The developmental dynamics of the human male germline.

Male germ cells undergo a complex sequence of developmental events throughout fetal and postnatal life that culminate in the formation of haploid gametes: the spermatozoa. Errors in these processes result in infertility and congenital abnormalities in offspring. Male germ cell development starts when pluripotent cells undergo specification to sexually uncommitted primordial germ cells, which act as precursors of both oocytes and spermatozoa. Male-specific development subsequently occurs in the fetal testes, resulting in the formation of spermatogonial stem cells: the foundational stem cells responsible for lifelong generation of spermatozoa. Although deciphering such developmental processes is challenging in humans, recent studies using various models and single-cell sequencing approaches have shed new insight into human male germ cell development. Here, we provide an overview of cellular, signaling and epigenetic cascades of events accompanying male gametogenesis, highlighting conserved features and the differences between humans and other model organisms.

Male fertility preservation and restoration strategies for patients undergoing gonadotoxic therapies†.

Medical treatments for cancers or other conditions can lead to permanent infertility. Infertility is an insidious disease that impacts not only the ability to have a biological child but also the emotional well-being of the infertile individuals, relationships, finances, and overall health. Therefore, all patients should be educated about the effects of their medical treatments on future fertility and about fertility preservation options. The standard fertility preservation option for adolescent and adult men is sperm cryopreservation. Sperms can be frozen and stored for a long period, thawed at a later date, and used to achieve pregnancy with existing assisted reproductive technologies. However, sperm cryopreservation is not applicable for prepubertal patients who do not yet produce sperm. The only fertility preservation option available to prepubertal boys is testicular tissue cryopreservation. Next-generation technologies are being developed to mature those testicular cells or tissues to produce fertilization-competent sperms. When sperm and testicular tissues are not available for fertility preservation, inducing pluripotent stem cells derived from somatic cells, such as blood or skin, may provide an alternative path to produce sperms through a process call in vitro gametogenesis. This review describes standard and experimental options to preserve male fertility as well as the experimental options to produce functional spermatids or sperms from immature cryopreserved testicular tissues or somatic cells.

The use of human artificial gametes and the limits of reproductive freedom.

Recent developments in generating gametes via in vitro gametogenesis (IVG) from induced pluripotent stem cells (iPSCs) and their successful use for reproductive purposes in animals strongly suggest that soon these methods could also be used in human reproduction. At least two questions emerge in this context: (a) if a legislator should permit their use and (b) if ethical claims emerge that support their provision, e.g., by public health care systems. This urges an ethical reflection of the new reproductive options this technique might offer. Since the concept of reproductive freedom is a key aspect for the ethical evaluation of artificial reproductive technologies (ARTs), it is necessary to analyze if the new possibilities emerging from IVG fall within the scope of this concept. The results may constitute a morally relevant difference between different imaginable applications of IVG and potentially justify differences in claims to access this technology.

Drawing the line on in vitro gametogenesis.

In vitro gametogenesis (IVG) might offer numerous research and clinical benefits. Some potential clinical applications of IVG, such as allowing opposite-sex couples experiencing infertility to have genetically related children, have attracted support. Others, such as enabling same-sex reproduction and solo reproduction, have attracted significantly more criticism. In this paper, we examine how different ethical principles might help us to draw lines and distinguish between ethically desirable and undesirable uses of IVG. We discuss the alleged distinction between therapeutic and non-therapeutic uses of assisted reproduction in the context of IVG, and show how it is both problematic to apply in practice and theoretically dubious. We then discuss how the ethical principles of reproductive justice and beneficence apply to IVG for opposite-sex reproduction, same-sex reproduction, and solo reproduction. We suggest that these principles generate strong reasons for the use of IVG for opposite-sex and same-sex reproduction, but not for solo reproduction.

In vitro gametogenesis: The end of egg donation?

This paper explores whether egg donation could still be ethically justified if in vitro gametogenesis (IVG) became reliable and safe. In order to do this, issues and concerns that might inform a patient's reasoning in choosing to use donor eggs instead of IVG are explored and assessed. It is concluded that egg donation would only be ethically justified in a narrow range of special cases given the (hypothetical) availability of IVG treatment and, further, that egg donation could itself be replaced by donation through IVG techniques. Two possible criticisms of this position are then considered: Ones based on respect for patient wishes, and on loss of donor benefit. It is concluded that whilst neither argument constitutes a strong enough reason to continue with programmes of egg donation, egg-sharing programmes could still be permitted come the advent of IVG; these could then provide a morally acceptable source of "natural" donor eggs.

