Definition and measurement of follicle stimulating hormone.

FSH has a key role in the development and function of the reproductive system and is widely used both diagnostically and therapeutically in developmental and reproductive medicine. The accurate measurement of FSH levels, in patients for diagnosis and monitoring and in therapeutic preparations for clinical use, is essential for safe and successful treatment. Historically, FSH was defined on the basis of classical in vivo endocrine activity, and early therapeutic preparations were calibrated using in vivo bioassays. There was early recognition that reference preparations were required for calibration if the results from different laboratories were to be comparable. In response to the perceived need, the World Health Organization established the first standard for such preparations in 1959. Subsequent developments in biotechnology have led to recognition that there is no single molecule that can be uniquely defined as FSH, and that FSH can induce a range of biological activities. Several highly purified standards for FSH are now available, but discontinuity and heterogeneity of estimates of FSH activity in terms of these standards made using in vitro assays and binding assays have been noted. It is thus essential that any measurement of FSH include specification both of the standard with which the measured FSH is compared and the assay method used for that comparison.

Characterisation, calibration and comparison by international collaborative study of international standards for the calibration of therapeutic preparations of FSH.

Therapeutic preparations of FSH, used primarily for treatment of infertility, are calibrated by in vivo bioassay against international standards (IS) derived from different sources deemed appropriate to their use according to pharmacopoeial monographs. Menotrophins, which have been used for several decades to treat infertility, have been calibrated against the IS for urinary FSH and LH (ISU) but are now being replaced by highly purified urinary FSH or rDNA-derived FSH (rFSH). The aim of this study was to evaluate two preparations of human rFSH and one preparation of highly purified urinary FSH as candidate WHO IS for bioassay in an international collaborative study by 27 laboratories in 12 countries, and to characterise them in a range of in vitro bioassays and immunoassays. The biological activity of the three candidate standards was confirmed by all laboratories using all assays contributed to the study. Dose-response relationships by in vivo bioassay for any of the candidate standards did not differ significantly from that for the ISU. Dose-response relationships obtained in in vitro bioassays and immunoassays were also broadly similar among these preparations although dose-response lines for some preparations appeared to be non-parallel in some immunoassays. For each of the three candidate IS, estimates of the relative potency in terms of ISU by in vivo bioassay did not differ significantly between laboratories. In contrast estimates by immunoassays and in vitro bioassays showed significant differences between laboratories. Estimates of relative potency of the highly purified candidate IS materials in terms of one another exhibited less inter-laboratory variability than estimates in term of ISU. Each of the candidate standards showed adequate stability to serve as an IS. On the basis of the results of this study rFSH (code 92/642) was established as the first IS for FSH, human, recombinant for bioassay with an assigned unitage of 138 IU per ampoule and urinary FSH (code 92/512) was established as the first IS for FSH, human, urinary (urofollitropin) for bioassay with an assigned unitage of 121 IU per ampoule, based on their respective calibration by in vivo bioassay in terms of ISU. These assignments of unitage maintain continuity of unitage for preparations in therapeutic use and also appear to be consistent with one another.

Follicle stimulating hormone international standards and reference preparations for the calibration of immunoassays and bioassays.

Follicle stimulating hormone is a dimeric glycoprotein hormone which is used widely in reproductive and developmental medicine both as a diagnostic analyte and as a therapeutic product. It is therefore a good example of a clinically important heterogeneous material for which a number of different assay methodologies have been developed. Immunoassays for follicle stimulating hormone (FSH) are used in the diagnosis of disorders of reproduction and development, whereas in vivo bioassays are used for calibration of therapeutic preparations. Different immunoassay systems, based on different formats, exhibit variability in their estimates of activity of FSH which arises from different specificities of antibodies for different forms of FSH which are encountered. In order to minimise between assay variation and to enable better between laboratory comparisons the World Health Organisation (WHO) has issued a series of ampouled preparations of FSH. The availability of these materials has been reviewed previously but on the completion of a recent collaborative study to evaluate candidate standards for rDNA-derived FSH and highly purified urinary FSH (urofollitropin) it is now appropriate to review the current status of these standards and to discuss the future of standardisation for FSH in particular and where appropriate to make reference to other materials.

