GDF15 linked to maternal risk of nausea and vomiting during pregnancy.

GDF15, a hormone acting on the brainstem, has been implicated in the nausea and vomiting of pregnancy, including its most severe form, hyperemesis gravidarum (HG), but a full mechanistic understanding is lacking1-4. Here we report that fetal production of GDF15 and maternal sensitivity to it both contribute substantially to the risk of HG. We confirmed that higher GDF15 levels in maternal blood are associated with vomiting in pregnancy and HG. Using mass spectrometry to detect a naturally labelled GDF15 variant, we demonstrate that the vast majority of GDF15 in the maternal plasma is derived from the feto-placental unit. By studying carriers of rare and common genetic variants, we found that low levels of GDF15 in the non-pregnant state increase the risk of developing HG. Conversely, women with ß-thalassaemia, a condition in which GDF15 levels are chronically high5, report very low levels of nausea and vomiting of pregnancy. In mice, the acute food intake response to a bolus of GDF15 is influenced bi-directionally by prior levels of circulating GDF15 in a manner suggesting that this system is susceptible to desensitization. Our findings support a putative causal role for fetally derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by prepregnancy exposure to the hormone, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

Energy status and HIF signalling in chorionic villi show no evidence of hypoxic stress during human early placental development.

Early human placental and embryonic development occurs in a physiologically low oxygen environment supported by histiotrophic secretions from endometrial glands. In this study, we compare the placental metabolomic profile in the first, second and third trimesters to determine whether the energy demands are adequately met in the first trimester. We investigated whether hypoxia-inducible factors, HIF-1α and/or HIF-2α, might regulate transcription during the first trimester. First and second trimester tissue was collected using a chorionic villus sampling-like (CVS) technique. Part of each villus sample was frozen immediately and the remainder cultured under 2 or 21% O2 ± 1 mM H2O2, and ±the p38 MAPK pathway inhibitor, PD169316. Levels of HIF-1α were assessed by western blotting and VEGFA, PlGF and GLUT3 transcripts were quantified by RT-PCR. Term samples were collected from normal elective Caesarean deliveries. There were no significant differences in concentrations of ADP, NAD(+), lactate, and glucose, and in the ATP/ADP ratio, across gestational age. Neither HIF-1α nor HIF-2α could be detected in time-zero CVS samples. However, culture under any condition (2 or 21% O2 ± 1 mM H2O2) increased HIF-1α and HIF-2α. HIF-1α and HIF-2α were additionally detected in specimens retrieved after curettage. HIF-1α stabilization was accompanied by significant increases in VEGFA and GLUT3 and a decrease in PlGF mRNAs. These effects were suppressed by PD169316. In conclusion, our data suggest that first trimester placental tissues are not energetically compromised, and that HIF-1α is unlikely to play an appreciable role in regulating transcriptional activity under steady-state conditions in vivo. However, the pathway may be activated by stress conditions.

Fetally-encoded GDF15 and maternal GDF15 sensitivity are major determinants of nausea and vomiting in human pregnancy.

Human pregnancy is frequently accompanied by nausea and vomiting that may become severe and life-threatening, as in hyperemesis gravidarum (HG), the cause of which is unknown. Growth Differentiation Factor-15 (GDF15), a hormone known to act on the hindbrain to cause emesis, is highly expressed in the placenta and its levels in maternal blood rise rapidly in pregnancy. Variants in the maternal GDF15 gene are associated with HG. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We found that the great majority of GDF15 in maternal circulation is derived from the feto-placental unit and that higher GDF15 levels in maternal blood are associated with vomiting and are further elevated in patients with HG. Conversely, we found that lower levels of GDF15 in the non-pregnant state predispose women to HG. A rare C211G variant in GDF15 which strongly predisposes mothers to HG, particularly when the fetus is wild-type, was found to markedly impair cellular secretion of GDF15 and associate with low circulating levels of GDF15 in the non-pregnant state. Consistent with this, two common GDF15 haplotypes which predispose to HG were associated with lower circulating levels outside pregnancy. The administration of a long-acting form of GDF15 to wild-type mice markedly reduced subsequent responses to an acute dose, establishing that desensitisation is a feature of this system. GDF15 levels are known to be highly and chronically elevated in patients with beta thalassemia. In women with this disorder, reports of symptoms of nausea or vomiting in pregnancy were strikingly diminished. Our findings support a causal role for fetal derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to GDF15, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

Placental endoplasmic reticulum stress and oxidative stress in the pathophysiology of unexplained intrauterine growth restriction and early onset preeclampsia.

