Effects of urinary gonadotrophin preparations on human in-vitro immune function.

Among commercially available urinary human menopausal gonadotrophin (HMG) material, gonadotrophins comprise <5% of the total protein content. Thus, during a typical ovarian stimulation cycle with HMG, several milligrams of non-relevant proteins are administered that may lead to unwanted side effects, including allergic or other hypersensitivity reactions. The effects of two recombinant and four urinary gonadotrophin preparations of different purity upon the function of T cells from healthy blood donors were studied. Only one of the HMG preparations significantly enhanced the spontaneous proliferation of peripheral blood mononuclear cells. Phytohaemagglutinin-induced proliferation was not modified by any preparation, while two preparations significantly increased proliferation in the mixed lymphocyte reaction. Three of the HMG preparations induced the release of interleukin (IL)-1. Highly purified FSH, either urinary or recombinant, showed no effect. None of the preparations induced detectable IL-2 production, whereas only one HMG preparation tended to decrease IL-2 secretion. No major changes in CD25 expression were induced by any of the gonadotrophins. Cytokine measurement by immunoassays detected only IL-1beta in two commercially available preparations. The various effects exhibited by the crude urinary preparations were not a result of the gonadotrophin content and differed from product to product, suggesting that the contaminants present in these preparations are not identical. This could contribute to unpredictable clinical manifestations of allergic or other immune reactions.

Localization of human follicle-stimulating hormone in the testis.

Localization of the follicle-stimulating hormone (FSH) molecule and its receptor (FSHR), as well as the role of FSH in Sertoli cell mitosis and maturation, has been demonstrated by several investigators in human and murine testis by detecting the localization of anti-FSH antibodies or [(131)I]-labeled FSH and by detecting FSH receptor (FSHR) mRNA by in situ hybridization, or FSHR by anti-FSHR antibodies. The presence of FSH in germinal cells is controversial or, in humans, excluded. We have investigated the distribution of the human FSH molecule and its receptor in human and mouse testicular cells under different experimental conditions, at the submicroscopical level, by using a better antigenicity conservative procedure. Thus, the distribution of FSH and of the messenger RNA for its receptor in Sertoli cells has now been clarified. In germinal cells, our observations demonstrate the presence of FSH and the FSHR mRNA: the first on the plasma membrane and in endocytotic vesicles, and the second scattered in the cytoplasm. The cells presenting the higher amount of positivity ranged from spermatogonia to spermatocytes, including round spermatids. Penetration was by the endocytosis via membrane vesicles in which the FSHR is present, whereas its messenger is largely present in the cytoplasm and is responsible for the binding and subsequent internalization of the FSH molecule. As a control, human FSH was administered in vitro to the Y1 mouse cell line, which was stably transfected with cDNA for FSHR and devoid of endogenous FSH. The FSH molecule has been localized by monoclonal antibodies on plasma membranes and vesicles, and the FSHR mRNA was found scattered in the cytoplasm after in situ hybridization. We can now conclude that FSH is present in Sertoli cells and in round germinal cells, both expressing the FSHR. FSH penetrates in a similar way in both kinds of cells via endocytosis, and is therefore subsequently localized in the same membranous organelles.

Composition of commercial gonadotrophin preparations extracted from human post-menopausal urine: characterization of non-gonadotrophin proteins.

Gonadotrophin preparations extracted from post-menopausal urine are of low purity and the major protein components are not gonadotrophins. A study was undertaken to identify some of these non-gonadotrophin proteins present in the extracted human urinary gonadotrophin preparations that are commercially available, i.e. Humegon (Organon), HMG Massone (Massone), Metrodin (Serono), Metrodin HP (Serono), Pergonal (Serono) and Progonadyl (Elea). As revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining and Western blotting analysis, these products had electrophoretic protein profiles which differed in the amounts and species of proteins present. With the exception of Metrodin HP, all the other preparations tested contained tumour necrosis factor binding protein-I, transferrin, and immunoglobulin-related proteins. Some of the products contained in addition: urokinase, Tamm-Horsfall glycoprotein and epidermal growth factor. Recently, a highly purified human urinary follicle stimulating hormone (FSH) preparation (Metrodin HP) became available. In this preparation human FSH represents > 95% of the total proteins (approximately 10,000 IU of FSH/mg of protein). Metrodin HP was demonstrated to be the purest preparation tested, with none of the above-mentioned contaminants detected.

