Background/Aims: Fetal growth restriction is a complex problem of pregnancy arising from multiple etiologies. Key regulatory elements of growth are the insulin-like growth factor (IGF) axis, and estrogen and progesterone receptors. The aims were to determine the relations of expression of IGF-I, estrogen receptors α and β (ERα and ERβ, respectively), and progesterone receptor (PR), with maternal anthropometry, focusing on birth weight outcomes. Methods: Placental samples were obtained from 33 patients following delivery. mRNA expression was determined by a solution hybridization technique. Samples were divided into normal control (NC) and growth-restricted (GR) groups. Results: IGF-I expression was lower in the GR as compared to the NC group. PR levels correlated positively with IGF-I expression, infant anthropometry, and gestational age (GR). ERα correlated positively with PR expression (NC), and maternal BMI at delivery (GR). ERβ correlated positively with maternal delivery weight and gestational age (NC). Conclusion: The differences in placental expression of IGF-I emphasize its key role in birth weight outcomes. We further suggest the importance of PR expression in the pathogenesis of intrauterine growth restriction, as there were direct correlations of PR expression with both IGF-I expression and infant anthropometric parameters, as well as gestational age.

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.

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.

BACKGROUNDInflammatory cytokines, including interleukin (IL)-1, IL-6, IL-8 and tumour necrosis factor-alpha (TNF-alpha), are important in the pathogenesis of endometriosis. We assessed the efficacy of anti-TNF monoclonal antibody (mAb, c5N), known to prevent induced endometriosis in baboons, in reducing established endometriosis in baboons.METHODSThis prospective, randomized, blinded, controlled study was conducted in baboons at the Institute of Primate Research (IPR), Nairobi, Kenya. Endometriosis was induced in 18 adult female baboons (Papio anubis) with regular menstrual cycles and a normal pelvis; the extent of endometriosis was documented by videolaparoscopy 25 days later. The baboons were then randomly assigned to receive a single infusion of either placebo (n=7, 5 ml/kg) or c5N (n=11, 5 mg/kg). Follow-up laparoscopy was performed 25 days later to document any differences in the number, surface area and estimated volume of lesions between the two groups and between the first and the second laparoscopies in each group. Representative biopsies of at least one endometriotic lesion per baboon were obtained at the final laparoscopy.RESULTSSignificant reductions in total surface area, estimated total volume of endometriotic lesions and both number and surface area of red lesions were observed after treatment with c5N, but not after placebo treatment, when compared to the initial laparoscopy. Conversely, a significant increase in the number of typical and red lesions was observed after placebo treatment when compared to the initial laparoscopy. Neither c5N nor placebo treatment affected the menstrual cycle.CONCLUSIONIn baboons with induced endometriosis, anti-TNF-mAb (c5N) treatment significantly reduced the extent of endometriosis, mainly due to reducing both the number and surface area of red lesions. These findings suggest that anti-TNF-mAb therapy may have therapeutic potential for active peritoneal endometriosis.

In order to study estrogen effects on developing human neurons, we have established primary cultures of neurons and glia from 8-13-week human embryo cortex and spinal cord. The neuronal identity of the cultures was verified using the neuronal synaptic vesicle and neuronal endosomal membrane markers synaptotagmin, synapsin and synaptophysin, and the glial contribution to the mixed glial-neuronal cultures was verified using the glial marker glial fibrillary acidic protein (GFAP). We here report expression of estrogen receptor beta (ERbeta) in these cells using RT-PCR and sequencing, RNAse protection assay, immunohistochemistry and immunoblotting. We found that both neuronal and mixed glial-neuronal cultures expressed ERbeta. Treatment with 17beta-estradiol gave an increased expression of ERbeta in both types of cultures. These results suggest that ERbeta is expressed in fetal brain and thus may mediate effects of estrogen in the developing nervous system. Furthermore, the results suggest that expression of ERbeta in fetal brain may be regulated by estrogen.

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.

