Gonadotropin and free alpha-subunit secretion in patients during GnRH-agonist treatment.

In female patients gonadotropin and free alpha-subunit serum levels during GnRH-agonist (GnRH-a) treatment were investigated. Using two different immunoassays (RIA, IRMA) as well as LH in-vitro bioassay evidence was found for pituitary secretion of incomplete LH molecular forms which are detected false positive in RIA measurements but neither in IRMA nor in bioassay. In the GnRH-a induced desensitization state suppression of LH serum levels was more profound as compared to FSH. In contrast to gonadotropins free alpha-subunit serum levels were shown to increase in a biphasic course leading to ninefold higher levels persisting during GnRH-a treatment. A further increase was achieved by exogenous GnRH but not by estradiol. In conclusion these in-vivo data give evidence for a gradually different suppression of gonadotropic secretion by the use of GnRH-a. The mechanisms of inhibition are discussed to be directed to steps in the processing of beta-subunit synthesis but not of alpha-subunit.

Ovarian response and pregnancy rates in in vitro fertilization, gamete intrafallopian transfer, and in vivo fertilization therapies after combined gonadotropin-releasing hormone agonist/human menopausal gonadotropin stimulation.

Ovarian stimulation cycles were initiated using human menopausal gonadotropin (HMG) in 318 women (643 cycles) without pretreatment and in 341 women (525 cycles) after pituitary desensitization by pretreating with the gonadotropin-releasing hormone agonists (GnRH-A) buserelin acetate or triptorelin acetate. Significantly higher pregnancy rates were observed in the GnRH-A/HMG group (15%) as compared to the HMG group (7%) following in vitro (p less than 0.05) but not in vivo fertilization therapy (14 vs. 9%, respectively). After in vitro fertilization, the rates increased with increasing length of the active phase of follicular maturation. Gamete intrafallopian transfer, performed only in the GnRH-A/HMG group, led to a pregnancy rate of 34%. Overall, there was a clear trend to higher pregnancy rates in the GnRH-A/HMG group (16%) as compared to the HMG group (8%). Abortion rates were comparable in both groups (24 vs. 19%). The higher pregnancy rates in the GnRH-A/HMG group were attributable to enhanced follicular maturation and optimized ovarian stimulation produced by the hypogonadotropic state. However, an increased risk of the ovarian hyperstimulation syndrome was observed in these patients.

Induction of pharmacological hypogonadotropism using gonadotropin-releasing hormone agonists in patients undergoing controlled ovarian stimulation.

Pharmacological hypogonadotropism was induced in 167 women using the gonadotropin-releasing hormone agonists (GnRH-A) buserelin acetate or triptorelin acetate. 84 patients (group 1) began treatment using 1.2 mg/day buserelin acetate intranasally during the follicular phase (days 1-3); 41 patients (group II) began the same treatment, supported by 10 mg medroxyprogesterone acetate for 10 days, during the early luteal phase; 42 patients (group 3) received triptorelin acetate as an intramuscular depot injection, supported by 10 mg medroxyprogesterone acetate for 10 days, during the early luteal phase. Serum luteinizing hormone, follicle-stimulating hormone, oestradiol (E2), prolactin, and testosterone levels were monitored. Pituitary function was assessed by (1) measurement of endogenous luteinizing hormone fluctuation; (2) response to luteinizing hormone releasing hormone administration, and (3) response to oestradiol benzoate (E2 test). Complete pituitary desensitization was only assumed, if all three tests were negative. The LH-RH test and the E2 test were shown to be the most reliable indicator of pituitary function. E2 administration led to further reduction of gonadotropin secretion after pituitary desensitization. The desensitization time was 41.1 +/- 11.7 days in group 1 and was significantly reduced to 20.7 +/- 10.5 days in group 2 (p less than 0.01); a further, non-significant shortening to 15.1 +/- 3.0 days was observed in group 3. Changes in endocrine parameters demonstrated hypogonadotropic hypo-oestrogenism after initial pituitary stimulation.

Endocrine profiles and luteal function during GnRH-analogue/HMG therapy.

We have found a significant improvement of pregnancy rates after pretreatment with an agonist of gonadotrophin releasing hormone (GnRH-a). The pregnancy rate in patients treated with HMG/HCG was 17% per patient and 5.5% per cycle, in patients treated with buserelin, 25% per patient and 15% per cycle and in the triptorelin group 25% per patient and 22% per cycle. From 740 HMG/HCG cycles without GnRH-a only 66% were sufficient according to the analytical data. In 16% we found a premature LH discharge and in 18% an irregular LH fluctuation during stimulation. It is clear that gonadotrophin stimulation during pituitary suppression provokes a more intense ovarian reaction with respect to the number of follicles, as well as the endocrine activity. There are also some important practical advantages: ovarian stimulation can be started without any respect to a definite time of menstruation or of the cycle. Of further importance is the much greater flexibility in the timing of HCG administration. Finally, it will be favourable for all patients who need ovulation induction, especially for oocyte retrieval for IVF or GIFT, because no cycle has to be cancelled.

