Early screening for reprotoxicity now possible!

For a long time the dogma existed that there are no in vitro alternatives for reprotoxicity studies. Luckily, this holds no longer true!

Until now the standard for reprotoxicity testing are long-lasting in vivo studies  necessary to cover at least a complete life cycle and to explore the entire complexity of the hormonal driven interplay between the different organs of the reproductive system. They involve a large number of animals, are expensive and require substantial amounts of test compound. For these reasons the reprotox studies are often scheduled at the end of drug development, during the clinical phase. However, discovery of adverse effects at this stage causes a disastrous delay in the development of the new drug and financial loss for the company. Early detection is pivotal for cost saving, gives the opportunity to explore less toxic alternatives or define preventive measures, based on the mechanism of action, before entering the clinical phase.

The only solid way to approach early screening of the reproductive function is by in vitro methods, an array of in vitro tests, each representing an important target organ system. However, simple static cell culture methods, except for the receptor binding assays, which give no information on biological downstream effects of hormone-like compounds, are prone to give false results. In vivo, important target cells in the reproductive organs, especially of the gonads, differentiate continuously during their development, whereby their susceptibility to a certain compound constantly alters. Predictive in vitro methods need to be composed of the key elements of the target organ, which reflect the dynamics of its physiological processes. Only then a lifelike (in vitro) response to a test compound can be achieved.

For female fertility the central organ is the ovary, the production unit of oocytes and regulatory hormones. Oocytes are very peculiarly non-dividing cells; having a long strictly defined growth path to attain the capacity to be fertilizable and developmental competent. During this process they require intimate contact with granulosa cells enclosed in a growing follicle. Together with the surrounding theca cells they represent to the functional units of the ovary.

The growth and development of these units have been mimicked in an in vitro follicle culture system (Cortvrindt et al.: (1996) Human Reproduction 11, 2656-2666; (1998) Theriogenology 49, 845-859). Based on this follicle culture system, EggCentris has developed their Follicle Bioassay (FBA), which is a multi-parametric long-term 3D-culture method of single early pre-antral follicles up to the ovulatory stage. The FBA allows simultaneous evaluation of the interrelated processes of folliculogenesis, oogenesis and steroidogenesis by quantitative and qualitative parameters. The different endpoints of the bioassay are very reproducible (10% CV). Under control conditions, almost all follicles differentiate into pre-ovulatory follicles and more than 80% will release a mature oocyte. The steroid output has a physiological profile during follicle growth and differentiation, with a hundred fold progesterone increase upon the ovulatory stimulus.

The analysis of the results of these different outcome parameters allows predicting a compound's effect on the female reproductive cycle and its influence on the capacity to conceive. If a compound disrupts follicle growth and differentiation, it points to menstrual cycle disturbances, while alteration of the steroid output points to cycle disturbances, ovulation problems and alterations of the uterine receptivity. A reduced oocyte number or quality gives indications for impairment of conception or the risk of early abortion. Dose concentration studies will pinpoint the NOAELS at all functional levels.

If the FBA is extended with in vitro fertilisation (IVF) of the in vitro grown oocytes (exposed oocytes) and in vitro culture of the resulting embryos (IVEC- in vitro embryo culture) up to the blastocyst stage, an analogous exposure scenario as in the in vivo fertility studies for females is obtained in vitro.

In addition, detailed analysis of the daily steroid output of the FBA reveals information on the activity of all the enzymes involved in the steroidogenic pathway and allows detection and characterisation of endocrine disruptive compounds.

Male fertility can be approached in a similar way. Culture methods for Leydig cells, Sertoli cells and analysis of exposed sperm cells are already available. More complex culture systems are under development.

Some in vitro assays for embryo toxicity screening; the embryonic stem cell test (EST), the whole embryo culture (WEC) and micromass assay (MM) are already available for some time. EggCentris has optimized and down scaled the EST test to a 96-well format and is successfully used for ranking of lead compounds. Besides this assay EggCentris has developed also an in vitro assay to study the effect on pre- and peri-implantation embryos (MEPA; mouse embryo peri-implantation assay). Effects that are difficult to deduce from the secondary endpoints available upon an in vivo study. In this long term in vitro bioassay, mouse zygotes are cultured up to embryonic day 8 (= 2 days post hatching). Daily microscopic evaluation allows identification of the sensitive stage(s) of the pre-implantation embryo and the embryo survival capacity in the implantation window. From concentration response experiments the respective NOAEL values can be deduced for all sensitive stages.

This array of unique, complex three dimensional in vitro bioassays gives the opportunity to the pharmaceutical industry to test and select their NCEs (new chemical entities) in the discovery phase for their impact on the reproductive function and to come up with NDAs (new drug applications) that are characterised and safe for the reproductive life cycle.