Final Report Summary - GROWSPERM (Development of in vitro and in vivo strategies to avoid and cure male infertility)
Project reference: 603568
Funded under: FP7-PEOPLE-2013-ITN
In many European countries at least 20% of young men exhibit sperm parameters below the lower WHO reference level and this will affect their fertility.
Male infertility has a dramatic impact on the individual and/or couple’s psychological and social well-being and results in significant healthcare costs. Currently male patients that do not produce sperm have no therapeutic options to father children. Different therapeutic interventions for male infertility have to be developed depending on the type and severity of germ cell deficits in individual patients.
In this Network, nine early stage researcher (ESRs) and two experienced researchers (ERs) have been recruited. They were trained (WP4) within this Growsperm network (www.Growsperm.eu) that joins together the complementary knowledge and expertise of several public and private EU partners from disciplines of physiology, cell biology, molecular biology, chemistry and medicine in the field of male reproduction to investigate three strategies for sperm development (objectives): (WP1) propagation of human SSCs in vitro followed by their transplantation, (WP2) sperm development in vitro from stem cells or early germ cells, (WP3) sperm development in testis tissue grafts in vivo (Figure 1).
In WP1, normative reference values of spermatogonia during prepuberty have been established and subsequently used for evaluating the effects of cancer therapies on spermatogonial quantities and hence spermatogenic recovery in prepubertal boys with acute lymphoblastic leukaemia treated with either alkylating agents or non-alkylating agents. The results showed long-term depletion of the spermatogonial pool in survivors treated with alkylating agents, while spermatogonial quantity after therapy with non-alkylating agents remains within normative reference values for prepubertal boys. This knowledge will be used to develop recommendations for inclusion criteria for fertility preservation options in prepubertal boys diagnosed with cancer. This includes those already treated with non-alkylating agents, which are now in some European countries excluded from testicular tissue cryopreservation for fertility preservation.
Furthermore, we have improved the cryopreservation protocol to allow storage of testicular cells and found that adding an anti-apoptotic component to the cryo-protecting medium enhances the survival of spermatogonial stem cells. This is an important improvement towards bringing spermatogonial stem cell propagation and transplantation closer to clinical implementation.
In WP2, we obtained more insight into the process of in vitro differentiation of male germ cells and the influence of somatic cells in this process. More precisely, we investigated the molecular mechanisms and factors involved in cord formation and interaction between different somatic cell types and interaction between somatic and germ cells in various culture approaches. In doing so, we achieved cord formation in vitro and generated the world’s first testicular organoid. Further characterisation of the organoids reveals that Sertoli cells form tight junctions and are nicely layered in spheres surrounded by spermatogonia, thereby mimicking in vivo seminiferous cord structure. This organoid culture system is an excellent tool for further studies in the field of basic biology, medicine, repro-toxicology and pharmacology, as currently no in vitro testicular models are available. Furthermore, by adjusting the mouse liquid-air testicular organ culture, we achieved full spermatogenesis in a rodent testicular tissue culture. In addition, we investigated the role of laminins in stem-ness maintenance in pluripotent stem cells as well as male germ cells. We showed that laminin 521 leads to a stabilization of stem-ness related genes in various stem cell lines, allowing for more controlled culture condition.
In WP3, we developed strategies to generate sperm in immature testis tissue by grafting of immature testicular tissue fragments. We developed this system for human testis tissue and a non-human primate model. For that, we first confirmed the similarities between human and non-human primate germ cell characteristics. As model for prepubertal tissue development, we successfully established long-term (12 months) human foetal testis tissue xenografting in mice and achieved testicular cord structure development and maintenance. When prepubertal human testis tissue fragments were pre-treatment with VEGF prior to xenografting to host mice, increased vascularisation and a higher percentage of intact seminiferous tubules was identified. Nine months of xenografting resulted in de development of spermatogonia to spermatocytes in these grafts. Using non-human primate prepubertal testicular tissue in orthotopic xenografting in mice, we were able to achieve full development from spermatogonia to sperm at nine month after xenografting. These tissue xenografting models are reliable methods to predict the development of human prepubertal testis by testis tissue grating. These preclinical results are an important step towards bringing testicular tissue grafting to the clinic.