Toward a standard experiment for studying post-treatment return of fear.

Fear sometimes returns after successful fear attenuation via in vivo exposure to fear signals. Post-treatment return of fear is of considerable interest both practically and theoretically, but factors associated with return of fear are poorly understood due to conflicting results from procedurally diverse experiments. This paper reports two very similar experiments in which fear of animal specimens was weakened then allowed to return so that factors associated with return of fear could be studied. In each experiment attentional focus versus distraction during exposure served as a between-subjects independent variable. In each case, attempts also were made to predict return of fear via several nonmanipulated variables: initial fear, initial avoidance during voluntary exposure, initial heart rate during voluntary exposure, and speed of fear reduction during repeated exposure trials. With the sample sizes used there was only suggestive evidence that return of fear was associated with distraction during exposure, and with relatively rapid fear decline during exposure. More importantly, the experiments are offered as standard, replicable models for research that will permit procedurally homogeneous investigations of variables with which return of fear is associated.

Serological diagnosis of ovine enzootic abortion by comparative inclusion immunofluorescence assay, recombinant lipopolysaccharide enzyme-linked immunosorbent assay, and complement fixation test.

Since the 1950s, serological diagnosis of ovine enzootic abortion (OEA), caused by strains of Chlamydia psittaci, has been based mainly on the complement fixation test (CFT), which is neither particularly sensitive nor specific since antibodies to other chlamydial and enterobacterial pathogens may be detected. In this study. a recombinant enzyme-linked immunosorbent assay (rELISA) (medac, Hamburg, Germany), based on a unique chlamydial genus-specific epitope of Chlamydia trachomatis L2 lipopolysaccharide, was evaluated for sensitivity and specificity as a primary screening assay for OEA by comparison with the CFT. A comparative inclusion immunofluorescence assay (IFA), in which antibody titers to C. psittaci and Chlamydia pecorum were examined, was used as the reference test for 573 serum samples from four flocks. Reactivity to C. pecorum was measured since inapparent intestinal infections by C. pecorum are believed to be common in British flocks. In detecting positive sera from an abortion-affected flock, in which a C. pecorum infection was also suggested by IFA, the rELISA outperformed the CFT with significant evidence for increased sensitivity (P = 0.003). In two flocks in which C. pecorum infections alone were suggested by IFA, the rELISA and CFT were prone to detect low levels of false-positive results, but the values were not significant. The rELISA provided results in one flock in which sera that were anticomplementary could not be resolved by the CFT. In another flock in which abortion had not occurred but infection by both chlamydial species was suspected, no significant difference was found between the sensitivities of the rELISA and CFT. The rELISA could not differentiate ovine C. psittaci and C. pecorum infections but was shown to be a more sensitive primary screening test for OEA than was the CFT, particularly where abortion had occurred and even when antibodies due to additional inapparent infection(s) by C. pecorum were present.

International collaborative study by in vitro bioassays and immunoassays of the First International Standard for Inhibin, Human Recombinant.

The First International Standard for Inhibin, Human Recombinant, (ISI), a lyophilized preparation of rDNA-derived human 32 kDa Inhibin A in ampoules coded 91/624, was evaluated by international collaborative study for its suitability to serve as an International Standard. This study, which involved 15 laboratories in nine countries, included a variety of in vitro bioassays and immunoassays. The ISI was compared with two other lyophilized preparations of human recombinant inhibin, the International Standard for Porcine inhibin (ISP) and preparations of human follicular fluid inhibin. Predicted loss of activity based on estimates of potency of contents of ampoules which had been stored under conditions of accelerated thermal degradation indicated that the ISI has satisfactory stability. On the basis of the results of this study, the ISI was deemed suitable to serve as a standard for in vitro bioassays and immunoassays and was established by the Expert Committee on Biological Standardization of the World Health Organization as the First International Standard for inhibin, recombinant human, with an assigned unitage of 150,000 International Units per ampoule. This unitage maintains an approximate continuity of units with the ISP.

