Bibliography in Assisted Reproductive Technology (ART)

In an ever-evolving field marked by rapid scientific progress and increasing clinical complexity, it is essential to remain updated on key innovations, best practices, and the most relevant publications.

Compiled and reviewed by the clinical team at the xxxxxx—one of Paris’ most established and active ART centers—this summary offers a concise yet rigorous overview of recent advances in the field. Drawing from high-impact international journals, the work highlights emerging techniques, evolving protocols, and critical findings in ART.

With this bibliographical review, we aim to offer an original tool to aid in scientific monitoring, positioned between the conventional abstract and the often lengthy and time-consuming full reading of the original article. This hybrid format ensures substantial time savings by enabling the rapid and targeted identification of publications that, based on their content, warrant an in-depth reading.

Regular updates will follow to keep pace with the dynamic landscape of ART.

In vitro gametogenesis

Dr Cécile François

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Dr François durand

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Overview

Spermatogenesis is the process coupled with spermiogenesis that leads to the production of spermatozoa from spermatogonial stem cells (SSCs). SSCs undergo mitosis to produce a self-renewing daughter cell and another daughter cell that differentiates. The differentiated cell then enters meiosis I and II as spermatocytes, resulting in four haploid round spermatids. These spermatids then undergo spermiogenesis, involving morphological changes like DNA compaction and cytoplasm reduction, ultimately forming mature spermatozoa. This entire process occurs within the specialized microenvironment of the testes, specifically the seminiferous tubules. The delicate nature of this testicular microenvironment means it can be susceptible to disruption by factors such as diseases, drugs, injuries, and extreme environments, which can negatively impact normal sperm development.

Achieving complete spermatogenesis and spermiogenesis in vitro is a complex and highly coordinated process. Despite the challenges, researchers are exploring various in vitro approaches, which offer advantages such as negating the risk of contaminating a patient with cancer cells, avoiding xeno-exposures by using xeno-free media, and allowing for precise spatial and temporal control of cellular exposures. The ultimate goal of IVS is to generate a sufficient number of functional sperm cells in vitro for clinical applications.

Advances in regenerative medicine have introduced novel techniques for in vitro spermatogenesis, including ex vivo tissue culture and technologies like bioreactors, microfluidic systems, organoids, 3D bioprinting, and scaffolds. These techniques aim to replicate the physiological conditions and structural support necessary for cell growth and tissue function.

Culture techniques for IVS can be broadly categorized into 2D and 3D systems. While 2D culture systems involve cells grown as a monolayer, they struggle to fully replicate the natural microenvironment due to limited cellular communication and interaction. In contrast, 3D culture systems are better able to mimic physical cell interactions, tissue organization, communication, and the function of testicular cells. In a 3D framework, cells can attach to each other, facilitating their specific functions like adhesion, proliferation, and apoptosis.

  • 3D Scaffolds: These use natural or synthetic matrices to provide structural support for cell growth. They are important for developing the extracellular matrix (ECM), which is crucial for vasculature and spatial arrangements between Sertoli and germ cells, thus mimicking seminiferous tubules in vitro. Examples of materials include Matrigel, hydrogels, hard supports, lyophilization, electrospinning, stereolithography, decellularized extracellular matrix (dECM) from testicular tissue, platelet-rich plasma (PRP), collagen gel, agarose, and laminin-coated protein. Scaffolds like alginate hydrogel with Sertoli cells have shown promise in increasing SSC markers and improving transplantation efficiency in mice.
  • Organoids: These are scaffold-free techniques where organoids are suspended in culture. They are designed to model organ function and can be generated from pluripotent or primary niche cells. Aggregation of these cells leads to self-organization into 3D masses that aim to represent the organ’s functional morphology.
  • 3D Bioprinting: This technology allows for increased precision in cellular layer placement and utilizes bioinks that replicate the ECM and natural microenvironment. It can be used to fabricate complex biological constructs, including the tube-shaped architecture of seminiferous tubules. Studies have shown that 3D bio-printed seminiferous tubules can lead to an increase in certain stem cell and spermatocyte markers compared to organoids.
  • Organ-on-a-Chip / Microfluidic Systems: These systems aim to replicate physiological conditions by incorporating physical elements like vascular flow, shear stress, oxygen gradients, and nutrient distribution. A microfluidic device incorporating these elements has shown potential to maintain the artificial niche environment throughout a complete cycle of mouse spermatogenesis.

The evolution of ex vivo culture conditions has moved from basic monolayer cultures to static organ culture systems (like air–liquid interface and hanging drop setups) and then to dynamic 3D systems such as organoids and organ-on-a-chip. Static setups are similar to the SSC niche in collecting spermatogenic and accessory cells and facilitating cell-to-cell contact and communication, but they lack crucial dynamic physical elements.

Significant progress has been made in mouse models, with the production of spermatids and oocytes entirely in culture from pluripotent stem cells (PSCs) that were potent enough to support the development of healthy mice after fertilization. Organ culture methods have been successful in inducing in vitro spermatogenesis from mouse germ cells, leading to mature spermatozoa capable of generating healthy offspring through intracytoplasmic sperm injection (ICSI).

