madman
Super Moderator
Human in vitro spermatogenesis as a regenerative therapy — where do we stand? (2023)
Meghan Robinson, Sydney Sparanese, Luke Witherspoon & Ryan Flannigan
Abstract
Spermatogenesis involves precise temporal and spatial gene expression and cell signaling to reach a coordinated balance between self-renewal and differentiation of spermatogonial stem cells through various germ cell states including mitosis, and meiosis I and II, which result in the generation of haploid cells with a unique genetic identity. Subsequently, these round spermatids undergo a series of morphological changes to shed excess cytoplast, develop a midpiece and tail, and undergo DNA repackaging to eventually form millions of spermatozoa. The goal of recreating this process in vitro has been pursued since the 1920s as a tool to treat male factor infertility in patients with azoospermia. Continued advances in reproductive bioengineering led to the successful generation of mature, functional sperm in mice and, in the past 3 years, in humans. Multiple approaches to studying human in vitro spermatogenesis have been proposed, but technical and ethical obstacles have limited the ability to complete spermiogenesis, and further work is needed to establish a robust culture system for clinical application.
Introduction
Spermatogenesis is the foundation of male reproduction. Following the pubertal transition, males typically produce tens to hundreds of millions of motile sperm1. During natural conception, these sperm will navigate the female reproductive tract to find and fertilize the oocyte2. However, ~15% of couples globally experience challenges with natural conception, and approximately half of these instances can be ascribed to male factors3. Advances in assisted reproductive technologies have enabled the reproductive community to overcome most forms of male infertility, and pregnancies and live births can be achieved with techniques such as in vitro fertilization–intracytoplasmic sperm injection (IVF-ICSI) using as few as one viable and functional sperm4,5. However, IVF-ICSI is not a suitable solution in patients with no detectable sperm, a condition known as azoospermia.
Azoospermia is diagnosed when a complete absence of sperm is observed in at least two semen samples, although microscopic evaluation of a centrifuged sample is performed. Azoospermia ascribed to abnormalities in sperm production is termed non-obstructive azoospermia (NOA). Amongst European men, NOA with an intrinsic etiology (due to genetic abnormalities) occurs in <25% of patients6,7. Therapeutic options in these patients are limited to microdissection testicular sperm extraction, which leads to surgical sperm retrieval in ~50% of patients and, in combination with IVF-ICSI, might result in a live birth in 10–25% of couples8–10. However, the remaining patients have no therapeutic options to father a biological child. NOA might also be ascribed to extrinsic factors such as gonadotoxic therapies including chemotherapy and radiotherapy11,12. Sperm banking might be offered to patients who undergo these iatrogenic insults after puberty13. With regard to patients with prepubertal iatrogenic insults, some centers offer testicular biopsy and cryopreservation, with the hope that future technologies will be developed to facilitate complete differentiation of spermatogonial stem cells (SSCs) into mature sperm capable of producing offspring14. Thus, regenerative treatment strategies for patients with NOA are strongly needed, particularly for patients with no retrievable sperm or prepubertal cancer survivors15–22. A possible approach is in vitro spermatogenesis and spermiogenesis (IVS), in which spermatozoa are generated from SSCs in a laboratory setting. Considering ethical concerns and the scarcity of human fetal tissue, animal models have provided an invaluable tool to optimize technologies that can be applied to support human spermatogenesis. Approaches developed in animal models have laid the foundation for the potential use of in vitro spermatogenesis for infertility treatment. Successful ex vivo production of mature spermatids with fertilization capacity from immature germ cells using mouse testicular organotypic culture was first described in 2011 (ref. 23). In this study, fertile, haploid sperm were obtained with similar efficiency from fresh and cryopreserved testicular samples from neonatal mice. Moreover, using a similar culture method, differentiated spermatogonia were produced from testis tissue obtained from adult mice with spermatogenic defects24
*In this Review, we comprehensively discuss the approaches used to date in developing human IVS, emerging technologies that might be integrated to advance this field, and safety considerations necessary before implementing future IVS technologies into regular clinical use.
Human spermatogenesis
Human spermatogenesis is a complex process in which precise temporal and spatial events are coordinated by local support from somatic cells through direct contact, and juxtacrine and paracrine signalling25–28 (Fig. 1). The entire process of spermatogenesis occurs in cycles of 74 days, with new waves of SSCs entering differentiation every 16 days29
Approaches for human in vitro spermatogenesis
IVS requires spermatogenesis and spermiogenesis to occur outside the body. Preliminary findings have suggested that partial differentiation can occur in vitro47–51, but the entire process has been shown to be challenging. To date, several approaches have been attempted, spanning 2D and 3D culture systems (Fig. 2), and a variety of media and biomaterials (Table 1). These approaches have been tested in numerous animal models with impressive results17,23,52–73. Owing to the increasing breadth of research in model systems, this Review focuses on human IVS.