The pregnancy complications of unexplained intrauterine growth restriction and early onset preeclampsia are thought to share a common aetiology in placental malperfusion secondary to deficient maternal spiral artery conversion. A key question is whether the contrasting clinical manifestations reflect different placental pathologies, or whether they are due to altered maternal responses to a common factor derived from the placenta. Recently, molecular evidence of protein synthesis inhibition secondary to endoplasmic reticulum stress has provided an explanation for the small placental phenotype in both conditions. However, other pathways activated by more severe endoplasmic reticulum stress are only observed in placentas from pregnancies associated with early onset preeclampsia. Here, we review the literature and conclude that there is evidence of greater maternal vascular compromise of the placenta in these cases. We speculate that in cases of normotensive intrauterine growth restriction the placental pathology is centred predominantly around endoplasmic reticulum stress, whereas in cases complicated by preeclampsia oxidative stress is further superimposed. This causes the release of a potent mix of pro-inflammatory cytokines, anti-angiogenic factors and trophoblastic aponecrotic debris into the maternal circulation that causes the peripheral syndrome. Maternal and fetal constitutional factors may modulate how the placenta responds to the maternal vascular insult, and how the mother is affected by the placental factors released. However, the principal conclusion is that the difference between these two conditions lies in the severity of the initiating deficit in spiral arterial conversion, and the relative degrees of endoplasmic reticulum stress and oxidative stress induced in the placenta as a result.

Regulation of vascular growth and function in the human placenta.

During the course of 9 months, the human placenta develops into a highly vascular organ. Vasculogenesis starts during the third week post-conception. Hemangioblastic cell cords differentiate in situ from mesenchymal cells in the villous cores, most probably under the influence of vascular endothelial growth factor (VEGFA) secreted by the overlying trophoblast. The cords elongate through proliferation and cell recruitment, and connect with the vasculature of the developing fetus. A feto-placental circulation starts around 8 weeks of gestation. Elongation of the capillaries outstrips that of the containing villi, leading to looping of the vessels. The obtrusion of both capillary loops and new sprouts results in the formation of terminal villi. Branching and non-branching angiogenesis therefore play key roles in villous morphogenesis throughout pregnancy. Maternal circulating levels of VEGFA and placental growth factor vary across normal pregnancy, and in complicated pregnancies. Determining the impact of these changes on placental angiogenesis is difficult, as the relationship between levels of factors in the maternal circulation and their effects on fetal vessels within the placenta remains unclear. Furthermore, the trophoblast secretes large quantities of soluble receptors capable of binding both growth factors, influencing their bioavailability. Villous endothelial cells are prone to oxidative stress, which activates the apoptotic cascade. Oxidative stress associated with onset of the maternal circulation, and with incomplete conversion of the spiral arteries in pathological pregnancies, plays an important role in sculpting the villous tree. Suppression of placental angiogenesis results in impoverished development of the placenta, leading ultimately to fetal growth restriction.

Effect of low-dose mifepristone administration on day 2 after ovulation on transcript profiles in implantation-stage endometrium of rhesus monkeys.