Effects of relaxation training on fear and arousal during in vivo exposure to a caged snake among DSM-III-R simple (snake) phobics.

Eight pairs of DSM-III-R snake phobic subjects (Ss) were exposed to a caged snake while seated in front of a package-conveyor apparatus during eight 4-minute trials. Heart rates and skin-conductance levels were recorded before and during each of the eight trials. Self-reports of fear were obtained after each trial. One S in each pair controlled the conveyor on alternating trials. One subject (S) in each pair had received a representative regimen of relaxation training beforehand. Heart-rate decreased more in Ss controlling the conveyor than in their yoked partners. Ss who had received relaxation training showed lower heart-rate change, lower skin-conductance change, and lower self-reports of fear after the exposure trials. Relaxed Ss also moved the snake closer to themselves than did unrelaxed subjects on some trials.

Control and attention during exposure influence arousal and fear among insect phobics.

Heart beats, skin conductance, and subjective fear levels were recorded among eight pairs of DSM-III-R spider-phobic subjects (Experiment 1) and among eight pairs of DSM-III-R cockroach-phobic subjects (Experiment 2) who were exposed simultaneously to an approaching specimen during eight 4-minute trials. Control over the approach of the specimen alternated between subjects over trials. On different trials, both subjects were instructed either to attend closely to the features of the specimen or to attend closely to their bodily fear reactions. Among spider-phobic subjects (Experiment 1), Self-Control over the specimen produced higher skin conductance during exposure than did Partner-Control over the specimen; instructions to attend closely to the features of the specimen produced higher skin-conductance than did instructions to attend closely to one's bodily fear reactions. Among cockroach-phobic subjects (Experiment 2), Self-Control over the specimen produced higher skin conductance and higher self-reported fear than did Partner-Control over the specimen during the early exposures. Instructions to attend closely to the specimen produced higher skin conductance and higher self-reported fear throughout the experiment and higher heart rates early during the experiment than did instructions to attend to one's bodily reactions. Empirical generalizations based on these data are intended as contributions toward a fund of experimental information that, in due course, will be used to conceptualize the means by which exposure to feared stimuli leads to fear reduction.

Effects of oestrogen on progesterone synthesis and arachidonic acid metabolism in human luteal cells.

OBJECTIVE: Locally produced oestrogens and prostaglandins (PGs) are implicated in the regulation of luteal lifespan in the human ovary. This study (1) assesses direct effects of these factors on progesterone synthesis in isolated luteal cells, and (2) explores interactions between luteal age and treatment with gonadotrophin or oestrogen on the metabolism of arachidonic acid (prostaglandin precursor) by steroidogenic luteal cells in vitro. DESIGN: Primary monolayer cultures of human luteal cells obtained at different stages of the luteal phase were used to investigate the effect of oestradiol, catechol oestrogens (2- and 4-hydroxyoestradiol), diethylstilboestrol, PGE2 and PGF2 alpha on basal and human chorionic gonadotrophin (hCG) stimulated progesterone production in vitro. The role of PGs as modulators of luteal cell function was further investigated by studying the metabolic fate of radioactively labelled arachidonic acid in hormone treated (oestradiol and hCG) and control cultures, assessed by high performance liquid chromatography. PATIENTS: Corpora lutea were enucleated from nine women with regular ovulatory cycles undergoing microsurgical reversal of tubal sterilization. Granulosa cell aspirates were obtained from three patients undergoing in-vitro fertilization treatment. RESULTS: PGE2 and PGF2 alpha at various concentrations did not have a consistent effect, whereas oestradiol, diethylstilboestrol (and 2-hydroxyoestradiol in early luteal cell cultures) significantly inhibited basal and hCG stimulated progesterone biosynthesis. Evidence for direct inhibition of 3 beta-hydroxysteroid dehydrogenase enzymic activity by oestradiol was obtained. Both major metabolic pathways of arachidonic acid (lipoxygenase and cyclo-oxygenase) were operative in steroidogenic luteal cells recovered throughout the luteal phase. The ratio of PGE2 to PGF2 alpha synthesis in vitro by human luteal cells from endogenously incorporated arachidonic acid did not change significantly with corpus luteum age, with PGE2 tending to predominate. Oestradiol treatment shifted arachidonic acid metabolism from the lipoxygenase towards the cyclooxygenase pathway in cells isolated from ageing corpora lutea. CONCLUSIONS: Oestradiol, at relatively high concentrations, is a potent inhibitor of basal and hCG induced luteal cell steroidogenesis in vitro. No support is provided for the concept that luteolysis is mediated by local production of PGF2 alpha. The putative luteolytic effect of oestradiol may entail reduced metabolism of arachidonic acid to lipoxygenase derived products by luteal cells rather than direct stimulation of prostaglandin production by itself.

