Inhibition of progesterone secretion in cultured human granulosa cells by a low molecular weight fraction of human follicular fluid.

Previous studies demonstrated that a low molecular weight peptide fraction (G10-3) from human follicular fluid (FFl) could inhibit steroidogenesis by rat granulosa cells in vitro. In the present study this FFl fraction was tested upon human granulosa cells. The G10-3 fraction (less than 1000 mol wt) was obtained by sequential gel filtration on Sephadex G50 and G10 of a steroid-free extract of a pool of human FFl collected from various-sized follicles at different stages of the menstrual cycle. Human granulosa cells were obtained from large, healthy follicles in the mid- to late follicular phase of eight patients, and cultured for 4-6 days in the absence or presence of G10-3. In all cases G10-3 produced a dose-dependent inhibition of basal progesterone secretion and, when tested, also of FSH-stimulated progesterone secretion. Inhibition of progesterone secretion appeared to be greater in cells obtained from less mature follicles as compared to cells obtained from follicles that were periovulatory. The results suggest the presence of a low molecular weight luteinizing inhibitor in human FFl.

Isolation of luteinising hormone receptor binding inhibitor from bovine corpus luteum.

A luteinising hormone receptor binding inhibitor (LHRBI) has been purified from bovine corpus luteum (CL). Steroid-free extract of the CL was subjected to successive chromatographies on Sephadex G-50, Q-Sepharose, Orange A dye and metal chelate affinity columns followed by high performance-reverse phase and gel filtration columns. Purification was monitored by the ability of the fractions to inhibit the binding of 125I-human chorionic gonadotropin (hCG) to porcine granulosa cells in vitro. The final isolate showed an 8000-fold enrichment of activity. It was also capable of inhibiting porcine granulosa cell secretion of estradiol and progesterone (P) in vitro. Administration of LHRBI into follicle-stimulating hormone (FSH)-stimulated, immature rats strongly inhibited the ovarian ovulatory response to hCG as revealed by decreased P levels and the number of ova released. The M(r) of LHRBI as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was ca. 15 kDa and the pI was between 5.0 and 5.5.

Regulators of steroid secretion and inhibin activity in human ovarian follicular fluid.

Inhibin activity from human follicular fluid was purified by successive chromatographies on Sephadex G-50, DEAE-Biogel A and orange A dye matrix. Inhibin activity was associated only with the protein(s) that bound to orange A (OrA-2). Daily injection of OrA-2, 1 h prior to hMG into 10-day-old female rats for 4 days caused a significant inhibition of hMG-induced estradiol secretion. In vitro, OrA-2 dose-dependently inhibited the amounts of estradiol secreted by porcine granulosa cells during a 3-h incubation. Orange A-unbound proteins, on the other hand, induced a dose-dependent increase in estradiol as well as progesterone secretion by porcine granulosa cells in vitro. Separation of stimulator from the inhibitor by orange A chromatography led to an increase in the relative inhibin activity (25-50-fold) as well as aromatisation-suppressing activity (60-fold). The results indicate a possible local action of hFF inhibin to regulate aromatisation activity.

Oocyte maturation as regulated by follicular factors.

Follicular fluid (FFl), obtained from 24 women treated with clomiphene/hCG in an in vitro fertilization program, was characterized with respect to steroid hormone levels and oocyte maturation inhibitor (OMI) activity. Three FFl samples apparently were derived from cystic follicles and contained low steroid levels and no OMI activity in an in vitro rat oocyte assay. The remaining 21 follicles contained normal preovulatory steroid levels and mature and generally fertilizable oocytes. In 7 of these follicles the FFl (at 50% concentration) significantly inhibited rat oocyte meiosis, while 14 exerted no OMI activity. The results confirm earlier work on porcine and human FFl, suggesting that the putative OMI activity declines with follicular maturation.

Transvaginal hysterosalpingo-contrast sonography for the assessment of tubal patency with gray scale imaging and additional use of pulsed wave Doppler.

