Analysis of oestrogen regulation of alpha-, beta- and gamma-secretase gene and protein expression in cultured human neuronal and glial cells.

BACKGROUND: A key event in Alzheimer's disease pathology is the proteolytic processing of amyloid precursor protein (APP), whereby ß-amyloid (Aß) peptide is produced. Oestrogen is acknowledged to influence cognitive function and has been shown to regulate the secretory metabolism of APP and decrease the production of Aß, thereby protecting the brain from neurodegeneration. The mechanism for this effect is unknown. OBJECTIVE: To investigate possible oestrogen regulation of the expression of the APP processing enzymes at the gene and/or protein level. METHODS: The effects of oestrogen on gene and protein expression of α- (TACE and ADAM10), ß- (BACE) and γ- (presenilin 1) secretases in cultured human fetal neurons and glial cells were studied. The RNase protection assay, gene expression microarray analysis and relative quantitative real-time PCR were used to analyse gene expression, while the protein expression and cellular localization of the secretases were studied by immunoblot and confocal microscopy. RESULTS: Oestrogen is involved in the regulation of the gene expression of TACE and presenilin 1. Most importantly, BACE protein expression was downregulated by oestrogen treatment in mixed neuronal/glial cell cultures. Our results also show the cellular localization of the secretases in human neurons and glial cells, which has been thoroughly discussed in the light of the localization of APP processing. CONCLUSION: Our results indicate that oestrogen may affect APP processing directly by regulating the expression of the involved enzymes.

Fertility preservation in girls with turner syndrome: prognostic signs of the presence of ovarian follicles.

CONTEXT: Many girls with Turner syndrome have follicles in their ovaries at adolescence. OBJECTIVE: Our objective was to study which girls might benefit from ovarian tissue freezing for fertility preservation. DESIGN: Clinical and laboratory parameters and ovarian follicle counts were analyzed among girls referred by 25 pediatric endocrinologists. SUBJECTS AND SETTING: Fifty-seven girls with Turner syndrome, aged 8-19.8 yr, were studied at a university hospital. INTERVENTIONS: Ovarian tissue was biopsied laparoscopically, studied for the presence of follicles, and cryopreserved. Blood samples were drawn for hormone measurements. MAIN OUTCOME MEASURES: Presence of follicles in the biopsied tissue related to age, signs of spontaneous puberty, karyotype, and serum concentrations of gonadotropins and anti-Müllerian hormone were assessed. RESULTS: Ovarian biopsy was feasible in 47 of the 57 girls. In 15 of the 57 girls (26%), there were follicles in the tissue piece analyzed histologically. Six of seven girls (86%) with mosaicism, six of 22 (27%) with structural chromosomal abnormalities, and three of 28 with karyotype 45X (10.7%) had follicles. Eight of the 13 girls (62%) with spontaneous menarche had follicles, and 11 of the 19 girls (58%) who had signs of spontaneous puberty had follicles. The age group 12-16 yr had the highest proportion of girls with follicles. Normal FSH and anti-Müllerian hormone concentrations for age and pubertal stage were more frequent in girls with follicles. CONCLUSIONS: Signs of spontaneous puberty, mosaicism, and normal hormone concentrations were positive and statistically significant but not exclusive prognostic factors as regards finding follicles.

Transcriptional analysis of estrogen effects in human embryonic neurons and glial cells.

Estrogen exerts many effects in the central nervous system which extend beyond regulating sexual behavior and gonadal function in reproduction. It has been shown that estrogen has a range of both neuroprotective and neuromodulatory effects, but little is known about the molecular mechanisms underlying estrogen actions in the human brain. We therefore analyzed the transcriptional response to estrogen by microarray technology in primary cell cultures of neurons and glial cells derived from 8- to 12-week human fetal brain tissue. Estrogen treatment of the cell cultures during a 7-day culture period revealed a number of genes with significantly altered expression. Fourteen genes were selected for further analysis by quantitative real-time PCR in order to confirm the gene expression changes observed by the microarray analysis. Six genes were identified with important roles in the nervous system that have not been described before to be estrogen-regulated. Three genes, IGFBP7, Transgelin and Cyr61, were further analyzed by immunostaining, and were found to be expressed in both primary neurons and glial cells. Our results indicate that estrogen may influence developing human neurons and glia through regulating expression of genes that are important for the development of the nervous system.

Endometriosis and genetic polymorphisms.