Direct effect of arachidonic acid on protein kinase C and LH-stimulated steroidogenesis in rat Leydig cells; evidence for tonic inhibitory control of steroidogenesis by protein kinase C.

The role of arachidonic acid in the regulation of steroidogenesis in rat Leydig cells was studied. A dose- and time-dependent biphasic effect on maximal and submaximal LH- and dibutyryl-cAMP-stimulated testosterone production was found. The locus of the inhibition, which occurred during 3 h incubation, was prior to the side chain cleavage of cholesterol and after cAMP production. The same inhibitory effect was found with the protein kinase C (PKC) activators, phorbol-12-myristate, 13-acetate (PMA) and oleic acid, also with no change in LH-stimulated cAMP production. Arachidonic acid, PMA, and diolein, all stimulated PKC activity in a dose-dependent fashion in partially purified Leydig cell homogenates. When the cells were incubated for 5 h, arachidonic acid potentiated LH- and dibutyryl-cAMP-stimulated testosterone production. Similarly, incubation with PMA for 5 h, potentiated subsequent basal and dibutyryl-cAMP-stimulated testosterone production. PKC was down-regulated over 5 h (but not during 3 h) by pretreating Leydig cells with PMA or arachidonic acid in the presence of LH. Lipoxygenase and cyclooxygenase inhibitors did not alter the stimulatory effects of arachidonic acid. We conclude that the short-term inhibitory effect of arachidonic acid (and PMA) is via activation of PKC, but when protein kinase C (PKC) is down-regulated by these ligands, steroidogenesis is enhanced. These results suggest that steroidogenesis is normally under tonic inhibitory control by PKC.

Induction of lutropin receptors by lutropin and cyclic AMP in cultured mouse tumour (MA10) Leydig cells.

The role of cyclic AMP in the regulation of lutropin (luteinizing hormone, LH) receptors has been investigated in cultured mouse tumour (MA10) Leydig cells. The LH receptors were quantified by measuring the binding of 125I-labelled human chorionic gonadotropin (hCG). LH (0.03 nM) in the presence of 1 mM-dibutyryl-cyclic AMP [(Bu)2cAMP] caused a 3-8-fold increase in subsequent 125I-hCG binding. (Bu)2cAMP (1 mM), cholera toxin (11.9 nM) and forskolin (1 microM) each caused a 2-4-fold increase in binding. In the presence of translation (cycloheximide) and transcription (actinomycin D) inhibitors, there was a loss of detectable binding sites. (Bu)2cAMP increased the rate of recovery of binding sites after trypsin treatment of MA10 cells, with a concomitant 2-fold increase in the level of binding sites. Under conditions where receptor levels were increased by 3-8-fold there was also a significant increase in pregnenolone production. It is concluded that LH and cyclic AMP have positive regulatory effects on LH receptors in MA10 cells by inducing the synthesis of new receptors. These induced receptors are functionally coupled to steroidogenesis.

Pathways of arachidonic acid metabolism in human amnion cells at term.

