Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.

Artificial oocyte activation has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). In a multicentre setting artificial oocyte activation was applied to 101 patients who were diagnosed with fertilization abnormalities (e.g. less than 50% fertilized oocytes) in a previous conventional ICSI cycle. Female gametes were activated for 15 min immediately after ICSI using a ready-to-use Ca(2+)-ionophore solution (A23187). Fertilization, pregnancy and live birth rates were compared with the preceding cycle without activation. The fertilization rate of 48% in the study cycles was significantly higher compared with the 25% in the control cycles (P < 0.001). Further splitting of the historical control group into failed (0%), low (1-30%) and moderate fertilization rate (31-50%) showed that all groups significantly benefitted (P < 0.001) in the ionophore cycle. Fewer patients had their embryo transfer cancelled compared with their previous treatments (1/101 versus 15/101). In total, 99% of the patients had an improved outcome with A23187 application resulting in a 28% live birth rate (35 babies). These data suggest that artificial oocyte activation using a ready-to-use compound is an efficient method.

Lipid metabolism in Norplant-2 users--a two-year follow-up study. Total cholesterol, triglycerides, lipoproteins and apolipoproteins.

Changes in lipid metabolism in 25 healthy female volunteers during a 24-month application of Norplant-2 were evaluated in an open clinical trial. Total serum cholesterol decreased significantly (p less than 0.05/p less than 0.05) by 10%/9% after 12 months and by 3%/7% (n.s./n.s.) after 24 months of Norplant-2 use (all subjects/subjects completing 24 cycles). Serum triglycerides decreased by 34%/28% (n.s./p less than 0.05) after 12 months and by 29%/25% (p less than 0.05/p less than 0.05) after 24 months of Norplant-2 use (all subjects/subjects completing 24 cycles). HDL-cholesterol decreased significantly by 18%/12% (p less than 0.01/p less than 0.05) after 12 months and by 12%/12% (p less than 0.05/p less than 0.05) after 24 months of Norplant-2 use (all subjects/subjects completing 24 cycles). No statistically significant difference between serum levels of LDL-cholesterol prior to and after 12 and 24 months of Norplant-2 use could be found. VLDL-cholesterol levels decreased significantly by 38%/38% (p less than 0.05) after 12 and by 25%/25% after 24 months of Norplant-2 application (p less than 0.01) (all subjects/subjects completing 24 cycles). Apolipoprotein Al decreased significantly by 23%/23% (p less than 0.001/p less than 0.01) after 12 and by 21%/22% after 24 months of Norplant-2 application (p less than 0.01/p less than 0.01) (all subjects/subjects completing 24 cycles). No statistically significant difference between apolipoprotein All levels prior to and after 12 and 24 months of Norplant-2 implantation could be found. Apolipoprotein B decreased significantly by 27%/17% (p less than 0.05/p less than 0.05) after 12 months of Norplant-2 application (all subjects/subjects completing 24 cycles). The decline after 24 months of Norplant-2 use was not significant. Changes in lipid metabolism caused by oral hormonal contraceptives differ in the various clinical trials; however, most investigators found that serum levels of total cholesterol and triglycerides increase under the application of OCs. Contrary to this, a decrease of total cholesterol and triglycerides under Norplant-2 use was noted. Furthermore, we found a significant decrease of lipoproteins and apolipoproteins--with the exception of LDL-cholesterol and apolipoprotein All, which did not show any significant modifications. Thus, Norplant-2 seems to be non-contributory to cardiovascular risk and might even provide protection against such risks.

[Future prospects in contraception]. / Zukunftsperspektiven der Empfängnisverhütung.