OBJECTIVE: To determine whether the additional use of pulsed wave (PW) Doppler can improve the tubal diagnosis reached with gray scale imaging in doubtful cases. DESIGN: The study is an open, uncontrolled clinical trial of women of childbearing age. SETTING: Clinical environment. PATIENTS: Seventeen female patients (23 to 37 years of age) with diagnosed sterility problems. INTERVENTIONS: The contrast agent SH U 454 was administered transcervically during transvaginal gray scale and PW Doppler sonography. MAIN OUTCOME MEASURES: Hysterosalpingo-contrast sonography by gray scale and by PW Doppler and follow-up chromolaparoscopy (n = 16) or hysterosalpingography (HSG, n = 1) were performed. The diagnostic efficacy of gray scale and PW Doppler were compared with each other and against a conventional control procedure (chromolaparoscopy or HSG). RESULTS: The gray scale findings were confirmed by PW Doppler in 5 cases on one side; confirmed by PW Doppler in 7 cases on both sides; corrected by PW Doppler in 4 cases on one side; and corrected by PW Doppler in 1 case on one side and confirmed on the other side by PW Doppler. In all 17 cases, the tubal findings after PW Doppler were confirmed by chromolaparoscopy or HSG. CONCLUSION: The additional use of PW Doppler in hysterosalpingo-contrast sonography is recommended as a supplement to gray scale imaging (1) in cases of suspected tubal occlusion and (2) in the event of intratubal flow demonstrable only over a short distance.

Influence of gestodene and desogestrel as components of low-dose oral contraceptives on the pharmacokinetics of ethinyl estradiol (EE2), on serum CBG and on urinary cortisol and 6 beta-hydroxycortisol.

A randomized controlled clinical trial was undertaken over a 6-month treatment period with two low-dose combined oral contraceptives (OC) to investigate whether the metabolism and elimination of ethinyl estradiol (EE2) is differently influenced by the two progestational components gestodene (G) and desogestrel (D), an issue which has been very controversial recently. The two formulations contained 30 micrograms EE2 each, together with either 75 micrograms G or 150 micrograms D. Of the 40 young women recruited for each formulation, 31 of each group were available for statistical evaluation. The pharmacokinetics of serum EE2 were studied on day 1, 10 and 21 of cycle 1, 3 and 6. There were no significant differences between the two groups in any cycle with respect to parameters measured. This was true for the distinct intracyclical rise in the mean EE2 serum levels from day 1 to day 10 and the smaller further increase between day 10 and day 21, with no change in this respect between the cycles studied. Respective changes were seen with regard to the area under the EE2 serum concentration curve up to 4 and 24 hours (AUC0-4 and AUC0-24), cmax and tmax of serum EE2. The estrogen-dependent corticoid-binding globulin (CBG) increased similarly in the two groups intracyclically and slightly also intercyclically at all times tested. Except for the first treatment cycle, urinary excretion of cortisol and 6 beta-hydroxycortisol displayed a tendency to lower values intracyclically as well as intercyclically, again with no differences between the two groups. Also, the 6 beta-hydroxycortisol-to-cortisol ratio was not different between the groups, showing a slight tendency to rise from about 4 at the beginning of the medication to around 5.5 at the end of the 6th treatment cycle in both groups. It is concluded that G and D as components of low-dose OCs exert comparable effects on the metabolism and elimination of EE2.

PIP: The question of whether the pharmacokinetics of ethinyl estradiol (EE2) is affected differently by the progestins in low-dose combined oral contraceptives containing gestodene or desogestrel was revisited. 80 randomly allocated women took 30 mcg EE2 and either 75 mcg gestodene or 150 mcg desogestrel for the first 21 days of each cycle for 6 months. Blood samples taken on days 1, 10, and 21 of the 1st, 3rd and 6th cycle, at frequent times for 24 hours after pill intake, were analyzed for EE2, corticosteroid binding globulin, cortisol and 6beta-hydroxycortisol. 31 women in each group completed the study. Minor side effects such as headache, breast tension, acne, and nausea occurred in each group; 1 subject dropped out because of headache, nausea, and hypermenorrhea and 1 because of a hematoma. No significant differences were seen in serum EE2 levels including the rise in mean EE2 on days 1-10, or the smaller rise between days 10-21, or the pharmacokinetic parameters Cmax, tmax, area under the curve (AUC) at 0-4 hours, or AUC at 0-24 hours. There was a maximal variation of 11% in intracyclical increases in serum EE2, but no change in intercyclical variations. There were also no significant differences between groups in the expected estrogen-induced increase in corticosteroid binding globulin. Urinary hydroxycortisol increased slightly over each cycle, somewhat more in the 1st cycle, and a bit more in the desogestrel cycles than in gestodene cycles, but not significantly. This study was contrasted in detail with the reports that prompted the controversy over pharmacokinetics of estradiol during intake of the involved combined pills. The import of the assays for cortisol metabolites is the fact that estradiol and cortisol are metabolized by the same liver cytochrome P450 isoenzyme.