While human IVS is still progressing, some studies have reported the differentiation of haploid cells from human germline stem cells using 3D systems, although with limited efficiency. Comparing 2D and 3D cultures of human SSCs from patients with non-obstructive azoospermia (NOA), 3D soft agar conditions increased both SSCs and haploid germ cell numbers compared to 2D monolayers.

Future directions for IVS include developing more reliable and efficient culture systems that are devoid of animal-derived components and substrates. Bioengineered organoids supported by smart bio-printed tubules and microfluidic organ-on-a-chip systems are seen as promising, personalized platforms for autologous PSC sources to undergo the spermatogenetic cycle.

IVS also serves as a valuable model system for studying processes currently difficult to examine in vivo, such as human gametogenesis, the mechanisms of normal spermatogenesis, infertility, testicular disorders, and the effects of drugs and toxic compounds on testicular cells. Ultimately, IVS could potentially enhance male fertility outcomes.

Oocytes are essential for fertility, and in addition to transmitting their genome, they contain a large cytoplasm with factors that direct embryonic development. Reconstituting mouse oocyte and embryonic development in culture has provided significant experimental opportunities and enhanced understanding of molecular mechanisms.

The journey of in vitro culture systems for oocyte development has evolved over nearly a century. Recent progress in stem cell biology, particularly the use of PSCs, has led to impressive advances in generating functional cell types in culture, including oocytes. A robust protocol for generating PSC-derived oocytes has been reported, particularly in mice.

The process of in vitro oogenesis typically involves several steps, mimicking the in vivo development: in vitro PGC differentiation (IVP) to generate PGC-like cells (PGCLCs), followed by co-culture with gonadal somatic cells for in vitro oocyte differentiation (IVD), leading to follicle formation. Subsequent culture of these follicles for in vitro growth (IVG) and then in vitro maturation (IVM) results in mature MII oocytes.

In mouse models, remarkable advances have been achieved:

  • Reconstitution of the entire life cycle of the mouse female germ line in vitro.
  • Production of oocytes entirely in culture from PSCs that are potent to support the development of healthy mice after fertilization.
  • Generation of offspring from oocytes derived from in vitro primordial germ cell-like cells.
  • PSC-derived MII oocytes have been successfully fertilized and developed into full-term mice after embryo transplantation. These PSC-derived MII oocytes were described as almost indistinguishable from those grown in vivo.

Despite these successes, applying in vitro gametogenesis, including IVO, to reproductive medicine or infertility treatment remains challenging. A significant concern is the quality of in vitro-derived oocytes. Challenges in the in vitro culture system that affect oocyte competence include developmental gaps between PGCLCs and somatic cells, issues with meiosis (e.g., meiotic chromosome asynapsis), premature activation of certain genes, insufficient cell-cell interactions, improper accumulation of maternal transcripts/proteins essential for early embryo development, and lack of oocyte dormancy. Aberrant expression during oocyte development can lead to abnormal transcript accumulation.

Interventions to improve oocyte quality are being explored, such as utilizing healthy ooplasm via somatic cell nuclear transfer (SCNT) to potentially address issues with cytoplasmic components crucial for fertilization and subsequent embryogenesis.

Applying IVG to species other than mice remains challenging because the mechanisms underlying germ cell development differ between species.

Infertility is a global health issue affecting a significant portion of the population. Male infertility can result from various factors, including genetic diseases, trauma, congenital disorders, and gonadotoxic treatments, which can cause malfunction in spermatogenesis. These causes can be pre-testicular, testicular, or post-testicular. Conditions like non-obstructive azoospermia (NOA), where there is an absence of sperm in ejaculate not due to blockage, are a target for IVS research.

IVG holds promise as a future therapeutic tool for infertility. For men with complete germ cell aplasia or those who have lost their germ cells due to treatments like chemotherapy, obtaining a novel germ cell source from their own somatic cells, reprogrammed into induced pluripotent stem cells (iPSCs), is a major research focus. Differentiating human ESCs and iPSCs into SSCs and haploid germ cells is being investigated to potentially provide a source of gametes for these patients.

In summary, in vitro gametogenesis, encompassing both in vitro spermatogenesis and in vitro oogenesis, is a rapidly advancing field leveraging stem cell technology and sophisticated 3D culture systems to replicate complex biological processes outside the body. While significant progress has been made, particularly in mouse models leading to viable offspring, challenges remain in translating these achievements to humans, especially concerning the efficiency and quality of the resulting gametes. The development of more advanced bioengineered platforms and a deeper understanding of the intricate cellular and molecular requirements of gametogenesis are crucial next steps towards realizing the full potential of IVG for treating infertility.