In Vitro Gametogenesis and the Creation of 'Designer Babies'.

Research into the development of stem cell-derived (SCD) gametes in humans, otherwise known as in vitro gametogenesis (IVG), is largely motivated by reproductive aims. Especially, the goal of establishing genetic parenthood by means of SCD-gametes is considered an important aim. However, like other applications in the field of assisted reproduction, this technology evokes worries about the possibility of creating so-called 'designer babies.' In this paper, we investigate various ways in which SCD-gametes could be used to create such preference-matched offspring, and what this would mean for the acceptability of IVG, if it is premised that it is morally problematic to 'design' offspring. We argue that IVG might facilitate the creation of preference-matched offspring, but conclude that this should not undermine the moral acceptability of IVG altogether-even if one concedes the premise that creating 'designer babies' is morally problematic. In the light of this, we also point at a possible inconsistency for a position that condemns the creation of 'designer offspring,' while accepting the various endeavors to have genetically related offspring.

Relevance, strategies, and added value of mouse models in androgenetics.

BACKGROUND: Male Infertility is a prevalent condition worldwide, and a substantial fraction of cases are thought to have a genetic basis. Investigations into the responsible genes is limited experimentally, so mice have been used extensively to identify genes required for fertility and to understand their functions. OBJECTIVES: To review the progress made in reproductive genetics based on experiments in mice, the impact upon clinical fertility genetics, and discuss how evolving technologies will continue to advance our understanding of human infertility genes. RESULTS AND DISCUSSION: Gene knockout studies in mice have shown that several hundreds of genes are required for normal fertility and that this number is much higher in males than in females. In addition to gene discovery, the mouse is a powerful platform for functionally dissecting genetic pathways, modeling putative human infertility variants, identifying contraceptive targets, and developing in vitro gametogenesis. CONCLUSION: These ongoing studies in mice have made an enormous contribution to our understanding of the genetics of human reproduction in the sense that the "parts list" of genes for mammalian gametogenesis is being elucidated. This would have been impossible to do in humans, and in vitro systems are not yet adequate to associate genes with andrological phenotypes, especially in the germline.

Making human eggs in a dish: are we close?

In the space of 50 years, we have seen incredible achievements in human reproductive medicine. With these leaps forward, it is no wonder that there is a major interest in women's reproductive health research, including extension of reproductive lifespan. Substantial effort is currently being made to address this challenge, including from the commercial sector. In vitro gametogenesis (IVG) in mice is a spectacular breakthrough and has the potential to offer hope to women with intractable infertility. However, with such lofty goals, some reflection may be called for: mastering all of the techniques required for complete and safe IVG in women is likely to be extraordinarily difficult.

Artificial Gametes and Human Reproduction in the 21st Century: An Ethical Analysis.

Artificial gametes, derived from stem cells, have the potential to enable in vitro fertilization of embryos. Currently, artificial gametes are only being generated in laboratory animals; however, considerable efforts are underway to develop artificial gametes using human cell sources. These artificial gametes are being proposed as a means to address infertility through assisted reproductive technologies. Nonetheless, the availability of artificial gametes obtained from adult organisms can potentially expand the possibilities of reproduction. Various groups, such as same-sex couples, post-menopausal women, and deceased donors, could potentially utilize artificial gametes to conceive genetically related offspring. The advent of artificial gametes raises significant bioethical questions. Should all these reproductive scenarios be accepted? How can we delineate the range of future reproductive choices? A normative bioethical framework may be necessary to establish a consensus regarding the use of human artificial gametes. This review aims to present the current state of research on the biological roadmap for generating artificial gametes, while also summarizing proposed approaches to establish a normative framework that delineates ethically acceptable paths for reproduction.

Source cell-type epigenetic memory persists in induced pluripotent cells but is lost in subsequently derived germline cells.