Allergenic potential of gonadotrophic preparations in experimental animals: relevance of purity.

Local reactions have been frequently reported following repeated injections of human menopausal gonadotrophins (HMG) for the treatment of infertility. Also immunoglobulin (Ig) E-mediated systemic reactions have sporadically been observed. Since most HMG preparations contain significant amounts of non-hormonal urine-derived proteins, it was suggested that these contaminating proteins are responsible for the various allergic reactions. In order to verify this hypothesis, different human follicle stimulating hormone (HFSH) and HMG preparations (Metrodin and Pergonal from Ares-Serono, and Humegon from Organon), were compared with a highly purified preparation (Metrodin HP from Ares-Serono) for the frequency and severity of allergic reactions induced in laboratory animals. The occurrence of anaphylactic shock or related symptoms was studied in sensitized guinea-pigs. The production of specific IgE was evaluated in serum from mice sensitized with the test drugs by the induction of passive cutaneous anaphylaxis in rats. In both models, two different schedules of sensitization were used. Severe allergic reactions were found in 20 of 7% of the guinea-pigs receiving highly purified FSH (Metrodin HP) in the two schedules, respectively, compared to 90 and 88% with the other preparations. Similarly significantly lower IgE titres were induced by highly purified FSH in respect to the other preparations. It can be concluded that the elimination of contaminating proteins significantly reduces the allergenicity of urine-derived HFSH preparations.

Binding kinetics of the long-acting gonadotropin-releasing hormone (GnRH) antagonist antide to rat pituitary GnRH receptors.

The GnRH antagonist Antide has been shown to produce prolonged inhibition of gonadotropin secretion in ovariectomized monkeys and other animal models. The reasons for such a long duration of action have not yet been clarified. To understand the mode of action of this new antagonist, we have performed association and dissociation binding kinetics using either crude rat pituitary homogenates as source of GnRH receptors or dispersed pituitary cells in culture. The binding characteristics of the radioiodinated Antide analog 125I-labeled[D-Tyr0] Antide to GnRH receptors in rat pituitary homogenates were comparable to those of the first generation GnRH antagonist 125I-labeled [Ac(3)Pro1,pFD-Phe2,D-Trp3,6]GnRH or the GnRH agonist 125I-labeled [D-Trp6,(N-Et)Pro9,Des,Gly10]GnRH, with an affinity constant (Ka) in the 10(10) M-1 range. The maximum binding capacity was consistently higher with the antagonist tracers than with the [125I]GnRH agonist. Both antagonists dissociated at a slower rate at 4 C (approximately 4 times) than the [125I]GnRH agonist. Incubation at 23 C of 125I-labeled [D-Tyr0] Antide previously bound at 4 C resulted in complete dissociation within 8 h after the addition of an excess amount of any of the GnRH analogs; in addition, simple dilution of the incubation medium produced spontaneous dissociation at this temperature. Using rat pituitary cells, Antide was found to inhibit the LH response to native GnRH (10(-8) M) in a dose-related manner. To test whether the binding of Antide is normally reversible at 37 C, Antide (10(-7) M) was added to the culture medium 3 days after cell plating, and the initial preincubation was resumed for 24 h. Cells were then washed twice, and dissociation was allowed to take place. Bound Antide was shown to dissociate rapidly at 37 C, as cells previously treated with Antide produced a full LH response within 24 h if challenged with native GnRH. In conclusion, the binding kinetics of 125I-labeled [D-Tyr0]Antide to GnRH receptors, which should reflect those of Antide, did not present abnormal features. Although this antagonist, similar to other GnRH antagonists, dissociated from pituitary receptors at a slower rate than GnRH analogs, rapid and spontaneous dissociation was achieved at 23 C with simple dilution, and dissociation of unmodified Antide occurred at 37 C. Taken together, our results support the concept that the long duration of action of Antide is not due to any toxic effect of Antide at the receptor site and could derive only marginally from the slow dissociation rate of this antagonist.