FAQ

The process of IVO typically involves several steps mimicking in vivo development: in vitro differentiation of primordial germ cell-like cells (PGCLCs) from PSCs, co-culture with gonadal somatic cells for in vitro oocyte differentiation (IVD) and follicle formation, subsequent culture of these follicles for in vitro growth (IVG), and then in vitro maturation (IVM) to obtain mature metaphase II (MII) oocytes.

Achieving complete spermatogenesis and spermiogenesis in vitro is a complex and highly coordinated process. Challenges include recreating an appropriate microenvironment that mimics the physiological conditions of the testis, as well as obtaining functional sperm in sufficient quantity.

Unlike 2D monolayer cultures, 3D culture systems better mimic the physical cell-cell interactions, tissue organization, communication, and function of testicular cells. Within a 3D framework, cells can attach to each other, facilitating their specific functions like adhesion, proliferation, and apoptosis.

Various 3D culture models are being explored, including 3D scaffolds (using natural or synthetic matrices), organoids (scaffold-free techniques where cells self-organize into 3D masses), 3D bioprinting (allowing precise cell placement and the use of bioinks), and organ-on-a-chip or microfluidic systems (aiming to replicate physiological conditions with dynamic elements). Studies have used methods like soft agar culture systems and platelet-rich plasma (PRP)-based tissue scaffolds for culturing human SSCs in 3D.

Significant progress has been made in mice, allowing the production of spermatids and oocytes entirely in culture from pluripotent stem cells capable of supporting the development of healthy mice after fertilization. Organ culture methods have been successful in inducing in vitro spermatogenesis from mouse germ cells, leading to mature sperm capable of generating healthy offspring via ICSI.

Human IVS is still progressing. Some studies have reported the differentiation of haploid cells from human germline stem cells using 3D systems, although with limited efficiency. Recent studies in various 3D culture systems with human testicular cells have shown the presence of spermatocytes and spermatids, but achieving high yields and full functionality remains a challenge.

Remarkable advancements have been made in mice, including the in vitro reconstitution of the entire life cycle of the female germline. Oocytes derived from pluripotent stem cells (PSCs) have been produced entirely in culture and were capable of supporting the development of healthy mice after fertilization. MII oocytes derived from PSCs have been successfully fertilized and developed into full-term mice after embryo transfer.

A major challenge is the quality of in vitro derived oocytes. Current in vitro culture systems can lead to developmental deficiencies, meiotic issues, premature gene activation, insufficient cell-cell interactions, inappropriate accumulation of maternal transcripts/proteins essential for early embryonic development, and a lack of oocyte dormancy. Aberrant expression can lead to abnormal accumulation of transcripts.

Future directions for IVS include developing more reliable and efficient culture systems devoid of animal-derived components. Promising platforms being considered are bioengineered organoids supported by smart bio-printed tubules and microfluidic organ-on-a-chip systems, offering personalized platforms for autologous PSC sources to undergo the spermatogenetic cycle. IVS also serves as a valuable model for studying processes difficult to examine in vivo, such as human gametogenesis and mechanisms of infertility.

Bibliography

The introduction highlights that infertility is a common issue, affecting approximately 17% of adults worldwide. Of these cases, 20–30% result directly from male infertility. Understanding male infertility is crucial for addressing reproductive health challenges, as it directly impacts the ability to conceive and can have a significant emotional and psychological impact on the affected individual and their partner.

One specific cause of male infertility discussed is Non-Obstructive Azoospermia (NOA). This condition, where no sperm are present in the ejaculate, can result from factors including single gene mutations that disrupt protein production and functionality. A comprehensive review of cellular dysfunction and genetic underpinnings of NOA has been performed by Piechka et al. (2023).

Currently, for men with NOA, advanced reproductive therapies offer the potential for sperm extraction from the affected individual. A common procedure for this is microdissection testicular sperm extraction (microTESE), first described by Schlegel (1999). Successful sperm retrieval using microTESE occurs in approximately 50% of patients. However, once sperm are retrieved, options are limited. For couples experiencing NOA, the available options typically involve in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI), which ultimately leads to live birth rates of 10–25%. Given these limitations, increasing efforts are being directed towards finding regenerative therapies to induce spermatogenesis either in vivo or in vitro.

The review article aims to provide an overview of the physiology of spermatogenesis, current treatments for male infertility (like microTESE and IVF/ICSI), advances in IVS, and the implications of IVS. It notes that while IVS technology is promising, further work is needed to develop successful, replicable, and safe IVS for humans. The development of IVS involves the intersection of tissue engineering, molecular biology, and reproductive medicine, allowing for multidisciplinary involvement to overcome challenges and realize regenerative therapies as a viable option.

Beyond the technical challenges, the article highlights important ethical considerations associated with IVS. It is deemed essential to continuously monitor the viability and functionality of sperm generated through IVS, as well as factors that contribute to success and potential risk factors that could result in congenital disabilities or other harms. Additional ethical considerations include ensuring that germ cells are sourced with ongoing consent, that services are offered to preserve future fertility, and that patients are given the opportunity to exercise their fundamental right to reproduce. Crucially, clear information regarding risks and benefits must be provided before beginning any IVS treatment.