Progesterone is essential for endometrial receptivity in primates. In studies previously performed using global gene profiling based on microarray technology, attempts have been made to identify changes in gene expression between early luteal-phase and mid-luteal-phase endometria. However, the issue of the putative impact of preimplantation embryo-derived signal in the process of endometrial receptivity was missing in the previous studies. In the present study, an attempt has been made to delineate the transcripts profile in implantation-stage endometrium under combinatorial regulation of progesterone and embryo-derived signal in the rhesus monkey. To this effect, we have compared transcript profiles for 409 known genes between control receptive stage (n=13), and mifepristone-induced desynchronized and non-receptive stage (n=12) monkey endometrial samples collected on days 4 (n=12) and 6 (n=13) after ovulation from mated, potential conception cycles, using cDNA arrays containing sequence-verified clones. Statistical analysis of correlation of estimated transcript abundance between arrays and qRT-PCR for nine selected gene products yielded significant (P<0.05) concordance. Of 409 genes, a total of 40 gene transcripts were seen to be affected, nine gene transcripts in endometrial samples were found to progressively increase between days 4 and 6 following mifepristone treatment, while an additional five genes showed differential expression profile depending on the day after treatment. Additionally, different sets of 12 and 14 gene products showed changes in days 4 and 6 post-ovulation samples respectively. A new cohort of 28 gene products in implantation-stage endometrium was seen to be affected by luteal-phase mifepristone.

Birth weight to placenta weight ratio and its relationship to ultrasonic measurements, maternal and neonatal morbidity: A prospective cohort study of nulliparous women.

INTRODUCTION: Birth weight to placenta weight (BWPW)-ratio is an indicator of the ability of the placenta to maintain adequate nutrient supply to the fetus. We sought to investigate the relationship between BWPW-ratio with fetal growth, utero-placental Doppler and neonatal and maternal morbidity. METHODS: We studied a group of 3311 women recruited to a prospective cohort study of nulliparous women (Rosie Hospital, Cambridge, UK) who delivered a live born infant at term and whose placental weight and birth weight were known. Ultrasonic indices and BWPW ratio were converted to gestational age adjusted z scores. Analysis of continuous variables was by multivariable linear regression. BWPW ratio was also categorized (lowest or highest quintile, both referent to quintiles 2 to 4) and associations with adverse outcomes analyzed using multivariable logistic regression. RESULTS: Lowest quintile of BWPW-ratio was associated (adjusted odds ratio [95% CI], P) with both neonatal morbidity (1.55 [1.12-2.14], 0.007) and maternal diabetes (1.75 [1.18-2.59], 0.005). Highest quintile of BWPW ratio was associated with a reduced risk of maternal obesity (0.71 [0.53 to 0.95], 0.02) and preeclampsia (0.51 [0.31 to 0.84], 0.008), but higher (adjusted z score [95% CI], P) uterine artery Doppler mean pulsatility index (PI) at 20 weeks of gestation (0.09 [0.01-0.18], 0.04) and umbilical artery Doppler PI at 36 weeks of gestation (0.16 [0.07-0.25], <0.001). CONCLUSION: BWPW-ratio is related to ultrasonic measurements and both neonatal and maternal morbidity. Therefore, this ratio may be an indicative marker of immediate and longer term health risks for an individual.

Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids.

Angiogenesis is important in the pathophysiology of endometriosis, a condition characterized by implantation of ectopic endometrium in the peritoneal cavity. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in physiological and pathological angiogenesis, and elevated levels of VEGF are found in peritoneal fluid of patients with endometriosis. Our aim was to investigate the site of expression and regulation of VEGF in endometriosis. VEGF immunoreactivity was found in tissue macrophages present in ectopic endometrium and in activated peritoneal fluid macrophages. Macrophage activation was highest in women with endometriosis, and media conditioned by peritoneal fluid macrophages from these women caused a VEGF-dependent increase in endothelial cell proliferation above that seen from normal women. Peritoneal fluid macrophages secreted VEGF in response to ovarian steroids, and this secretion was enhanced after activation with lipopolysaccharide. Peritoneal fluid macrophages expressed receptors for steroid hormones. VEGF receptors flt and KDR (kinase domain receptor) were also detected, suggesting autocrine regulation. During the menstrual cycle, expression of flt was constant but that of KDR was increased in the luteal phase, at which time the cells migrated in response to VEGF. KDR expression and the migratory response were significantly higher in patients with endometriosis. This study demonstrates that activated macrophages are a major source of VEGF in endometriosis and that this expression is regulated directly by ovarian steroids.