Neural tube defects after infertility treatment: a review.

OBJECTIVE: To investigate whether an association exists between ovulation induction and neural tube defects (NTDs). MATERIALS AND METHODS: Risk estimations in the medical literature were identified through Medline, and validity and power were assessed. Large in vitro fertilization-embryo transfer (IVF-ET) registries represent another source of information. The total number of NTDs and the total number of fetuses were computed from five registries. These data were expressed as proportions and compared with data from the general population. RESULTS: Only one study could be identified as both valid and powerful, through literature review. This case-control study concluded there was no association between ovulation induction and NTDs. The pool of IVF-ET registry data represents another powerful epidemiologic tool. Analysis of the registry data confirms the findings of the case-control study. CONCLUSIONS: Ovulation induction does not seem to represent a risk factor for NTDs in the offspring.

Combination ultrafiltration and 6 M urea treatment of human growth hormone effectively minimizes risk from potential Creutzfeldt-Jakob disease virus contamination.

Although genetically engineered human growth hormone (hGH) is now commercially available, native pituitary-derived hGH is still used by physicians in many countries for the treatment of hormone deficiency states. We describe a method using ultrafiltration and 6 M urea that reduced infectivity in human pituitary tissue that had been deliberately contaminated with scrapie virus (an animal analogue of human Creutzfeldt-Jakob disease virus) from an initial level of 10(9.7) infectious units to just 5 infectious units. Based on estimates of the frequency of contamination and infectivity levels in batches of human pituitaries, the use of this protocol to prepare GH from cadaveric human glands yields a calculated probability of exposure to a contaminated vial of not greater than 1 in 3.2 million recipients; therefore, native hormone prepared by this method may be considered to be essentially risk-free. The same methodology may be useful in the preparation of other hormones, such as prolactin, for which no synthetic substitutes are currently available, as well as biological products derived from sheep or cattle, that may be infected with scrapie or bovine spongiform encephalopathy.

Effects of uncomplicated acute myocardial infarction on biochemical parameters of stress and sexual function.

The blood levels of several "stress indicators" (prolactin [PRL], growth hormone [hGH], total catecholamines, and adenosine 3',5'-cyclic monophosphate [cAMP]) were measured in men during the first 2 weeks of uncomplicated acute myocardial infarction (AMI) and during a 3-month follow-up period. PRL levels were significantly elevated during the first day, and hGH was elevated during the week after the AMI. The levels returned to the control baseline values thereafter. The levels of the total catecholamines and cAMP in blood remained normal throughout the study period. Sexual function during the 3-month follow-up was measured in 29 patients, using a specially designed questionnaire. Impotence was found in five patients and decreased libido in four. No correlation was noted between PRL values, the other stress indicators, and sexual dysfunction. A trend toward increased incidence of sexual dysfunction was found among patients with recurrent AMI.

Growth hormone (GH) deprivation induced by passive immunization against rat GH-releasing factor delays sexual maturation in the male rat.

