CSH RNA Interference Reduces Global Nutrient Uptake and Umbilical Blood Flow Resulting in Intrauterine Growth Restriction.

Deficiency of the placental hormone chorionic somatomammotropin (CSH) can lead to the development of intrauterine growth restriction (IUGR). To gain insight into the physiological consequences of CSH RNA interference (RNAi), the trophectoderm of hatched blastocysts (nine days of gestational age; dGA) was infected with a lentivirus expressing either a scrambled control or CSH-specific shRNA, prior to transfer into synchronized recipient sheep. At 90 dGA, umbilical hemodynamics and fetal measurements were assessed by Doppler ultrasonography. At 120 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies with the 3H2O transplacental diffusion technique at 130 dGA. Nutrient uptake rates were determined and tissues were subsequently harvested at necropsy. CSH RNAi reduced (p ≤ 0.05) both fetal and uterine weights as well as umbilical blood flow (mL/min). This ultimately resulted in reduced (p ≤ 0.01) umbilical IGF1 concentrations, as well as reduced umbilical nutrient uptakes (p ≤ 0.05) in CSH RNAi pregnancies. CSH RNAi also reduced (p ≤ 0.05) uterine nutrient uptakes as well as uteroplacental glucose utilization. These data suggest that CSH is necessary to facilitate adequate blood flow for the uptake of oxygen, oxidative substrates, and hormones essential to support fetal and uterine growth.

Development of ovine chorionic somatomammotropin hormone-deficient pregnancies.

Intrauterine growth restriction (IUGR) is a leading cause of neonatal mortality and morbidity. Chorionic somatomammotropin hormone (CSH), a placenta-specific secretory product found at high concentrations in maternal and fetal circulation throughout gestation, is significantly reduced in human and sheep IUGR pregnancies. The objective of this study was to knock down ovine CSH (oCSH) expression in vivo using lentiviral-mediated short-hairpin RNA to test the hypothesis that oCSH deficiency would result in IUGR of near-term fetal lambs. Three different lentiviral oCSH-targeting constructs were used and compared with pregnancies (n = 8) generated with a scrambled control (SC) lentiviral construct. Pregnancies were harvested at 135 days of gestation. The most effective targeting sequence, "target 6" (tg6; n = 8), yielded pregnancies with significant reductions (P ≤ 0.05) in oCSH mRNA (50%) and protein (38%) concentrations, as well as significant reductions (P ≤ 0.05) in placental (52%) and fetal (32%) weights compared with the SC pregnancies. Fetal liver weights were reduced 41% (P ≤ 0.05), yet fetal liver insulin-like growth factor-I (oIGF1) and -II mRNA concentrations were reduced (P ≤ 0.05) 82 and 71%, respectively, and umbilical artery oIGF1 concentrations were reduced 62% (P ≤ 0.05) in tg6 pregnancies. Additionally, fetal liver oIGF-binding protein (oIGFBP) 2 and oIGFBP3 mRNA concentrations were reduced (P ≤ 0.05), whereas fetal liver oIGFBP1 mRNA concentration was not impacted nor was maternal liver oIGF and oIGFBP mRNA concentrations or uterine artery oIGF1 concentrations (P ≥ 0.10). Based on our results, it appears that oCSH deficiency does result in IUGR, by impacting placental development as well as fetal liver development and function.

Placental lactogen is expressed but is not translated into protein in breast cancer.

INTRODUCTION: Several studies reported that the pregnancy-specific hormone placental lactogen (hPL) is expressed at both mRNA and protein levels in breast cancer. The overall objective was to establish hPL, the product of the CSH1 and CSH2 genes, as a biomarker for breast cancer. METHODS: CSH expression was determined at the mRNA level in breast cancer cell lines (BCC) and primary carcinomas by real-time and conventional PCR and the products verified as CSH1 by sequencing. Expression of hPL protein was examined by western blots and immuno-histochemistry, using commercial and custom-made polyclonal and monoclonal antibodies. RESULTS: Variable levels of CSH mRNA were detected in several BCC, and in some primary tumors. We detected a protein, slightly larger than recombinant hPL by western blotting using several antibodies, leading us to postulate that it represents an hPL variant ('hPL'). Furthermore, some monoclonal antibodies detected 'hPL' by immunohistochemistry in breast carcinomas but not in normal breast. However, further examination revealed that these antibodies were non-specific, as efficient suppression of CSH mRNA by shRNA did not abolish the 'hPL' band. Custom-made monoclonal antibodies against recombinant hPL detected hPL of the correct size in placental lysate and hPL-overexpressing BCC, but not in unmodified cells or primary carcinomas. hPL protein was detected only when mRNA was increased several thousand fold. CONCLUSIONS: We call into question previous reports of hPL expression in breast cancer which relied on mRNA levels as surrogates for protein and/or used improperly validated antibodies to measure hPL protein levels. Our data suggests that an inhibitory mechanism(s) prevents translation of CSH mRNA in breast cancer when not highly expressed. The mechanism by which translation of CSH mRNA is inhibited is intriguing and should be further investigated.

Absence of human placental lactogen and placental growth hormone (HGH-V) during pregnancy: PCR analysis of the deletion.

Human placental lactogen (HPL) is produced in large amounts in normal pregnancies. We report a pregnancy with complete lack of HPL and the placental variant of the human growth hormone HGH-V. The pregnancy resulted in a severely growth-retarded but otherwise normal male baby. PCR analysis of DNA extracted from the placenta showed that the HPL encoding genes hPL-4 and hPL-3 were deleted along with the human growth hormone variant gene (hGH-V), which is located between these two active hPL genes and also expressed in the normal placenta. Of the five members of this multigene family, hGH-N, which is expressed in the pituitary gland, and hPL-1, a presumed pseudogene, were left intact. The latter (hPL-1) was expressed as RNA transcripts only at very low levels as is usually reported in normal pregnancies. Analysis of the parents' DNA showed that both of them carried a different heterozygous deletion at the 3' end of the hGH/hPL locus.