BACKGROUND:A prospective, semi-randomized, open, clinical study was conducted to compare ovarian response, pregnancy outcome and delivery rates using two recombinant FSH preparations (Gonal-F and Puregon) for IVF.METHODS:We analysed stimulation parameters and outcome data in 812 initiated treatment cycles where 292 women used Gonal-F and 200 used Puregon. Embryo transfer was carried out in 676 cycles. In the two preparation groups we also compared 170 women previously treated with FSH for IVF with 266 previously untreated.RESULTS:The pregnancy rate with Gonal-F was 26% and with Puregon 28%. Delivery rates were identical, 22%. Clinical pregnancy and delivery rates per cycle with embryo transfer in earlier untreated women were 29.0 and 24.0%, whereas in previously treated women they were 23.5 and 18.8% respectively. After repeated cycles, delivery frequencies consecutively decreased, independent of the FSH preparation used.CONCLUSION:Gonal-F and Puregon seem to be equally potent in achieving follicular development and equally effective, in terms of delivery rates, for use in an IVF programme. Repeated cycles resulted in equally large consecutive decreases in delivery rates, regardless of preparation choice, but were considered worthwhile for up to three stimulation cycles in selected patients.

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.

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.

Objective: Inhibin B has in previous studies been found to be not predictive of IVF treatment outcome, but has appeared to be correlated to ovarian function and to the development of follicles. We hypothesized that inhibin B might be useful as an indicator of ovarian reserve, and thus might be correlated to ovarian response after stimulation. We have therefore performed a retrospective analysis of inhibin B, insulin-like growth factor I (IGF-I) and insulin-like growth factor binding protein 1 (IGFBP-1) in 62 women who underwent ovarian stimulation for IVF in our centre. Design: Retrospective randomized study in a university teaching hospital. Materials/Methods: The study population consisted of 62 age-matched patients treated in our clinic in 1996–1997, and encompasses a total of 62 cycles. Patients were selected into three groups based on 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. Inhibin, IGF-I and IGFBP-1 were analyzed in follicular fluid and in serum obtained two days before oocyte retrieval, and correlated to a number of clinical parameters including estradiol levels, progesterone levels, oocyte number and follicle size and number. Results: Patients were grouped according to number of oocytes (low, normal and high). There was a significant difference in inhibin levels in the three groups, with lower levels correlating to low number of oocytes. Inhibin levels in follicular fluid and in serum was thus strongly correlated to number of oocytes retrieved (p = 0.007, ∗∗). The number of oocytes retrieved were in addition to inhibin also correlated to total FSH dose (p = 0.02, ∗), estradiol 2 days before OPU (p = 0.018, ∗), estradiol at OPU (p = 0.037, ∗∗), progesterone at OPU (p < 0.0001, ∗∗∗), progesterone at ET (p = 0.038,∗) and to number of follicles (12–15 mm diameter) before OPU (p = 0.0027,∗∗). Inhibin in serum also correlated to inhibin in follicular fluid (p = 0.039, ∗∗) and to IGF-1 in follicular fluid (p = 0.0164, ∗). Inhibin was not correlated to pregnancy. Conclusions: The results show that serum inhibin predicts number of oocytes retrieved. Serum inhibin was as expected correlated to follicular fluid inhibin. Inhibin does not predict pregnancy outcome. Inhibin was weakly correlated to IGF-1 in follicular fluid, but neither IGF-I nor IGFBP-1 appeared to be useful as markers of ovarian function or capacity. In contrast, inhibin B in serum before OPU in ovarian hyperstimulation appears to be strongly predictive of the number of oocytes that will be retrieved, and therefore we suggest it is a strong marker for ovarian response. Supported by: This work was supported by MFR 14X-07164 and Karolinska Institutets Fonder.

