Angiogenesis and the placental environment.

Rapid growth and vascularization of the human placenta are characteristic of early pregnancy and are accomplished in an unusually hypoxic environment. Stimulation of placental growth through hypoxia-induced angiogenesis may therefore be of particular importance. We have previously found that several varieties of vascular endothelial growth factor (VEGF) mRNA, including VEGF165, are present in cultured placental fibroblasts. We hypothesized that hypoxia would increase the transcription and translation of VEGF by these cells and provide one mechanism linking placental development with its environment. Placental fibroblasts were grown in aerobic or anaerobic atmospheric conditions for 72 h. By 24 h the oxygen tension of the anaerobic culture media was significantly less than that of the aerobic cultures. RNA was extracted from the cells at 24, 48 and 72 h. Following reverse transcription polymerase chain reaction (RT-PCR) stronger signals for VEGF were always found in the anaerobic cultures and this was confirmed by competitive PCR. mRNA for VEGF165 was represented most strongly but the anaerobic cultures also showed clearly mRNA for VEGF121, VEGF189 and VEGF206. The VEGF protein was also measured in the aerobic and anaerobic culture medium. By 72 h the average concentration of VEGF was significantly higher (P = 0.01) in the anaerobic culture medium. VEGF production is one mechanism through which oxygen supply may influence placental development. Examples of this may include the compensatory placental hypertrophy associated with maternal anaemia and with reproduction at high altitude.

Influence of hypoxia on vascular endothelial growth factor and chorionic gonadotrophin production in the trophoblast-derived cell lines: JEG, JAr and BeWo.

Growth of trophoblast tissue in early pregnancy is rapid and accomplished in an unusually hypoxic environment. Hypoxia has been reported to upregulate mRNA production of vascular endothelial growth factor (VEGF), and VEGF receptors have been found on trophoblast cells. These observations suggest that VEGF may have an important role in early placentation. This study examines the influence of hypoxia on both the production of the VEGF message and protein and on the production of human chorionic gonadotrophin (hCG) protein by the cell lines JEG, JAr and BeWo. Cells were grown under normoxic and hypoxic conditions for 72 h. The average oxygen tension in the culture media of the hypoxic cultures (6-7 kPa) was significantly less than in the normoxic cultures (19-21 kPa). RNA was extracted and message for VEGF(121), VEGF(165) and VEGF(189) found in all cell lines by reverse transcription and the polymerase chain reaction (RT-PCR). These messages were upregulated by hypoxia; findings confirmed by competitive PCR for VEGF and expression of the house keeping gene GAPDH. hCG and VEGF were measured by immunoassay. Hypoxia resulted in an increase in VEGF production (P<0.05) but had inconsistent effects on hCG production. In some experiments the absolute concentrations of hCG and VEGF in the culture media were noted to be significantly correlated (r>0.5, P<0.05). In addition to its role in angiogenesis, VEGF may have direct effects on trophoblast cells encouraging proliferation and invasion. These effects may be regulated in part through oxygen supply and hCG.

Short report: identification of a specific pattern of vascular endothelial growth factor mRNA expression in human placenta and cultured placental fibroblasts.

Reverse transcription and the polymerase chain reaction (PCR) were used to detect vascular endothelial growth factor (VEGF) mRNA in human placental tissue and cultured placental fibroblasts obtained during the first trimester of pregnancy. The primers for VEGF corresponded to areas in exon 4 and exon 8 of the VEGF gene. After one round of PCR three products, equivalent to VEGF121, VEGF165 and VEGF189, were detected within placental tissue and cultured placental fibroblasts. A further round of PCR revealed the presence of two more products equivalent to VEGF206 and VEGF145. Thus, in addition to the production of readily secreted forms of VEGF (VEGF121 and VEGF165), the placenta produces several transcripts expected to increase the growth factor pool of the extracellular matrix.

Pregnancy-specific-beta 1-glycoprotein: effect on lymphocyte proliferation in vitro.