UNLABELLED: Endometriosis is a benign gynecological disease with an unclear pathophysiology characterized by ectopic endometrium causing endometrium-like inflammatory lesions outside the uterine cavity. Recently, a number of studies have investigated genetic polymorphisms as a possible factor contributing to the development of endometriosis. In this review, we have summarized current data regarding genes with nucleotide polymorphisms investigated with regard to endometriosis. We searched PubMed for publications on endometriosis and polymorphism and found 108 publications between January 1979 and September 2005. These were classified according to the type of genetic polymorphism investigated and whether the result favored or did not favor association with endometriosis. We found a strikingly large amount of conflicting results. About 50% of the reviewed studies demonstrated positive correlations between different polymorphisms and endometriosis. This relation is most clearly seen in groups 1 (cytokines and inflammation), 2 (steroid-synthesizing enzymes and detoxifying enzymes and receptors), 4 (estradiol metabolism), 5 (other enzymes and metabolic systems), and 7 (adhesion molecules and matrix enzymes). Group 8 (apoptosis, cellcycle regulation, and oncogenes) seemed to be negatively correlated with the disease, whereas group 3 (hormone receptors), 6 (growth factor systems), and especially 9 (human leukocyte antigen system components) showed a relatively strong correlation. The review indicates that polymorphisms may have a limited value in assessing possible development of endometriosis. TARGET AUDIENCE: Obstetricians & Gynecologists, Family Physicians. LEARNING OBJECTIVES: After completion of this article, the reader should be able to recall the complexity of attempting to link endometriosis to single nucleotide polymorphisms (SNPs), explain that the literature is varied on results and recommendations and is population specific, and state that there are some SNP relationships that are clinically stronger than others.

Culture and expansion of the human embryonic stem cell line HS181, evaluated in a double-color system.

An approach of using RFP-transfected human foreskin fibroblasts (hFS-RFP) to support the growth of GFP expressing human embryonic stem cells (hES; HS181-GFP) is reported. The two-color system was applied to detect interactions between hFS and human embryonic stem cells (hES). After overnight culture, the hES cell colonies showed a behavior of "pushing away" the underlying feeder cells. This phenomenon occurred with both a low and high density of feeders. The density of the feeder cell layer, however, influenced the growth pattern of hES cell colonies. At a high feeder cell density, the hES colonies were more pointed and aligned with the direction of the fibroblasts, whereas less dense feeder layers allowed a more rounded and flat hES colony formation. Not surprisingly, a small fraction of mitotically inactivated feeder cells reattached after passage and remained viable in the cultures for up to four subsequent passages. The prospect of using the two-color system for detection of possible fusion events between hES cells and feeder cells was assessed by screening a large number of cell cultures for double RFP/EGFP expressing cells. The results indicate that fusion events are extremely rare (<10(-6)), or alternatively that after fusion the dual expression of both EGFP and RFP is not easily detected for other reasons. In summary, a two-color system allows analysis of colony formation and also helps to identify and follow the differentiation of cells.

Transforming growth factor-beta1 in fetal serum correlates with insulin-like growth factor-I and fetal growth.

OBJECTIVE: To estimate whether transforming growth factor-beta1 in fetal serum obtained by umbilical cord sampling at delivery is correlated with fetal growth. We also estimated whether transforming growth factor-beta1 is correlated with insulin-like growth factor-I and insulin-like growth factor binding protein-1, which have been shown to correlate with fetal growth. METHODS: The active form of transforming growth factor-beta1 was analyzed in serum from cord blood from 68 fetuses by the enzyme-linked immunosorbent assay technique. Of the 68 pregnant women, 12 had preeclampsia, 14 had preeclampsia and intrauterine growth restriction, 15 had intrauterine growth restriction alone, and seven had fetuses that were large for gestational age (LGA). Twenty pregnancies with fetuses appropriate for gestational age (AGA) served as controls. RESULTS: Transforming growth factor-beta1 concentrations were significantly correlated with birth weight. The average transforming growth factor-beta1 concentration in the following groups were: intrauterine growth restriction, 22.4 +/- 2.7 microg/L; intrauterine growth restriction plus preeclampsia, 22.9 +/- 2.0 microg/L; preeclampsia without intrauterine growth restriction, 28.8 +/- 2.1 microg/L; LGA, 30.3 +/- 4.3 microg/L; and AGA, 36.8 +/- 2.0 microg/L. Transforming growth factor-beta1 levels were significantly lower in pregnancies complicated by intrauterine growth restriction and showed a positive correlation with birth weight (r = 0.48, P <.001). Furthermore, there was a positive correlation between insulin-like growth factor-I levels and birth weight (r = 0.36, P <.01) and a negative correlation between insulin-like growth factor binding protein-1 and birth weight (r = -0.32, P <.01). There was also a correlation between transforming growth factor-beta1 and insulin-like growth factor-I (r = 0.29, P <.05) and between transforming growth factor-beta1 and insulin-like growth factor binding protein-1 (r = -0.25, P <.05). CONCLUSION: Transforming growth factor-beta1 might be related to fetal growth in pregnancy. The results also support previous data showing that insulin-like growth factor-I and insulin-like growth factor binding protein-1 are related to fetal growth.