Introduction: Retention of source cell-type epigenetic memory may mitigate the potential for induced pluripotent stem cells (iPSCs) to fully achieve transitions in cell fate in vitro. While this may not preclude the use of iPSC-derived somatic cell types for therapeutic applications, it becomes a major concern impacting the potential use of iPSC-derived germline cell types for reproductive applications. The transition from a source somatic cell type to iPSCs and then on to germ-cell like cells (GCLCs) recapitulates two major epigenetic reprogramming events that normally occur during development in vivo-embryonic reprogramming in the epiblast and germline reprogramming in primordial germ cells (PGCs). We examined the extent of epigenetic and transcriptomic memory persisting first during the transition from differentiated source cell types to iPSCs, and then during the transition from iPSCs to PGC-like cells (PGCLCs). Methods: We derived iPSCs from four differentiated mouse cell types including two somatic and two germ cell types and tested the extent to which each resulting iPSC line resembled a) a validated ES cell reference line, and b) their respective source cell types, on the basis of genome-wide gene expression and DNA methylation patterns. We then induced each iPSC line to form PGCLCs, and assessed epigenomic and transcriptomic memory in each compared to endogenous PGCs/M-prospermatogonia. Results: In each iPSC line, we found residual gene expression and epigenetic programming patterns characteristic of the corresponding source differentiated cell type from which each was derived. However, upon deriving PGCLCs, we found very little evidence of lingering epigenetic or transcriptomic memory of the original source cell type. Discussion: This result indicates that derivation of iPSCs and then GCLCs from differentiated source cell types in vitro recapitulates the two-phase epigenetic reprogramming that normally occurs in vivo, and that, to a significant extent, germline cell types derived in vitro from pluripotent cells accurately recapitulate epigenetic programming and gene expression patterns corresponding to equivalent endogenous germ cell types, suggesting that they have the potential to form the basis of in vitro gametogenesis as a useful therapeutic strategy for treatment of infertility.

Unexpected absence of exo-erythrocytic merogony during high gametocytaemia in two species of Haemoproteus (Haemosporida: Haemoproteidae), including description of Haemoproteus angustus n. sp. (lineage hCWT7) and a report of previously unknown residual bodies during in vitro gametogenesis.

Neglected avian blood parasites of the genus Haemoproteus (Haemoproteidae) have recently attracted attention due to the application of molecular diagnostic tools, which unravelled remarkable diversity of their exo-erythrocytic (or tissue) stages both regarding morphology and organ tropism levels. The development of haemoproteids might result in pathologies of internal organs, however the exo-erythrocytic development (EED) of most Haemoproteus species remains unknown. Seven individual birds - Curruca communis (1) and Phylloscopus trochilus (6) - with high gametocytaemia (between 1% and 24%) of Haemoproteus angustus n. sp. (hCWT7) and Haemoproteus palloris (lineage hWW1) were sampled in Lithuania, and their internal organs were examined extensively by parallel application of histology and chromogenic in situ hybridization methods. Tissue stages were apparently absent, suggesting that the parasitaemia was not accompanied by detectable tissue merogony. Haemoproteus angustus n. sp. was described and characterized morphologically and molecularly. Sexual process and ookinete development of the new species readily occurred in vitro, and a unique character for Haemoproteus parasites was discovered - the obligatory development of several tiny residual bodies, which were associated with intracellular transformation of both macrogametocytes and microgametocytes before their escape from the host cells and formation of gametes. A DNA haplotype network was constructed with lineages that cluster in one clade with the lineage hCWT7. This clade consists of lineages mostly found in Curruca birds, indicating specificity for birds of this genus. The lineage hCWT7 is mainly a parasite of C. communis. Most reports of this lineage came from Turkey, with only a few records in Europe, mostly in birds wintering in Africa where transmission probably occurs. This study highlights unexpected difficulties in the research of EED even when using sensitive molecular diagnostic tools and extends information about transformation in early stages of gametogenesis in haemosporidian parasites.

Anticipating in vitro gametogenesis: Hopes and concerns for IVG among diverse stakeholders.

In vitro gametogenesis (IVG), the reconstitution of germ cell development in vitro, is an emerging stem cell-based technology with profound implications for reproductive science. Despite researchers' long-term goals for future clinical applications, little is currently known about the views of IVG held by the stakeholders potentially most affected by its introduction in humans. We conducted focus groups and interviews with 80 individuals with lived experience of infertility and/or LGBTQ+ family formation in the US, two intersecting groups of potential IVG users. Respondents expressed hope that IVG would lead to higher reproductive success than current assisted reproductive technology (ART), alleviate suffering associated with ART use, and promote greater social inclusion, while expressing concerns predominantly framed in terms of equity and safety. These findings underscore the importance of sustained engagement with stakeholders with relevant experience to anticipate the implications of IVG for research and clinical translation.