*2D culture systems
*Sertoli cell feeder systems
*Vero cell feeder systems
*Pluripotent stem cell-derived systems
*2D IVS systems — considerations
Organotypic culture systems
A possible approach to IVS is in vitro culture of the complete testicular niche (known as organotypic culture (Fig. 2c)), with the idea that spermatogenesis can resume in vitro with the support of the natural microenvironment. A major challenge with this approach has been to keep the tissues viable and functional without bodily support, including delivery of oxygen, vitamins, nutrients, and trophic factors through diffusion from the local vascular system.
*Air–liquid interface
*Bioreactors
*Organotypic systems — considerations
3D scaffolds, 3D organoids and 3D bioprinted systems
An alternative approach to organotypic systems is to bioengineer the testicular niche (Fig. 2d–f). With this method, physiological tissue structures can be recreated in vitro, as intercellular signaling, diffusion, and local concentration gradients of cell-secreted factors are recapitulated in the 3D histoarchitecture104. Thus, this approach could mimic in vivo tissue organization, but, differently from organotypic culture systems, cellular alterations or treatments can be performed before establishing the 3D cytoarchitecture. Moreover, the issue of cell viability is solved by the diffusion of nutrients from the medium and oxygen through porous scaffolding.
*3D scaffolds
*3D organoids
*3D bioprinting
*3D scaffolds, 3D organoids and 3D bioprinted IVS systems — considerations
*Lessons on successful IVS from animals
*Medium considerations
Future directions
An impressive body of work has been dedicated to developing complete human IVS; however, the establishment of a fully characterized system for clinical use remains an open challenge. Additional research is required to generate a robust model to recreate the native testicular microenvironment and spermatogenic functionality.
*3D-bioprinting, bio-inks, and microspheres
*In vitro rescue of somatic cell functions in patients with NOA
*Ethical and safety measures
Conclusions
Restoring sperm production as a potential therapeutic strategy for patients with infertility is urgently needed. To date, several approaches have been used to model human IVS. Organotypic and organoid cultures highlighted the importance of the testicular niche and, perhaps, have been the most promising approaches to support testicular microenvironment assembly and spermatogenesis94,97,111. However, in most studies to date, robust completion of spermiogenesis was not shown and was limited in part by the need to optimize medium factors for the delivery of physiological nutrients in vitro, and to recreate the testicular histological architecture and cell–cell signaling required for differentiation. Several promising technologies such as 3D organoid and bio-printed IVS systems are emerging with the potential to bring IVS technology close to clinical translation. However, numerous safety considerations and evaluations including the use of xenofree materials and the establishment of safety checkpoints to confirm the generation of genetically, epigenetically, and morphologically normal sperm will be necessary to achieve the direct application of these technologies to the clinical sphere.
Meghan Robinson, Sydney Sparanese, Luke Witherspoon & Ryan Flannigan
Abstract
Spermatogenesis involves precise temporal and spatial gene expression and cell signaling to reach a coordinated balance between self-renewal and differentiation of spermatogonial stem cells through various germ cell states including mitosis, and meiosis I and II, which result in the generation of haploid cells with a unique genetic identity. Subsequently, these round spermatids undergo a series of morphological changes to shed excess cytoplast, develop a midpiece and tail, and undergo DNA repackaging to eventually form millions of spermatozoa. The goal of recreating this process in vitro has been pursued since the 1920s as a tool to treat male factor infertility in patients with azoospermia. Continued advances in reproductive bioengineering led to the successful generation of mature, functional sperm in mice and, in the past 3 years, in humans. Multiple approaches to studying human in vitro spermatogenesis have been proposed, but technical and ethical obstacles have limited the ability to complete spermiogenesis, and further work is needed to establish a robust culture system for clinical application.
Introduction
Spermatogenesis is the foundation of male reproduction. Following the pubertal transition, males typically produce tens to hundreds of millions of motile sperm1. During natural conception, these sperm will navigate the female reproductive tract to find and fertilize the oocyte2. However, ~15% of couples globally experience challenges with natural conception, and approximately half of these instances can be ascribed to male factors3. Advances in assisted reproductive technologies have enabled the reproductive community to overcome most forms of male infertility, and pregnancies and live births can be achieved with techniques such as in vitro fertilization–intracytoplasmic sperm injection (IVF-ICSI) using as few as one viable and functional sperm4,5. However, IVF-ICSI is not a suitable solution in patients with no detectable sperm, a condition known as azoospermia.
