[In vitro fertilization of human ova].
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Biomedical subjects
Publications and source records attributed to L Hamberger.
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The present four-center investigation involves 516 never-pregnant women in whom a copper-7-intrauterine device (Cu-7-IUD) was inserted. Out of these patients 173 were teenagers at the time of insertion. A "small-sized" Cu-7-IUD (Cu-7-Nullip) - designed for nulliparous women - was randomely used instead of the commercially available Cu-7-IUD (GravigardR) in 76 - 44 of whom were teenagers - out of the 516 women. The incidence of pelvic inflammatory disease (PID) of 9,080 woman-months was reviewed. In the present study the incidence of PID varied from one center to another between 0.9 and 1.4 per cent/year in women wearing Cu-IUDs. When comparing these figures with the estimated incidence (1.2%) in the population of fertile-aged women, it was concluded that the risk for PID was not increased in never-pregnant women using Cu-IUDs, including teenagers as well as women of 20 years of age or more. No difference in the incidence of PID was observed between the GravigardR and the Cu-7 Nullip.
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Human corpora lutea of defined ages were excised at operation, cut into pieces and incubated in the presence of HCG, PGF2 alpha and PGE2 alone or in combination. Following incubation cAMP formation in tissue and medium was determined. HCG-stimulated tissue cAMP content was most pronounced at a corpus luteum age of 7-10 days after ovulation. This stimulation was antagonized by PGF2 alpha in corpora lutea older than 6 days. PGE2 stimulated cAMP formation per se and this effect was more pronounced when HCG and PGE2 were combined. A possible role for PGF2 alpha as a luteolytic substance in the human is suggested.
The isthmic portion of human Fallopian tubes was excised during surgery and the ampullary-isthmic junction was identified. The external, longitudinal and the inner, circular muscle layers of this structure were then mechanically separated. The contractile activity of thin strips from these layers was studied isometrically in organ bath experiments. Administration of various adrenoceptor agonists and antagonists revealed that the responses of the longitudinal smooth muscle are primarily mediated by alpha-adrenoceptors while there is a marked dominance of beta-adrenoceptor-mediated effects in the circular layer. Transmural nerve stimulation to those caused by exogenous noradrenaline, although nerve-mediated inhibition of circular muscle activity was not abolished entirely by propranolol, atropine or guanethidine, but by tetradotoxine, indicating the existence of a hitherto unknown non-adrenergic, non-cholinergic, inhibitory neurogenic mechanism. The results support the concept of a specific sphincter-like function controlling ovum transport at the human oviductal isthmus, and that this sphincter may be controlled by both adrenergic nerves and nerves liberating an unidentified transmitter substance.
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A technique for the mechanical isolation of granulosa cells from the rat ovary is described. Cyclic AMP formation by the isolated granulosa cells of the follicles in various stages of development was studied in response to the administration in vitro of gonadotrophins. In granulosa cells from small to medium-sized follicles FSH but no LH stimulated cAMP formation, while in cells from pre-ovulatory follicles both gonadotrophins had a stimulatory effect. The effects of both gonadotrophins were transient with a maximal response after 15 to 60 min of incubation. In the presence of the phosphodiesterase inhibitor, 3-isobutyl-methylxanthine, the action of FSH was potentiated and prolonged while the response to LH was unaffected. These data indicate that both gonadotrophins activate the adenylate cyclase system of the isolated granulosa cells while FSH in addition stimulates the phosphodiesterase activity. Consecutive determinations of cAMP during and after the pre-ovulatory LH-FSH surge, demonstrated a rise of cAMP levels in granulosa cells from the pre-ovulatory follicles following endogenous gonadotrophin release. cAMP levels remained high or increased until the time of ovulation.
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The contraceptive action of a copper-containing intrauterine device (Cu IUD) is mainly associated with the copper ions released, thus its efficiency varies with the surface area of the copper wire. It is of considerable clinical interest to know the usful life-span of a Cu IUD. Therefore, the concentration of copper in the uterine secretion of women wearing a Cu-7 IUD (Gravigard) with a surface area of the copper wire of 200 sq mm was determined by atomic absorption spectroscopy. No decrease in the concentrations of copper was found in a series of women followed for 4 years after the insertion of a Cu IUD.
Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.
Experiments were designed to examine whether one contraceptive action of the copper intrauterine contraceptive devices (Cu-IUD's) could be associated with an influence on the smooth muscle activity of the Fallopian tube. Sections of the isthmic portion of human oviducts were removed during operation. Preparations from the circular muscle layer were mounted for isometric recording of contractile activity. Subsequently, spontaneous rhythmic contractions appeared within one to five minutes after the preparation procedure was completed. Transmural nerve stimulation or addition of norepinephrine or prostaglandin F2alpha to the Tris buffer medium caused an increase in tone and frequency. Addition of CuCl2 caused a concentration-dependent increase in frequency and a concomitant decrease in amplitude. High concentrations of CuCl2 produced a marked initial increase in frequency, but the rhythmic activity was soon abolished. Metallic copper in the incubation medium caused the same qualitative effects on the smooth muscle preparation as CuCl2. Influences of copper on contractions of rat portal vein preparations were studied in the same way, and similar effects were found. The copper concentration of the secretion in the Fallopian tube was measured by atomic absorption in seven patients wearing CU-IUD's until operation. The levels found were of the same order of magnitude as the concentration of free copper ions which in vitro caused effects on the smooth muscle. It is concluded that copper can affect smooth muscle activity. An influence of copper on the motility of the human oviduct has to be considered as a possible factor contributing to the contraceptive action of the Cu-IUD.
The effect of luteinizing hormone (LH) on the respiration of isolated cumulus cell complexes (the oocyte surrounded by cumulus granulosa cells) obtained from immature Sprague-Dawley rats injected with 10 IU pregnant mare's serum gonadotropin on day 30 was investigated. The cell complexes were isolated from preovulatory follicles of rats killed at specific time intervals on the day preceding ovulation, i.e., on day 32. The samples were incubated in Eagle's tissue culture medium. Oxygen uptake was recorded either with the Cartesian diver technique or with a recently described microspectrophotometric technique using hemoglobin as an indicator. Exposure to exogenous bovine LH in vitro or in vivo or to endogenous LH, i.e., the preovulatory LH-surge, resulted in a marked decrease in respiratory activity of the cumulus cell complex, as revealed by both techniques. The cumuli exposed to LH showed an oxygen uptake of approximately 40-65% of the control cumuli. The results suggest that LH has a direct effect on this cell complex resulting in a decreased oxidative metabolism.
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In order to study substrate dependent respiration oocytes were isolated from antrum follicles obtained from ovaries of prepubertal rats. The oocytes used were either surrounded by 1 to 3 layers of granulosa cells (oocyte + corona radiata), or mechanically denuded from surrounding cells (denuded oocytes). By use of Cartesian micro-diver technique the respiratory rate could be determined in single oocyte + corona radiata cell complexes or in samples of a few denuded oocytes. The oocytes were incubated either in an isotonic Tris-HCL buffer or in a hypotonic phosphate buffer. The rate of respiration was determined without addition of exogenous substrate and with addition of different substrates in varying concentrations. Cellular respiration was linear 2-4 h both in absence and presence of substrate. Both types of oocyte preparations were unable to utilize glucose as substrate. Lactate stimulated slightly the respiration of the denuded oocyte while pyruvate and oxalcetate increased markedly the respiratory activity of both types of oocyte preparations. Succinate had an influence on the oocyte + corona radiata only when the hypotonic medium was used while it was easily utilized in both media by the denuded oocyte. In a preliminary series of experiments the in vitro influence of bovine lutienizing hormone (LH) on the cellular respiration was studied. Both in presence and absence of corona radiata a significant decrease in oxygen uptake was registered, consistently appearing within 30 min after the addition of the hormone. As the relationship of the phenomenon to ovum maturation was not studied, the possible biological meaning remains to be elucidated.