GH deprivation after passive immunization against rat GRF (rGRF) markedly affects somatic growth in male rats. Since it has been postulated that GH and probably insulin-like growth factor-I (IGF-I) might have a permissive role on sexual maturation, the effects of GH deprivation on the course of sexual maturation were tested. Male rats were treated with a potent anti-rGRF serum between 15 and 39 days of life (0.25 ml administered sc every second day). Body weight of treated rats averaged 62% of that of control (normal rabbit serum-treated) rats at 40 days of life (d), and 64% at 50 d after which age, treated rats started to grow normally. At 40 and 50 d, pituitary GH content was very much depressed (representing approximately 20% of control values at both ages), plasma GH was undetectable, and plasma IGF-I levels averaged 30% of those of control rats. At 70 d, 30 days after cessation of treatment, pituitary GH content, and IGF-I secretion were almost normal. At 40 d, testes and seminal vesicles of treated rats were small-for-age in agreement with significantly decreased plasma levels of FSH and delayed spermatogenesis characterized by the presence of only few or no spermatozoa. At 50 d, 10 days after cessation of anti-rGRF injections, progress of sexual maturation was found to be consistent with age and coincided with normalization of growth rate. At 40 and 50 d, pituitary contents of FSH and LH were severely decreased but became normal at 70 d. In conclusion, GH deprivation which markedly affected somatic growth induced a transient delay of sexual maturation. GH deficiency seems to have affected mostly the synthesis and secretion of FSH, thus producing a delay in testes growth and in the differentiation of the germinal cells. The low levels of IGF-I might also have been the cause for the delay of maturation at the pituitary and/or the gonadal levels.

Adrenal response to adrenocorticotropin stimulation in unexplained infertile women.

The response to a rapid intravenous administration of adrenocorticotropin (ACTH, cortrosyn) was compared in 24 unexplained infertile women and 13 fertile women. There was no significant difference in the response to a rapid ACTH stimulation as expressed by the slope of testosterone (T) response in both groups. However, the individual and mean T values prior and following the stimulation were significantly higher in the infertile group (P less than 0.001; P less than 0.005, respectively). There was no significant difference in individual values as well as in mean basal values or in mean values of 17-hydroxyprogesterone (17-OHP) after the stimulation between the two study groups (P less than 0.3; P less than 0.9, respectively). There was no significant difference in individual values as well as in mean values of dehydroepiadnrosterone (DHEA) before or 90 min after the stimulation between the two study groups (P less than 0.1; P less than 0.2, respectively). It can therefore be concluded that the elevated basal testosterone values in the infertile group originated from the ovary. Despite the fact that no attempt was made to reduce androgen values 7 (46.66%) of 15 infertile women who were available for follow-up and treatment conceived following ovulation induction therapy.

Plasma growth hormone (GH) response to intravenous GH-releasing factor (GRF) in adult rats: evidence for transient pituitary desensitization after GRF stimulation.

The ability of human (h)GRF-(1-29)NH2 to stimulate GH secretion was studied in cannulated adult rats. In order to suppress endogenous GRF secretion and the inhibitory action of hypothalamic somatostatin (SRIF), rats were anesthetized with sodium pentobarbital. Intravenous administration of hGRF-(1-29)NH2 elicited a dose-dependent response of plasma GH, with 250 ng/kg being the smallest effective dose in male rats. In female rats, for each dose tested (250 to 70,000 ng/kg), the GH response represented only about 60% that of male rats. Repeated iv stimulations with hGRF-(1-29)NH2 at short time intervals (45 min) produced transient desensitization of pituitary responsiveness to GRF: a blunted GH response to the second and third stimulations was observed both in male and in female rats and for each dose tested. Similar blunted responses were also obtained with repeated injections of native hGRF-(1-44)NH2. The possibility that these blunted responses could be due to incomplete suppression of hypothalamic SRIF secretion by sodium pentobarbital was excluded by the use of rats that were passively immunized against SRIF; in these rats, it was shown that at least 65% of the inhibition of the GH response after the second GRF stimulation was unrelated to SRIF action. Similar transient desensitization to repeated hGRF-(1-29)NH2 stimulations was also observed in conscious rats that were passively immunized against SRIF. This occurrence of blunted responses was shown to be related to the length of the time interval between GRF stimulations, with longer intervals resulting in less or no desensitization. It appears thus that modulation of pituitary responsiveness to the action of GRF is mediated by at least two independent mechanisms in the rat: in addition to the inhibitory action imposed by hypothalamic SRIF, which induces periods of refractoriness to the action of GRF, it was shown in this study that in the pituitary level each GRF stimulation also induces a transient desensitization of somatotrophs for about 1 h. This period of refractoriness might not be due to excessive stimulation with GRF, since it was also observed with the lowest dose of hGRF-(1-29)NH2 that gave a significant release of GH. Finally, a sex difference was confirmed for the response of anesthetized adult rats to stimulation with hGRF-(1-29)NH2, reflecting a sex steroid-induced modification of pituitary responsiveness to GRF stimulation.