An ultrasound-based approach to the assessment of infertility, including the evaluation of tubal patency.

An optimal initial infertility investigation protocol would be a process that is diagnostically accurate, expeditious, cost-effective, reliable and as minimally invasive as possible. In addition, the investigation should provide the clinician with useful prognostic information regarding possible future treatment. At present, extensive use of invasive procedures such as diagnostic hysteroscopy and laparoscopy is the standard at many fertility centres. Recent advances in gynaecological ultrasonography have shown that ultrasound can replace routine invasive investigative procedures. An ultrasound-based approach would make the basic infertility investigation less time-consuming and less expensive, but at the same time more acceptable to the majority of patients. This chapter describes an ultrasound-based approach to the assessment of infertility. In addition, the role of ultrasonography for assessment of the pelvic organs as a basic part of the initial investigation of an infertile couple is discussed and compared to more traditional invasive methods.

Effects of anti-TNF-mAb treatment on pregnancy in baboons with induced endometriosis.

OBJECTIVE: Hormonal suppressive therapy is not effective for endometriosis-associated subfertility and can even prevent conception. Medical inhibition of TNFalpha, which has been shown to improve conception, is effective in the prevention and treatment of endometriosis in baboons. DESIGN: Prospective, placebo-controlled fertility trial. SETTING: Animal research and laboratory facility. ANIMAL(S): Sixteen adult female baboons with induced endometriosis. INTERVENTION(S): All animals received a single IV dose of the anti-TNFalpha monoclonal antibody c5N (n = 9) or placebo (n = 7) at four different time points. The animals were then exposed to timed mating up to nine completed cycles or until pregnancy was achieved. MAIN OUTCOME MEASURE(S): Pregnancy rate (PR), cycle fecundity rate (CFR), time to pregnancy (TTP), and cumulative pregnancy rate (CPR). RESULT(S): Inhibition of TNFalpha did not result in a significant improvement in PR (100% c5N vs. 86% placebo), CFR (18% c5N vs. 30% placebo), median TTP (5 cycles c5N vs. 2 cycles placebo), or CPR (100% c5N vs. 80% placebo). The duration of the menstrual cycle was unchanged in both groups before and after the study. Two nonpregnant baboons in the c5N-group died during the study. CONCLUSION(S): Medical inhibition of TNFalpha allowed for normal conception but did not improve fecundity in baboons with induced endometriosis when compared with placebo. Larger studies with clinically available TNFalpha blockers in baboons with moderate to severe endometriosis are needed to further test the potential of these agents in the prevention or treatment of endometriosis-associated subfertility.

Follicle-stimulating hormone receptor polymorphisms in a population of infertile women.

BACKGROUND: There are two known polymorphisms of clinical relevance in the follicle-stimulating hormone (FSH) receptor exon 10, alanine or threonine at position 307, and asparagine or serine at position 680, giving rise to two discrete allelic variants: Thr307/Asn680 and Ala307/Ser680. At position 680, three FSH receptor variants are possible: Asn/Asn, Asn/Ser, and Ser/Ser. We hypothesized an association between FSH receptor polymorphisms and ovarian reserve. METHODS: FSH receptor polymorphisms at position 680 were studied in a population of 68 infertile women. We used serum FSH level at cycle day 3 as a screening for ovarian reserve. DNA was extracted from peripheral leukocytes in whole blood by using PCR and DNA sequencing in order to determine the type of FSH receptor. RESULTS: The distribution of FSH receptor variants was Asn/Asn (AA) 35%, Asn/Ser (AS) 24%, and Ser/Ser (SS) 41%. In women with normal ovarian reserve, FSH levels at cycle day 3 were 5.6 +/- 1.9 (AA group), 6.7 +/- 1.3 (AS group), and 5.7 +/- 1.7 (SS group) (non-significant). Corresponding FSH levels at cycle day 10 were 6.9 +/- 1.9, 6.3 +/- 1.7, and 8.3 +/- 2.8 (P < 0.01, AA and AS vs. SS group). In the SS group, FSH levels at cycle day 10 were significantly higher than they were at cycle day 3 (P < 0.05). CONCLUSIONS: The results show that Ser/Ser-680 predominates in the studied infertile population. Furthermore, women with normal ovarian reserve and the Ser/Ser FSH receptor variant had significantly higher FSH levels, compared to women with Asn/Asn and Asn/Ser variants. FSH receptor genotyping may, thus, be interesting as an adjunct indicator of ovarian reserve for infertile women undergoing assisted reproduction, and may be helpful in the determination of the starting dosage of FSH in in vitro fertilization.