We have compared the metabolism of (3H) arachidonic acid by monolayers of human amnion, cells obtained prior to or following labor at term. Radiolabel was either added exogenously or previously incorporated into cellular phospholipid pools to compare metabolism of arachidonic acid from different substrate sources. Cells obtained both prior to and following labor synthesized metabolites co-chromatographing on HPLC with di- and mono-HETEs and also a metabolite with polarity corresponding to a epoxyeicosatrienoic acid. Both types of cells were able to synthesize PGE2 when (3H) arachidonic acid was added exogenously. However, only those cells obtained following labor synthesized PGE2 from (3H) arachidonic acid incorporated into intracellular pools. These findings suggest that the cyclooxygenase and PGE2 isomerase enzymes are present in amnion prior to delivery but that exogenous arachidonic acid would be required for PGE2 synthesis at that time as the enzymes do not appear to be linked to a source of endogenous arachidonic acid. At the time of parturition, there may be a switching on of an enzyme system to generate arachidonic acid from intracellular pools specifically for PGE2 synthesis or alternatively coupling of such a system to a cyclooxygenase-PGE2 isomerase system resulting in PGE2 synthesis. These findings raise intriguing new possibilities for the regulation of eicosanoid synthesis in amnion which may include membrane topography, substrate pool-enzyme linking and regulation of specific phospholipase enzymes.

Mechanisms of hormone-induced desensitization of adenylate cyclase.

Luteinizing hormone (LH) interacts with its plasma membrane receptor to activate the formation of cyclic AMP via the regulatory GTP binding protein (Gs). This is followed by a desensitization of that same hormonal response which is caused by an uncoupling of the LH receptor from Gs. The coupling between Gs and the adenylate cyclase catalytic unit remains intact. Treatment of Leydig and other cell types with phorbol esters mimics hormone-induced desensitization. However, differences between hormone- and phorbol ester-induced desensitization have been found. In testis Leydig cells phorbol esters, as well as uncoupling the LH receptor from Gs, also inactivates the subunit of the inhibitory GTP binding protein (Gi). These studies suggested that activation of protein kinase may be involved in the hormone-induced desensitization of adenylate cyclase. Paradoxically, it has also been found that two inhibitors of protein kinase C, sphingosine and psychosine also inhibited LH-induced cyclic AMP production. These effects were mainly found during the initial stimulatory period with LH. It is suggested that activation of adenylate cyclase may require a protein kinase C-mediated phosphorylation step which is followed by further phosphorylation resulting in uncoupling of the receptor from Gs. No direct stimulation of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), diacylglycerol and/or activation of protein kinase C by LH in Leydig cells has been demonstrated. An alternative mechanism of protein kinase C activation has been proposed for brain cells, i.e. that involving arachidonic acid activation of protein kinase C instead of diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

Preterm labor: stimulation of arachidonic acid metabolism in human amnion cells by bacterial products.

There is a strong association between preterm labor and infection. Some potentially pathogenic bacteria have phospholipase activity, and it has been suggested that release of phospholipase from these organisms may increase prostaglandin E2 synthesis in amnion cells and hence initiate preterm labor. In this study we established monolayer amnion cell cultures from tissue collected at elective cesarean section at term before labor. Cells were prelabeled with tritiated arachidonic acid and then further incubated after addition of 2%, 5%, or 10% (vol/vol) filtered medium in which either group B beta-hemolytic streptococcus, Streptococcus viridans, Escherichia coli, Bacteroides fragilis, or Lactobacillus had been growing. Tritiated arachidonic acid and its metabolites released by the amnion cells in these or control incubates were extracted from culture medium and separated by high-performance liquid chromatography. Addition of conditioned medium from each of the organisms with the exception of Lactobacillus caused an increase in overall arachidonic acid metabolism. There was an increase in the ratio of cyclooxygenase to lipoxgenase metabolism and in prostaglandin E2 production in particular when compared to controls. The profile of arachidonic acid metabolism in amnion cells following addition of filtered bacterial medium resembled that obtained from amnion cells cultured following spontaneous labor. We suggest that abnormal bacterial colonization of the genital tract may lead to an increase in arachidonic acid metabolism in amnion cells with an increase in prostaglandin E2 production and the consequent initiation of preterm labor.