Absence of human chorionic somatomammotropin during pregnancy associated with two types of gene deletion.

Complete absence of human somatomammotropin (hCS) was demonstrated in two patients experiencing an otherwise uneventful pregnancy. After delivery, DNA was prepared from the neonate blood or from the placenta and the integrity of the hCS-hGH gene cluster was investigated by Southern blotting and hybridization with an hCS cDNA probe. Patient 1 was found to be homozygous for a deletion involving hCS-A, hGH-V, and hCS-B. Patient 2 was a double heterozygote, with one chromosome bearing the same deletion as that of patient 1, while in the other, only the hCS-A gene was missing. Considerations relative to the frequency of the defect are derived from the present results.

An effect of gene dosage on production of human chorionic somatomammotropin.

The gene deletions responsible for isolated partial deficiency of fetal human chorionic somatomammotropin (hCS) production were characterized by restriction endonuclease analysis of genomic DNA prepared from the leukocytes of an affected child. The phenotypically normal child was the product of a 38-week pregnancy characterized by peak maternal hCS levels of 1.1 micrograms/microliter (normal, 3-9.2 micrograms/ml) and normal levels of other pregnancy-associated hormones. Two genes, termed hCS-A and hCS-B, specify the same mature hCS peptide and are responsible for fetal hCS production. Digestion of the child's DNA with the enzymes Hind III, Eco RI, Bam HI, Bgl II, Hinc II and Msp I disclosed absence of the restriction fragments that normally contain the hCS-A gene. However, the hCS-B gene was present in the child's DNA. The child's DNA digests contained an abnormally large Eco RI fragment of 10.0 kb containing the hCS-L gene. This abnormal fragment is a marker for a deletion that is responsible for complete deficiency of hCS when present in the homozygous state. The child's DNA restriction patterns were consistent with heterozygosity for two different deletions involving hCS genes. The paternal hGH gene cluster lacked the hCS-A, human GH variant, and hCS-B genes, while the maternal cluster lacked only the hCS-A gene. Thus, the child's DNA contained only one of the normal complement of four functional hCS genes per diploid genome. Material hCS levels approximately one fourth as great as those present at comparable stages of normal pregnancies indicated that there was no compensatory increase in expression of the remaining hCS gene.

Human placental lactogen (hPL) deficiency in a normal pregnancy.

A case of human placental lactogen (hPL) deficiency together with normal oestriol levels associated with a normal pregnancy in a woman in her second pregnancy is reported. The woman gave birth to a healthy male infant. The placenta was normal. Extremely low hPL levels may be compatible with the delivery of a healthy infant.

Plasma human chorionic somatomammotropin deficiency in a normal pregnancy is the consequence of low concentration of messenger RNA coding for human chorionic somatomammotropin.

Human chorionic somatomammotropin (hCS) is important in the hormonal monitoring of human pregnancies. Presented is the case of a clinically normal pregnancy in which a very low plasma level of hCS was detected. The concentration of messenger ribonucleic acid (mRNA) coding for hCS was evaluated to determine the level on which the deficiency occurred.

A gene deletion is responsible for absence of human chorionic somatomammotropin.

We have examined the human growth hormone (hGH) and human chorionic somatomammotropin (hCS) family of genes in genomic DNA from an individual with complete antenatal deficiency of hCS. Following digestion with a variety of bacterial restriction endonucleases, the DNA from this individual produced fewer fragments with homology to a radiolabeled hCS cDNA probe than did control DNA specimens. The patterns indicated that his DNA contained the normal hGH gene and an "hGH-like" gene, but lacked the hCS gene, a variant hGH gene, and another gene or genes with structural homology to hGH and hCS, which were present in all control DNA specimens. The findings were consistent with homozygosity for a gene deletion with a minimum length of 18.5 kb. Analysis of polymorphic restriction site variation related to the hGH and hCS gene cluster indicated that both parents and three older siblings were heterozygous for the deletion. The association between gene deletion and a normal growth pattern in this individual indicates that hCS and any other peptide hormones encoded by the variant hGH and the other related gene(s) that are deleted in this individual are not required for fetal or extrauterine growth.

hPL deficiency with normal estriol levels in a normal pregnancy.

The third published case of human placental lactogen (hPL) deficiency in a normal pregnancy is reported. The results of the 3 cases are discussed. In all cases, estriol levels were normal and hPL levels were either unmeasurable or below 1 microgram/ml. The placenta showed no obvious abnormalities. Growth hormone and prolactin determinations did not contribute to the understanding of the deficiency; neither did the glucose levels in the maternal blood. All 3 infants were male.

Isolated defect in human placental lactogen synthesis in a normal pregnancy. Case report.

A deficiency in the synthesis of human placental lactogen (hPL) was found in a woman in her second pregnancy. Other placental hormone levels were normal. The woman gave birth to a healthy female infant. hPL deficiency is rare and a survey of the literature has revealed only one previous case report which described the birth of a male infant. The present report of a hPL deficiency is the first associated with the birth of a normal female infant.

Absence of human placental lactogen in an otherwise uneventful pregnancy.

Human placental lactogen (hPL) is commonly used in surveying the placental function during normal and pathologic pregnancies. This report describes a pregnancy where hPL could not be found in maternal serum or placental tissue. The pregnancy was in all other respects completely normal, ending with the birth of a normal baby. Some possible reasons and consequences of this unique event are discussed.