The effects of three different preparations of pregnancy-specific-beta 1-glycoprotein (SP1) were investigated in parallel with progesterone, oestradiol and human chorionic gonadotrophin (hCG) on the proliferative response of lymphocytes in the mixed lymphocyte reaction (MLR), and to the mitogens phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). A dose-related inhibition of the MLR was obtained with SP1 at 2.5-10 mg/l. SP1 also inhibited the PHA response at 10 mg/l, but had no effect on the PWM response at these concentrations. Progesterone and oestradiol inhibited all systems at 10-20 mg/l while hCG inhibited all systems at 1500-3000 IU/ml. The observations suggest that SP1 selectively inhibits the proliferative responses of T lymphocytes at the concentrations studied.

Placental protein 14 secretion during in vitro fertilization cycles with and without human chorionic gonadotropin for luteal support.

OBJECTIVE: To investigate levels of placental protein 14 (PP14) in in vitro fertilization (IVF) patients with and without exogenous human chorionic gonadotropin (hCG) for luteal support. DESIGN, PATIENTS: Thirty-one women undergoing IVF were studied. For 18 women, hCG was administered in the luteal phase, and 12 became pregnant. Five pregnancies occurred in 13 women not receiving exogenous hCG. SETTING: All the patients attended the University of Southampton/Chalybeate Hospital IVF program. RESULTS: There was no change in PP14 levels 2 days after embryo transfer (ET), but small significant rises were noted by day 8 in all patients. Thereafter, levels rose further in pregnant subjects but showed no change in nonpregnant patients. The highest level of PP14 was seen in the group of women on hCG support, but there was no overall statistical difference between those on support and those not. In the nonpregnant group, there was no significant correlation between progesterone (P) and PP14 8 days from ET, whereas a highly significant correlation was noted in the pregnant group. CONCLUSIONS: Neither hCG nor P are primary factors in the control of endometrial PP14 secretion, but PP14 and P may have common underlying control mechanisms.

The relation between immunoglobulin G antibodies to Chlamydia trachomatis and poor ovarian response to gonadotropin stimulation before in vitro fertilization.

OBJECTIVE: To determine whether a relation exists between previous exposure to Chlamydia trachomatis and impaired ovarian response to gonadotropin stimulation. DESIGN: Controlled clinical study. SETTING: Two university IVF centers. PATIENT(S): Two hundred forty-two patients receiving IVF treatment and 81 control patients. Ninety-four patients with a poor response to IVF, defined by cycle cancellation in response to a daily stimulation dose of 300 IU of FSH, and 148 patients with a good response were matched for age. Twenty-eight pregnant controls and 53 controls of proven fertility also were included. INTERVENTION(S): Serum samples were obtained from patients and controls. Serum levels of immunoglobulin (Ig) G antibodies to C. trachomatis were determined by ELISA. MAIN OUTCOME MEASURE(S): The prevalence of serum IgG antibodies to C. trachomatis in critically defined poor responders was compared with that of age-matched good responders. RESULT(S): A significantly higher proportion of poor responders had serum IgG antibodies to C. trachomatis compared with good responders (44.7% and 30.4%, respectively). Patients undergoing IVF had a significantly higher prevalence of IgG antibodies to C. trachomatis (36%) than did either pregnant or nonpregnant controls (12%). CONCLUSION(S): A significantly higher prevalence of serum IgG antibodies to C. trachomatis was observed in critically defined poor responders, suggesting a possible detrimental effect of C. trachomatis on subsequent ovarian function.

Progesterone up-regulates WT1 mRNA and protein, and alters the relative expression of WT1 transcripts in cultured endometrial stromal cells.

