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In Germany the application of assisted reproductive techniques (ART) is regulated by federal legislation. Compared with the international situation the "German Embryo Protection Law" is very "restrictive" and various methods of ART are prohibited, e.g. oocyte/embryo donation, embryo cryopreservation and Preimplantation Genetic Diagnosis (PGD). Furthermore, in Germany only 1 to 3 fertilized oocytes may be cultured to embryo. All these embryos then have to be transferred into the uterus of a particular patient. Additional fertilized oocytes can only be cryopreserved in a pronuclear state. The success rate of ART has increased significantly over the past few years owing to the introduction of blastocyst cultures and the selection of 1 to 2 good quality blastocysts for embryo transfer. Furthermore, the transfer of only 1 to 2 blastocysts effectively reduces the risk of high rank multiple pregnancies. In Germany, however, the selection of only a few good quality blastocysts for transfer is prohibited by law. New laboratory techniques, e.g. pronuclear scoring and polar body biopsy screening for aneuploidy are in accordance with German law. The application of these methods provides a selection of "good quality oocytes" and seems to increase the overall success rate. Further studies are required, however. The success rate, quality and cost effectiveness of ART in Germany appears compromised when compared with many other countries. What is more, in contrast to the international situation research and development in ART in Germany has been decreasing constantly over the past few years, due to the inappropriate regulations of the German health care system and the insufficient support given to university-based centers.

This communication describes the in vitro maturation and in vitro fertilization of bovine follicular oocytes. Further development of the embryos was achieved by using a granulosa cell culture system. The in vitro development of oocytes to morula/blastocyst stages obtained from individual cows was compared to the results of pooled simultaneous cultured oocytes and to our over-all results of this method. While there were no statistical differences in the developmental rates between these three groups (individual cows: 28.1%, simultaneous pool: 34.0%, over-all results: 32.7%) marked differences were found between the 22 animals investigated separately. These results indicate that there were great individual variabilities due to the oocyte population comparable to the variations in ovarian response to superovulation.

Experimental studies on animal models are necessary for the maintenance of practice and experience on IVF and ET, before starting an IVF program. Only so the handling skill of gametes zygote and human embryo culture can be improved. We have started for this reason with IVF on mouse oocytes of CB6F1 Han hybrid strains and used modified Ham's F-10 with 0.75 BSA as culture medium. Until December 1988, 3652 oocytes were collected by the superovulation of 178 oocyte donor mice. The fertilization rate was 67.94%. After the transfer of 62 hybrid embryos obtained from 4 other oocyte donor mice to 4 Balb/c recipient females, 1 recipient mouse delivered 10 living hybrid mice.

The effect of the cumulus oophorus on fertilisation was investigated by 478 in vitro maturated bovine oocytes. Culturing revealed that the amount of defects on the pellucid zone and in ooplasm of cumulus-free oocytes was higher than that in oocytes cultured in the cumulus-oocyte complex (38 percent or 77/203 versus eleven percent or 30/275). Insemination of the cumulus-oocyte complex (COC) seemed to increase the fertilisation capability of bovine oocytes (63 percent or 70/111), though its penetration rate was only 45 percent (111/245) and was thus below that of cumulus-free oocytes (55 percent or 69/126). Use of COC is recommended as basic material for in vitro maturation and fertilisation.

The ovarian follicles of six normocyclic women were laparoscopically punctured and aspirated on the eleventh or twelfth postmenstrual day of the cycle. Ovocytes and follicular fluid are characterised. Endometrial biopsies were taken of the patients concerned, after two days or so had been allowed to pass.--On histochemical evaluation, the abraded material seemed to exhibit early signs of beginning secretion as a "normal" endometrial response to artificial follicular rupture. The findings are discussed with reference to unanswered questions regarding in vitro fertilisation of human probands.

As a part of an IVF project, "superfluous" ova were frozen, then thawed and fertilized under in-vitro conditions. Altogether 133 oocytes of varying quality, from 22 patients, were frozen. To date, 57 ova have been thawed and 47 oocytes have been found. The survival rate after thawing was 34%, the in-vitro fertilization rate 75%. Out of a total of seven embryo transfers performed there was one pregnancy following transfer of a six-cell embryo.