Objective: The amino acid gamma aminobutyric acid (GABA) acts as an inhibitory neurotransmitter in the CNS. GABA is transported across the plasma membrane into cells by transporter proteins. Four such proteins have been cloned and sequenced (GAT 1-4). A protein different from GAT 1-4, the vesicular GABA transporter (VGAT) has been identified, mediating GABA transport from cytoplasm into storage vesicles called small synaptic vesicles in neurons. Recently, a role for GABA has been proposed in reproductive medicine. Specific GABAergic proteins have been identified in human sperm and oocytes and a possible role for GABA in the initiation of the acrosome reaction has been proposed. We have previously reported the presence of specific GABA transport proteins in human and rat spermatozoa and testis. In this study we have identified VGAT in rat testis by immunoblotting immunohistochemistry and RT-PCR. Design: Experimental design in laboratory setting Materials/Methods: Immunohistochemistry: Male Sprague-Dawley rats were sacrificed and testis were removed and fixed in formalin/picric acid. Fixed testis were rinsed and finally cut for indirect fluorescence. Sections of rat testis and spermatozoa were incubated in rabbit antiserum to VGAT for 18-22 hours. The tissues were then washed and incubated with fluorescin isothiocyanate conjugated donkey anti rabbit secondary antibodies. Sections were examined in a laser scanning confocal imaging system equipped with a krypton/argon mixed gas laser. SDS-PAGE and Immunoblotting: Homogenized samples were denatured in SDS-PAGE sample buffer. Equal amounts of protein were loaded and subjected to analysis on a 7.5% SDS-PAGE gel. Proteins were transferred to nitrocellulose and following blocking, the blots were probed overnight with rabbit anti VGAT. Immunoreactive protein bands were visualized following incubation with peroxidase-conjugated goat-anti-rabbit IgG. Reverse transcriptase PCR: Messenger RNA was prepared from rat testis and the mRNA was transcribed into cDNA using a commercially available kit. Primers used for PCR were designed on basis of sequences for cloned vesicular GABA transport. The validity of the PCR procedure was tested by gel electrophoreses and DNA sequencing. Results: Immunohistochemistry: Confocal images of rat testis sections demonstrated strong immunofluorescence in the cytoplasm of germ cells. Peritubular cells did not exhibit staining. Confocal images of rat sperm cells incubated with antibodies to VGAT demonstrated immunofluorescence in the head region. SDS-PAGE: The testis VGAT protein reacted with the rabbit anti VGAT antibody. The immunoreactivity was observed as a band in the 60 kDa region RT-PCR: With the oligonucleotide primer pair based on the cDNA sequence from the rat VGAT, a PCR product matched the size predicted from the location of the primers. This PCR product was sequenced and found identical to rat VGAT. Conclusions: The results from both immunohistochemistry and immunoblots in this study strongly indicate the presence of the vesicular GABA transporter in rat testis. The identification of mRNA expression of VGAT in rat testis confirms this finding. Several GABAergic proteins have now been identified in testis and spermatozoa, this is suggestive of a role for GABA in male reproductive organs and/or spermatozoa. Supported By: This project was supported by grants from the Swedish Medical Council 14X-07164 and the Karolinska Institutes Research Funds.

We have recently found that values of the transforming growth factor (TGF)beta1 in human ovarian follicular fluid obtained during ovarian stimulation for IVF were higher in women who subsequently became pregnant following embryo transfer. We therefore postulated that TGFbeta1 may have a beneficial effect on the preimplantation embryo and improve the chances of a successful implantation. We have used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry to investigate the presence in human oocytes and preimplantation embryos of the essential components of the TGFbeta signalling pathway, TGFbeta receptors type I and II and the substrate proteins Smad 2 and 3. We found that both receptors, as well as Smad 2 and 3, were present in the unfertilized oocyte, whereas only the type I receptor and Smad 2 and 3 were present at the blastocyst stage. At the 4-cell and 8-cell stages neither of the receptors was present, but Smad 2 and 3 were present at both stages. These findings support our hypothesis that the TGFbeta1 in follicular fluid may interact with the oocyte and preimplantation embryo via TGFbeta receptors, and that TGFbeta signalling may be important for the development of the oocyte and the preimplantation embryo.

Exocytosis is mediated by high-affinity interactions between different SNARE proteins. The existence of several variants of each SNARE protein suggests that the specificity of fusion may be directed by unique combination of SNARE family members. We examined if two alternatively spliced variants of synaptosomal-associated protein of 25 kD, SNAP-25a and SNAP-25b, possessed distinct cellular distribution if coexpressed within the same neuroblastoma cell. Double-labelling immunofluorescence histochemistry in combination with confocal laser microscopy of individual cell clones revealed a different subcellular localisation pattern for the two SNAP-25 variants. Sucrose density gradient centrifugation of cell homogenates followed by Western blotting showed that the SNAP-25 protein variants associated with intracellular organelles of different density. Taken together, this study shows that two alternatively spliced variants of SNAP-25, differing in only nine amino acids, possess distinct properties at the level of intracellular trafficking, suggesting that the cellular localisation of SNAP-25 protein is regulated at the level of mRNA splicing.