Human early placental development: potential roles of the endometrial glands.

There is strong evidence that the endometrial glands play a key role in regulating placental development in many domestic species, but their contribution in the human has largely been ignored once implantation is complete. Here we re-evaluate their role during the first trimester. Connections between the glands and the intervillous space have been observed from day 17 post-conception through to the end of the first trimester. In the absence of a maternal arterial supply to the early placenta it is believed that the carbohydrate- and lipid-rich secretions represent an important source of nutrients during the first trimester, and possibly the beginning of the second trimester. The secretions also contain a variety of growth factors that may regulate placental morphogenesis since their receptors are present on villous and extravillous trophoblast, and villous endothelial cells. Other components of the secretions may modulate immune responses and trophoblast invasion at the materno-fetal interface. We speculate that lactogenic hormones secreted by decidual cells and the syncytiotrophoblast may act in concert with human chorionic gonadotropin to stimulate the secretory activity of glandular epithelial cells during the first trimester. There is circumstantial evidence, but as yet no conclusive proof, that deficient glandular activity is associated with pregnancy failure in the human.

Expression of vascular endothelial growth factor and its receptors flt and KDR in ovarian carcinoma.

BACKGROUND: Two thirds of patients with ovarian carcinoma have advanced disease at diagnosis and have poor prognoses because of the presence of highly invasive carcinoma cells and rapidly accumulating ascitic fluid. Vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells, is produced in elevated amounts by many tumors, including ovarian carcinomas. The known human receptors for VEGF, flt and KDR, are both cell surface tyrosine kinases and are expressed predominantly on endothelial cells. Acting through these receptors, VEGF may stimulate angiogenesis and promote tumor progression. PURPOSE: We aimed to clarify the function of VEGF in tumor development by identifying the cells in ovarian carcinoma tissue that express VEGF and its receptors. METHODS: VEGF, flt, and KDR expression was localized by in situ hybridization and immunohistochemistry in frozen sections of primary tumors from five patients with ovarian carcinoma and from metastases of ovarian carcinoma from three different patients. Reverse transcription followed by polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay were used to analyze VEGF, flt, and KDR expression in six epithelial cell lines derived from ovarian carcinoma ascites from five additional patients. RESULTS: Messenger RNAs (mRNAs) encoding VEGF, flt, and KDR were detected in primary ascitic cells and in three of four ovarian carcinoma cell lines examined by RT-PCR. Two novel complementary DNAs that may encode truncated, soluble forms of flt were cloned from one primary source. VEGF levels of 20-120 pM were found in culture media conditioned by the cell lines. Elevated expression of VEGF mRNA was found in all primary tumors and metastases, especially at the margins of tumor acini. VEGF immunoreactivity was concentrated in clusters of tumor cells and patches of stromal matrix. flt immunoreactivity was confined to tumor blood vessels, but flt mRNA was not detected by in situ hybridization. In contrast, KDR mRNA was detected not only in vascular endothelial cells but also in tumor cells at primary malignant sites. CONCLUSIONS: VEGF is expressed by tumor cells in primary and metastatic ovarian carcinoma and accumulates in the stromal matrix. Its receptors, flt and KDR, are expressed by some tumor cells that coexpress VEGF. This is the first localization of KDR expression in nonendothelial cells. IMPLICATIONS: Coexpression of VEGF and KDR by tumor cells in ovarian carcinoma raises the possibility of autocrine stimulation and of therapeutic strategies targeting this receptor-ligand interaction.

Computational modeling of the structure-function relationship in human placental terminal villi.