PIP: Only 40% of the 1.2 billion couples in reproductive age have access to effective contraceptive methods, although only $3.0 per couple per year would suffice for contraception worldwide. Abortions are performed for 40-60 million women annually. More than 200,000 women die as a result of abortions, and another 500,000 die due to labor complications. Contraception for women comprises the following: 1) agents that prevent ovulation; prolonged breast feeding (98% safe contraception within the first 6 months); oral contraceptives containing estrogens and gestagens (60-80 million women use them worldwide; in 1968 the 50 g estrogen containing pill, in 1972 the micropill with 30 g of ethinyl estradiol [EE], and in 1992 the ultra-low-dose pill with 20 g of EE were introduced); and future developments (third generation progestagens, antigestagens, nonsteroidal natural substances, melatonin, the combination of gonadotropin-releasing hormone analogs and natural estrogens); 2) prevention of fertilization: mechanical methods (diaphragm, sterilization methods by laparoscopy or chemical means); chemical methods (spermicides such as nonoxynol); behavioral methods (temperature methods using refined measurement of the body temperature, cervical mucus resistance); hormonal methods (implants such as Norplant containing levonorgestrel [LNG], Implanon containing 3-ketodesogestrel, the vaginal ring [the WHO-ring and the Organon ring], the minipill with pure gestagen, one-month injection with Cyclofem), IUDs (copper-containing IUDs, LNG-containing IUDs with a Pearl Index of 0.2-0.5 and reduction of dysmenorrhea); and immunological contraception (ovum and spermatozoon antigens); 3) the prevention of implantation: hormonal methods (the morning-after pill with high-dose EE or the combination of estrogen and gestagen); insertion of an IUD up to the 6th day after coitus; immunological methods (human chorionic gonadotropin antibodies, antibodies against the zona pellucida glycoproteins, implantation inhibition through interaction with interleukin IL-1 receptor, and antibodies against specific proteins of the endometrium influencing implantation). Contraception for men consist of the condom, vasectomy, coitus interruptus, and medical inhibition of spermiogenesis (testosterone ester and gossypol).^ieng

The efficacy and tolerability of norgestimate/ethinyl estradiol (250 micrograms of norgestimate/35 micrograms of ethinyl estradiol): results of an open, multicenter study of 59,701 women.

The efficacy and tolerability of a new oral contraceptive, norgestimate/ethinyl estradiol (250 micrograms of norgestimate/35 micrograms of ethinyl estradiol; Cilag GmbH Research, Sulzbach, Germany) were examined in an open-label study of 59,701 women who were evaluated during 342,348 menstrual cycles; 42,022 women completed the planned treatment regimen of six cycles. A use-efficacy (overall) Pearl index of 0.25 pregnancies per 100 woman-years was calculated based on 342,348 cycles. Tolerability was assessed for all women who completed six treatment cycles. Reductions in mean cycle length and duration of bleeding were noted; 32% of the women experienced reductions in the intensity of bleeding by the end of cycle 6. After six cycles of use, amenorrhea occurred in 1%, spotting in 4%, and breakthrough bleeding in 3% of the participating women. Treatment with norgestimate/ethinyl estradiol had minimal effects on weight, blood pressure, pulse, lipid metabolism, and blood glucose. Adverse effects (acne, nausea, or headaches) occurred at low frequencies and in many cases, were reduced compared with pretreatment levels. The results of this large-scale open trial were comparable with results from two other multicenter trials of the same formulation.

Marked effects of salt on estrogen receptor binding to DNA: biologically relevant discrimination between DNA sequences.

Avidin-biotin complexed with DNA (ABCD) assays were employed to determine the binding affinity of estrogen receptor (ER) to DNA under various salt conditions. Type and concentration of salt in the reaction buffer dramatically affected the ability of the ER to discriminate between DNA sequences. Under appropriate salt conditions, ER was able to bind to the estrogen response element from the Xenopus vitellogenin A2 gene with at least 3 orders of magnitude greater affinity than a two base pair mutant sequence, and 5 orders of magnitude greater affinity than plasmid DNA. In these studies, the best discrimination was observed under conditions of salt type and concentration that more closely approximated intracellular conditions, i.e., 100-150 mM potassium salts. Analysis of the binding affinities for ER to all three types of DNA over a range of KCl concentrations indicated that the ionic interactions upon ER binding were the same for the three DNA molecules tested. Therefore, the additional stability of ER binding to target DNA sequences was contributed by nonionic interactions.

[Serum hormones before and during therapy with cyproterone acetate and spironolactone in patients with androgenization]. / Serumhormone vor und unter Therapie mit Cyproteronazetat und Spironolacton bei Patientinnen mit Androgenisierungserscheinungen.

