Interaction of newly synthesized antiprogesterone ZK98299 with progesterone receptor from human myometrium.

We have undertaken characterization of binding of the newly synthesized progesterone receptor (PR) antagonist ZK98299 in the cellular fractions of human myometrium. Specific [3H]progesterone and [3H]ZK98299 binding was observed in the cytosol and the nuclear fractions, and could be competitively replaced by either of the steroids in their radioinert form. Although PR occupied by both steroids exhibited nuclear uptake, the extent of nuclear binding was lower with [3H]ZK98299-receptor complexes. The binding of both ligands to PR was a function of the duration of incubation and the protein concentration: it was saturable at 3-6 nM steroids with a dissociation constant of approximately 2 nM. However, the number of ZK98299 binding sites (72 fmoles/mg protein) was lower compared to that of progesterone (322 fmoles/mg protein). The relative binding affinity (RBA) of ZK98299 for the nuclear PR was about 33% that of progesterone. The results of our study suggest that ZK98299 binds to PR in the cytosol and the nuclear fractions. The antiprogestin effects of ZK98299 reported in the literature are PR-mediated and may result from suboptimal nuclear binding/retention of antiprogestin-receptor complexes. Since this study did not involve isolation and study of individual PR isoforms, PR-A and PR-B, the present data should be viewed as representing an average of contributions by the two receptor forms.

Treatment with a progesterone antagonist ZK 98.299 delays endometrial development without blocking ovulation in bonnet monkeys.

The effects of an antiprogestin ZK 98.299 (onapristone) on serum levels of estradiol and progesterone, and on the endometrial morphology were studied in adult bonnet monkeys. Twelve animals having menstrual cycles of normal duration (24 to 30 days) were randomly distributed into 4 equal groups. The animals in Group 1 were treated (s.c.) with the vehicle (benzyl benzoate: castor oil, 1:10), and in Groups 2, 3 and 4 with 5 mg, 10 mg, or 20 mg ZK 98.299 once-a-week, respectively. Treatment was initiated on day 1 of the menstrual cycle and each animal in Groups 1, 2 and 3 was treated for two consecutive cycles. Since the treatment cycle length of animals in Group 4 was considerably prolonged, they were treated for one menstrual cycle only. Endometrial biopsy was taken around day 20 of the second treatment cycle of first three groups and around day 50 of the 4th group of animals. Treatment with vehicle or 5 mg ZK 98.299 had no significant effect on the menstrual cycle length. Treatment with 10 mg dose had no effect in two animals and prolonged the cycle length in one, whereas, further increase in the dose to 20 mg prolonged the cycle length in all the animals. The duration of menses was generally reduced. Treatment with vehicle or different doses of ZK 98.299 had no effect on ovulation. In animals treated with 5 or 10 mg dose, the pattern of mid cycle rise in serum estradiol levels and progesterone levels during the luteal phase of both treatment cycles were comparable to those of vehicle-treated animals and were suggestive of normal ovulatory cycles. On the other hand, in animals treated with the higher dose (20 mg/week), progesterone levels during the luteal phase were significantly reduced and were indicative of luteal insufficiency. The hormonal data during the treatment period of this group of animals was suggestive of two distinct ovarian cycles indicating that the menstrual bleeding during the treatment period was probably very scanty. Treatment with ZK 98.299 impaired the endometrial development in a dose-dependent manner. In vehicle-treated animals, the endometrium had large and tortous glands with secretions. Treatment with ZK 98.299 caused atrophic changes in the glands as well as in the stroma. The height of the epithelial cells was markedly decreased and they became small and inactive. This study, therefore, suggests that treatment with low doses of antiprogestin ZK 98.299 at weekly intervals does not block folliculogenesis or ovulation, but has an inhibitory effect on the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiprogestin ZK-98.299 and progesterone display differential binding characteristics in the human myometrial cytosol.