Maintenance of Human Primordial Germ Cell-Like Cells in a Long-Term Culture System.

Primordial germ cells (PGCs) are the earliest form of mammalian germ lineage. In humans, PGCs are present during a very early and limited window in development, limiting the ability to study fundamental developmental steps in human reproductive biology. However, recent advancements in generating in-vitro models of gametogenesis have allowed the field to generate human primordial germ cell-like cells (hPGCLCs). In this chapter, we will review the generation of hPGCLCs using the incipient mesoderm-like cell (iMeLC) protocol and the subsequent expansion of hPGCLCs in a long-term culture system.

Modelling in vitro gametogenesis using induced pluripotent stem cells: a review.

In vitro gametogenesis (IVG) has been a topic of great interest in recent years not only because it allows for further exploration of mechanisms of germ cell development, but also because of its prospect for innovative medical applications especially for the treatment of infertility. Elucidation of the mechanisms underlying gamete development in vivo has inspired scientists to attempt to recapitulate the entire process of gametogenesis in vitro. While earlier studies have established IVG methods largely using pluripotent stem cells of embryonic origin, the scarcity of sources for these cells and the ethical issues involved in their use are serious limitations to the progress of IVG research especially in humans. However, with the emergence of induced pluripotent stem cells (iPSCs) due to the revolutionary discovery of dedifferentiation and reprogramming factors, IVG research has progressed remarkably in the last decade. This paper extensively reviews developments in IVG using iPSCs. First, the paper presents key concepts from groundwork studies on IVG including earlier researches demonstrating that IVG methods using embryonic stem cells (ESCs) also apply when using iPSCs. Techniques for the derivation of iPSCs are briefly discussed, highlighting the importance of generating transgene-free iPSCs with a high capacity for germline transmission to improve efficacy when used for IVG. The main part of the paper discusses recent advances in IVG research using iPSCs in various stages of gametogenesis. In addition, current clinical applications of IVG are presented, and potential future applications are discussed. Although IVG is still faced with many challenges in terms of technical issues, as well as efficacy and safety, novel IVG methodologies are emerging, and IVG using iPSCs may usher in the next era of reproductive medicine sooner than expected. This raises both ethical and social concerns and calls for the scientific community to cautiously develop IVG technology to ensure it is not only efficacious but also safe and adheres to social and ethical norms.

Efficient and scalable generation of primordial germ cells in 2D culture using basement membrane extract overlay.

Current methods to generate human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) can be inefficient, and it is challenging to generate sufficient hPGCLCs to optimize in vitro gametogenesis. We present a differentiation method that uses diluted basement membrane extract (BMEx) and low BMP4 concentration to efficiently induce hPGCLC differentiation in scalable 2D cell culture. We show that BMEx overlay potentiated BMP/SMAD signaling, induced lumenogenesis, and increased expression of key hPGCLC-progenitor markers such as TFAP2A and EOMES. hPGCLCs that were generated using the BMEx overlay method were able to upregulate more mature germ cell markers, such as DAZL and DDX4, in human fetal ovary reconstitution culture. These findings highlight the importance of BMEx during hPGCLC differentiation and demonstrate the potential of the BMEx overlay method to interrogate the formation of PGCs and amnion in humans, as well as to investigate the next steps to achieve in vitro gametogenesis.

In vitro gametogenesis from embryonic stem cells in livestock species: recent advances, opportunities, and challenges to overcome.