In this network, all nine ESRs and two ERs, have been recruited within a short time interval. Each fellow was supervised by 2 PIs from different universities, which were both involved in the training and quality of the PhD supervision process. All fellows have followed the full training program and attended many courses inside and outside the network (WP4). All fellows participated in many international conferences and workshops with abstracts, posters or oral presentations and several prestigious prizes for best presentations have been awarded. All MSCA person months have been completed. Both ERs found a job shortly after finishing their 24 months MSCA contract and both stayed in academic science in Europe. Two of the ESRs already defended their thesis shortly after their 36 months MSCA contract. All other ESRs continued for a fourth year (mandated by most universities) on personal grants obtained by fellows or supervisors. They have already generated enough data for additional publications to prepare and obtain their PhD degree by spring 2019. By coaching these young scientists in this inter-sectorial and multidisciplinary network, we have trained the next generation of researchers within the EU. They are excellently equipped to become leaders in basic science and translational research leading to novel interventions and clinical applications in academic and/or private biological and medical sectors.
The outcome of the work from this project is the development of outstanding novel methods that deliver a very important step forward in the understanding of human germ cell development and the influence of the somatic testicular cells on this process. This is not only of interest from the basic science perspective, but is also a prerequisite for developing future clinical applications in vivo and in vitro to treat male infertility problems. Currently, several centres in Europe, including those of partner’s clinics, are cryopreserving testicular tissue from prepubertal boys diagnosed with cancer or other diseases requiring gonadotoxic therapies. The establishment of age-related spermatogonial reference quantities will serve as a clinical tool to determine the size and quality of the biopsy. Furthermore, many novel testicular models established in this network are of broader interest in the field of basic biology, medicine, repro-toxicology and pharmacology as currently no in vitro testicular models are available and repro-toxicology and pharmacological studies are still performed on animals. The highly relevant output of the program shows the outstanding collaborations between fellows and supervisors and among supervisors and is reflected in co-authorships in a substantial part of the published papers.
Contact
Website: www.growsperm.eu
Remark:
In this tekst box, it was not possible to include figure and logo in the publishable summary. Therefore, we have attached the publishable summary with logo and figure as a PDF document.
Funded under: FP7-PEOPLE-2013-ITN
In many European countries at least 20% of young men exhibit sperm parameters below the lower WHO reference level and this will affect their fertility.
Male infertility has a dramatic impact on the individual and/or couple’s psychological and social well-being and results in significant healthcare costs. Currently male patients that do not produce sperm have no therapeutic options to father children. Different therapeutic interventions for male infertility have to be developed depending on the type and severity of germ cell deficits in individual patients.
In this Network, nine early stage researcher (ESRs) and two experienced researchers (ERs) have been recruited. They were trained (WP4) within this Growsperm network (www.Growsperm.eu) that joins together the complementary knowledge and expertise of several public and private EU partners from disciplines of physiology, cell biology, molecular biology, chemistry and medicine in the field of male reproduction to investigate three strategies for sperm development (objectives): (WP1) propagation of human SSCs in vitro followed by their transplantation, (WP2) sperm development in vitro from stem cells or early germ cells, (WP3) sperm development in testis tissue grafts in vivo (Figure 1).
In WP1, normative reference values of spermatogonia during prepuberty have been established and subsequently used for evaluating the effects of cancer therapies on spermatogonial quantities and hence spermatogenic recovery in prepubertal boys with acute lymphoblastic leukaemia treated with either alkylating agents or non-alkylating agents. The results showed long-term depletion of the spermatogonial pool in survivors treated with alkylating agents, while spermatogonial quantity after therapy with non-alkylating agents remains within normative reference values for prepubertal boys. This knowledge will be used to develop recommendations for inclusion criteria for fertility preservation options in prepubertal boys diagnosed with cancer. This includes those already treated with non-alkylating agents, which are now in some European countries excluded from testicular tissue cryopreservation for fertility preservation.
Furthermore, we have improved the cryopreservation protocol to allow storage of testicular cells and found that adding an anti-apoptotic component to the cryo-protecting medium enhances the survival of spermatogonial stem cells. This is an important improvement towards bringing spermatogonial stem cell propagation and transplantation closer to clinical implementation.
