It is well established that erythropoiesis occurs first in the yolk sac, then in the liver, subsequently moving to the bone marrow and, in rodents, the spleen during development. The origin of the erythropoietic precursors and some factors suggested to be important for the changing location of erythropoiesis are discussed in this review. Until recently, the major site of erythropoietin (Epo) production in the fetus was thought to be the liver, but studies have shown now that the Epo gene is expressed strongly in the fetal kidney, even in the temporary mesonephros. The metanephric Epo mRNA is upregulated by anemia, downregulated by glucocorticoids, and contributes substantially to circulating hormone levels in hemorrhaged ovine fetuses. Other sites of Epo and Epo receptor production, likely to have important actions during development, are the placenta and the brain.

Developmental regulation of erythropoietin and erythropoiesis

The present invention relates to at least one novel human EPO mimetic hinge core mimetibody or specified portion or variant, including isolated nucleic acids that encode at least one EPO mimetic hinge core mimetibody or specified portion or variant, EPO mimetic hinge core mimetibody or specified portion or variants, vectors, host cells, transgenic animals or plants, and methods of making and using thereof, including therapeutic compositions, methods and devices.

Human EPO Mimetic Hinge Core Mimetibodies, Compositions, Methods and Uses

Circulating levels of maternal erythropoietin (EPO) rise during gestation due to increased biosynthesis of the hormone. Our objective was to investigate the human placenta as a potential extrarenal site of EPO production. Using two monoclonal antibodies recognizing different antigenic determinants, we identified immunoreactive EPO associated with villous cytotrophoblast, endovascular and intravascular cytotrophoblast, cytotrophoblast cell columns, and syncytiotrophoblast of first and second-trimester placenta as well as syncytiotrophoblast and extravillous cytotrophoblast of normal third-trimester and preeclamptic placenta. In addition, cultured JAR (trophoblast-derived) choriocarcinoma cells, cytotrophoblasts isolated from term placenta, villous core cells, and possibly other nontrophoblast cells within the decidual basal plate expressed immunoreactive EPO. Using reverse transcription-polymerase chain reaction and EPO-specific primers, a 378 bp DNA product was amplified from placental tissues of various gestational ages, cytotrophoblasts isolated from term placenta, and JAR choriocarcinoma cells. The amplified product yielded restriction enzyme fragments of predicted sizes. On Southern analysis, hybridization was observed for two of these fragments in which the radiolabeled EPO cDNA probe did not overlap with the primer sequences. Finally, the JAR choriocarcinoma cells elaborated EPO into the culture medium as determined by enzyme-linked immunosorbant assay and expressed EPO mRNA as determined by Northern analysis, both of which were stimulated by hypoxia (15-20 torr). Taken together, these results suggest a new site of EPO expression: the trophoblast cell of the human placenta.

Expression of erythropoietin by the human placenta.

During past decades, knowledge on placental drug metabolism and mechanisms of placental transfer has increased significantly. Most pharmaceutical drugs administered during pregnancy cross the placenta to some extent. The important properties determining the placental transfer by passive diffusion are molecular weight, pKa, lipid solubility and protein binding. In addition to passive diffusion, compounds may cross the placenta via active transfer, facilitated diffusion, phagocytosis and pinocytosis. This review gives an update of efflux transporter proteins and xenobiotic-metabolizing enzymes that modify the fate and effects of drugs in the placenta.

The fate and effects of xenobiotics in human placenta.

Background

Hematocrit falls after birth in preterm infants due to physiological factors and blood letting. Low plasma levels of erythropoietin (EPO) in preterm infants provide a rationale for the use of EPO to prevent or treat anemia.



Objectives

Primary objective: To assess the effectiveness and safety of late initiation of EPO (initiated at 8 days after birth or later) in reducing the use of red blood cell transfusions in preterm and/or low birth weight infants.



Secondary objectives: Subgroup analyses of low ( 500 IU/kg/week) doses of EPO and within these subgroups analyses of the use of low ( 5 mg/kg/day) doses of supplemental iron, in reducing the use of red blood cell transfusions in these infants.



Search strategy

MEDLINE, EMBASE, CINAHL, abstracts from scientific meetings published in Pediatric Research and reference lists of identified trials and reviews were searched in November 2005/April 2006 and the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 2, 2006). No language restrictions were applied.



Selection criteria

Randomised or quasi-randomized controlled trials of late initiation of EPO treatment (started at eight days of age or later) vs. placebo or no intervention in preterm (< 37 weeks) and/or low birth weight (< 2500 g) neonates. For inclusion the studies needed to provide information on at least one outcome of interest.