In terms of research, the excerpts indicate the review touches upon various aspects relevant to IVS, including different types of 3D culture systems (like bioprinting), the composition of testicular tissue and extracellular matrix, and the complexities of genetics and azoospermia.

The article’s structure, as outlined in the introduction, suggests a progression from fundamental understanding (physiology) to current clinical practices, and then to the cutting-edge research in IVS and its broader societal impacts (implications). The detailed authorship contributions listed show the roles of each author in the creation of the review, including writing, validation, methodology, data curation, formal analysis, visualization, and conceptualization.

In summary, the article provides a comprehensive review of the state of human in vitro spermatogenesis research, emphasizing the context of male infertility and the limitations of current ART, outlining the promising yet challenging path of IVS development, and highlighting the crucial ethical considerations that must accompany this scientific progress. It represents a collaborative effort across multiple disciplines and institutions to chart the course for future regenerative therapies for male infertility.

Despite researchers’ long-term goals for future clinical applications of IVG, the authors note that little is currently known about the views of IVG held by the stakeholders who might be most affected by its introduction in humans.

To address this gap, the researchers conducted a qualitative study using focus groups and semi-structured interviews. They utilized a methodology well-suited for exploring novel considerations raised by a future technology. Participants included individuals experiencing involuntary childlessness. Specifically, the study involved self-identified cisgender-heterosexual and LGBTQ+ individuals who were either currently undergoing or had previously undergone fertility treatment, as well as fertile LGBTQ+ individuals interested in family formation. Follow-up interviews were conducted with 17 focus group participants, and an additional 8 interviews were conducted with key informants in these communities. In total, the study involved data collected over 11 focus groups and 25 individual interviews.

Participants were first introduced to the scientific concept of IVG and how it could potentially be used for assisted reproduction. They were then asked for their views about IVG. The focus groups lasted approximately 90 minutes, while interviews ranged from 1 hour to 90 minutes. Both focus groups and interviews were conducted via Zoom video conferencing with participants located across the United States. Participants provided consent and were financially compensated for their time.

The analysis of the 42 hours and 44 minutes of recordings from interviews and focus groups followed a grounded theory approach. This involved independent readings of transcripts, generation of interpretative categories, resolving discrepancies, and jointly creating a codebook to identify and analyze core themes. Representative quotes were selected to illustrate participant views in their own words.

The study reports on participant views regarding potential applications of IVG to human reproduction, focusing on dominant themes that emerged from the data. These dominant themes included:

  • Systemic frustrations with existing reproductive care and family formation. Participants highlighted the limitations and challenges they faced with current assisted reproductive technologies (ART).
  • Hope that IVG could increase the success of ART while limiting its potential to cause pain and trauma.
  • Hope that IVG could increase reproductive justice for LGBTQ+ families.

Concerns voiced by participants primarily centered on access and safety. Figure 1 in the sources provides a visual representation of the total number of discrete times key topics were discussed across all focus groups and interviews.

Regarding hopes (Figure 1A):

  • Gamete Scarcity was a major topic, discussed a total of 106 times. IVG holds potential for people who cannot use their own gametes to have a biological child, including those whose bodies do not produce gametes, individuals in same-sex relationships, or those unable to undergo egg retrieval.
  • Avoiding Egg Retrieval was another significant hope, discussed 61 times. Participants frequently highlighted the potential of IVG to facilitate having a biological child while avoiding the pain associated with egg retrieval. They described egg retrieval as “horrible” and “excruciating,” even when the procedure went as planned. The authors note that the potential of IVG to avoid the pain and trauma of egg retrieval is rarely discussed in the literature, where it is typically framed medically as avoiding the rare but dangerous complication of ovarian hyperstimulation. In contrast, participants saw this as a major benefit.
  • LGBTQ+ Reproductive Equality was discussed 65 times. Participants expressed hope that IVG could increase reproductive justice for LGBTQ+ families. They pointed out that current legal restrictions and uncertainties regarding parental rights for non-biological parents in same-sex couples, varying by state, present additional burdens and fears for LGBTQ+ parents. IVG could potentially offer new avenues for family formation for these individuals.

Regarding concerns (Figure 1B):

  • Financial Barriers were the most frequently discussed concern, totaling 179 mentions. Within this category, “Health insurance” was discussed 127 times and “Cost inequity” was discussed 52 times. Access and cost were central themes for concerns.
  • Safety was discussed 116 times. Specific safety concerns included “Pregnancy health” (45 mentions), “Gamete and embryo integrity” (32 mentions), “Acceptable risks” (28 mentions), and “Child health” (11 mentions). Monitoring the viability and functionality of sperm generated through IVG, as well as potential risk factors, is considered essential.
  • Ethically Questionable Uses were discussed 77 times. Subtopics included “Ethical guidelines” (27 mentions), “Stealing cells and abuses” (21 mentions), “Eugenics; designer babies” (18 mentions), and “Solo reproduction” (11 mentions). Ethical considerations noted in the context of related technologies include ensuring germ cells are sourced with ongoing consent and providing clear information regarding risks and benefits.