In WP2, we obtained more insight into the process of in vitro differentiation of male germ cells and the influence of somatic cells in this process. More precisely, we investigated the molecular mechanisms and factors involved in cord formation and interaction between different somatic cell types and interaction between somatic and germ cells in various culture approaches. In doing so, we achieved cord formation in vitro and generated the world’s first testicular organoid. Further characterisation of the organoids reveals that Sertoli cells form tight junctions and are nicely layered in spheres surrounded by spermatogonia, thereby mimicking in vivo seminiferous cord structure. This organoid culture system is an excellent tool for further studies in the field of basic biology, medicine, repro-toxicology and pharmacology, as currently no in vitro testicular models are available. Furthermore, by adjusting the mouse liquid-air testicular organ culture, we achieved full spermatogenesis in a rodent testicular tissue culture. In addition, we investigated the role of laminins in stem-ness maintenance in pluripotent stem cells as well as male germ cells. We showed that laminin 521 leads to a stabilization of stem-ness related genes in various stem cell lines, allowing for more controlled culture condition.
In WP3, we developed strategies to generate sperm in immature testis tissue by grafting of immature testicular tissue fragments. We developed this system for human testis tissue and a non-human primate model. For that, we first confirmed the similarities between human and non-human primate germ cell characteristics. As model for prepubertal tissue development, we successfully established long-term (12 months) human foetal testis tissue xenografting in mice and achieved testicular cord structure development and maintenance. When prepubertal human testis tissue fragments were pre-treatment with VEGF prior to xenografting to host mice, increased vascularisation and a higher percentage of intact seminiferous tubules was identified. Nine months of xenografting resulted in de development of spermatogonia to spermatocytes in these grafts. Using non-human primate prepubertal testicular tissue in orthotopic xenografting in mice, we were able to achieve full development from spermatogonia to sperm at nine month after xenografting. These tissue xenografting models are reliable methods to predict the development of human prepubertal testis by testis tissue grating. These preclinical results are an important step towards bringing testicular tissue grafting to the clinic.
In this network, all nine ESRs and two ERs, have been recruited within a short time interval. Each fellow was supervised by 2 PIs from different universities, which were both involved in the training and quality of the PhD supervision process. All fellows have followed the full training program and attended many courses inside and outside the network (WP4). All fellows participated in many international conferences and workshops with abstracts, posters or oral presentations and several prestigious prizes for best presentations have been awarded. All MSCA person months have been completed. Both ERs found a job shortly after finishing their 24 months MSCA contract and both stayed in academic science in Europe. Two of the ESRs already defended their thesis shortly after their 36 months MSCA contract. All other ESRs continued for a fourth year (mandated by most universities) on personal grants obtained by fellows or supervisors. They have already generated enough data for additional publications to prepare and obtain their PhD degree by spring 2019. By coaching these young scientists in this inter-sectorial and multidisciplinary network, we have trained the next generation of researchers within the EU. They are excellently equipped to become leaders in basic science and translational research leading to novel interventions and clinical applications in academic and/or private biological and medical sectors.
The outcome of the work from this project is the development of outstanding novel methods that deliver a very important step forward in the understanding of human germ cell development and the influence of the somatic testicular cells on this process. This is not only of interest from the basic science perspective, but is also a prerequisite for developing future clinical applications in vivo and in vitro to treat male infertility problems. Currently, several centres in Europe, including those of partner’s clinics, are cryopreserving testicular tissue from prepubertal boys diagnosed with cancer or other diseases requiring gonadotoxic therapies. The establishment of age-related spermatogonial reference quantities will serve as a clinical tool to determine the size and quality of the biopsy. Furthermore, many novel testicular models established in this network are of broader interest in the field of basic biology, medicine, repro-toxicology and pharmacology as currently no in vitro testicular models are available and repro-toxicology and pharmacological studies are still performed on animals. The highly relevant output of the program shows the outstanding collaborations between fellows and supervisors and among supervisors and is reflected in co-authorships in a substantial part of the published papers.
Contact
Website: www.growsperm.eu
Remark:
In this tekst box, it was not possible to include figure and logo in the publishable summary. Therefore, we have attached the publishable summary with logo and figure as a PDF document.