To investigate whether the synthetic progesterone antagonist ZK-98.299 binds to progesterone receptor or also has distinct binding sites, the binding characteristics of ZK-98.299 were compared with those of progesterone in the human myometrial cytosol. [3H]ZK-98.299 and [3H]progesterone showed specific binding in the myometrial cytosol and the binding of each radiolabelled ligand could be displaced with the respective ligand in a dose-response manner. However, while the binding of [3H]progesterone could be completely blocked with progesterone or ZK 98.299, the binding of [3H]ZK-98.299 could not be displaced more than 50%. The non-specific binding of [3H]ZK-98.299 was very high as compared to that of [3H]progesterone. Using [3H]progesterone, the relative binding affinity (RBA) of progesterone was more than that of ZK 98.299, whereas using [3H]ZK-98.299 the RBA of ZK 98.299 exceeded that of progesterone. Treatment of myometrial cytosol with increasing concentrations of -SH-modifying agents (iodoacetamide (IA) 0-10 mM or N-ethylmaleimide (NEM) 0-1000 nM) decreased the binding of progesterone by over 80%, whereas similar treatment did not have appreciable effect on the binding of [3H]ZK-98.299. Although both preformed ligand-receptor complexes were relatively stable in the presence of IA and NEM, the [3H]progesterone-receptor complex was more sensitive as compared to the [3H]ZK-98.299-receptor complex. The addition of 20 mM molybdate in the cytosol had a protective effect against the -SH-modifying agents. [3H]ZK-98.299 and [3H]progesterone-receptor complexes also showed differential stability when incubated at elevated temperatures (25 degrees C and 37 degrees C), [3H]ZK-98.299-binding sites being more thermolabile as compared to [3H]progesterone binding sites. Prior occupation of the receptor by the two ligands gave the complexes the ability to resist an elevated temperature of 25 degrees C. Moreover, molybdate stabilized both the liganded and unoccupied receptors at 25 degrees C. When the ligand-receptor complexes were applied onto a prefocused polyacrylamide gel, the progesterone and ZK-98.299-receptor complexes were resolved and focused at pH 7.2 and 8.4, respectively. The results of this study suggest that although progesterone and ZK-98.299 are mutually competitive for binding to progesterone receptor, ZK-98.299 also has distinct binding sites.

Volume of semen as a parameter of its quality.

A total of 1655 semen samples obtained from men who underwent initial semen analysis before enrolling for IVF-ET programme were analysed. The semen samples were categorized arbitrarily on the basis of volume as high (> 4.5 ml), normal (1.5-4.5 ml) and low (< 1.5 ml). The semen parameters studied vis-a-vis volume of the sample were viscosity, osmolality, motility, sperm count and morphology. The different volume groups were also compared to the overall quality of the sample. The study showed that high and low volume semen were associated with high incidence of spermatozoa exhibiting subfertile characteristics. Normal volume per se did not indicate the quality of the sample, however high and low volumes were indicators of semen of subfertile quality. There was an inverse correlation between osmolality and volume of semen sample. Thus the parameter 'volume' gives a rough estimate of the quality of the sample.

Relevance of plasma fibrinogen estimation in obstetric complications.

We present a study of fibrinogen levels in 133 patients who were prone to develop disseminated intravascular coagulation as a result of an underlying complication of pregnancy such as abruptio placentae, pregnancy-induced hypertension, missed abortion, septic abortion, intrauterine fetal death, vesicular mole and amniotic fluid embolism. A high incidence of hypofibrinogenemia was found in cases of abruptio placentae (43.9%) and pregnancy-induced hypertension (25%). Hypofibrinogenemia occurred in 10% cases of intrauterine fetal death within 4 weeks of fetal demise. The use of this simple investigation makes possible the diagnosis of hemostatic failure and also helps to guide replacement therapy during the fibrinopenic state. There were 4 maternal deaths and 12 perinatal losses in this study.

Localization and detection of ovarian follicular fluid protein in follicles of human ovaries.