The article also highlights perspectives from individuals from underserved communities. These participants sometimes expressed skepticism towards IVG and biomedical research due to historical abuses, which they linked to “the medical industrial complex, Western science, and colonialism”. Furthermore, some noted that they could not dedicate mental, emotional, or organizational resources to thinking about IVG when facing more pressing concerns like high rates of pregnancy-related death among Black and Indigenous birthing people.

In conclusion, the study emphasizes that the perspectives of potential beneficiaries, grounded in lived experience, provide valuable insights into the future real-world uses and concerns surrounding IVG and related embryo and germ-line research. The data show that issues of equity and access to reproductive care and family formation are central rather than secondary for potential IVG users and should be foregrounded in discussions about the social and ethical implications of IVG. The study highlights existing gaps between the views of potential users and key themes in scholarly and media debates, underscoring the importance of sustained public engagement through empirical studies and other means to ensure the inclusion of all IVG stakeholders in this debate.

The authors state that human conception leading to a live birth is naturally an inefficient process, and assisted reproductive technologies have been developed to address this. Recent experimental work, primarily using the mouse model, has demonstrated a significant breakthrough: the capability of developing eggs and sperm in vitro.

This IVG process in mice has been achieved by deriving gametes from induced pluripotent stem cells (iPSCs). The technology involves selecting each step of the IVG process when using mouse iPSCs. A notable success in the mouse model is that the resulting oocytes display high fertilizability, and the embryos derived from these iPSC gamete procedures are capable of implantation and result in live births.

The development of IVG technology forces a re-evaluation of what is possible in the field of fertility treatment. The authors suggest that IVG includes the potential for same-sex conception, the possibility of expanded fertility for patients experiencing abnormal ovarian development, and even the prospect of obtaining eggs from fibroblasts of postmenopausal women.

However, despite these remarkable possibilities and successes in the mouse model, a significant challenge remains: the efficiency of this technology is currently unacceptably low. Specifically, approximately only 4% of transferred embryos derived from iPSC gamete procedures in mice result in a live birth. The authors explicitly state that these rates are low when compared to the outcomes observed in traditional in vitro fertilization procedures also performed in the mouse model. This significant disparity raises critical questions about what might be missing in the current state-of-the-art technologies and successes.

The article posits that the ability to modulate epigenetic reprogramming is a key factor in improving IVG outcomes. Epigenetic reprogramming involves changes in gene expression that do not alter the underlying DNA sequence but are crucial for proper development and function, including gamete development. The authors express hope that being able to modulate epigenetic reprogramming increases the potential for future eggs and sperm derived from in vitro approaches to be of high fidelity. Achieving this “high fidelity” is seen as essential, such that at some point, IVG may become a possibility in humans.

Furthermore, the article notes a limitation regarding the use of mice as a model for studying the germ line in this context. It points out that many of the epigenetic changes and mechanisms leading to the epigenome differ between mice and humans. This suggests that findings related to epigenetics in mouse IVG may not directly translate to human IVG, highlighting a need for further research specifically focused on human cells.

In summary, this article highlights the exciting potential of IVG, demonstrated by the ability to produce functional gametes from iPSCs in mice that can lead to live births. It points to the technology’s broad potential applications for various individuals facing infertility or seeking alternative reproductive options. Crucially, it identifies the currently low efficiency of live births from IVG-derived embryos in mice as a major hurdle and emphasizes the importance of understanding and modulating epigenetic reprogramming as a key strategy to improve the fidelity and ultimately the success of IVG, particularly as researchers work towards its application in humans, acknowledging the limitations of relying solely on mouse models for epigenetic insights.

The authors aim to review the current understanding and identify limitations in generating oocytes in vitro, and from this foundation, explore opportunities for future improvements of the in vitro approach for generating high-quality oocytes.

The article highlights that recent progress in stem cell biology has led to impressive advancements in generating tissues and fully functional cell types in culture. These approaches are based on pluripotent stem cells (PSCs).

The process of female germ cell development in vivo in mice is described as follows: After embryo implantation, primordial germ cell (PGC) precursors emerge in the proximal epiblast and migrate to the gonads. Upon sex determination, female PGCs develop as oogonia, forming germ cell cysts through proliferation. These cysts subsequently break down, leading to the formation of primordial follicles housing non-growing oocytes. A subset of primordial follicles undergoes folliculogenesis, progressing to antral follicles. Eventually, the cumulus-oocyte complex (COC) is released from an antral follicle, resulting in the emergence of mature metaphase II (MII) oocytes. Key biological events such as embryonic development, germ cell characterization, meiosis, and oocyte development occur along a specific timeline in vivo, accompanied by corresponding epigenetic events like DNA methylation, X-chromosome inactivation, and histone modification.