Fertility and pregnancy outcome after myomectomy in sterility patients.

OBJECTIVES: To examine total pregnancy rate, pregnancy rate in relation to pretreatment with GnRH-analogues, the frequency of myoma recurrencies and the influence of size, number and localization of removed myomata on pregnancy rate and outcome in infertility patients after myomectomy. STUDY DESIGN: A comparative, retrospective non-randomized clinical study involving 67 patients with desire for children and no other recognizable infertility factor. Myomectomy had been performed between 1985 and 1993. Most patients had been operated by laparotomy using microsurgical instruments and techniques. Thirty-three patients had been treated with a GnRH agonist for usually 3 months, and in 34 patients the operation was performed without pretreatment. Patients were followed up to June, 1994. All patients were mailed a questionnaire and invited to an ultrasound examination. RESULTS: Thirty-nine of the 67 patients (58.2%) became pregnant, and a total of 51 pregnancies were observed. Of the women who actually conceived, 61.5% did so within the first year. There was no significant difference in pregnancy rates between patients who had been pretreated with GnRH agonists and those who had not. However, 1 year after the operation the group of GnRH-treated women was significantly overrepresented among those already pregnant (P = 0.02). Sonografical examination revealed in 31 out of the 67 patients (46.3%) recurrent myomata > 1 cm in diameter. There was no statistically significant difference in the pregnancy rates between patients with and without recurrencies. However, there was a significant tendency toward a loss or short duration of the pregnancy due to spontaneous abortion and premature delivery in patients with recurrent or persistent myomata (P < 0.01). Pregnancy rate was significantly lower in patients with more than five myomata removed (P < 0.001). In the group with a larger myoma volume the pregnancy rate was significantly higher than in the group with the smaller one (P < 0.01), possibly indicating that the size on removal of myomata is an important factor for infertility patients. Concerning the localization of the removed myomata, no statistically significant difference was found in the pregnancy rates between various localizations. Of the 51 pregnancies, 31 (60.8%) led to a delivery, vaginal in 13 cases (41.9%) and 18 times by Caesarean section (58.1%). Of the pregnancies that were lost, 39.2% were due to spontaneous abortion or ectopic pregnancy. CONCLUSIONS: Our study supports reports on the benefits of myomectomy, performed with the appropriate technique, in patients with otherwise unknown cause of infertility. It shows, additionally, that characteristics of myomata, such as number and size, may influence postmyomectomy pregnancy rates.

Influence of modern low-dose oral contraceptives on hemostasis.

Several reports have appeared on the increased risk of thromboembolic diseases associated with the use of oral contraceptives (OCs). The increased risk of thromboembolism has been related to increased circulating blood levels of certain factors of both the clotting and fibrinolytic systems seen in OC users. These changes have been associated primarily with the estrogen component of the OC preparations. The two new oral contraceptives under study contain reduced levels of ethinyl estradiol, 30 micrograms and each utilizes a new progestogen--75 micrograms gestodene or 150 micrograms desogestrel. A prospective randomized study was performed with 50 women over one year in which several factors of the hemostatic system were investigated; blood samples were taken in treatment cycles 1, 3, 6 and 12 and 6-8 weeks after cessation of therapy. During treatment with both preparations, factors II, VII, IX, X, XI, XII, VIII clotting activity, and prekallikrein were elevated; factor V was not elevated. Antithrombin III which controls these factors, was decreased by 8-10% after 12 months; Protein C which controls factors V and VIII was not changed. Markedly elevated levels of plasminogen and its unaffected inhibitor alpha antiplasmin were seen in the first and all subsequent treatment cycles; this represents increased potency of the lytic system, which can be looked upon as a compensatory mechanism. There were no differences seen between the gestodene and desogestrel preparations regarding changes in the hemostatic system. As with all other low-dose pills, a history of thromboembolism is a contraindication to their use.