Human ovarian follicular fluid protein has been partially purified and the active fraction designated as hGF2. Using specific polyclonal antiserum to hGF2, it was observed to be localized immunohistochemically in the granulosa cells of medium but not large follicles of human ovary. The hGF2 levels were estimated by ELISA in serum and follicular fluid of 10 gonadotropin-stimulated women recruited for IVF-ET programme. The results revealed a 3-fold increase in the concentration of hGF2 in follicular fluid compared to that in serum of these patients. These data indicate that the protein is secreted by granulosa cells and plays an important role in the regulation of follicular maturation and ovulation.

In vitro decondensation of nuclear chromatin of human spermatozoa: assessing fertilizing potential.

Spermatozoan nuclear chromatin is in a highly condensed state prior to fertilization. In vivo decondensation occurs in the ooplasm and is essential for successful fertilization and the formation of male pronucleus and the zygote to occur. The chromatin of spermatozoa and nucleus can undergo in vitro decondensation with sodium dodecyl sulfate (SDS) and 6 mM ethylene diamine tetraacetic acid (EDTA). The ability of sperm to decondense in vitro was compared with their ability to fertilize human oocytes in vitro. Spermatozoa from normal samples were studied for their decondensation ability as regards their fertilizing performance in an in vitro fertilization (IVF) program. Fertilization occurred when the decondensation percentage of sperm nuclear chromatin was more than 70%. The effective sperm count was significantly (p less than 0.05) lower in the unfertilized group. This is a new diagnostic technique to assess sperm-fertilizing potential at the initial evaluation of the male.

Ectopic pregnancy after tubal sterilization.

We report 13 cases of ectopic pregnancy following tubal ligation out of 287 ectopic pregnancies seen during a six year period (1984-1989). These findings suggest that tubal sterilization does not invariably confer infertility. Ectopic pregnancy must not be disregarded in women who have undergone tubal ligation, especially if two or more years have elapsed since the sterilization.

PIP: This study of ectopic pregnancy reports an incidence of 13 (4.53%) out of 287 ectopic pregnancies between 1984-89 in a Bombay hospital which occurred after tubal sterilization. 6 had a ruptured ectopic pregnancy, 5 unruptured, and 2 with a chronic ectopic gestation. Bilateral salpingectomy should be performed when exploring for an ectopic pregnancy after sterilization. Although the failure of voluntary sterilization is small, 15-20% are likely to be ectopic pregnancies. In this study, 84.6% occurred 1 year after sterilization. Physicians should be alerted to the possibility of ectopic pregnancy, particularly if 2 or more years have elapsed, because of the maternal mortality risk. The risk of ectopic pregnancy is 12.5% for women undergoing tubal ligation had they continued previous contraception, which is less than observed among normal women. An uncorrected incidence rate of .64/10,000 sterilizations has been reported and 7.7/100 pregnancies conceived after tubal ligation. The explanation given is that recanalization or formation of a tuboperitoneal fistula occurs. The opening is small enough for sperm to pass through but too small for the fertilized ovum, which results in implantation in the distal tubal segment. Another possibility is the formation of blind pouches or slitlike spaces in the abnormal reconstitution of the tubal lumen, which increases the likelihood of ectopic implantation. There may also be development of tuboperitoneal fistulas associated with endometriosis, particularly when the site is within 4 cm of the cornua, which is a probable point of implantation. Fluid movement within the remaining tubal segments may influence implantation. Sterilization was performed in 92.3% of the study patients in conjunction with medical termination of pregnancy or puerperium. When sterilization is performed during the postpartum period, the incidence of ectopic pregnancy is higher because of incomplete occlusion of the tubal lumen due to the edematous, friable, and congested fallopian tubes. A recommended procedure in laparoscopic sterilization to avoid failure of silastic band application is to "milk" the fallopian tube several times by drawing it in and out of the applicator sheath before applying the band. Then, examine carefully for a vertical crease between the 2 limbs of the loop and blanching of the banded tubal segment. Extra caution and meticulous technique are required to avoid failure. Physician and patients awareness about the possibility and risk of extra- and intrauterine gestation following all methods of sterilization is necessary.