The effect of cyproterone acetate (CPA) and spironolactone (SPL) on the serum androgen concentrations of premenopausal women with symptoms of hyperandrogenism were investigated in a total of 39 women. The observation period was 12 months. CPA was administered according to the Hammerstein regimen: cyproterone acetate (CPA) [Androcur] 100 mg/die 5.-14. day of the cycle; ethinylestradiol (EE) [Progynon C]: 40 mg/die 5.-25. day of the cycle; Spironolactone (SPL) was given in a dosage of 100 mg/die from day 1.-21. of the cycle. During the therapy with CPA a significant decrease of total testosterone (61%), free testosterone (78%), LH (48%) and 17 alpha-Hydroxyprogesterone (72%) was observed; during the medication with spironolacton only a significant decrease of 5 alpha-dihydrotestosterone (81%), which could not be seen during CPA use, was observed. Serum concentrations of total testosterone, free testosterone, LH and 17 alpha-Hydroxyprogesterone remained unchanged. DHA and DHAS did not change during neither medication. Since peripheral androgens were not suppressed by SPL the positive therapeutical effect of SPL can be explained by the antiandrogenic effect at the level of the receptor. A disadvantage of spironolacton is the lack of contraceptive efficacy. In cases where contraindication for oral contraceptives are present SPL can be considered as a good alternative to CPA. The suppressive effect of CPA/EE on total testosterone, LH addition to the antivulatory effect makes it the preferable medication for hyperandrogenemic patients with polycystic changes of the ovaries (PCOD).

[Liver tumors in women using oral hormonal contraceptives]. / Lebertumoren bei Frauen unter Anwendung von oralen hormonalen Kontrazeptiva--Kurzmitteilung.

The association between the use of oral contraceptives and the induction of liver tumors was first discussed by Baum et al. in 1973 [4]. Since the introduction of the pill a slight increase in the incidence of benign liver tumors has been found. In recent studies this could not be shown for liver cell carcinomas. In the world literature (1971-1994) 637 cases of patients with liver tumors who had taken oral contraceptives at any time of their lives have been reported. 233 liver cell adenomas, 210 focal nodular hyperplasia and 194 liver carcinomas were found. The incidence of liver tumors was not influenced by the type of progestin used.

[Diagnosis, incidence and clinical significance of premature LH rise/peak in patients of an in vitro fertilization and gamete transfer program]. / Diagnose, Häufigkeit und klinische Bedeutung des vorzeitigen LH-Anstiegs/-Gipfels bei Patientinnen eines In vitro-Fertilisierungs- und Gametentransfer-Programmes.

In this study we analysed the prevalence and the clinical relevance of premature (related to the day of ovulation induction with HCG) LH rise (LH greater than or equal to 180%, but less than 300% of the mean of all previous values) or peak (LH greater than or equal to 300% of the mean of all previous values): 12% (2/16) of the conception cycles showed a premature LH peak, 44% (7/16) a premature LH rise and 44% (7/17) showed neither a premature LH rise nor a premature LH peak. The pregnancy rate of cycles with premature LH peak was found to be 10% (2/21), without premature LH peak 37% (7/19). These results indicate, that the premature LH peak represents an unfavourable condition for achievement of pregnancy; the cancellation of such stimulations before oocyte retrieval is recommended. On the other hand, cycles with premature LH rise need not be cancelled. The fertilisation rate was independent of an LH rise/peak (no LH rise/peak: 55%, LH rise: 62%, LH peak: 46%): it is supposed, that the premature LH peak causes submicroscopical injury of the oocyte and although fertilisation is possible, further embryonic development will be hindered. The use of the GnRH analogue Buserelin in combination with HMG leads to a significantly (p less than 0.001) lower prevalence of the premature LH peak (16%; 7/44 versus 51%; 48/95); the combined Buserelin + HMG stimulation is recommended for treatment of premature LH peak.

[Effect of the GnRH analog buserelin on sex hormone serum level in relation to treatment onset and duration]. / Wirkung des GnRH-Analogons Buserelin auf die Serum-Spiegel von Sexualhormonen in Abhängigkeit von Behandlungsbeginn und Behandlungsdauer.