Azoospermia is diagnosed when a complete absence of sperm is observed in at least two semen samples, although microscopic evaluation of a centrifuged sample is performed. Azoospermia ascribed to abnormalities in sperm production is termed non-obstructive azoospermia (NOA). Amongst European men, NOA with an intrinsic etiology (due to genetic abnormalities) occurs in <25% of patients6,7. Therapeutic options in these patients are limited to microdissection testicular sperm extraction, which leads to surgical sperm retrieval in ~50% of patients and, in combination with IVF-ICSI, might result in a live birth in 10–25% of couples8–10. However, the remaining patients have no therapeutic options to father a biological child. NOA might also be ascribed to extrinsic factors such as gonadotoxic therapies including chemotherapy and radiotherapy11,12. Sperm banking might be offered to patients who undergo these iatrogenic insults after puberty13. With regard to patients with prepubertal iatrogenic insults, some centers offer testicular biopsy and cryopreservation, with the hope that future technologies will be developed to facilitate complete differentiation of spermatogonial stem cells (SSCs) into mature sperm capable of producing offspring14. Thus, regenerative treatment strategies for patients with NOA are strongly needed, particularly for patients with no retrievable sperm or prepubertal cancer survivors15–22. A possible approach is in vitro spermatogenesis and spermiogenesis (IVS), in which spermatozoa are generated from SSCs in a laboratory setting. Considering ethical concerns and the scarcity of human fetal tissue, animal models have provided an invaluable tool to optimize technologies that can be applied to support human spermatogenesis. Approaches developed in animal models have laid the foundation for the potential use of in vitro spermatogenesis for infertility treatment. Successful ex vivo production of mature spermatids with fertilization capacity from immature germ cells using mouse testicular organotypic culture was first described in 2011 (ref. 23). In this study, fertile, haploid sperm were obtained with similar efficiency from fresh and cryopreserved testicular samples from neonatal mice. Moreover, using a similar culture method, differentiated spermatogonia were produced from testis tissue obtained from adult mice with spermatogenic defects24
*In this Review, we comprehensively discuss the approaches used to date in developing human IVS, emerging technologies that might be integrated to advance this field, and safety considerations necessary before implementing future IVS technologies into regular clinical use.
Human spermatogenesis
Human spermatogenesis is a complex process in which precise temporal and spatial events are coordinated by local support from somatic cells through direct contact, and juxtacrine and paracrine signalling25–28 (Fig. 1). The entire process of spermatogenesis occurs in cycles of 74 days, with new waves of SSCs entering differentiation every 16 days29
Approaches for human in vitro spermatogenesis
IVS requires spermatogenesis and spermiogenesis to occur outside the body. Preliminary findings have suggested that partial differentiation can occur in vitro47–51, but the entire process has been shown to be challenging. To date, several approaches have been attempted, spanning 2D and 3D culture systems (Fig. 2), and a variety of media and biomaterials (Table 1). These approaches have been tested in numerous animal models with impressive results17,23,52–73. Owing to the increasing breadth of research in model systems, this Review focuses on human IVS.
*2D culture systems
*Sertoli cell feeder systems
*Vero cell feeder systems
*Pluripotent stem cell-derived systems
*2D IVS systems — considerations
Organotypic culture systems
A possible approach to IVS is in vitro culture of the complete testicular niche (known as organotypic culture (Fig. 2c)), with the idea that spermatogenesis can resume in vitro with the support of the natural microenvironment. A major challenge with this approach has been to keep the tissues viable and functional without bodily support, including delivery of oxygen, vitamins, nutrients, and trophic factors through diffusion from the local vascular system.
*Air–liquid interface
*Bioreactors
*Organotypic systems — considerations
3D scaffolds, 3D organoids and 3D bioprinted systems
An alternative approach to organotypic systems is to bioengineer the testicular niche (Fig. 2d–f). With this method, physiological tissue structures can be recreated in vitro, as intercellular signaling, diffusion, and local concentration gradients of cell-secreted factors are recapitulated in the 3D histoarchitecture104. Thus, this approach could mimic in vivo tissue organization, but, differently from organotypic culture systems, cellular alterations or treatments can be performed before establishing the 3D cytoarchitecture. Moreover, the issue of cell viability is solved by the diffusion of nutrients from the medium and oxygen through porous scaffolding.
*3D scaffolds
*3D organoids
*3D bioprinting
*3D scaffolds, 3D organoids and 3D bioprinted IVS systems — considerations
*Lessons on successful IVS from animals
*Medium considerations
Future directions
An impressive body of work has been dedicated to developing complete human IVS; however, the establishment of a fully characterized system for clinical use remains an open challenge. Additional research is required to generate a robust model to recreate the native testicular microenvironment and spermatogenic functionality.
*3D-bioprinting, bio-inks, and microspheres
*In vitro rescue of somatic cell functions in patients with NOA
*Ethical and safety measures
Conclusions
Restoring sperm production as a potential therapeutic strategy for patients with infertility is urgently needed. To date, several approaches have been used to model human IVS. Organotypic and organoid cultures highlighted the importance of the testicular niche and, perhaps, have been the most promising approaches to support testicular microenvironment assembly and spermatogenesis94,97,111. However, in most studies to date, robust completion of spermiogenesis was not shown and was limited in part by the need to optimize medium factors for the delivery of physiological nutrients in vitro, and to recreate the testicular histological architecture and cell–cell signaling required for differentiation. Several promising technologies such as 3D organoid and bio-printed IVS systems are emerging with the potential to bring IVS technology close to clinical translation. However, numerous safety considerations and evaluations including the use of xenofree materials and the establishment of safety checkpoints to confirm the generation of genetically, epigenetically, and morphologically normal sperm will be necessary to achieve the direct application of these technologies to the clinical sphere.
