[Secretory protein pattern and implantation readiness of the endometrium].
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Biomedical subjects
Publications and source records attributed to H M Beier.
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In this study, the uterine protein patterns of greater than 200 patients of the sterility treatment programme of the Department of Obstetrics and Gynaecology, University of Ulm were analysed by the technique of uterine secretion electrophoresis (USE) of minute quantities. These analyses reveal three protein patterns, typical for the equivalent phases of the menstrual cycle: intermediate phase-, proliferative phase- and secretory phase. There appear up to 63 protein bands, most of which represent proteins less than 68 kd. Those separation areas below 68kd show characteristic 'families' of protein bands, which typically constitute the three phase patterns. As a result of our study, a secretory phase pattern lasting 10 days is demonstrated, suggesting a remarkably long 'receptive phase' for the initiation of implantation. The fully expressed secretory phase pattern appears from day 15 to day 25 of the ideal menstrual cycle (28 days length). The 'implantation window' of the endometrium seems to remain open for a surprisingly long period of time. This insight sheds light on the success of embryo transfer after IVF when the cleavage stage embryo arrives in the uterine cavity several days earlier than after normal fertilization. Such early exposure to the uterine milieu nevertheless does not preclude implantation.
In this study, we present further experiences with the intravaginal culture (IVC) of oocytes and cleavage stages. IVC is a new technique for in-vitro fertilization, its principle consisting of fertilization of oocytes in an air-free plastic capsule which is placed into the maternal vagina. In a total of 45 patients, 22 were treated by IVC and 23 by a conventional in-vitro fertilization technique (IVF). The pregnancy rates for IVC and IVF amounted to 22.7% (5/22) and 17.4% (4/23), respectively. Our results indicate that IVC is a valuable new method for treatment of special cases of sterility. Furthermore, extracorporal stress factors such as light and low temperature can be minimized by IVC. We also consider the psychological factor for the patients as very important since the mother can actively participate in the early development of the conceptus.
The effects of the progesterone antagonist (AG) onapristone ZK 98.299 on the uterine cervix were investigated by electron microscopic examination in guinea-pigs during late pregnancy. Treatment with the AG led to dissolution, splitting up and dissociation of collagen fibres as well as expansion of the inter-fibrillar spaces due to oedema. This was associated with an increased number of polymorphonuclear granulocytes, macrophages and mast cells as well as with the appearance of highly active fibroblasts. The possible roles of these cells in the dissociation of collagenous fibres are discussed. Comparing the AG-induced prominent transformation of the uterine cervix with the morphological signs characteristic of cervical ripening in untreated guinea-pigs at term pregnancy, no significant differences could be observed. This suggests that AG brings about ripening and dilatation of the cervix in a physiological manner and may be, after thoughtful toxicological screening, an extremely useful agent for obstetrical indications.
Compared to in vivo development, in vitro culture of mammalian embryos results in developmental retardation. To study the potential of reversibility of growth retardation we investigated ultrastructurally day 3 rabbit embryos after 1 day in vitro, and cultured embryos that were transferred after culture into recipient rabbits for 1 day. Morphology was compared with ultrastructure of noncultured controls. The noncultured embryos were compacted morulae; the characteristic ultrastructure is described in detail. After 1 day in culture, morulae had developed into early blastocysts. However, unlike the results in vivo, expansion of the blastocysts did not occur and some of the cultured embryos developed trophoblast herniations. In all cultured embryos morphological signs of degeneration were seen with swollen mitochondria, with a dense, granular appearance of the cytoplasm, and with an increase in number of lysosomes. Transfer of cultured blastocysts into uteri of day 3 pseudopregnant recipients resulted in ultrastructurally intact and expanded blastocysts. Transfer into uteri of day 4 pseudopregnant recipients and into uteri of nonpregnant recipients, however, did not yield reversibility of the unphysiological features suffered during the previous time in culture. Although blastocysts were well expanded, distinct signs of injury to the blastomeres were present, proceeding from loss of complete blastomeres to structural changes such as large lamellar structures, dilation of smooth endoplasmic reticulum and Golgi complexes, and clumping of mitochondria. We conclude that developmental retardation during in vitro culture is accompanied by distinct morphological changes. As soon as 24 hr after transfer, these changes can be reversed. This compensation, however, is achieved only if embryos are transferred into recipients that are adapted to the embryo's developmental stage, which is not identical to the embryo's chronological age. Our findings demonstrate that the period of 24 hr of in vitro development matches only a few hours of in vivo development.