Placental oxygen transport takes place at the final branches of the villous tree and is dictated by the relative arrangement of the maternal and fetal circulations. Modeling techniques have failed to accurately assess the structure-function relationship in the terminal villi due to the geometrical complexity. Three-dimensional blood flow and oxygen transport was modeled in four terminal villi reconstructed from confocal image stacks. The blood flow was analyzed along the center lines of capillary segments and the effect of the variability in capillary diameter, tortuosity and branching was investigated. Additionally, a validation study was performed to corroborate the simulation results. The results show how capillary variations impact motion of the fetal blood, and how their bends and dilatations can decelerate the flow by up to 80%. Vortical flow is also demonstrated not to develop in the fetal capillaries. The different geometries are shown to dictate the transport of gases with differences of over 100% in the oxygen flux between samples. Capillary variations are key for efficient oxygen uptake by the fetus; they allow the blood to decelerate where the villous membrane is thinnest allowing for a better oxygenation, but also by reducing the vessel diameter they carry the oxygenated blood away fast. The methodology employed herein could become a platform to simulate complicated in-vivo and in-vitro scenarios of pregnancy complications.

The relationship between human placental morphometry and ultrasonic measurements of utero-placental blood flow and fetal growth.

INTRODUCTION: Ultrasonic fetal biometry and arterial Doppler flow velocimetry are widely used to assess the risk of pregnancy complications. There is an extensive literature on the relationship between pregnancy outcomes and the size and shape of the placenta. However, ultrasonic fetal biometry and arterial Doppler flow velocimetry have not previously been studied in relation to postnatal placental morphometry in detail. METHODS: We conducted a prospective cohort study of nulliparous women in The Rosie Hospital, Cambridge (UK). We studied a group of 2120 women who had complete data on uterine and umbilical Doppler velocimetry and fetal biometry at 20, 28 and 36 weeks' gestational age, digital images of the placenta available, and delivered a liveborn infant at term. Associations were expressed as the difference in the standard deviation (SD) score of the gestational age adjusted ultrasound measurement (z-score) comparing the lowest and highest decile of the given placental morphometric measurement. RESULTS: The lowest decile of placental surface area was associated with 0.87 SD higher uterine artery Doppler mean pulsatility index (PI) at 20 weeks (95% CI: 0.68 to 1.07, P < 0.001). The lowest decile of placental weight was associated with 0.73 SD higher umbilical artery Doppler PI at 36 weeks (95% CI: 0.54 to 0.93, P < 0.001). The lowest decile of both placental weight and placental area were associated with reduced growth velocity of the fetal abdominal circumference between 20 and 36 weeks (both P < 0.001). CONCLUSION: Placental area and weight are associated with uterine and umbilical blood flow, respectively, and both are associated with fetal growth rate.

Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT-1) pre-mRNA is important for the regulation of VEGF activity.

Angiogenesis is essential for normal mammalian development and is controlled by the local balance of pro- and antiangiogenic factors. Here we describe a novel mouse cDNA sequence encoding sFLT-1 that is a potent antagonist to vascular endothelial growth factor (VEGF) and show for the first time its in vivo production. In situ hybridization and Northern blot analysis with probes specific for sFLT-1 or FLT-1 showed that the relative abundance of their mRNAs changed markedly in spongiotrophoblast cells in the placenta as gestation progressed. On day 11 of pregnancy, sFLT-1 mRNA was undetectable but FLT-1 readily apparent, and by day 17 sFLT-1 mRNA was abundant but FLT-1 barely detectable. sFLT-1 was identified in conditioned medium of cultured placenta from day 17 pregnant mice and likely to be present in the circulation, as there is a substantial increase of VEGF-binding activity in the serum from day 13 of pregnancy, which coincides with the abundant sFLT-1 expression in placenta. Expression of sFLT-1 was also observed in adult lung, kidney, liver, and uterus. These data suggest a novel mechanism of regulation of angiogenesis by alternative splicing of FLT-1 pre-mRNA. Treatment of pregnant mice with exogenous VEGF from day 9 to 17 of pregnancy, which alters the ratio of VEGF to sFLT-1, resulted in an increase in the number of resorption sites and fibrin deposition in the placenta of ongoing pregnancies. These findings have important implications for understanding placental function and may be relevant in a range of disease states.