The article then details an established culture system for germ cell development from pluripotent stem cells (PSCs) in vitro, referencing the work by Hikabe et al.. In this system, PSCs (either Embryonic Stem Cells – ESCs, or Induced Pluripotent Stem Cells – iPSCs) are differentiated into primordial germ cell-like cells (PGCLCs) through in vitro PGC differentiation (IVP). The sorted PGCLCs are then co-cultured with somatic cells collected from fetal gonads, leading to the formation of a reconstituted ovary (rOvary). Primary or secondary follicles appear in the rOvary during in vitro oocyte differentiation (IVD). The isolation and subsequent culture of these follicles lead to the development of fully grown oocytes and MII oocytes through in vitro growth (IVG) and in vitro maturation (IVM), respectively. MII oocytes generated from PSCs using this culture system can proceed to preimplantation development in vitro after fertilization with sperm. This advancement of in vitro culture systems for oocyte development has been a significant journey, evolving for nearly a century to the cutting-edge methodologies of today, primarily aimed at understanding the mechanism of germ cell development and offering insights into developmental and reproductive biology.

Despite the progress, the article highlights key challenges (indicated in brown in one figure) in the in vitro culture system that affect the competence of oocytes for preimplantation development and embryogenesis. These challenges are listed along the timeline of in vitro oocyte development. While the specific details of these challenges are not fully elaborated in the provided excerpts, the source identifies them as crucial issues to address for improving oocyte quality.

The authors discuss the potential applications and opportunities that obtaining fully functional oocytes through in vitro culture opens up for research and medicine, particularly in infertility treatment. Using donor oocytes can help compare the quality of cytoplasmic components between natural oocytes and PSC-derived oocytes, aiding in evaluating the competence of embryogenesis. Furthermore, the source mentions that haploid ESC genomes can be directly modified in culture due to their self-renewal capacity, making them a promising tool for genetic investigations, particularly in biological studies such as genetic screening.

In summary, this review article provides an overview of generating oocytes in vitro from pluripotent stem cells, outlining the steps involved in an established mouse model system. It emphasizes the historical progression of in vitro oocyte culture and, importantly, points out the limitations and challenges that currently hinder the generation of highly competent oocytes suitable for preimplantation development, while also highlighting the potential benefits for research and medicine, including the use of haploid ESCs for genetic studies. The authors’ future improvements will focus on addressing these limitations to produce high-quality oocytes in vitro.

The authors highlight that sperm is a crucial male gamete essential for sexual reproduction, and it is naturally generated through the intricate processes of proliferation, differentiation, and morphological transformations of spermatogonial stem cells (SSCs) within the specialized microenvironment of the testes. Replicating this complex biological environment artificially presents a significant challenge. However, they note that advancements in various interdisciplinary fields, including physics, materials science, and cell engineering, have facilitated the utilization of innovative materials, technologies, and structures to achieve in vitro sperm production.

The article categorizes the techniques developed for inducing in vitro sperm production into two major systems: matrix-based approaches and non-matrix-based approaches. The review aims to offer researchers in this field a comprehensive view by providing detailed discussions on these systems, including aspects like matrix formulation, culture medium composition, methods for detecting markers or cell characteristics, and whether the techniques have successfully generated sperm cells or resulted in offspring. By comparing the similarities and differences among these technical systems and outlining their research progress, the authors intend to provide a broad panorama and share their own perspectives and prospects for the future.

Matrix-Based Approaches

Within the matrix-based category, the article discusses Biomaterial-Based Scaffold Induced Spermatogenesis. The most commonly utilized testicular scaffold in this approach is the decellularized testicular matrix (DTM). DTM is prepared by treating testicular tissue with a substance like SDS to remove the cellular components while preserving the essential three-dimensional structure and the major constituents of the natural tissue scaffold. These conserved components include type I and IV collagen, fibronectin, laminin, and glycosaminoglycans. Furthermore, proteomic analysis of DTM has revealed the presence of numerous other extracellular matrix proteins, indicating its intricate composition.

Studies using DTM have shown promising results. Culturing mouse SSCs on DTM hydrogel scaffolds containing the stimulant D-serine demonstrated that these scaffolds are suitable for SSC culture and actively promote their proliferation, as indicated by an upregulation in Plzf expression levels. Further research indicated that DTM hydrogels, also containing D-serine, could enhance both the proliferation and differentiation of SSCs, leading to a significant increase in the expression of the pre-meiotic gene Plzf, the meiotic gene Sycp3, and the post-meiotic gene Tnp1.

The application of DTM extends to human cells as well. Human spermatogonial cells cultured on decellularized testicular matrix derived from sheep testes exhibited significantly enhanced expression of pre-meiotic genes such as OCT4 and PLZF, meiotic genes like SCP3 and Boule, and post-meiotic genes including Crem and Protamine2 after 6 weeks of culture, when compared to two-dimensional (2D) culture systems. The expression levels of differentiation genes were observed to increase with longer culture periods. In another experiment, sheep DTM was used as ink for 3D printing hydrogel scaffolds to cultivate mouse testicular cells. This approach resulted in improved cell viability and engraftment capacity, along with increased expression of pre-meiotic markers such as Plzf, Gfrα1, and Id4. Importantly, spermatogonial cells were observed to differentiate into sperm-like cells on the DTM scaffold in this study. Human spermatogonial cells isolated and cultured on sheep DTM also showed significantly increased expressions of pre-meiosis genes OCT4, Plzf, SCP3, BOULE, and post-meiosis genes CREM and Protamine2 compared to the 2D group.