Progesterone withdrawal causes endothelin release from cultured human uterine microvascular endothelial cells.

BACKGROUND: Current theories on the physiology of menstrual bleeding in humans offers an explanation for the shedding of the endometrium as a result of a breakdown of the extracellular matrix due to an inflammatory reaction. The link between the fall in progesterone levels and these events is not clear. Neither has an explanation been presented for the vasoconstriction in the coiled arteries occurring during menses. We have hypothesized a chain of events where the fall in progesterone levels induces an upregulation of the thrombin receptor in the small uterine arteries leading to an increased thrombin response and subsequent endothelin release. METHODS: Endothelial cells from human umbilical cord (HUVECs) and from human small uterine arteries (UtMVECs) were cultured under conditions partly resembling the female hormonal cycle with progesterone withdrawal. RESULTS: Following progesterone increase and subsequent withdrawal, we found an increased production of thrombin receptor and an increased release of endothelin from UtMVECs compared with HUVECs. CONCLUSION: Endothelin release in response to progesterone withdrawal in UtMVECs can offer an explanation for the vasoconstriction seen in the coiled arteries during menses in humans.

Predictive power of clomiphene citrate challenge test for failure of in vitro fertilization treatment.

BACKGROUND: To evaluate the impact of ovarian reserve on the outcome of in vitro fertilization (IVF) treatment in 140 women, in a total of 279 treatment cycles. METHODS: All women underwent a clomiphene citrate (CC) challenge test to assess their ovarian reserve before IVF treatment. One hundred and eighteen women (84%) had normal basal follicle stimulating hormone (FSH) levels (3.1-10.0 IU/l) and 22 women (16%) had elevated FSH levels (> 10.0-24.0 IU/l). The FSH levels measured on cycle day 10 showed that 106 (76%) of the women could be regarded as having a normal ovarian reserve and 34 (24%) a diminished ovarian reserve. RESULTS: In the group with diminished ovarian reserve, pregnancies and live births were dramatically lower than in the group with normal ovarian reserve. Counting only the first cycle (n = 140), the number of ongoing pregnancies and live birth rate were highly different between the two groups: 3% vs. 36% (1/33 vs. 28/78). Counting all treatment cycles (n = 210 + 69) the clinical pregnancy rate in the diminished ovarian reserve group was 6%-31% compared with the normal woman (4/69 compared 65/210). The number of started treatment cycles per woman were similar in the two groups. The length of the ovarian stimulations were slightly longer in the group with elevated FSH compared with the group with normal FSH levels. The number of cancellations resulting from insufficient ovarian response was significantly higher in the group with diminished ovarian reserve (n = 38, 55%) compared with the normal women (n = 32, 15%) (p < 0.0001). In addition, the average E2 levels before oocyte pick up were significantly lower in the group of women with diminished ovarian reserve compared with normal women (p < 0.0001). Calculation of the sensitivity and specificity of the CC test showed that an abnormal test has a high probability for a negative treatment outcome. The number of retrieved, fertilized oocytes, the number of divided oocytes, and the number of embryo transfers in the first as well as in all cycles differed significantly between the two of groups women (p < 0.001-0.009). CONCLUSIONS: We found that the CC challenge test is a useful tool in assessing a woman's ovarian capacity before infertility treatment. The predictive value of the test for a negative outcome of IVF treatment was strong. We recommend performing the test before infertility treatment. This may prevent unnecessary treatment trials and unrealistic expectations from both patients and doctors.

Endothelin-1 and macrophage colony-stimulating factor are co-localized in human amnion membrane cells and secreted into amniotic fluid.