Morphological and biochemical changes occurring in rabbit endometrial epithelial cells when placed in culture were investigated. Cells were examined by scanning- and transmission electron microscopy and freeze-fracture. Morphologically, cultured cells are shorter and broader than the columnar epithelial cells in vivo, but retain their polarity as indicated by the presence of apical microvilli and a well-developed junctional belt. To study changes in biochemical function, proteins synthesized by cells in primary culture were analyzed by two-dimensional gel electrophoresis. Proteins were labeled during a 24-h incubation with 35S-methionine and gels examined by fluorography. The pattern of proteins changed after cells had been in culture for 48 h. On day 3 new proteins were synthesized and several protein species labeled during days 1 or 2 of culture, including uteroglobin, no longer appeared. On days 3-8 of culture the protein patterns were similar. Addition of progesterone, estradiol, prolactin, or combinations of these hormones to the culture medium for 24-144 h failed to elicit consistent changes in the pattern of labeled proteins established after 3 days of culture. Minor differences in protein patterns among unrelated cultures appear to have been derived from the original cells of the culture. These results indicate that after 48 h in primary culture, cells grown in vitro resemble endometrial epithelial cells morphologically, but no longer reflect functionally the character of epithelial cells in the uterus.
Adverse effects of simultaneous exposure to visible light and room temperature were investigated in rabbit early cleavage stages and morulae. Routine laboratory conditions were mimicked as close as possible. Embryonic development was assessed by cell proliferation via incorporation of tritiated thymidine, by gross morphology, and by electron microscopy. Damage was detectable in both stages at 1-hour exposure by statistically significant impaired cell proliferation. Compared with single exposure to each individual stressor, combined exposure to light and room temperature amplified detrimental effects. Ultrastructural analysis of light-exposed cleavage stages revealed no indication of cell injury at 4-hour exposure. Gross morphology proved to be too inaccurate to evaluate damage imposed by exposure toward both physical factors investigated.
We present in this study our first experience with intravaginal culture (IVC) of human oocytes as a modified technique for in-vitro fertilization. Up to 4 oocytes and washed, pre-incubated spermatozoa (final concentration: 0.1-0.2 x 10(5)/ml) are pipetted into a plastic capsule. The capsule is closed, avoiding any air inclusion. It is placed into the maternal vagina for up to 50 hours, held in place by a diaphragm. The first 15 patients treated by IVC achieved a fertilization rate of 58% (35/60). Following transfer of the cleavage stages, 3 intact clinical pregnancies were established: 2 in the group with tubal sterility factors and 1 in the group of patients with endometriosis. In the group with andrological causes of sterility, one beta-hCG-positive reaction was detected. However, no intact clinical pregnancy was achieved. Our preliminary results show IVC to be a promising alternative in particular cases of sterility treatment. In contrast to the conventional technique, IVC minimizes exposure of gametes to factors which may affect their early development, such as light or low temperature. Furthermore, there is an important psychological factor to be considered, as the patient actively participates in the incubation period of in-vitro fertilization.
Corpora lutea and ovarian stromal tissue were analysed for prostaglandin (PG) concentrations and activities of enzymes involved in PG metabolism at 8, 10, 12, 13 and 15 days after induction of ovulation. In CL of pseudopregnant rabbits, the PGE-2-9-ketoreductase (PGE-2-9-KR) was highly active on Days 10, 12 and 15 when compared with Day 8 (P less than 0.01; P less than 0.001; P less than 0.05). In pregnant animals PGE-2-9-KR activity was only increased on Day 12 (P less than 0.05) but declined to basal levels on Days 13 and 15. Comparing PGE-2-9-KR activity of pseudopregnant and pregnant animals, a significant elevation was found on Day 15 of pseudopregnancy (P less than 0.025). Activities of PG-15-hydroxydehydrogenase did not exhibit any significant changes with time in pseudopregnant or pregnant rabbits. PGE-2 concentrations were increased on Days 12, 13 and 15 (P less than 0.025) when compared with Day 8. Changes in PGF-2 alpha concentrations paralleled those of PGE-2-9-KR. The concentrations of PG metabolites 13,14-dihydro-15-keto-PGE-2 and -PGF-2 alpha were lower than those of the primary PGs and did not show stage-specific changes in pseudopregnant and pregnant animals. These results demonstrate that the rabbit CL possesses enzymes to convert PGE-2 to PGF-2 alpha and to metabolize both PGs. PGE-2-9-KR may be involved in regulating the PGF-2 alpha/PGE-2 ratio and possibly in controlling the life-span of the corpus luteum.