Changes of serum estradiol, progesterone, LH, FSH, prolactin, testosterone, androstendione, DHEA and DHEAS levels during a GnRH-analogue (buserelin) treatment have been analysed retrospectively taking account of effectiveness of treatment and its relation to the beginning and duration of treatment. 1200 micrograms/day buserelin were administered intranasally from the first day of the menstrual cycle (n = 30) or the 7th hyperthermic day of the cycle (n = 22). The results proved, that the administration of buserelin to sterile women inhibits the ovarian (estradiol, progesterone) and pituitary (LH, FSH) hormone secretion during the first 10-14 days of treatment. The adrenal hormone secretion (DHEA, DHEAS) remained unaffected, whereas the androgens of ovarian origin (testosterone, androstendione) were suppressed during the GnRH analogue treatment. The serum prolactin level increased during the first two weeks of treatment and returned to pretreatment values within the following two weeks. On the basis of the faster suppression of estradiol secretion with buserelin treatment, beginning in the middle of the luteal phase, this therapy is recommended for ovarian suppression.

High preovulatory serum luteinizing hormone level is unfavorable to conception.

Serum estradiol, progesterone and luteinizing hormone (LH) levels of 16 pregnant and 58 non-pregnant stimulated in vitro fertilization-embryo transfer (IVF-ET) or gamete intrafallopian transfer (GIFT) cycles have been compared with regard to their predictive value for achievement of pregnancy. Serum estradiol and progesterone pattern of the pregnant and non-pregnant group did not show any significant difference. Around the time of ovulation induction by human chorionic gonadotropin (hCG) the serum LH values proved to be higher in the non-pregnant group than in the pregnant one. In spite of having a permissive function, preovulatory serum estradiol and progesterone seem not to have a predictive value with regard to pregnancy. Elevated preovulatory serum LH is detrimental for pregnancy, therefore the measurement of serum LH beyond hCG administration also, and the cancellation of cycles with high serum LH levels shortly before oocyte retrieval is recommended.

[Combined treatment with a GnRH-analog and gonadotropins in IVF-ET/GIFT patients after previously unsuccessful pure HMG stimulation]. / Kombinierte Behandlung mit einem GnRH-Anologon und Gonadotropinen bei IVF-ET/GIFT-Patientinnen nach vorangegangenen erfolglosen reinen HMG-Stimulationen.

Clinical results of a combined GnRH-analogue (buserelin, long protocol) + HMG stimulation of 44 IVF-ET or GIFT patients with previously failed pure HMG stimulation have been analysed. The cancellation rate and the incidence of premature LH surge was found to be lower in the buserelin + HMG group than in the control HMG group (30% vs. 40% and 16% vs. 51%). There was no significant difference between the buserelin + HMG and the control HMG group, neither in the number of retrieved and fertilized oocytes and transferred embryos nor in the fertilization and pregnancy rate. The combined GnRH-analogue and gonadotropin therapy led to a decrease of the cancellation rate and of premature LH surge in patients with previously failed pure HMG stimulation. In this group of patients the use of buserelin leads to treatment results comparable to results of pure HMG stimulated patients without previously failed stimulation cycles. Thus the GnRH-analogue + HMG stimulation is recommended for patients with previously unsuccessful pure HMG stimulation.

Action of leukotriene B4 in human granulosa cells in vitro.

Leukotriene B4 directly enhanced progesterone release from superfused human granulosa cells. This secretory effect was observed in concentrations from 10(-12) to 10(-10) M. Lower and higher concentrations failed to affect progesterone release. When we analysed the high performance liquid chromatography profile of supernatant from human granulosa cell cultures, we detected a leukotriene B4 peak. In conclusion, these data support the hypothesis that leukotriene B4 may participate in the intracellular mechanism of progesterone release in human granulosa cells.

Aneuploidy in human granulosa lutein cells obtained from gonadotrophin-stimulated follicles and its relation to intrafollicular hormone concentrations.