[The corpus luteum in the ultrasound image and its endocrine function]. / Das Corpus luteum im Ultraschallbild und seine endokrine Funktion.

The corpora lutea of 38 patients with sterility problems were sonographically measured. In a total of 51 cycles the corpus luteum values were compared with the estradiol (E2) and progesterone (P) plasma levels on the day of sonographic examination (SE). In 49 SEs performed immediately following ovulation the corpus luteum was cystic in 26 cases (means = 14.9 mm) and solid in 23 cases (means = 12.8 mm, p less than 0.05). The postovulatory differences between the corresponding E2 and P values, respectively, were not significant. In the midluteal phase the corpora lutea were cystic in 22 cases (means = 18.2 mm) and solid in 14 (means = 13.4 mm, p less than 0.001). Both E2 (means = 362.8 pg/ml) as well as P (means = 27.7 ng/ml) were higher in the women with cystic than in those with solid corpora lutea (E2 means = 222 pg/ml; P means = 19.4 ng/ml; p less than 0.01 in each case). When the patient collective was split into two groups, with and without stimulation therapy, a significantly higher E2 was only found in the "with therapy" group in patients with cystic corpus luteum in the midluteal phase. The size of the midluteal corpus luteum correlated in particular with the E2 plasma concentrations in all patients (p less than 0.005). Thus, in addition to sonographic measurement of the endometrium, sonographic demonstration of the corpus luteum is also useful in assessing the luteal phase.

Long arm deletions of the X chromosome and their symptoms: a new case (bp q24) and a short review of the literature.

The clinical and cytogenetic data from a 26-year-old female with del(X)(q24----ter) are reported. This breakpoint has not been described yet. Besides this report we give a comparative summary of 24 cases from the literature with different deletions of Xq.

Short-arm deletion of an X chromosome (45,XO/46,XX p--).

A 31-year-old female patient with short stature, signs of gonadal dysgenesis, and slight Turner signs is described with a mosaic 45,XO/46,XX del (X) (qter leads to p11) determined with trypsin Giemsa-banding and C-staining. BUdR incorporation indicated the deleted X to be late replicating.

Ectopic production of placenta-"specific" tissue proteins (PP5 and PP11) by malignant breast tumors.

With use of an enzyme-bridge immunoperoxidase (PAP) technique, an immunohistochemical localization of the two new placenta-"specific" tissue proteins, PP5 and PP11, was investigated in 16 cases of breast cancer. PP5 was localized in the cytoplasm of breast cancer cells in 62.5% of cases examined and PP11, in 46.7%. Thus PP5 and PP11 may be markers for breast cancer and the detection of these proteins in sera from breast cancer patients may be more reliable and useful in the detection and monitoring of breast cancer patients than the determination of SP1, PP10, or PP12, other pregnancy "specific" proteins.

The sonographic and endocrinologic evaluation of the endometrium in the luteal phase.

Follicular development and the endometrial thickness were determined sonographically in 19 outpatients with different causes of sterility, treated during natural or stimulated cycles. The estimates of the endometrial thickness, assessed by ultrasound in the mid-luteal phase, and the levels of 17 beta-oestradiol and progesterone in the same patients sampled on the day of sonography were compared. Five patients became pregnant (group 1) and showed good progesterone values. Eight patients who were not pregnant showed progesterone values above 15 ng/ml in the mid-luteal phase (group 2). The progesterone values of group 3 were below 15 ng/ml by definition. The mean endometrial thickness in group 1 (means = 11.3 mm) and group 2 (means = 11.8 mm) were significantly higher than that of group 3 (means = 8.3 mm). The sonographic measurement of the mid-luteal endometrium thickness serves as an additional criterion for the evaluation of the secretory phase of the menstrual cycle.