[GnRH agonist/hMG stimulation in gamete intrafallopian transfer (GIFT)]. / GnRH-Agonist/hMG-Stimulation für den intratubaren Gametentransfer (GIFT).

During a period of 17 months 61 couples without pathological tubal factors were treated by follicular puncture and gamete intra-Fallopian transfer (GIFT) at the department of Obstretrics and Gynaecology, University of Hamburg. The combination GnRH-antagonist (GnRH-A)/hMG was administered in 69 stimulation cycles. Two different GnRH-A application forms were used (daily intranasal spray/monthly depot injection). In all cases mature oocytes were collected after ovulation induction, and gamete transfer was performed. None of the cycles had to be cancelled. Twenty-two clinical pregnancies were achieved (32% by stimulation cycle). The highest pregnancy rate was observed in the group of cervical infertility (58%), lowest rate in cases of pathological male factors (15%). In addition, pregnancy rate correlated with the number and maturity of transfered oocytes. The combined GnRH-A/hMG stimulation therapy allows for a prolonged active follicular development without the occurrence of endogenous, premature luteinization. Besides a more flexible and effective strategy of ovarian stimulation, the number of follicles/oocytes was L, increased which provided a better condition for GIFT.

Endocrine parameters of human follicular fluid and fertilization capacity of oocytes.

The induction of multiple follicular growth during ovarian stimulation for in vitro fertilization (IVF) implies follicular asynchrony. As a consequence oocytes of different quality are obtained. The maturity and fertilizability of oocytes cannot sufficiently be predicted by their morphological appearance under the light microscope. Looking for additional parameters of oocyte quality, FSH, hCG, estradiol (E2), progesterone (P), testosterone (T), prolactin (PRL) and cAMP were analysed in human follicular fluid (FF) containing a morphologically mature oocyte. The evaluation of the relationship between FF values and oocyte fertilization showed the following results: no differences of FSH, hCG, E2, P and T concentrations in FF between the group of fertilized and not fertilized ova. However, significant differences were detected for PRL and cAMP. In FF of fertilized oocytes PRL content was higher (38.8 +/- 2.2 vs 29.7 +/- 2.3 ng/ml, P less than 0.01) and cAMP level was lower (32.7 +/- 1.9 vs 59.8 +/- 7.4 pmol/ml, P less than 0.01) as compared with FF of unfertilizable oocytes. In conclusion PRL- and cAMP concentration of FF might be additional parameters of oocyte maturation and fertilizability.

[Gonadotropin stimulation and in vitro fertilization following selective hypophyseal suppression by LH/RH analogs]. / Gonadotropin-Stimulation und In-vitro-Fertilisation nach selektiver Hypophysen-Suppression durch LH/RH-Analogon.

Out of 109 hMG stimulations for in vitro fertilization (IVF) 41 cycles (38%) had to be cancelled because of premature endogenous luteinization. Pretreatment with the LH/RH-agonist Buserelin induces selective pituitary suppression and prevents spontaneous LH-surge during hMG stimulation. The pharmacologic hypogonadotropism allows considerable flexibility with respect to the timing of starting gonadotropin stimulation and ovulation induction. In 74 Buserelin/hMG/IVF-cycles no premature luteinization occurred and all started stimulations yielded in follicular puncture. In addition increased oocyte recovery rate, significant higher fertilization rate and significant better pregnancy rate could be achieved as compared with hMG/IVF-cycles. In conclusion pharmacologic hypogonadotropism by administration of LH/RH-analogue reliably prevents endogenous luteinization and improves gonadotropin stimulation as well as IVF results.

Polymorphism of human pituitary lutropin (LH). Effect of the seven isohormones on mouse Leydig cell functions.

The effect of the 7 different molecular forms of human pituitary lutropin (LH) on mouse Leydig cells as a model of a target organ were studied. The hormone functions have been characterized by receptor binding, cAMP accumulation and testosterone production. One important finding was the similar intrinsic in vitro biological activity for all isohormones. Quantitatively, however, the potencies of the 7 hormone forms did not correlate with the activities obtained by radioimmunoassay: there was a dramatic decrease of receptor binding activity and biological activity compared to immunoactivity from the more alkaline to the more acidic LH isohormones.

Polymorphism of human pituitary lutropin (LH). Isolation and partial characterization of seven isohormones.

The complete microheterogeneous system of human pituitary lutropin was demonstrated by gel isoelectric focusing. In addition 7 LH isohormones could be isolated by preparative column focusing and characterized with respect to physicochemical, biological and immunological properties. The in vivo biopotencies ranged from 4.50-11.50 IU/mg, the in vitro bioactivity was from 1.20-10.10 IU/mg, and the immunological activity was from 3.10-7.55 IU/mg. The sialic acid content was found to be 1.8-3.2%. Treatment with neuraminidase resulted in a shift of all bands to the alkaline region, however the 7 LH forms were still present.