Despite these successes, the article points out a significant challenge associated with DTM: inherent variability. Due to differences in the production process and variations in age and condition among tissue donors, each batch of DTM can exhibit variations, potentially leading to inconsistencies in experimental outcomes across different groups.

Non-Matrix-Based Approaches

The review also discusses Non-Biomaterial-Based Scaffold Induced Spermatogenesis, where the culture scaffolds are derived from materials other than biological matrices like DTM. A commonly utilized organic scaffold in this category is derived from agarose. A study culturing pig SSCs in a 0.2% agarose 3D hydrogel demonstrated significant increases in the transcript levels of NANOG, EPCAM, UCHL1, GFRA1, and Plzf. Additionally, notable elevations were observed in the protein levels of Plzf, OCT4, SOX2, and TRA-1-81. The transcription of OCT4 and THY1 was upregulated, while the translation of NANOG and TRA-1-60 was also upregulated. Conversely, the transcription level of the germ cell differentiation marker C-KIT exhibited significant downregulation. These findings suggest that a three-dimensional (3D) culture microenvironment using agarose can more effectively sustain the self-renewal of pig SSCs compared to a 2D culture microenvironment.

Further research has shown that scaffolds for cultivating SSCs can be composed of various materials. Examples include:

  • An agarose and laminin-coated protein scaffold was successfully used to cultivate human spermatogonial cells, resulting in the presence of cells positive for Plzf, SCP3, PRM2, and Acrosin, as well as the formation of sperm-like and elongated sperm cells.
  • A scaffold created using SACS along with laminin-coated protein and supporting cells was used to cultivate human SSCs, which showed expression of Plzf, α6-Integrin, Bcl2, and c-KIT genes.
  • Co-cultivating mouse spermatogonial cells with alginate hydrogel and supporting cells led to significantly increased levels of integrin alpha-6, integrin beta-1, Nanog, Plzf, Thy-1, Oct4a, and Bcl2 expression. This type of scaffold was effective in promoting proliferation and maintaining the self-renewal capacity of SSCs, while also improving the efficiency of SSC transplantation.
  • Purified pig SSCs were cultivated on poly-L-lysine (PLL) coated dishes along with laminin coating for 28 days without compromising their undifferentiated germ cell phenotype.
  • Mouse spermatogonial cells cultured on laminin-coated protein (LCP) and poly-L-lysine (PLL) demonstrated expression of VASA, GPR125, Uchl1, GFR-A1, and DAZL genes, indicating that LCP and PLL-based in vitro culture systems are efficient for the long-term maintenance of stable SSCs with self-renewal ability.
  • A gelatin-based hydrogel known as Dynamic gelatin-based hydrogels was utilized to mimic the inherent structural dynamics of the extracellular matrix for the cultivation of primordial germ cells. Following cultivation, notable expression of pluripotency markers like NANOG and OCT3/4 was observed, along with the presence of nestin-positive, alpha-fetoprotein-positive, and alpha-SMA-positive cells, representing differentiated cells from all three germ layers. These findings suggested that mouse embryonic stem cells (mESCs) obtained after 2 months of 3D cultivation in this hydrogel possessed functional pluripotency, and the hydrogel served as an effective 3D platform supporting their long-term proliferation and self-renewal.
  • Cryopreserved mouse testicular tissue cultured in an agarose hydrogel containing VEGF nanoparticles demonstrated maintenance of seminiferous tubule integrity and the presence of Plzf- and KI67-positive cells, suggesting that this gel formulation can enhance the primordial germ cell recovery rate.

Under the umbrella of in vitro induced spermatogenesis, the article also details the use of organ culture methods. This method involves placing small pieces of testicular tissue onto a gel block (such as agar) and culturing it in a medium that partially submerges the block. When employed with mouse spermatogonial cells, the organ culture method resulted in the observation of GFP-positive cells and the formation of round spermatozoa. Using this method, rat testicular tissue was successfully cultured for 52 days, showing the expression of Acrosin and Crem positive proteins and testosterone production after 3 days. The addition of adipose tissue from the epididymis even led to spontaneous contraction of the cultured seminiferous tubules after 21 days. Matsumura et al. demonstrated that organ culture techniques could effectively induce in vitro spermatogenesis from mouse germ cells, leading to the development of mature spermatozoa that could be sustained for over 70 days within the cultured tissue. Notably, Sato et al. successfully cultivated mouse germ cells using organ culture methods and obtained viable mouse sperm capable of generating healthy offspring through intracytoplasmic sperm injection (ICSI).