Antagonism by ETYA of the effects of leukotrienes on ileum and lung parenchymal strips independent of effects on arachidonic acid metabolism.

5,8,11,14-Eicosatetraynoic acid (ETYA), a compound which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism, antagonized the contraction of segments of guinea-pig ileal longitudinal muscle produced by SRS-A (IC50 = 2.73 microM). This activity was unaffected by pretreatment of the tissues with 10 microM indomethacin. Phenidone, another mixed cyclooxygenase-lipoxygenase inhibitor, was inactive. FPL-55712, an SRS-A antagonist, was a very potent inhibitor (IC50 = 0.011 microM). BW755C and NDGA nonselectively inhibited the contractions of the guinea-pig ileal longitudinal muscle induced by SRS-A or histamine. ETYA antagonized the contraction of the guinea-pig ileal strip produced by 6 nM synthetic LTC4 (IC50 = 9.3 microM). FPL-55712 demonstrated an IC50 of 0.3 microM in a similar series of experiments. ETYA, 1, 3 or 10 microM did not inhibit the contractions elicited by 0.5 microM of histamine. This was not a tissue-selective effect since 100 microM ETYA antagonized the LTC4-induced contraction of the guinea-pig lung parenchymal strip preparation. These data demonstrate that ETYA antagonized the contractile effect of the leukotrienes on tissues from the gastrointestinal tract and lung. Furthermore, the inability of indomethacin or phenidone to inhibit the contractile response suggests that antagonism by ETYA may occur by a mechanism independent of cyclooxygenase and lipoxygenase enzymes.

The clinical use of a tubular compression bandage, Tubigrip, for burn-scar therapy: a critical analysis.

Control of burn-scar hypertrophy remains a priority in the care of the burn patient. However, because of the problems associated with traditional compression therapy methods a study of the clinical utility of a tubular compressive bandage (TCB) was initiated. The clinical effectiveness of TCB was determined by studying 210 separate anatomic burn sites in 88 burn patients with a mean age of 25 years and a mean burn size of 21 per cent of the total body surface area (TBSA). To facilitate analysis of the results the patients were divided into two groups, the first group consisted of 71 patients who received prophylactic pressure therapy and a second group of 17 patients who received therapeutic pressure therapy after the establishment of hypertrophic scars. Mean follow-up of the entire group of patients was 11 months. The anatomic area involved by the burn was the most important factor in determining the effectiveness of TCB in this study. Failures primarily occurred at sites of mobility where pressure could not be consistently delivered or maintained, including the digits of the hand, axilla, groin and the head/neck regions. Overall, 85 per cent of the anatomic sites treated had good or satisfactory results. Based on the results of this study, we use TCB on all burn patients, who are at risk of developing burn-scar hypertrophy, immediately after the burn wound has healed or been surgically closed.

Hypertrophic burn scars: analysis of variables.

A major problem in patients surviving thermal injury is the development of hypertrophic burn scars. The current study was performed to determine the factors associated with an increased risk of the development of hypertrophic burn scars. Fifty-nine children (mean age, 3 years; mean TBSA, 14%) and 41 adults (mean age, 37; mean TBSA, 21%) followed from 9 to 18 months formed the study group. The location as well as time required for the burns to heal were recorded in addition to the age and race of the patients. Sixty-three (26%) of the 245 burn areas, in these 100 patients, became hypertrophic. No correlation between patient age and the development of wound problems was found. Blacks had more wound problems than others, if the burn wound took longer than 10 to 14 days to heal. The most important indicator of whether wound problems would occur, in our series, was the time required for the burn to heal. If the burn wound healed between 14 and 21 days then one third of the anatomic sites became hypertrophic; if the burn wound healed after 21 days then 78% of the burn sites developed hypertrophic scars. Based upon these results we have developed a selective, individualized protocol for the use of prophylactic pressure therapy in patients with spontaneously healing burn wounds.