OBJECTIVE: To determine the change in expression of the Wilms tumor suppressor gene product, WT1, by progesterone alone in endometrial stromal cell culture and to study its relationship with prolactin, a marker of decidualization. In addition, to examine the change in ratio of WT1 isoforms with and without exon 5 message. METHODS: Endometrial biopsies were taken from eight patients who had hysterectomy. Stromal cells were isolated and cultured in the presence of progesterone alone (12 days) or progesterone and 8-bromo-cyclic adenosine monophosphate (cAMP) (6 days). RNA was extracted from cells, and reverse transcription, real-time polymerase chain reaction (PCR), and conventional PCR were done to analyze WT1 mRNA expression. Immunocytochemistry was performed on equivalent cells to study WT1 protein expression. Decidualization was identified by increased prolactin concentrations in the media and immunocytochemical markers IGFBP-1 and collagen IV. RESULTS: Reverse transcription and real-time PCR revealed a significant increase in WT1 mRNA with increasing progesterone concentrations when decidualization was occurring (n = 6, P =.002). Increasing progesterone concentrations also increased the proportion of the WT1 transcript containing a 17-amino-acid insert (+ exon 5 expression); changes in WT1 exon 5 expression have been shown to be involved in control of proliferation and differentiation. Significant correlations between WT1 message and prolactin existed at physiologic progesterone concentrations (6.25, 12.5, 25, and 50 nM; P <.05) until prolactin concentrations reached a plateau at 100 nM. At concentrations of progesterone alone (> 25 nM) and progesterone with 8-bromo-cAMP, WT1 protein was localized to the nuclei of many of the decidualized stromal cells. CONCLUSION: The changing expression of WT1 isoforms in endometrial stromal cells caused by progesterone may be important for differentiation into the decidualized phenotype.

Variation in detection of VEGF in maternal serum by immunoassay and the possible influence of binding proteins.

Using radioimmunoassay (RIA) and a polyclonal antibody we have shown that maternal serum vascular endothelial growth factor (VEGF) is elevated during pregnancy. In contrast, a commercial VEGF ELISA utilizing a sandwich two-site immunoassay was unable to detect VEGF in 19 of the 20 maternal serum samples analysed. In addition, the recovery of exogenous VEGF added to the pregnancy samples was low or not recordable with the ELISA. Using RIA, 82-101% of the added VEGF was recovered. These differing results could be explained by the formation of VEGF-protein complexes that are detectable using RIA but undetectable with the ELISA. Our data imply that there is a substantial increase in circulating VEGF binding proteins during pregnancy. The increase in VEGF and its binding proteins during pregnancy may reflect important physiological events in the mother and feto-placental unit.

Suppression of ovarian function by Microgynon 30 in day 1 and day 5 "starters".

The suppression of ovulation during the first treatment cycle with Microgynon 30 (150 micrograms levonorgestrel and 30 micrograms ethinyl oestradiol) for nine subjects starting the "pill" on day 1 of their cycle and five subjects on day 5 was investigated. Serum oestradiol and progesterone levels throughout the cycle and midcycle urinary LH levels were reliably suppressed in all day 1 "starters". Serum progesterone levels and urinary LH levels were also suppressed in day 5 "starters" but one subject produced oestradiol levels within the normal range of ovulatory cycles. Mean oestradiol levels of day 5 "starters" were found to be significantly higher than those of day 1 "starters" (p less than 0.05).

Trial of support treatment with human chorionic gonadotrophin in the luteal phase after treatment with buserelin and human menopausal gonadotrophin in women taking part in an in vitro fertilisation programme.

OBJECTIVE: To evaluate the effect of support with human chorionic gonadotrophin in the luteal phase in women taking part in an in vitro fertilisation programme after buserelin and human menopausal gonadotrophin were used to hyperstimulate their ovaries. DESIGN: Controlled group comparison. SETTING: Outpatient department of a private hospital. PATIENTS: 115 Women with indications for in vitro fertilisation, all of whom had at least one embryo transferred. INTERVENTIONS: After suppression of the pituitary with buserelin the ovaries of all the women were stimulated with human menopausal gonadotrophin on day 4 of the luteal phase. Human chorionic gonadotrophin (10,000 IU) was given to induce ovulation, and oocytes were recovered 34 hours later. Embryos were transferred 46 to 48 hours after insemination. Women who had received the 10,000 IU of human chorionic gonadotrophin on a date that was an uneven number (n = 61) were allocated to receive support doses of 2500 IU human chorionic gonadotrophin three and six days after that date. The remaining 54 women did not receive hormonal support. END POINT: Determination of the rates of pregnancy. MEASUREMENTS and main results--Support with human chorionic gonadotrophin did not significantly alter the progesterone or oestradiol concentrations in the early or mid-luteal phase. The mean (range) progesterone concentrations in the late luteal phase in women who did not become pregnant were, however, significantly higher in those who received support (16(9-110) nmol/l nu 8(4-46) nmol/l), and the luteal phase was significantly longer in this group (14 days nu 12 days). The rate of pregnancy was significantly higher in the women who received support than in those who did not (25/61 nu 8/54). CONCLUSIONS: When buserelin and human menopausal gonadotrophin are used to hyperstimulate ovaries support with human chorionic gonadotrophin in the luteal phase has a beneficial effect on in vitro fertilisation.