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Day 4 rabbit blastocysts were cultured in Ham's F-10 medium supplemented either with homologous serum or uterine flushings. Development was assessed by leucine and methionine incorporation at 24 h and 48 h, respectively, after initiation of culture and compared with day 4 and day 5 non-cultured controls. After 24 h in culture, incorporation data were similar for both media groups. After 48 h, a significantly higher protein synthesis activity was found in blastocysts which had access to uterine secretions in vitro. However, incorporation levels of day 5 controls were not reached. Modes of incorporation of leucine and methionine revealed to be very similar in all experimental groups but ranged on a different level. Leucine was incorporated on a 5.5-fold higher level than methionine. We conclude that (i) supplementation of culture media with uterine secretions is beneficial for blastocyst development, and (ii) further studies should focus on non-serum components within the uterine fluid and their significance for embryonic development.
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The effect of the synthetic steroid ZK 98.734, an anti-progesterone with high affinity for the progesterone receptor, on uteroglobin distribution in the rabbit endometrium has been studied by means of immunocytochemistry. Rabbits were treated with ZK 98.734 during the second, third and fourth day of pseudopregnancy. From the fifth up to the eighth day of pseudopregnancy the uteri were processed for immunocytochemistry using the peroxidase--antiperoxidase (PAP) and protein A--gold techniques. Uteroglobin synthesis and release could be inhibited by the anti-progesterone treatment. On day 5 and 6 there was no labelling of the uterine secretions and only a few diffusely labelled non-ciliated cells could be seen in the surface and glandular epithelium. The inhibition was reversible in so far as on day 7 and day 8 the rabbit endometrium exhibits a clear labelling of the uterine secretion as well as an increase in positive reaction in the epithelial cells lining the glands. In all treated animals the intracellular uteroglobin labelling was confined to the Golgi complex and secretory vesicles with a significant increase from the fifth to the eighth day of pseudopregnancy. Together with the described morphological changes these results indicate that ZK 98.734 is capable of inducing a delayed secretion in the rabbit endometrium, which is comparable to the delay in secretion caused by post-coital oestradiol treatment. However, the antigestagen effect is probably due to a different mechanism of endocrine interference with pre-implantation. The most exciting consequence, so far, is the prolongation of progesterone action after the anti-progesterone treatment had ended.
In an effort to characterize the effect of clomiphene citrate (CC) on the human endometrium, we took biopsy specimens of the endometrium 24 to 48 hours after CC treatment (100 to 250 mg/day for 5 consecutive days). Nineteen biopsy specimens were taken from 19 patients. Fifteen of the patients suffered from anovulatory infertility associated with oligomenorrhea or normal cycle length. The other four patients were amenorrheic, two in association with hypogonadotropic hypogonadism and two with hypergonadotropic hypogonadism. The histopathology of all samples was evaluated with the use of light microscopy, including periodic acid-Schiff (PAS) and PAS-diastase staining for glycogen demonstration. All samples were also examined with the use of scanning electron microscopy (SEM). Serum levels of estradiol (E2), progesterone (P), luteinizing hormone, and follicle-stimulating hormone were determined on the day of biopsy. In 10 of the 19 biopsy specimens, local or diffuse signs of early secretory events were demonstrated by the presence of subnuclear vacuolization and glycogen in the glandular epithelial cells. SEM corroborated these findings of advanced secretory changes by demonstrating apical protrusions at luminal epithelial cells and secretory products within the glands' openings. The E2 levels ranged between 110 and 1500 pg/ml (mean, 371 pg/ml) and P levels were either undetectable or less than 1.1 ng/ml. The two patients with hypogonadotropic hypogonadism both exhibited the same phenomena; those with primary ovarian failure had atrophic endometrium even after high-dose CC treatment. This observation, together with the low P levels detected, indicating the lack of luteinization, suggests a possible direct effect of CC on the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)
The effects of various concentrations of nicotine on the in vitro development of 1-cell rabbit preimplantation embryos and on the DNA-synthesis of 4-day-old rabbit blastocysts are investigated. Exposure of rabbit preimplantation embryos to concentrations of nicotine higher than 1 X 10(-3) M results in a marked decrease in the in vitro development and in DNA-synthesis. Concentrations of nicotine below 1 X 10(-3) M have no effect on these early embryos. It can be concluded that the concentrations of nicotine which exert a direct embryotoxic effect are higher than the concentrations that may be expected in the blood circulation of humans considered to be "normal smokers".