We have examined the cellular localization and human amniotic fluid content of endothelin-1 (ET-1) and macrophage colony-stimulating factor (M-CSF). The study material consisted of amniotic fluid from 20 patients referred for amniocentesis, and placental samples from normal deliveries. ET-1 and M-CSF were analysed by radioimmunoassay and enzyme-linked immunosorbent assay respectively. The cellular localization of ET-1 and M-CSF in the amnion membranes was analysed by double-labelling immunocytochemistry using fluorescein isothiocyanate- and Cy3-labelled secondary antibodies. Release of ET-1 and M-CSF was studied in cultured amniocytes. We found that the mean +/- SD concentrations of ET-1 and M-CSF in fetal amniotic fluid were 45.6 +/- 17.3 pmol/l (range 16.8-85.5) and 7323 +/- 3415 ng/l (range 2640-12 110) respectively. Double-labelling immunocytochemistry showed that both M-CSF and ET-1 were co-localized in the same cells to a high extent. Further analysis revealed that levels of M-CSF, but not ET-1, were significantly correlated with pregnancy length. Both M-CSF and ET-1 were released from cultured amniocytes in response to interleukin-1. These findings show that ET-1 and M-CSF are partly co-localized to specific cells in the human amniotic membrane. As both M-CSF and ET-1 were released from cultured amniocytes in vitro, this suggests that they both may be secreted into fetal amniotic fluid in vivo as well.

Inhibin B predicts oocyte number and the ratio IGF-I/IGFBP-1 may indicate oocyte quality during ovarian hyperstimulation for in vitro fertilization.

PURPOSE: To perform a retrospective analysis of 62 age-matched IVF-treated women in order to investigate whether levels of inhibin B, IGF-I, and IGFBP-1 in serum 2 days before oocyte retrieval and in follicular fluid at the day of oocyte retrieval might be useful as indicators of the ovarian ability to produce oocytes (ovarian reserve). METHODS: Patients were allocated into three groups on the basis of the number of oocytes retrieved. Group 1 ("low responders") had 0-3 oocytes, group 2 ("normal responders") had 6-11 oocytes, and group 3 ("high responders") had 12 oocytes or more. Levels of inhibin B, IGF-I, and IGFBP-1 in follicular fluid and in serum obtained 2 days before oocyte retrieval were analyzed and correlated to clinical parameters including estradiol levels, progesterone levels, follicle size, follicle number, and oocyte number. RESULTS: We found significant differences in inhibin B levels in the three groups. Inhibin B levels in follicular fluid and serum was strongly correlated to the number of oocytes retrieved (p < 0.01). The number of oocytes retrieved were also correlated to total FSH dose (p < 0.05), to estradiol 2 days before and at ovum pick-up (p < 0.05), to progesterone at ovum pick-up (p < 0.0001), to progesterone at embryo transfer (p < 0.05), and to the number of follicles (size 12-15 mm, p < 0.001, size > 15 mm, p < 0.01). Serum inhibin B also correlated to follicular fluid inhibin B (p < 0.01). Inhibin B was not correlated to pregnancy. In contrast, the ratio IGF-I/IGFBP-1 in serum as well as in follicular fluid was significantly higher in women who became pregnant (p < 0.05). CONCLUSIONS: The results show that inhibin B in serum 2 days before oocyte retrieval predicts number of oocytes retrieved. Since inhibin B in serum before oocyte retrieval in ovarian hyperstimulation was strongly predictive of the number of oocytes retrieved, it appears useful as a marker for ovarian response. Inhibin B did not predict treatment outcome, whereas the ratio IGF-I/IGFBP-1 in serum and follicular fluid was significantly higher in women who became pregnant. The ratio IGF-I/IGFBP-1 may thus reflect oocyte quality.

Cyclin-dependent kinase 5 associated with p39 promotes Munc18-1 phosphorylation and Ca(2+)-dependent exocytosis.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine protein kinase that requires association with a regulatory protein, p35 or p39, to form an active enzyme. Munc18-1 plays an essential role in membrane fusion, and its function is regulated by phosphorylation. We report here that both p35 and p39 were expressed in insulin-secreting beta-cells, where they exhibited individual subcellular distributions and associated with membranous organelles of different densities. Overexpression of Cdk5, p35, or p39 showed that Cdk5 and p39 augmented Ca(2+)-induced insulin exocytosis. Suppression of p39 and Cdk5, but not of p35, by antisense oligonucleotides selectively inhibited insulin exocytosis. Transient transfection of primary beta-cells with Munc18-1 templates mutated in potential Cdk5 or PKC phosphorylation sites, in combination with Cdk5 and the different Cdk5 activators, suggested that Cdk5/p39-promoted Ca(2+)-dependent insulin secretion from primary beta-cells by phosphorylating Munc18-1 at a biochemical step immediately prior to vesicle fusion.