Proliferation of granulosa cells is inversely related to differentiation and hormone production. The purpose of this study was to evaluate the intrafollicular and serum steroid concentrations and to compare these results to granulosa cell proliferation as measured by DNA flow cytometry. Human granulosa lutein cells in follicular fluid of in-vitro fertilization (IVF) patients were investigated with regard to ploidy, percentage of S-phase cells and proliferation index (PI: percentage of cells in the S- and G2/M-phase). The study was originally designed to indicate an additional marker for the outcome of IVF treatment by DNA flow cytometric measurements of granulosa lutein cells. Follicular fluids of 160 follicles (45 patients) were evaluated; 45.6% (n = 73) of the follicles showed aneuploid granulosa lutein cells and 5.6% (n = 9) of the follicles contained multiploid granulosa cells, defined as at least two aneuploid populations of cells with different DNA indices. A total of 48.8% (n = 78) of the follicles had only diploid cells. Thus >50 % of the investigated follicles showed aneuploidy. In all, 73% (33 of 45) of patients had at least one follicle containing aneuploid granulosa lutein cells. The PI of the aneuploid cell populations significantly exceeded that of the diploid cell populations (median: aneuploid: 15.5; diploid: 7.4; P < 0.0001). The intrafollicular concentrations of testosterone, progesterone and dehydroepiandrosterone sulphate (DHEA-S) were significantly lower in follicles with aneuploid granulosa cell populations. Luteinizing hormone concentration was significantly higher in follicles with aneuploid granulosa cells. Intrafollicular concentrations of oestradiol, follicle stimulating hormone and the serum concentrations of all steroid hormones did not show any significant correlation to ploidy. Although aneuploidy has been reported for oocytes (in approximately 17% of the oocytes), no study, to our knowledge, has observed such a high incidence of aneuploidy in granulosa lutein cells after gonadotrophin stimulation. Except for aneuploidy found in tissues with some characteristics of neoplastic growth (colon adenoma, borderline tumours, endometriosis with atypic cells, etc.), it is unique for non-malignant human cells. The correlation with intrafollicular steroid concentrations points to a possible pathophysiological or physiological relevance of these findings. However, it was impossible to correlate the outcome of IVF with DNA flow cytometry results.

Elevated serum inhibin levels and suppressed luteinizing hormone surge in young patients stimulated with gonadotropins.

The physiological role of inhibin and its relation to other sex hormones (estradiol, progesterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH)) has been investigated during gonadotropin-stimulated cycles of 38 in vitro fertilization-embryo transfer/gamete intrafallopian transfer (IVF-ET/GIFT) patients. Human menopausal gonadotropin (hMG) was given from day 3 of the cycle until 1 day before ovulation induction with human chorionic gonadotropin (hCG). Blood samples were taken twice daily and hormone measurements performed by radioimmunoassay or enzyme immunoassay. Patients were divided into two groups: Group A comprised patients < 35 years of age (n = 20) and Group B included patients > or = 35 years of age (n = 18). The pregnancy rate was significantly higher in Group A. During the follicular phase, serum inhibin level rose gradually in both groups but the values were higher in Group A (significantly between days -2 and 0). During the early luteal phase serum inhibin concentrations were similar in both groups. Estradiol pattern did not differ in the two groups. Estradiol pattern did not differ in the two groups. Whilst serum estradiol level did not increase significantly after day 0, serum inhibin concentration reached its peak value 1 day later, on day +1. Serum progesterone was higher in Group A between days +1 and +4 (significantly on days +1, +3 and +4). Serum FSH increased slowly in both groups and did not correlate with serum inhibin concentration. Basal LH concentrations were similar between days -6 and -2 in both groups. Around the time of ovulation induction (day -1, 0 and +1) serum LH was lower in Group A (significantly on day 0).(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy of human granulosa cells in follicular fluids from in vitro fertilization patients.