Developmental exposure to bisphenol A leads to cardiometabolic dysfunction in adult mouse offspring.

Bisphenol A (BPA) is a chemical compound that has adverse health outcomes in adults when exposed during the perinatal period. However, its effect on cardiovascular function remains to be elucidated. In this study, we examined the effects of daily administration of BPA to pregnant mice from gestational days 11 to 19 on cardiometabolic outcomes in the adult offspring. Prenatal BPA exposure resulted in altered growth trajectory and organ size, increase adiposity and impaired glucose homeostasis in male and female offspring. In addition, these BPA offspring exhibited raised systolic blood pressure, and in the males this was accompanied by impaired vascular tone. The aortas in females, but not in males, from the BPA group also showed reduced estrogen receptor gene expression. These results indicate that prenatal exposure to BPA increased susceptibility of the offspring to developing cardiovascular and metabolic dysfunction later in life.

Effect of a low-protein diet during pregnancy on expression of genes involved in cardiac hypertrophy in fetal and adult mouse offspring.

Gene markers for cardiomyocyte growth, proliferation and remodeling were examined in mouse fetuses and adult male offspring exposed to maternal low-protein (LP) diet during pregnancy. Whole heart volume, measured by magnetic resonance imaging, was smaller in day 15 LP fetuses v. those from chow-fed dams (C), whereas heart volume was greater in adult LP v. C offspring. These LP offspring were hypertensive and had larger cardiomyocytes v. C animals. The mRNA levels of cyclin G1, a marker for cell growth, were lower in LP fetal hearts v. C hearts, but similar in the left ventricle of adult LP and C offspring. Opposite trends were found in brain natriuretic peptide levels (a marker of cardiac hypertrophy). Thus, maternal LP during pregnancy results in smaller fetal hearts and is accompanied by changes in expression of genes involved in cardiomyocyte growth, which are associated with cardiac hypertrophy and hypertension in adulthood.

The selection criteria on an IVF program can remove the association between maternal age and implantation.

BACKGROUND: 1190 consecutive in vitro fertilization (IVF) treatment cycles from the Southampton University/BUPA Chalybeate unit, spanning a four year period, were studied retrospectively in order to assess the relationship between maternal age and implantation. Our aim was to evaluate the hypothesis that the number of transferred embryos can be determined by age alone. METHOD: The cases were allocated to two age groups, Group 1 was composed of patients of less than or equal to 35 years of age and Group 2 of patients greater than 35 years of age. RESULTS: We found that the selection criteria used in our programme for abandoning treatment cycles led to significantly more older patients being excluded from oocyte collection (p < 0.001). The patients from both groups that progressed to oocyte collection and embryo transfer showed no significant difference in embryo implantation. The overall implantation rate (12.4%) and clinical pregnancy rate per embryo transfer (22.8%) were achieved by being able to transfer comparable numbers of embryos in both age groups and in spite of the younger age group having a significantly better quality of transferred embryos. CONCLUSION: Although advancing maternal age predisposes to a reduced chance of success from IVF treatment, maternal age alone was not a useful predictor of embryo implantation or endometrial receptivity in completed IVF treatment cycles.

Progesterone inhibits insulin-like growth factor binding protein-1 (IGFBP-1) production by explants of the Fallopian tube.