Gamete intrafallopian transfer: A preliminary experience.

BACKGROUND: After in vitro fertilizationand embryo transfer for tubal infertility, gamete intrafallopian transfer has been introduced for patients with non-tubal infertility. However, the gametes need to be transferred in 2 to 5 minutes and the distance between the operating theatre and tissue culture laboratory delayed its introduction at our hospital. METHODS AND PATIENTS: To overcome this problem we designed a box in which gametes could be stored. Using gametes taken from this box and employing the standard technique, we achieved 5 pregnancies in 39 infertile women. RESULTS: From 41 treatment cycles, 39 women underwent oocyte retrieval. Five pregnancies were achieved of which 4 delivered live births at full term and 1 ended in abortion. Our first gamete intrafallopian transfer baby was born on 6 January 1988. CONCLUSION: The gamete intrafallopian transfer technique can be successfullyadapted for India.

Semen characteristics of asymptomatic males affected by Trichomonas vaginalis.

Trichomonas vaginalis is a sexually transmitted anaerobic parasite which causes vaginitis and cervicitis. The present study was carried out to determine the incidence of Trichomonas infection in semen samples of asymptomatic men and also to understand the changes of semen characteristics in the infected individuals before treatment and after treatment with metronidazole (Flagyl, 400 mg x 3x a day for 10 days). The semen obtained from 1131 men was examined for different characteristics and Trichomonas were detected in wet smear preparations in 50 cases (4.42%). The characteristics of semen in them was compared with 52 normal semen samples. Statistical analysis showed that the seminal fluid viscosity and percentage particulate debris was increased significantly in the infected group (P less than 0.001). There was no significant change in the pH of semen. Spermatozoan motility and morphologically normal forms were decreased significantly (P less than 0.001), spermatozoan viability was altered, and there was a significant change in membrane integrity (P less than 0.001) as indicated by the hypoosmotic swelling test. The significant improvement in semen characteristics were seen in 25 cases after a single course of treatment. Therefore, it is possible that in some cases, the infertility seen in asymptomatic individuals may be due to infection by Trichomonas.

Ultrastructure of the cortex in the human egg.

Two distinct types of cortical granules were discerned in the human oocyte. The first type, G1 granules, had a mean diameter of 350 nm, contained a uniformly compacted, electron-dense substance; these granules were probably synthesized even before the oocyte was aspirated from mature follicles and their contents were never found to be secreted in any of the preovulatory (immature), unfertilized and fertilized eggs examined. The G2 granules measured 450 nm (mean diameter), had a granular content and were found to be synthesized and secreted at all the stages of egg development studied. Endocytosis was also evident in the unfertilized and fertilized eggs. The secretion of the G1 cortical granules even before fertilization suggests that they may have an additional role, rather than merely contributing to the blockade of polyspermy.

Binding characteristics of progesterone and antiprogestin ZK 98.299 in human endometrial and myometrial cytosol.

The binding of ZK 98.299, a synthetic progesterone antagonist, with human endometrium and myometrium cytosol was studied and compared with that of progesterone. Progesterone showed specific saturable binding to its receptors in both endometrium and myometrium. ZK 98.299 and progesterone were mutually competitive for binding to progesterone receptors; however, the relative binding affinity of ZK 98.299 was 16% that of progesterone. ZK 98.299 exchanged the progesterone-labelled receptor sites. [3H]ZK 98.299 showed specific binding which was linearly related to the cytosol protein concentration. The binding was not saturable at 15 nM of ligand. The binding capacity and binding affinity of ZK 98.299 receptor was less than that of progesterone. Progesterone also partially displaced the binding of [3H]ZK 98.299. This study suggest that ZK 98.299 and progesterone both bind to the same protein. However, whether ZK 98.299 binds to progesterone receptors alone or even to other functionally related sites is not known. It appears that ZK 98.299 when present in higher concentration than progesterone would be an effective receptor ligand.