Cloning, expression and genomic organization of human placental protein disulfide isomerase (previously identified as phospholipase C alpha).

Phosphoinositol-specific Phospholipase C plays an important role in transducing receptor generated signals to the rest of the cell. A cDNA encoding a phospholipase has been described (Bennett et al., 1988, Nature 334, 268-270). However it is probable that this cDNA in fact encodes a protein disulfide isomerase. Since the original work suggested that this enzyme was important in the reproductive tract we sort to clone, sequence, express and characterize the recombinant protein isolated from the placenta. We have cloned and sequenced the cDNA encoding the human homolog of this cDNA from human placenta, although the mRNA was widespread in the female reproductive tract. We have transiently expressed it in both COS cells and also 1BR fibroblasts. Cell lysates were assayed for increased phospholipase activity and protein disulfide activity. We describe the entire cDNA sequence which is highly conserved between species. We have also cloned a portion of the genomic gene and described the intron/exon boundaries. In vitro translation of this cDNA showed that it encoded a protein of 61 kD with a cleavable signal peptide. Transient expression showed the protein produced had no phospholipase activity but did show protein disulfide isomerase activity. The expression work shows that this cDNA indeed encodes a protein disulfide isomerase and not a phospholipase. The nucleotide sequence shows marked conservation of the coding and regulatory regions which may suggest that this enzyme has evolved to perform a highly specialized function.

Quantification of messenger ribonucleic acid for epidermal growth factor in human myometrium and leiomyomata using reverse transcriptase polymerase chain reaction.

Epidermal growth factor (EGF) mRNA was quantified in samples of human myometrium, untreated leiomyomata, and leiomyomata from patients treated with a GnRH analog. Quantitative reverse transcriptase-polymerase chain reaction, using a synthetic internal standard, was applied to determine levels of EGF mRNA. In myometrium from uteri with no leiomyomata, levels of EGF mRNA did not differ between the proliferative and secretory phase of the cycle. Leiomyomata from women who had received no drug therapy had significantly higher amounts of EGF mRNA than myometrium from a normal uterus, but only in the secretory phase of the cycle. In the proliferative phase, leiomyomata did not have different amounts of EGF mRNA compared to normal myometrium. Untreated leiomyomata in the secretory phase of the cycle, but not those in the proliferative phase, had significantly more EGF mRNA than leiomyomata from women who had received treatment with a GnRH analog. These findings suggest that EGF is important in leiomyomata development, but imply that its production is only increased during the secretory phase of the cycle. This challenges the hypothesis that EGF production in leiomyomata is mediated by estrogen and raises the possibility that progesterone may be the more important hormone in fibroid growth.

Presence of messenger ribonucleic acid for endothelin-1, endothelin-2, and endothelin-3 in human endometrium and a change in the ratio of ETA and ETB receptor subtype across the menstrual cycle.

The aim of the present study was to determine whether mRNA for the three endothelin peptides (endothelin-1, endothelin-2, and endothelin-3) and the two known receptor subtypes (ETA and ETB) was present in human endometrium at different stages of the menstrual cycle (menstrual, early and mid-proliferative, and early, mid-, and late secretory). Endometrium was obtained from women undergoing surgery for benign disease, and total RNA was extracted using a guanidinium isothiocyanate method. mRNA for endothelin peptide and receptor was detected using the reverse transcriptase-polymerase chain reaction with nested oligonucleotide primers. mRNA for endothelin-1, endothelin-2, and endothelin-3 was demonstrated throughout the menstrual cycle, and three splice variants of mRNA encoding endothelin-3 were found in all samples. The ratio of ETA to ETB receptor mRNA was found to change throughout the menstrual cycle. In the proliferative phase, amplified cDNA product was almost exclusively confined to the ETA receptor, whereas an increase in the amplified product of the ETB receptor cDNA was seen in the secretory and menstrual phases. These studies show that mRNA for endothelin-1, endothelin-2, and endothelin-3 is present in human endometrium at all stages of the menstrual cycle and suggest that different physiological actions of the endothelin peptides may be mediated through changes in the ratio of the ETA and ETB receptor subtypes.