Primary culture of normal pituitary cells of carp (Cyprinus carpio) for the study of gonadotropin release.

A method for preparing enzymatically dispersed pituitary cell cultures of carp (Cyprinus carpio) is described. The cultures have been used to assay a synthetic analog of gonadotropin releasing hormone (GnRH) and to determine the specificity of steroids able to affect gonadotropin (GtH) release in vitro. Time course secretion studies indicated that by 48 h incubation, in the presence of 500 pM GnRH, cumulative secretion of gonadotropin (719 ng +/- 90/2.5 X 10(5) cells) had exceeded that of controls (446 ng +/- 106/2.5 X 10(5) cells). Estradiol-17 beta, progesterone, testosterone, and 11-ketotestosterone showed different inhibitory effects on pituitary basal GtH release. Based on the results, it was concluded that carp pituitary cell cultures can be applied to investigations of several aspects of the hypothalamo-hypophysial-gonadal system.

Biological and immunological potencies of lutropin (LH) in human serum: comparative studies using different standard preparations.

The mouse leydig cell in vitro-bioassay for LH, first reported by van Damme, Robertson and Diczfalusy (1974) was modified and applied to female and male serum. Non-parallelism of dose-response curves between serial dilutions of individual male as well as female sera and LH standards was caused by damage to incubated interstitial cells in the presence of human serum. The extent of cell damage- paralleled by an inhibition of testosterone production - was a characteristic of individual human sera rather than a general protein effect. This inhibition could be completely avoided by mild heating of the serum for 15 min at 50 degree C prior to the assay. Using this pretreatment, reliable LH values were obtained for normal males, cycling females and postmenopausal women. Biological LH measurements were compared with RIA-LH potencies. The following LH value were obtained by both methods (mean +/- sd) in terms of mlU 2nd IRP HMG/ml serum: males (n = 35), BIO: 23.3 +/- 9.0, IMMUNO: 11.0 +/- 4.3; cycling females (n = 30), BIO: 30.9 +/- 14.6, IMMUNO: 17.4 +/- 5.9; postmenopausal women (n = 12), BIO: 324 +/- 138, IMMUNO: 117 +/- 43. It could be shown that the use of different reference preparations caused great differences in the ratios of biological to immunological potencies e.g. for male serum 0.9, female serum 0.7-5.6 and postmenopausal female serum 1.1 - 8.8. Irrespective of the standard used, significant differences between the B:l ratios of male, female and postmenopausal female sera were found.

A low molecular weight substance obtained from serum which has luteinizing hormone like activity ("mini LH").

A low molecular weight substance of 1000-2000 daltons has been isolated in dialysates of normal human serum which, on testing in a radio-immunoassay, a radioligand receptorassay and an in-vitro bioassay exhibited LH activity comparable to the range of LH activity found in normal serum. The substance was heat labile. Its reactivity in a RIA suggests peptide nature. No "mini LH" activity could be derived from LH-free serum samples of hypophysectomized patients.

Existence of big and little forms of luteinizing hormone in human serum.

Serum fractions from normal subjects obtained by gel chromatography have been investigated using three different assay systems: radioimmunoassay (RIA), radioligand receptor assay (RRA), and testosterone production assay (TPA). The bulk of immunoassayable and "bioassayable" LH-activity was found in two fractions differing widely in their molecular size. The slower moving component, designated as "little" LH, migrated identical to the radioiodinated pituitary hormone (LER 960) with a molecular weight of about 30,000, while "big" LH appeared in an elution volume consistent with a molecular weight range between 140,000 and 180,000. Concordance was seen between the LH-activities measured in all three assay systems. The RRA/RIA ratio varied between 1.6 and 8.9, the RRA/TPA ratio was close to unity. Treatment with 6 M urea and 0.1% mercaptoethanol and also, exposure to different pH values and salt concentrations did not change the elution position of the two LH components. Also, "big" and "little" LH appeared unaltered after re-filtration and no conversion each other could be found. In another experiment injection of gonadotrophin releasing hormone (Gn-RH) into a male induced a profound shift of LH towards the low molecular weight species. Kinetic uptake studies with "big" and "little" LH using RRA showed identical affinities to the receptor preparation. Ion exchange chromatography of serum, however, did not give two LH components, indicating no major differences in charge properties. This finding could be confirmed by preparative gel isoelectric focusing. The RRA potencies following gel filtration were in good agreement with that applied to the column, however, the immunological activities exceeded that of loaded by a factor 3-4. A new aspect of serum LH heterogeneity is the finding of a low molecular substance (mol. weight approximately 1000) in the outer dialysate of serum, which has LH like activity in all three assay systems.