Purification of "inhibin" from human ovarian follicular fluid.

Following the earlier demonstration of inhibin-like activity in human ovarian follicular fluid a method for its purification to apparent homogeneity is described. The fluid was converted to acetone powder and subjected sequentially to ammonium sulphate fractionation, gel chromatography on Sephadex G-200, continuous gradient ion-exchange chromatography on DEAE-cellulose, first with a pH gradient from 8.0 to 4.0 and then with a NaCl gradient to 1 M at pH 5.2. The active fraction from this step was subjected to gel filtration on Sephadex G-100 and finally passed through an Amicon Centriflo membrane CF-25 (cut off point: 25,000 m.w.). The ultrafiltrate was homogeneous by SDS-polyacrylamide gel electrophoresis, had a molecular weight of the order of 23 000 and was capable of suppressing serum gonadotrophin levels in the castrated male rats in as low a dose as 25 microgram/rat.

Inhibition of follicle stimulating hormone binding to granulosa cells in vitro by human follicular fluid.

Evidence for the existence of a low molecular weight (less than 10,000 daltons) factor in human follicular fluid that inhibits binding in vitro of FSH to ovarian granulosa cells is presented.

Short arm deletion of an X chromosome, 46,XXp-.

A 27-year-old patient of short stature with primary amenorrhea and other slight Turner signs showed a 46,XX,del(X) (qter leads to p11:) karyotype, identified by a combination of fluorescence and giemsa-banding technique. By BUdR incorporation the deleted X chromosome was shown to be the late replicating one.

[Studies on the dependence of intrafollicular pressure on the pressure in the intraovarian-vascular-system and tissue, carried out in vitro on human ovaries (author's transl)]. / Untersuchungen über das Verhalten des intrafollikulren Druckes in Abhängigkeit vom Druck im ovariellen Gefässystem und Gewebe. Durchgeführt an in vitro perfundierten menschlichen Ovarien.

The intrafollicular, intraovarian and intraarterial hydrostatic pressures were measured in vitro on human ovaries in the vollicular ripening phase. It has been established that in the vascular system as well as in the intraovarian tissues pressure variations occur spontaneously. Thus for example under, the influence of epinephrine, norepinephrine, prostaglandine F2 alpha and oxytocine, the tonicity of the vascular system increases and does so the number of spontaneous contractions rather noticeably, particularly under the influence of prostaglandine F2alpha, and under the various catecholamines an increase of frequency and amplitude has been observed. Any increase of pressure in the intraovarian vascular system and tissues will effect all the follicles not yet in the preovulatorian phase, in which they cause a similar pressure increase. However, follicles imminently preovulatorian have not shown such pressure increase because the increase of the "liquor folliculi" runs parallel to the increasing elasticity of the follicle walls. The findings are discussed in this article with a particular view of the biophysical aspects of ovulation.

[Transvaginal contrast hysterosonography. A new diagnostic procedure for the differentiation of intrauterine and myometrial findings]. / Die transvaginale Hysterokontrastsonographie (HKSG). Ein neues diagnostisches Verfahren zur Differenzierung intrauteriner und myometraler Befunde.

In 30 patients with sterility problems, menstrual irregularities, and/or suspected tumor the uterine wall was evaluated by transvaginal hystero-contrast-sonography (CoSy). These findings were compared with those of hysterosalpingography (HSG) or chromo-laparoscopy which were done during the same anesthesia. In 6 patients there was no coincidence, and additional information of relevance could be achieved by the other procedure. One case of submucous and an other of intramural fibroid were exactly differentiated by the transvaginal CoSy. This was not possible by the HSG. In addition two large intramural fibroids were exactly localized by CoSy, where as this couldn't be demonstrated endoscopically. Two uteri arcuati have not been seen by CoSy, but were recognized by HSG. So we recommend the transvaginal CoSy of the uterus as a pretherapeutic procedure for the differentiation and localization of intracavital and myometrial findings. At least at moment it seems to be that the image of light uterine malformations could be better demonstrated by conventional diagnostic methods.