Localization and quantification of vascular endothelial growth factor messenger ribonucleic acid in human myometrium and leiomyomata.

Vascular endothelial growth factor (VEGF) messenger RNA (mRNA) and VEGF peptide were studied in human myometrium and leiomyomata. Amplification of complementary DNA from myometrium and leiomyomata by the polymerase chain reaction revealed three species of mRNA encoding VEGF: VEGF189, VEGF165, and VEGF121. In situ hybridization demonstrated VEGF mRNA expression throughout the smooth muscle cells of myometrium and leiomyomata. VEGF-like immunoreactivity was also detected in these tissues by immunocytochemistry. Quantification of VEGF mRNA using an RNAse protection assay demonstrated that in normal myometrium, levels of VEGF mRNA are significantly higher in the secretory phase than in the proliferative phase of the cycle. Leiomyomata did not have significantly different levels of VEGF mRNA compared with normal myometrium. In untreated leiomyomata, there was no significant difference between VEGF mRNA levels in the proliferative and secretory phases of the cycle. Leiomyomata from women treated with a GnRH anlog did not have significantly different levels of VEGF mRNA from untreated leiomyomata.

Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia.

Endometrial growth and repair after menstruation are associated with profound angiogenesis. Abnormalities in these processes result in excessive or unpredictable bleeding patterns and are common in many women. It is therefore important to understand which factors regulate normal endometrial angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays an important role in normal and pathological angiogenesis. In this study we show that expression of VEGF is regulated by hypoxia in human endometrium. Culture in vitro for 24 h under hypoxic conditions resulted in a 2- to 6-fold increase in VEGF secretion by both stromal and epithelial cells isolated from human endometrium. Quantitative RT-PCR was used to measure VEGF messenger ribonucleic acid (mRNA) levels in these cells. After hypoxia, VEGF mRNA levels increased 1.8-fold in stromal cells and 3.4-fold in glandular epithelial cells. The mRNA for each VEGF splice variant increased to an equal extent. The increase in VEGF secretion by stromal and epithelial cells in response to hypoxia was not altered by treatment at the same time with estradiol or progesterone. In situ hybridization of human endometrium during menstruation, when steroid levels are low but the tissue is subject to ischemia, showed strong hybridization to VEGF mRNA in both stromal and glandular cells. These results show that local factors, such as hypoxia, can regulate VEGF expression in the endometrium. This may play an important part in normal endometrial repair after menstruation. The secretion of VEGF by endometrial cells under hypoxic conditions may also be important in the pathogenesis of endometriosis, because it would be predicted to assist revascularization of desquamated endometrial explants when they attach at ectopic sites.

Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells.

Angiogenesis is essential for endometrial growth and repair, and disruption of this process may lead to common disorders of women, including menorrhagia and endometriosis. In pregnancy, failure of the endometrial spiral arterioles to undergo remodeling leads to preeclampsia. Here we report that in addition to vascular endothelial growth factor A (VEGF-A), human endometrium expresses messenger ribonucleic acids (mRNAs) encoding VEGF-C, placenta growth factor (PlGF), the angiopoietins, angiopoietin 1 (Ang1) and Ang2, and the receptors VEGFR-3 (Flt-4), Tie 1, and Tie 2. Levels of VEGF-C, PlGF, and Tie 2 changed during the menstrual cycle. Intense hybridization for VEGF-C and PlGF mRNAs was found in uterine nature killer cells in secretory phase endometrium and for Ang2 mRNA in the same cells in the late secretory phase. Interleukin-2 (IL-2) and IL-15 up-regulated VEGF-C, but not PlGF or Ang2, mRNA levels in isolated NK cells. Conditioned medium from decidual NK cells did not induce human umbilical vein endothelial cell apoptosis. These results indicate that human endometrium expresses a wide range of angiogenic growth factors and that uterine nature killer cells may play an important role in the abnormal endometrial angiogenesis that underlies a range of disorders affecting women.