The Fallopian tube provides the environment for early embryo growth, a process which is influenced by insulin-like growth factors (IGFs) in the tubal fluid. Whether the bioavailability of tubal IGFs is modulated by locally produced IGF-binding protein (IGFBP-1) is not clear. An explant culture system from human Fallopian tube mucosa was, therefore, developed enabling the potential for IGFBP-1 production by this tissue to be examined directly. Initial characterization of the system established that the explants maintained responsiveness to steroids. Thus, oviduct-specific glycoprotein production, a major product of the oviduct in vivo, continued to be made via an estrogen-sensitive pathway in the culture. The presence of mRNA for IGFBP-1 was established within the explants by the use of quantitative RT-PCR and IGFBP-1 protein was measured by enzyme-linked immunosorbent assay. Although insulin and estradiol had no consistent effect on IGFBP-1, addition of progesterone had a significant inhibitory effect on IGFBP-1 production, both at the mRNA and protein levels. A dose-range of progesterone revealed an incremental inhibitory effect of progesterone on IGFBP-1 output (maximal effect, 25-50 nmol/l), consistent with physiological inhibition of this process during the luteal phase. We suggest that progesterone might, therefore, play a role in controlling the bioavailability of IGFs to the embryo during early development within the Fallopian tube.

The influence of ovarian follicular activity on late proliferative phase serum IGFBP-1 in down-regulated assisted conception cycles.

Serum insulin-like growth factor binding protein-1 (IGFBP-1) concentrations were measured at the end of the proliferative phase in infertility patients undergoing normal menstrual cycle frozen embryo transfer, exogenous hormone-supported frozen embryo transfer and in-vitro fertilization (IVF) treatment cycles. These patients were divided into five groups according to their ovarian follicular activity. The exogenous hormone-supported frozen embryo transfer group, who had no ovarian follicles, and the IVF groups (number of follicles ranging from 4-38) showed statistically higher serum IGFBP-1 concentrations when compared to the normal menstrual cycle group (P < or = 0.01). There was no significant difference in the serum IGFBP-1 concentrations between the exogenous hormone support frozen embryo transfer group and the poor or normal response IVF groups (number of follicles ranging from 4 to 16). An IVF group that displayed an excessive response to our standard human menopausal gonadotrophin stimulation (> 20 mature follicles or oestradiol > 10,000 pmol/l) showed a significantly higher serum IGFBP-1 concentration when compared with the other groups (P = 0.001). This subgroup was subsequently given a modified (follicle-stimulating hormone) stimulation regime which resulted in a significant reduction in serum IGFBP-1 concentrations (P < 0.05). There was no correlation between serum oestradiol and IGFBP-1 overall or within the patient groups. We conclude that serum IGFBP-1 concentrations in our down-regulated assisted conception cycles did not increase in line with ovarian follicular activity, unless an excessive response was displayed.

Persistent elevation of serum oestradiol levels by functional ovarian cysts despite effective pituitary desensitization with GnRH agonists.

OBJECTIVE: This study assessed whether functional ovarian cysts may prevent suppression of serum oestradiol levels even after pituitary desensitization had been achieved with buserelin. PATIENTS: Of 288 in-vitro fertilization (IVF) cycles studied 10 patients were found to have cysts with serum oestradiol levels > 200 pmol/l despite 3 weeks of buserelin. DESIGN AND MEASUREMENTS: The 10 patients with cysts were given 0.1 mg GnRH and serum gonadotrophins were measured at time 0, 30 and 60 minutes subsequently. Immediately following the GnRH stimulation test the cysts were aspirated transvaginally under ultrasound guidance. Serum oestradiol levels were again measured 3 days after cyst aspiration. RESULTS: Basal LH and FSH levels were < 3 U/l and there was no significant rise in response to GnRH. On the day of cyst aspiration serum oestradiol levels varied between 244 and 1127 pmol/l, and in all cases serum oestradiol levels fell to < 200 pmol/l within 3 days of cyst aspiration. CONCLUSION: In the presence of a functional ovarian cyst, failure to suppress the serum oestradiol level does not necessarily imply a failure of pituitary desensitization with a GnRH agonist.

Variations in concentrations of the major endometrial secretory proteins (placental protein 14 and insulin-like growth factor binding protein-1) in assisted conception regimes.