Hyperinsulinemia--a link between glucose intolerance, obesity, hypertension, dyslipoproteinemia, elevated serum uric acid and internal cation imbalance.

A representative sample (n = 1211) of the Jewish population in Israel age 40-70 (excluding known diabetics), underwent a glucose tolerance test. Insulin response was found to be independently and positively associated with the GOH conditions--glucose intolerance (p less than 0.001), obesity (p less than 0.001), and hypertension (p less than 0.01) and with elevated serum uric acid (p less than 0.001) after accounting for the effects of sex, age, serum creatinine and use of antihypertensive medications. In a representative subgroup of 542 individuals, total VLDL and LDL fractions were estimated by standardized values (based on the reference group--individuals free of the GOH conditions), of their cholesterol and triglyceride components. Hyperinsulinemia was characterized by jointly elevated VLDL and LDL with reduced HDL. The risk ratio for this pattern (adjusted for the effects of age, sex, smoking and presence of any of the GOH conditions) was 3.4 (p less than 0.001). There was no further association of this disturbed lipoprotein profile with the GOH conditions. Cation concentrations were determined in a stratified subsample (n = 89) of the study group. The subsample comprised 30 individuals in the reference group, and 59 representing each of the seven possible combinations of abnormal glucose tolerance, obesity and hypertension (GOH group). Rate of cation imbalance defined as presence of at least one of three cation concentration--red blood cell sodium greater than or equal to 7.0 mEq/l, red blood cell potassium less than 92.5 mEd/l or plasma potassium greater than or equal to 4.5 mEq/l was 88.1% in the GOH, compared to 40.0% in the reference group (p greater than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Excretion of total estrogens and estrone-3-glucuronide during treatment with human menopausal gonadotropins.

HMG treatment is usually monitored by the evaluation of the cervical mucus, the determination of plasma 17 beta-estradiol, total urinary estrogens, ultrasonographic evaluation or a combination of these. We evaluated the daily validity of estrone-3-glucuronide excretion in urine as an indicator of follicular growth and maturation in 28 infertile women who were treated with HMG (Pergonal 500). Total urinary estrogens and estrone-3-glucuronide were measured in 24 h urine collections, and 11 of the women collected the early-morning urine separately. This allowed comparison of the concentrations and excretion of total estrogens and estrone-3-glucuronide of the 24 h urine with that found in the urine collected overnight. This comparison was made on 83 urine samples. The correlation between either the total excretion per 24 h or the concentration per liter in the 24 h urine collection of the two systems of determination was good in all determinations. Also in the urine collected on the day prior to HCG administration, total estrogens measurement was in good correlation with the estrone-3-glucuronide. However, there was statistically a significant difference in the concentrations of total estrogens and estrone-3-glucuronide between the women who ovulated and those who did not. Estrone-3-glucuronide, when calculated as a percentage of the total estrogens, was 60.86% in the women who ovulated and 33.15% in those who did not. These results demonstrate that although estrone-3-glucuronide reflects ovarian function in women treated with HMG, it may serve as a better predictor to ovulation.

Clinical, endocrine and ultrastructural study of XY gonadal dysgenesis. A case report.

About 120 cases of XY gonadal dysgenesis have been reported on. We treated such a patient with bilateral gonadectomy. The gonadal tissue's capacity to respond to hormonal trophic stimulation was assessed. When the gonads were examined ultrastructurally, structures with the morphologic characteristics of stromal ovarian cells, Sertoli's cells and Leydig's cells were found. Because of the potential malignancy of the XY gonads, bilateral gonadectomy and hormonal substitution therapy are recommended for these patients. We prefer to use combined hormone replacement with sequential estrogen and progesterone rather than sequential unopposed estrogen because of the small but increased risk of endometrial hyperplasia and carcinoma after long-standing sequential therapy.