The Munc-18 protein (mammalian homologue of theunc-18 gene; also called nSec1 or rbSec1) has been identified as an essential component of the synaptic vesicle fusion protein complex. The cellular and subcellular localization and functional role of Munc-18 protein in pancreatic β-cells was investigated. Subcellular fractionation of insulin-secreting HIT-T15 cells revealed a 67-kDa protein in both cytosol and membrane fractions. Immunohistochemistry showed punctate Munc-18 immunoreactivity in the cytoplasm of rat pancreatic islet cells. Direct double-labeling immunofluorescence histochemistry combined with confocal laser microscopy revealed the presence of Munc-18 immunoreactivity in insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing cells. Syntaxin 1 immunoreactivity was detected in extracts of HIT-T15 cells, which were immunoprecipitated using Munc-18 antiserum, suggesting an intimate association of Munc-18 with syntaxin 1. Administration of Munc-18 peptide or Munc-18 antiserum to streptolysin O-permeabilized HIT-T15 cells resulted in significantly increased insulin release, but did not have any significant effect on voltage-gated Ca2+ channel activity. The findings taken together show that the Munc-18 protein is present in insulin-secreting β-cells and implicate Munc-18 as a negative regulator of the insulin secretory machinery via a mechanism that does not involve syntaxin-associated Ca2+ channels.

BACKGROUND:We have examined whether endothelin-1 (ET-1) and erythropoietin (EPO) in amniotic fluid, and EPO in fetal serum obtained by cordocentesis from fetuses with signs of intrauterine growth retardation (IUGR), were correlated to fetal growth and/or chronic fetal hypoxia. METHODS:Amniotic fluid and fetal serum were obtained by cordocentesis from 28 fetuses suspected to have IUGR and subsequently analyzed for EPO and ET-1 by ELISA. These data were correlated to blood gas results and fetal/maternal parameters at delivery. RESULTS:A novel finding was that ET-1 correlated to PO2 in amniotic fluid. The average level of ET-1 in amniotic fluid was 48.3+/-4.7 pmol/L. The results also showed a correlation between EPO levels in amniotic fluid and EPO in fetal serum. Furthermore, EPO correlated weakly to birth weight at delivery. Children with the lowest birth weights had the highest EPO levels. High EPO values, similarly to ET-1, correlated to low pO2 values. The level of EPO in amniotic fluid was 8.0+/-1.6 mIU/ml and in cord blood 29.5+/-9.6 mIU/ml. CONCLUSIONS:The results indicate that ET-1 levels may be a marker for short-term hypoxia, but not for fetal growth, since ET-1 in amniotic fluid was correlated to PO2 at the time of cordocentesis, but not to birth weight. The results also indicate that EPO levels in amniotic fluid and in fetal cord serum are highly correlated, and thus both can be used as markers for fetal growth and chronic hypoxia before the onset of labor.

Pre-eclampsia is one of the major contributors to perinatal morbidity. This study was performed to test a hypothesis which suggests that pre-eclampsia is associated with inadequate control by the thioredoxin system and other related reducing systems. Placental tissue from normal pregnancies (NC), severe pre-eclampsia with fetuses small for gestational age (SPE), mild pre-eclampsia with fetuses small for gestational age (MPE) and pregnancies with small fetuses for gestational age without any sign of pre-eclampsia (IUGR) was collected immediately after delivery. The mRNA levels for thioredoxin and glutaredoxin were determined using a solution hybridization method and the distribution of the proteins in a normal placenta was analysed by immunohistochemistry. Results showed that the thioredoxin mRNA level in the SPE group was decreased to one third of the level in the NC group. Also the IUGR group showed a significant decrease. The glutaredoxin mRNA level in the SPE group was one half of that seen in the NC group. There was significant correlation between the mRNA levels for thioredoxin and glutaredoxin, both in the normal and growth restricted pregnancies. We conclude that the thioredoxin and glutaredoxin reducing systems are affected in placenta from pregnancies with pre-eclampsia and/or growth restriction of fetuses, and that the decrease correlates to the severity of the condition.

Synaptotagmin is involved in Ca2+-regulated secretion and has been suggested to serve as a general Ca2+ sensor on the membrane of secretory vesicles in neuronal cells. Insulin exocytosis from the pancreatic beta-cell is an example of a Ca2+-dependent secretory process. Previous studies of pancreatic beta-cells were unable to show presence of synaptotagmin I. We now present biochemical and immunohistochemical data showing that synaptotagmin III is present in pancreatic beta-cells as well as in the insulin-secreting cell line HIT-T15 and in rat insulinoma. By subcellular fractionation, we found synaptotagmin III in high-density fractions together with insulin and secretogranin I, indicating colocalization of synaptotagmin III and insulin in secretory granules. We could also show that blockade of synaptotagmin III by a specific antibody inhibited Ca2+-induced changes in beta-cell membrane capacitance, suggesting that synaptotagmin III is part of the functional protein complex regulating beta-cell exocytosis. The synaptotagmin III antibody did not affect the activity of the voltage-gated L-type Ca2+-channel. These findings are compatible with the view that synaptotagmin III, because of its distinct localization in the pancreatic beta-cell, functionally modulates insulin exocytosis. This indicates that synaptotagmin may have a general role in the regulation of exocytosis not only in neuronal cells but also in endocrine cells.