Pluripotent stem cells (PSC) can be stabilized in vitro from pre-implantation stage embryos (embryonic stem cells, ESC) or by reprogramming adult somatic cells (induced pluripotent stem cells, iPSC). The last decade has seen significant advances in the livestock PSC field, particularly the development of robust methods for long-term culture of PSC from several livestock species. Along with this, considerable progress has been made in understanding the states of cellular pluripotency and what they mean for cell differentiation capacity, and significant efforts are ongoing to dissect the critical signaling pathways required for the maintenance of PSC in different species and distinct states of pluripotency. Among the cell types that can be generated from PSC, the germline holds special importance as they are the genetic link between generations; and devising methods to enable in vitro gametogenesis (IVG) and produce viable gametes could revolutionize animal agriculture, wildlife conservation, and human assisted reproduction alike. Within the last decade, many pivotal studies about IVG were published using rodent models, filling some critical knowledge gaps in the field. Most importantly, the entire female reproductive cycle was reproduced in vitro from mouse ESC. Although complete male gametogenesis in vitro has not yet been reported, significant advances were made showing the capacity of germline stem cell-like cells to generate healthy offspring. In this review, we provide an overview of PSC and advances in the establishment of livestock PSC; we present the breakthroughs made in rodents regarding IVG and the current progress towards livestock IVG, including the importance of a detailed understanding of fetal germline development. Finally, we discuss some key advances that will be critical to enable this technology at scale. Given the potential impact of IVG for animal agriculture, major efforts will likely continue to be employed by research institutions and industry towards the development of methods to achieve efficient generation of gametes in vitro.

In this review, we summarize the current state of livestock embryonic stem cell establishment and the advances in production of sperm and eggs in vitro in rodents and livestock. We also discuss the potential and challenges of developing systems that support in vitro gametogenesis in livestock and the opportunities for this new technology in the reproductive field.

Reconstitution of Human Prospermatogonial Development from Human-Induced Pluripotent Stem Cells.

There is a scarcity of information regarding the molecular mechanisms underlying human germ cell development due to limitations in obtaining the relevant materials. Reconstitution of human germ cell development from pluripotent stem cells in vitro would provide critical insight into the etiology of various reproductive conditions and disorders, including infertility.Recently, we reported the in vitro reconstitution of human prospermatogonial development from human-induced pluripotent stem cells through human primordial germ cell (PGC)-like cells (hPGCLCs) using long-term cultured xenogeneic reconstituted testes. Here, we describe a method to generate M-prospermatogonia-like cells (MLCs) and T1-prospermatogonia-like cells (T1LCs), which closely resemble M- and T1-prospermatogonia present in second-trimester human fetal testes in vivo.

Pluripotent Stem Cell-Derived In Vitro Gametogenesis and Synthetic Embryos-It Is Never Too Early for an Ethical Debate.

Recently, 2 branches of the wide area of synthetic biology-in vitro gametogenesis and synthetic embryo development-have gained considerable attention. Rodent induced pluripotent stem cells derived via reprogramming of somatic cells can in vitro be differentiated into gametes to produce fertile offspring. And even synthetic embryos with organ progenitors were generated ex utero entirely from murine pluripotent stem cells. The use of these approaches in basic research, which is rightfully accompanied by an ethical discussion, will allow hitherto unattainable insights into the processes of the beginning of life. There is a broad international consensus that currently the application of these technologies in human-assisted reproduction must be considered to be unsafe and unethical. However, newspaper headlines also addressed the putatively resulting paradigm shift in human reproduction and thereby raised expectations in patients. Due to unsolved biological and technological obstacles, most scientists do not anticipate translation of any of these approaches into human reproductive medicine, if ever, for the next 10 years. Still, whereas the usage of synthetic embryos for reproductive purposes should be banned, in the context of in vitro-derived human gametes it is not too early to initiate the evaluation of the ethical implications, which could still remain assuming all technological hurdles can ever be cleared.

In Vitro Gametogenesis in Oncofertility: A Review of Its Potential Use and Present-Day Challenges in Moving toward Fertility Preservation and Restoration.

Current fertility preservation options are limited for cancer survivor patients who wish to have their own biological children. Human in vitro gametogenesis (IVG) has the hypothetical ability to offer a unique solution to individuals receiving treatment for cancer which subsequently shortens their reproductive lifespan. Through a simple skin punch biopsy, a patient's fertility could be restored via reprogramming of dermal fibroblast cells to induced pluripotent stem cells, then from primordial germ cell-like cells into viable oocytes and spermatocytes which could be used for embryogenesis. Induced pluripotent stem cells could also be used to form in vitro environments, similar to the ovary or testes, necessary for the maturation of oogonia. This would allow for the entire creation of embryos outside the body, ex vivo. While this area in stem cell biology research offers the potential to revolutionize reproduction as we know it, there are many critical barriers, both scientific and ethical, that need to be overcome to one day see this technology utilized clinically.

Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells.

Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for induced pluripotent stem cell (iPSC) culture in the common marmoset (Callithrix jacchus [cj]), allowing stable propagation in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes, culminating in the establishment of a primate-specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.