We have previously shown that placental protein 14 (PP14) concentrations were depressed in two pregnancies that followed down-regulation of the anterior pituitary and exogenous hormone support prior to a frozen-thawed embryo transfer. We now report on a more comprehensive series of pregnancies following this form of treatment, in-vitro fertilization (IVF) and natural cycle frozen-thawed embryo transfer. Serum specimens were analysed for PP14 and insulin-like growth factor binding protein-1 12 days after embryo transfer and at 7 weeks gestation. At 12 days after embryo transfer, the mean serum PP14 concentrations in the IVF and natural cycle were significantly higher in those who conceived than those who did not (82 versus 23 and 107 versus 39 micrograms/l respectively, P < 0.001). Although the mean PP14 concentration in the hormone-supported pregnant patients was higher than in the non-pregnant patients, this had not reached statistical significance 12 days after embryo transfer (49 versus 31 micrograms/l). By 7 weeks gestation the PP14 concentrations in the hormone-supported pregnant patients were significantly higher than in the non-pregnant patients (152 versus 31 micrograms/l, P < 0.001). However, the PP14 concentrations for hormone-supported pregnant patients were significantly lower (P < 0.001) than those for pregnant IVF or natural cycle patients at 7 weeks gestation (152, 777 and 660 micrograms/l respectively). The PP14 concentrations in the pregnant patients, although lower than those in IVF and natural cycle pregnancies, were higher than those previously reported in ovarian failure and Turner's syndrome ovum donation cycles.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation of women with endometrial carcinoma using serum vascular endothelial growth factor (VEGF) measurement.

This study assessed whether serum VEGF measurement in women presenting with endometrial cancer could predict advanced stage disease. Preoperative sera from 37 women undergoing laparotomy for suspected endometrial cancer were assayed for VEGF, CA125 and platelet count. Significant positive correlation was shown between VEGF and platelet levels (P = 0.003, r = 0.477). However, no correlation was demonstrated between VEGF and stage overall, and no significant difference was shown between those with early (stage 1A/1B, n = 20) compared to those with advanced (stage >1B, n = 13) or disseminated (stage >2, n = 7) disease. Serum VEGF measurement was not beneficial in the preoperative assessment of stage in patients with endometrial carcinoma. Strong correlation with platelet levels suggests that this is one of the sources of VEGF measured.

A longitudinal study of maternal serum vascular endothelial growth factor in early pregnancy.

Using a competitive radioimmunoassay to measure total immunoreactive vascular endothelial growth factor (VEGF), we describe for the first time longitudinal changes in serum VEGF in early pregnancy. The measurements were obtained from 26 women following the transfer of cryopreserved embryos; 18 singleton and eight twin pregnancies were identified by ultrasound at 6 weeks gestation and subsequently delivered as live births. Subjects did not have corpora lutea and exogenous hormone support was provided for the first 70 days of pregnancy. Serum VEGF increased approximately 30 days after embryo transfer and thereafter continued to rise in both singleton and twin pregnancies over a period of 20-40 days after which concentrations remained elevated. The longitudinal profile of serum VEGF concentrations was characterized by a logistic curve for singleton and twin pregnancies; the profile of VEGF concentrations in the twin pregnancies was significantly higher than in the singleton pregnancies (P < 0.0001). Profiles of the longitudinal concentrations of serum human chorionic gonadotrophin (HCG), oestradiol and progesterone were created by polynomial regression for singleton and twin pregnancies. The VEGF profiles were positively correlated with the profiles of HCG (r = 0.44, P = 0.02) and oestradiol (r = 0.36, P = 0.07) but not progesterone (r = 0.16, P = 0.42). Serum VEGF concentrations in the singleton thawed embryo pregnancies were compared with gestation-matched normal singleton pregnancies with corpora lutea. Concentrations of VEGF were significantly (P = 0.004) greater in the pregnancies with corpora lutea although this difference became less marked with advancing gestation. In addition to its important role in angiogenesis, we speculate that VEGF is involved in mechanisms which control the maternal cardiovascular adaptation to pregnancy.