OBJECTIVES: To study the proliferative behavior of granulosa cells found in follicular fluids from patients after hormone stimulation in the framework of in vitro fertilization (IVF) with gonadotropins. STUDY DESIGN: The deoxyribonucleic acid ploidy and the proliferation indices of granulosa cells in fresh and unfixed follicles (n = 119) from gonadotropin-stimulated patients (n = 32) were analyzed by flow cytometry. RESULTS: Aneuploid cells were found in a large number of follicles (65/119) as well as patients (25/32). A small number of follicles (8/119) and patients (7/32) contained multiploid cells. There was no correlation between proliferation indices and ploidy. Granulosa cells were the predominant cells in follicular fluids. No malignant cells were found in any case. CONCLUSION: This is the first report concerning the high incidence rate of aneuploidy in ovarian granulosa cells in IVF patients. The clinical relevance of the phenomenon is not clear. There should be further study to determine whether there is any link to a previously discussed possible relation between gonadotropin stimulation in women attempting to become pregnant and the occurrence of ovarian cancer or granulosa cell tumors. Of further interest might be a possible relation between ploidy and proliferation indices of stimulated granulosa cells as well as side effects of gonadotropin therapy and biologic parameters, like maturity, fertilizability of oocytes and rates of pregnancy.

Analysis of hormonal changes during combined buserelin/HMG treatment.

Changes of serum oestradiol, LH and progesterone have been analysed in view of the effect of the GnRH analogue buserelin on the late follicular and early luteal phase of cycles stimulated with combined buserelin/HMG (n = 31) in an IVF-ET/GIFT programme. Patients undergoing cycles with HMG only (n = 57) served as the control group. With the use of the GnRH analogue buserelin, a significantly higher amount of HMG (25 versus 20 ampoules; P less than 0.001) for a significantly longer stimulation period (10 versus 8 days; P less than 0.001) was necessary to achieve the same oestradiol response as seen in HMG cycles. Serum progesterone levels during a three day period before ovulation induction tended to be lower in the combined buserelin/HMG cycles than in cycles with HMG stimulation only. We did not observe any significant difference in the luteal phase progesterone levels of the buserelin/HMG and the HMG group. On the other hand, we found that an inadequate luteal phase in buserelin/HMG cycles could be avoided by HCG administration during the luteal phase. Both the elevation of basal serum LH and a premature LH rise could also be avoided by the use of buserelin.

Estrogen receptor binding to a DNA response element in vitro is not dependent upon estradiol.

Gel shift assays were employed to distinguish between the contribution of 17 beta-estradiol (E2) and a short heating step to the ability of the rat uterine cytosolic estrogen receptor (ER) to bind to the estrogen response element (ERE) from the vitellogenin A2 gene (vitERE). Despite the popularity of models in which the ER is a ligand-activated DNA-binding protein, these studies find that estrogen does not significantly contribute to receptor-DNA complex formation. An avidin-biotin complex with DNA (ABCD) assay was utilized to obtain quantitative measurement of the affinities of the ER for the vitERE and a mutant sequence. Scatchard analysis gave a dissociation constant of 390 +/- 40 pM for the E2-occupied, heated ER to the vitERE. The data fit a one-site model and evidence for cooperatively was not observed. A dissociation constant of 450 +/- 170 pM was obtained for the unoccupied, heated ER, leading to the conclusion that estrogen was not necessary for specific binding to DNA. The percentage of ER capable of binding vitERE varied with each cytosol preparation, ranging from 60 to 100% and estrogen did not appear to affect this variation. Competition against the vitERE with a 2-bp mutant sequence showed a 250-fold lower relative binding affinity of the receptor for the mutant over the vitERE sequence. This ability of the ER to discriminate between target and nonspecific DNA sequences was also not dependent on the presence of estrogen.

Normal values for a short-time ACTH intravenous and intramuscular stimulation test in women in the reproductive age.