Data collection and analysis

Data were abstracted by the two authors on pre-tested data collection forms. Data were entered by one review author (AO) and checked for accuracy by the other (SA). Data were analysed using RevMan 4.2.8. The statistical methods included relative risk (RR), risk difference (RD), number needed to treat to benefit (NNTB), number needed to treat to harm (NNTH) for dichotomous outcomes and weighted mean difference (WMD) for continuous outcomes reported with their 95% confidence intervals (CI). A fixed effects model was used for meta-analyses. Heterogeneity tests including the I squared (I2) statistic were performed to assess the appropriateness of pooling the data.



Main results

Twenty-eight studies enrolling 1302 preterm infants in 21 countries were included. The quality of the trials varied. Most trials were of small sample size. Only one study clearly stated that infants were excluded if they had received red blood cell transfusion prior to study entry (Samanci 1996). A total of 19 studies including 912 infants reported on the primary outcome of “Use of one or more red cell transfusions”. The meta-analysis showed a significant effect [typical RR; 0.66 (95% CI; 0.59, 0.74); typical RD -0.21 (95% CI; -0.26, -0.16); typical NNTB of 5 (95% CI 4, 6)]. There was statistically significant heterogeneity [for RR (p < 0.00001), I2 = 74.0% and for RD (p = 0.0006), I2 =58.9%]. Similar results were obtained in secondary analyses based on different combinations of high/low doses of EPO and iron supplementation. There was a significant reduction in the total volume (ml/kg) of blood transfused per infant (four studies enrolling 177 infants) [typical WMD = -7 ml (95% CI -12, -3)] and in the number of transfusions per infant (nine studies enrolling 567 infants); [typical WMD -0.78 (-0.97, -0.59)]. The effect size was less in a post hoc analyses of high quality studies compared to studies in which the quality was uncertain and in studies that used strict guidelines for red blood cell transfusions vs. studies that did not. There were no significant differences in mortality, retinopathy of prematurity, sepsis, intraventricular haemorrhage, periventricular leukomalacia, necrotizing enterocolitis, bronchopulmonary dysplasia, SIDS, neutropenia, hypertension, or length of hospital stay. Long-term neurodevelopmental outcomes were not reported.



Authors' conclusions

Late administration of EPO reduces the use of one or more red blood cell transfusions, the number of red blood cell transfusions per infant and the total volume of red blood cell transfused per infant. The clinical importance of the results for the latter two outcomes is marginal (< 1 transfusion per infant and 7 ml/kg of transfused red blood cells). Any donor exposure is likely not avoided as most studies included infants who had received red cell transfusions prior to trial entry. Late EPO does not significantly reduce or increase any of many important neonatal adverse outcomes including mortality and retinopathy of prematurity. Further research of the use of late EPO treatment to prevent donor exposure is not indicated. Research efforts should focus on limiting donor exposure during the first few days of life in sick neonates, when red blood cell requirements are most likely to be required and cannot be prevented by late EPO treatment.



Plain language summary

The amount of circulating red blood cells (hematocrit) falls after birth in all infants. This is particularly true in preterm infants due to their poor response to anemia and to the amount of blood that is drawn for necessary testing. Low plasma levels of erythropoietin (EPO) (a substance in the blood that stimulates red blood cell production) in preterm infants provide a rationale for the use of EPO to prevent/treat anemia. More than 1300 infants born preterm have been enrolled in 28 studies of late administration of EPO (at 8 days of age or later) to reduce the use of red blood cell transfusions and to prevent donor exposure. The guidelines for use of red blood cell transfusions varied among studies. EPO reduces the risk of receiving red blood transfusion following initiation of EPO treatment. However, the overall benefit of EPO is reduced as many of these infants had been exposed to donor blood prior to entry into the trials. Treatment with late EPO did not have any important effects on mortality or common complications of preterm birth, including retinopathy of prematurity. Future studies should focus on efforts to reduce the amount of blood withdrawn from sick newborns and the use of satellite packs (dividing one unit of donor blood into many smaller aliquots) to reduce donor exposure.

Early versus late erythropoietin for preventing red blood cell transfusion in preterm and/or low birth weight infants

We administered tracer quantities of biologically active 125I-labelled recombinant human erythropoietin by intravenous bolus injection to seven late gestation pregnant ewes. Maternal and fetal blood was sampled over the subsequent six hours and assayed for erythropoietin-specific radioactivity. Despite the expected increase in maternal plasma immunoprecipitable 125I-labelled erythropoietin radioactivity, fetal plasma levels remained unchanged throughout the study. In addition, erythropoietin receptors were not detected in ovine and human placental tissue. We conclude that biologically active 125I-labelled erythropoietin does not cross the placenta from mother to fetus in measurable quantities in sheep, and likely in humans. Thus, these data indicate the levels of erythropoietin measured in fetal plasma are reflective of fetal, and not maternal, erythropoietin production and elimination.

Sawyer St

Papers

Lack of maternal to fetal transfer of 125I-labelled erythropoietin in sheep.

A sensitive and specific erythropoietin immunoprecipitation assay: application to pharmacokinetic studies.