Seventy-four ovocytes were obtained by puncturing 188 follicles. Several criteria were tasted for their applicability to accurate assessment of ovocyte maturity or possible degenerative changes. They were diameters of follicles and ovocytes, number of rows and structure of granulous cells and of the pellucid zone, contours, granulation, and optical properties of cytoplasm, and existence of a germinal vesicle. Only seven of the above 74 ovocytes were mature and normal in structure. Proposed is a method for isolation of ovocytes by sedimentation of follicular fluid.

The penetration rate of human spermatozoa in zona pellucida-free hamster oocytes was reduced by 4 monoclonal anti-sperm-antibodies (A-24, B-20, III-3, and VII-5) out of 6 tested. The other 2 (VI-1 and VI-16) left the penetration rates virtually unchanged. The extent of penetration rate reduction depended on the concentrations of antibodies A-24, B-20, III-3, and VII-5; if undiluted, they produced a significant reduction of penetration rates, the largest average reduction being almost 50% with undiluted antibody A-24. Decreasing concentrations of the monoclonal anti-sperm-antibodies resulted in significantly smaller reductions of sperm penetration rates. The reason for the reduced penetration rates observed may be a blockage of receptors on the sperm surface by the named monoclonal anti-sperm-antibodies. This can inhibit enzyme reactions of the spermatozoa, or interfere with the interaction between oocyte and spermatozoa membranes. It is noted that only the antibody III-3 caused agglutination of human spermatozoa in the micro-sperm-agglutination and immobilization tests. Therefore, the agglutination or immobilization of spermatozoa may not be held responsible for the reduced penetration rates after addition of monoclonar antibodies, as opposed to the findings with polyclonal antibodies (Steinberg - 1985&).

In this report the minimum requirements for ejaculates to be used for in vitro fertilization of human oocytes are defined. Based on the experimental results obtained a total concentration of at least 5 X 10(6) spermatozoa per ml are required, with at least 30% of the sperm showing normal motility and normal morphology in the differential spermio-cytogram. The total volume of the ejaculate should not be less than 1 ml. Samples for insemination of human oocytes must be proven to be free of pathological microorganisms. In more than 40% of the cases examined bacteria concentrations of more than 10(5)/ml were detected during routine bacteriological check-ups. Especially enterococci und mycoplasma were frequently found. To further establish the quality of human spermatozoa, 91 ejaculate probes were tested within the hamster system using zona-free oocytes. A statistically significant correlation between sperm quality and the penetration rates of human spermatozoa into zona-free hamster oocytes was shown. A positive effect on penetration rates was observed after addition of L-ascorbic acid and Douglas-aspirate to the incubation medium. Rates increased 12 and 17%, respectively. Addition of kallikrein showed no effect on the penetration rates. Application of ejaculates with high concentrations of leucocytes for insemination of hamster oocytes resulted in clearly reduced penetration rates. Leucocyte concentrations of more than 10% led to penetration rates of 0%. The same reduction was observed when ejaculates with high concentrations of pathological micro-organisms (more than 10(5)/ml) were tested. After addition of sperm antibody containing sera from female sterility patients to the incubation medium also clear reduction of penetration rates was evident depending on: the antibody concentration, the initial motility of the spermatozoa used, and on the level of the primary penetration rate obtained without addition of antiserum.

Knowledge of probable ovulation date is essential to the recovery of pre-ovulatory oocytes for in vitro fertilisation. The authors used a commercially available LH-RIA kit for blood and urine samples together with the HI-Gonavis test for urine assay for high-accuracy determination of luteinising hormone (LH) in the peri-ovulatory cycle phase. Follicle status and oocyte quality were laparoscopically described of seven patients, between the eleventh and 16th days p.m. Optimum timing for oocyte recovery has not even become known by knowledge of LH rise. Different recommendations by other authors are discussed, and reference is made to unanswered problems.

A review is given of the present knowledge of the development of follicles and oocytes, especially in the human. The first report deals with the course of development of follicles from the beginning in the fetal ovary to the mature Graafian follicle in the adult women. Questions of terminology, functional morphology and important aspects of the regulation of follicular growth are discussed. The last part of the paper summarizes the present day possibilities in monitoring growth and maturation of ovarian follicles in clinical practice.