Normal values in endocrine testing are the most important precondition for the recognition of disorders of the endocrine system. To establish a reference range for adrenocorticotropic hormone (ACTH) stimulation tests, an intravenous and intramuscular ACTH stimulation test was conducted in 29 female volunteers without hyperandrogenism. A total of 25 IU of ACTH were administered intravenously or intramuscularly and blood sampling was performed before, 1 h and 2 h after ACTH injection. The test was performed on days 3-5 of the menstrual cycle. The following steroid hormones were assessed in the serum: 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, dehydroepiandrosterone, testosterone, free testosterone and 5 alpha-dihydrotestosterone. The normal range was defined by the interval between the 5th and 95th percentiles; additionally the 1st, 25th, 50th, 75th and 99th percentiles are reported. A significant increase of serum hormone levels after ACTH administration could be observed for the following hormones: cortisol, 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. There was no rise after ACTH application for testosterone, 5 alpha-dihydrotestosterone and free testosterone. It could be shown for all hormones that there was no significant difference between the serum levels that were reached after intravenous and intramuscular ACTH injection. Neither could we find a significant difference in the relative increase of the serum hormones when stimulation values were related to basal values. Since in most studies with ACTH stimulation tests, only the serum values 1 h after ACTH application are measured, we investigated whether the measurement of steroid hormones 2 h after ACTH application gave further information. We could demonstrate that for most measured serum hormones the majority of the volunteers had the maximal response 2 h after ACTH application, no matter whether ACTH was injected intramuscularly or intravenously. As a conclusion, we recommend the measurement of the respective hormones not only 1 h but also 2 h after ACTH stimulation. Since there is no increase after ACTH stimulation for total testosterone, free testosterone and 5 alpha-dihydrotestosterone, it is sufficient to assess the basal values of these hormones. Excessive adrenal response is reflected by dehydroepiandrosterone, 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone and cortisol.

Serum inhibin levels in gonadotrophin stimulated in-vitro fertilization/gamete intra-fallopian transfer cycles.

Serum inhibin concentrations of 64 cycles of in-vitro fertilization--embryo transfer (IVF-ET) or gamete intra-Fallopian transfer (GIFT) have been analysed retrospectively. No significant difference was observed in serum inhibin levels of cycles stimulated with buserelin and human menopausal gonadotrophin (HMG) or HMG alone. During the late follicular phase, serum inhibin was higher in cycles resulting in pregnancy than in cycles without a pregnancy (peak values on day +1: 8.3 versus 6.4 IU/ml, respectively). The same difference was found between stimulation cycles resulting in a viable or a non-viable pregnancy (peak values on day +1: 8.3 versus 7.5 IU/ml). However, these differences were not significant. During the early luteal phase, serum inhibin values were similar in these groups of patients. Our results indicate that the use of the gonadotrophin-releasing hormone (GnRH) analogue buserelin, in combination with HMG, for ovarian stimulation does not affect inhibin production by granulosa cells in vivo. The late follicular and early luteal concentrations of serum inhibin have to be considered unsuitable as predictors in IVF/GIFT cycles with respect to pregnancy and pregnancy outcome.

Identification of a novel Arg-->Cys mutation in the LDL receptor that contributes to spontaneous hypercholesterolemia in pigs.

We previously carried out genetic and metabolic studies in a partially inbred herd of pigs carrying cholesterol-elevating mutations. Quantitative pedigree analysis indicated that apolipoprotein (apo)B and a second major gene were responsible for the hypercholesterolemia in these animals. In this study, we assessed LDL receptor function by three different methods: ligand blots of liver membranes using beta-very low density lipoprotein (VLDL) as a ligand; low density lipoprotein (LDL)-dependent proliferation of T-lymphocytes; and direct binding of 125I-labeled LDL to cultured skin fibroblasts. All three methods demonstrated that LDL receptor ligands bound with decreased affinity to the LDL receptor in these animals. In skin fibroblasts from the hypercholesterolemic pigs, the Kd of binding was about 4-fold higher than in cells from normal pigs. The cDNA of the pig LDL receptor from normal and hypercholesterolemic pigs was isolated and sequenced. We identified a missense mutation that results in an Arg'Cys substitution at the position corresponding to Arg94 of the human LDL receptor. The mutation is in the third repeat of the ligand binding domain of the receptor. By single-stranded conformational polymorphism (SSCP) analysis, we studied the relationship between LDL receptor genotype and plasma cholesterol phenotype. In contrast to humans, the hypercholesterolemia associated with the LDL receptor mutation in pigs was expressed as a recessive trait. The LDL receptor mutation made a far more significant contribution to hypercholesterolemia than did the apoB mutation, consistent with observations made in human subjects with apoB mutations. Within each genotypic group (mutated apoB or mutated receptor), there was a wide range in plasma cholesterol. As the animals were on a well-controlled low-fat diet, this suggests that there are additional genetic factors that influence the penetrance of cholesterol-elevating mutations.