance of human placental lactogen mRNA was 14 min. Conclusions: Placenta-derived mRNA can be found in maternal plasma from very early on in gestation, suggesting a possible role for early noninvasive prenatal diagnosis or monitoring. The rapid kinetics of circulating placental mRNA suggest that its plasma concentrations may be used to monitor recent physiologic or pathologic events.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

Optimisation of tactics of conducting pregnancy and labours at women after auxiliary reproductive technologies

2016 ◽  
pp. 160-164
Author(s):  
D.N. Maslo ◽  
Keyword(s):  
Pregnant Women ◽  
Resistance Index ◽  
First Trimester ◽  
Reproductive Technologies ◽  
Premature Delivery ◽  
Control Group ◽  
Uterine Arteries ◽  
Placental Dysfunction ◽  
Placental Blood ◽  
Two Stages

The objective: frequency decrease perinatal pathologies at women after ART on the basis of studying clinical-ehografical, endocrinological, biochemical, dopplerometrical, cardiotokografical and morphological researches, and also improvement of algorithm of diagnostic and treatment-and-prophylactic actions. Patients and methods. The work basis is made spent by us from 2012 on 2015 by complex inspection of 300 pregnant women from which 250 were after ART and 50 – firstlabours which pragnency without ART, and also their newborns. For the decision of an object in view of research spent to two stages. At 1 stage spent prosperctive research which included 150 pregnant women: з them 100 women pregnancy at which has come out ART (1 group) and 50 healthy women (control group). At 2 stage spent prospective randomization in which result of patients after ART have divided on two equal groups by therapy principle: 2 basic group - 75 pregnant women after ART at which used the algorithm improved by us; 3 group of comparison - 75 pregnant women after ART which have been spent on the standard treatment-and-prophylactic actions. Results. The results suggest that women after using ART is a high frequency of reproductive losses in the first trimester (10.0%), 3.0% of spontaneous abortion from 16 to 22 weeks, and 3.0% "early" premature delivery (22 to 28 weeks of pregnancy). The frequency of violations of the functional state of placenta in women after using IVF is 63.0%, which is the main cause of high levels of perinatal losses (40.0 ‰), and delivery by cesarean section (96.0%). Placental dysfunction in women after using ART characterized by retrohorialnyh hematoma (21.0%); size mismatch fruit (30.0%) and hypertonicity of the uterus (73.0%) against changes in fruit-placental blood flow - increased resistance index in umbilical artery and increased vascular resistance in the uterine arteries. Endocrinological and biochemical changes in placental dysfunction in women after using IVF starting from 28 weeks of pregnancy and are in significant reduction in progesterone, placental b1-microglobulin, B2-microglobulin of fertility and trophic в-glycoprotein. Conclusion. The received results: use of the algorithm of diagnostic and treatment-and-prophylactic actions improved by us allows to lower frequency of spontaneous interruption of pregnancy till 22 weeks – from 13.0% to 5.7%; «early» premature birth – from 3.0% to 1.0%; placentary dysfunction from 63.0% to 40.6%; cesarean sections – from 96.0% to 56.5%, and also perinatal losses – from 40.0‰ to 16.2‰. Key words: pregnancy, childbirth, auxiliary reproductive technologies.

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