The objective of this prospective study was to compare the outcome of ovarian hyperstimulation for in-vitro fertilization (IVF) using two different preparations of recombinant follicle stimulating hormone (FSH). The study was based on 296 consecutive IVF cycles in 1997, 199 performed using follitropin alpha (Gonal-F) and 97 performed using follitropin beta (Puregon). Outcome was compared regarding pregnancy rate, oestradiol and progesterone response, endometrial thickness, follicle number, number of retrieved oocytes, fertilized oocytes, sperm count and sperm motility. There was no significant difference in outcome of stimulation. Clinical pregnancy rate was similar, 29.1% for Gonal-F and 28.1% for Puregon. There was no difference in endometrial response, oestradiol response, number of smaller (12-15 mm) or larger (>15 mm) follicles, number of oocytes retrieved, fertilized, divided and replaced, in sperm counts or in sperm progressive motility. There was a lower follicle number in the Puregon group, but not statistically significant. The serum progesterone concentrations on the day of oocyte retrieval, however, were significantly lower in the Puregon group. In conclusion, it was not possible to find significant differences in the IVF programme with regard to stimulation outcome between Gonal-F and Puregon. The results of this study indicate that Gonal-F and Puregon may be equally suitable for use in ovarian stimulation for IVF.

Objective: To determine the effects of hMG and highly purified FSH on follicular production of the ovarian growth factors transforming growth factor-beta(1) (TGF-beta(1)), insulin-like growth factors I and II (IGF-I and IGF-II), and insulin-like growth factor binding proteins-1 and -3 (IGFBP-1 and IGFBP-3).Design: Controlled clinical study.Setting: University IVF program.Patient(s): One hundred twenty women who were <38 years old and had a >3-year duration of infertility in their present relationship participated in the study.Intervention(s): Follicular fluid and matched serum were collected at oocyte pick-up and analyzed for growth factors and E-2 with the use of ELISA and RIA.Main Outcome Measure(s): Levels of TGF-beta(1), IGF-I, IGF-II, IGFBP-1, and IGFBP-3 in follicular fluid and levels of E-2 in serum were measured.Result(s): Compared with highly purified FSH, ovarian hyperstimulation with hMG produced lower levels of TGF-beta(1) and IGF-I and higher levels of IGFBP-1. Levels of IGF-II and IGFBP-3 were similar with the 2 treatments.Conclusion(s): In patients undergoing IVF, the follicular expression of TGF-beta(1), IGF-I, and IGFBP-1 was regulated differently by highly purified FSH compared with a preparation containing FSH and LH in a 1:1 ratio (hMG). The results indicate that FSH and LH control ovarian production of these growth factors differentially. (Fertil Steril(R) 1998;70:129-34 (C) 1998 by American Society for Reproductive Medicine.).

The neuropeptides enkephalin (ENK), galanin (GAL) and neuropeptide Y (NPY) are abundantly expressed in the paraaortic body (PAB) and adrenal glands of the newborn rabbit. To examine whether these neuropeptides are affected by acute stress, we exposed neonatal rabbits to asphyxia, insulin-induced hypoglycemia, and reserpine. Asphyxia, caused by rebreathing for 60 min in an airtight box, reduced the content of catecholamines (CAs) in the adrenal glands and increased ENK-like immunoreactivity (-LI) in the PAB. Insulin-induced hypoglycemia reduced the content of CAs as well as ENK-LI in the adrenal glands. Reserpine caused a marked depletion of the CAs both in the PAB and in the adrenal glands. In contrast, reserpine did not cause any change in the contents of the neuropeptides in either organ. These data indicate that tissue levels of the neuropeptides GAL-LI and NPY-LI, coexisting with CA in the PAB and the adrenal glands, are not biochemically affected by asphyxia, hypoglycemia or reserpine, whereas tissue levels of ENK-LI are reduced by hypoglycemia and, to some extent, are increased by asphyxia. Furthermore, even the CAs in the PAB were unaffected by asphyxia and hypoglycemia. Also, while reserpine reduces CA content, peptide levels are unaffected.