Hyperinsulinemia. A link between hypertension obesity and glucose intolerance.

Hypertension and glucose intolerance, determined in a random population sample (n = 2,475), showed a highly significant (P less than 0.001) association from the mildest levels of both conditions, independent of the confounding effects of age, sex, obesity, and antihypertensive medications. Summary rate ratios for hypertension were 1.48 (1.18-1.87) in abnormal tolerance and 2.26 (1.69-2.84) in diabetes compared with normal tolerance. Altogether, 83.4% of the hypertensives were either glucose-intolerant or obese--both established insulin-resistant conditions. Fasting and post-load insulin levels in a representative subgroup (n = 1,241) were significantly elevated in hypertension independent of obesity, glucose intolerance, age, and antihypertensive medications. The mean increment in summed 1- and 2-h insulin levels (milliunits per liter) compared with nonobese normotensives with normal tolerance was 12 for hypertension alone, 47 for obesity alone, 52 for abnormal tolerance alone, and 124 when all three conditions were present. The prevalence of concentrations (milliequivalents per liter) of erythrocyte Na+ greater than or equal to 7.0, K+ less than 92.5, and plasma K+ greater than or equal to 4.5 in a subsample of 59 individuals with all combinations of abnormal tolerance obesity and hypertension was compared with those in 30 individuals free of these conditions. Altogether, 88.1% of the former vs. 40.0% of the latter group presented at least one of these three markers of internal cation imbalance (P less than 0.001). We conclude that insulin resistance and/or hyperinsulinemia (a) are present in the majority of hypertensives, (b) constitute a common pathophysiologic feature of obesity, glucose intolerance, and hypertension, possibly explaining their ubiquitous association, and (c) may be linked to the increased peripheral vascular resistance of hypertension, which is putatively related to elevated intracellular sodium concentration.

Diet and weight loss: their effect on norepinephrine renin and aldosterone levels.

The effect of low calorie diet on blood pressure, catecholamines, plasma renin activity (PRA) and aldosterone was examined in 22 obese subjects (19 hypertensives and 3 borderline hypertensives). A significant decrease in blood pressure (P less than 0.05) was observed in all patients after 10 days on the diet. Twenty subjects showed a significant decrease in norepinephrine (NE) levels (P less than 0.05) and two patients showed an extreme increase in NE level. On reexamination six months later these two patients presented with high NE levels and predict blood pressure levels despite having lost 15 and 18 kg respectively. The study group showed a significant decrease in mean PRA (P less than 0.05). A correlation was observed between changes in NE levels and PRA (r = 0.42, P less than 0.05) and between changes in PRA and aldosterone (r = 0.54, P less than 0.05). The reduction in blood pressure associated with caloric restriction in these obese patients may be a result of reduced sympathetic nervous system activity.

Enhanced response of plasma aldosterone to metoclopramide in essential hypertension.

Aldosterone responses to posture and the dopamine antagonist, metoclopramide, were studied in seven normotensive controls and 12 patients with essential hypertension. Both groups had similar basal supine plasma renin activity and aldosterone levels. Aldosterone levels of the hypertensive patients were greater than those of the controls 10 min after assuming an upright posture but indistinguishable at 120 min. Metoclopramide induced a peak fourfold increase above basal aldosterone levels in the hypertensive group as compared to a peak twofold increase observed in the normotensive controls. Mean 120-min integrated aldosterone response area for the hypertensives (237 +/- 44 10(-10) mol min/l) was greater (P less than 0.05) than that for normotensive subjects (106 +/- 32 10(-10) mol min/l). Simultaneous cortisol, plasma renin activity, and serum potassium levels were unaffected by metoclopramide. It is concluded that dopaminergic modulation of aldosterone secretion may be altered in essential hypertension.