In vitro maturation of oocytes from small follicles: a field for active research interaction between veterinary and human reproductive medicine

Although powerful assisted reproduction techniques (ART) have been developed over the last 35 years, access to treatment remains still problematic in most parts of the world due to its high financial burden on existing social security systems. Classical ART therapies and have a profound impact on women's professional life in part due to long hormonal treatments associated with physical discomfort and occasionally with severe complications. The high efficacy of IVF/ICSI has been obtained by improving the technologiesrelated to the treatment procedure (association of drug compounds, treatment monitoring, ICSI, longer embryo culture, embryo selection using genetic diagnosis, cryopreservation of embryos, etc.). While this chain of technologies is useful for the more complex cases of infertility, there might be a place for a first line ART as a "step-in" technology, where intervention on patients' normal life is kept minimal. In this context the ovarian follicular pool present throughout the menstrual cycle might is a source of immature oocyte-cumulus complexes which could be processed in-vitro up to developmentally competent oocytes. While the IVM technique has been pioneered by Bob Edwards, Alan Trounson and Carl Wood and further clinically developed by Scandinavianand Canadian Teams, it has not been broadly adapted in the ART clinics because of its lower efficacy in terms of implantation rate per transferred embryo compared to classical IVF/ICSI (10% vs 25% respectively). One of the reasons for the slow implementation of IVM as a first-line technique for infertile patientsis that it has been worked out in a population of PCOS patients, who have an altered folliculogenesis, a compromised intrinsic oocyte quality and who demonstrate a higher miscarriage rate. While there is no doubt that IVM is the only technique that can totally avoid clinical hyperstimulation syndrome in PCOS, clinicians have remained hesitant to perform retrieval of oocytes out of small follicles (2-10 mm diameter), and most embryologists did not have precise and validated procedures to deal with a population of COC of diverse maturity grade. In contrast, large breeding programs for several economically important animals are entirely based on the IVM technique and are already applied for several years. The source of immature oocytes are either slaughterhouse ovaries or ultrasound-guided retrievals in in-vivo monitored cycles. The embryos produced from IVM are cryopreserved and transported over continents to be transferred into receptive animals.The difference is that in animal breeding there is a large source of immature oocytes at the disposition of the lab and thus strong selection can be easily performed. The genetic background in animal breeding is also more homogenous than is the case between patients. In general in human IVM relatively few (5-15) COC are retrieved per cycle; and if HCG is injected the retrieved pool of oocytes is in a disparate stage of maturity (from GV stage with compact corona up to metaphase II oocyte with fully expanded cumulus). Having to deal with this large spectrum of oocyte development stages has implications in embryo culture practise, and the embryos obtained have to be replaced in the uterus of which endometrial receptivity is non-ideal due to the short growth span of the follicles, which have to provide sufficient estrogen and progesterone. Despite all difficulties encountered with the introduction of IVM we believe that a suitable workmode could be set to guide the ART teams to a less invasive ART treatment procedure with competitive efficacy figures. The IVM research program at the University Hospital (UZ Brussel) from the Free University Brussels (VUB) has been partnered by the Institute for early human development of the University of Adelaide (Adelaide, Australia) since 2009. The principal aim is to improve results obtained with currently commercialized IVM methods so that the efficiency gap with classical IVF/ICSI is narrowed, by improving the in-vitro culture procedure. This research has been supported over the last 5 years by IWT (the Flemish Institute for Innovation by Technology and Science) and FWO (Fund for Scientific Research Flanders) who are both governmental organisations. The principal axes of translational research are based on recent findings emanating from experimental work in mouse (VUB) and other large mammals (Adelaide University) related to influences on intracellular cAMP levels in the COC and the use of recombinant factors of the EGF and TGF beta family. The experimental conditions from animal models providing the most promising results are translated into experiments done on human oocytes from small follicles (2-10 mm). Patients and volunteers have signed informed consent and the project works under approval of the Federal Commission for research on human embryos. Our clinical results on nearly 500 patients have been confirming the published efficacy data from the pioneering teams (McGill University Team; The Family Federation Team in Helsinki; Biogenesi atIstitutiCliniciZucchi from Monza). The IVM methodology from VUB team is based on oocyte retrieval without prior HCG injection. This approach allows to obtain uniformly GV oocytes from 2-6 mm follicles for setting new culture approaches.With the non-HCG approach the need for adequate luteal supplementation has been emphasized. The vitrification of day 3 embryos and their subsequent replacement in standard artificial cycles has demonstrated an attractive approach with high implantation figures per single embryo transferred. Associated research on imprinting establishment in human oocytes with the InstitutfurHumangenetik in Wurzburg (Germany) has demonstrated that IVM technology per se is not associated with methylation alterations for 3 susceptible genes. An analysis by array CGH on 20 transferable good quality IVM embryos (all blastomeresanalysed) could not detect significant differences with control "classical" ICSI embryos obtained after conventional stimulation. The clinical results obtained from September 2013 to March 2014 (50 cycles in PCO-like patients) with the non-HCG approach in combination with 'conventional' IVM culture method (Origio) in PCO-like patients (our control group) yields an ongoing pregnancy rate of 28% per oocyte retrieval (mainly single embryo transfer). Although losses by early miscarriage are still high in our series (44%) this figure is not different from the early pregnancy loss observed in our clinic in 2013 in PCO-like patients after regular IVF/ICSI. In conclusion, IVM provides unique insights on early follicle development before luteinisationin human; our research should bring stepwise improvements in the performance of IVM to the benefit of our patients. Fine-tuning the culture conditions will allow women to have a more simplified, short and safe infertility treatment. With the most recent technologies at reach we hope to transform IVM into a robust ART treatment with a much lower treatment burden for patient and savings for social security.