Conclusions and Prospects

In their conclusions, the authors emphasize that an imperative future direction for in vitro-induced spermatogenesis systems is the development of culture systems that are devoid of animal-derived components and substrates and possess a clear chemical composition. They argue that the use of animal-derived components, particularly inconsistent and ambiguous ones like sera, introduces numerous uncertainties that hinder the establishment of stable in vitro-induced spermatogenesis systems. This lack of consistency across different research groups, which have proposed various in vitro systems, makes it challenging to establish a unified and stable technical approach and translate it for clinical application, thereby impeding progress in the field. Therefore, the authors strongly advocate for researchers to collaborate across interdisciplinary domains, such as biophysics, biochemistry, and molecular biology, to develop more reliable and efficient culture systems that are free from animal sources or matrices.

The authors highlight that malfunction in spermatogenesis is a significant cause of infertility in approximately 7% of males. This malfunction can result from various factors including genetic diseases, trauma, congenital disorders, or gonadotoxic treatments. Currently, treatment options for male infertility can include testicular sperm extraction (TESE). However, a need exists for alternative approaches, particularly regenerative therapies.

The process of generating sperm, known as spermatogenesis, involves the proliferation, differentiation, and morphological transformations of spermatogonial stem cells (SSCs). This complex process occurs within a specialized microenvironment in the testes. This microenvironment is described as a three-dimensional, multifactorial, and dynamic niche that is crucial for supporting spermatogenesis and maintaining male fertility. The niche includes testicular Sertoli cells and is regulated by various peripheral cells within the seminiferous tubules. The interaction between supporting cells and germ/sperm cells is facilitated by a substantial amount of extracellular matrix (ECM) substances and structural matrix components, which collectively form the niche environment.

The article reviews the evolution of experimental culture platforms developed to recreate this complex testicular microenvironment in vitro. Early approaches involved basic monolayer SSC cultures which contained only stem cells and an artificial chemical microenvironment created through supplementation, lacking crucial physical contact and crosstalk with neighboring cells within the SSC niche. These evolved towards static organ culture systems.

Different in vitro culture systems for human samples have been tested, including both 2D and 3D culture systems. These include:

  • Monolayer culture: A 2D system where cells grow on a flat surface.
  • Air-Liquid Interface (ALI): A static organ culture system where tissue is cultured at the interface between air and liquid medium. ALI permits homogenous diffusion through a biphasic compartment. Studies using human fetal gonad strips in ALI platforms resulted in differentiation of haploid cells, but with limited efficiency (ranging between 0.07% to 9.83%). Culturing human testicular biopsies from prepubertal cancer patients in ALI also yielded an inadequate or absent production of haploid germ cells.
  • Hanging drop setups: Another static organ culture system that avoids damage by immersion in growth factor containing media.
  • Soft agar: A 3D culture system. Culturing SSCs from post-pubertal non-obstructive azoospermia (NOA) patients in a 3D soft agar culture system increased hSSCs and haploid germ cells compared to 2D monolayer culture.
  • Testicular organoid: A 3D culture system designed to mimic organ function. Organoids involve the differentiation of pluri/multipotent stem cells within a dynamic 3D milieu. Studies comparing bio-printed seminiferous tubules to organoids noted advantages in the bio-printed structure. Primary human testicular cells have been shown to self-organize into organoids with testicular properties.
  • Microfluidic platforms / Organ-on-a-chip systems: These advanced 3D culture techniques aim to replicate physiological conditions and model organ function. They involve differentiation of pluri/multipotent stem cells within a dynamic 3D milieu. The authors’ group recently designed a microfluidic compartment mimicking cellular, chemical, and physical factors of the SSC niche, including a pumpless flow, controlled O2 gradient, and diffusion-based nutrient distribution. This allowed for an increased culture period of up to 42 days, maintaining an artificial niche environment throughout a complete cycle of mouse spermatogenesis.

The article highlights the use of Pluripotent Stem Cells (PSCs) as a potential source for generating male germ cells in vitro, due to their plasticity. Both Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs) are classified as PSCs.

  • ESCs are derived from the inner cell mass of blastocysts and have the potential to differentiate into cells from all three germ layers. Their differentiation potential makes them candidates for in vitro male germ cell differentiation, but their use is limited by ethical and allogenic restrictions.
  • iPSCs are reprogrammed from somatic cells and possess a pluripotent profile similar to ESCs. Reprogramming can be achieved using chemically induced supplements or vectors (integrating or non-integrating). While integrating viral vectors offer higher efficiency, they risk unpredictable and permanent mutagenesis. Non-integrating vectors present higher safety profiles and are considered promising for autogenic or allogeneic stem cell-based therapies, including male fertility approaches.

Despite advancements, the current methods for in vitro spermatogenesis face challenges. The yield and functionality of the resulting human iPSC-derived haploid spermatogenic germ cells are currently insufficient for direct clinical application as a therapeutic tool. Existing experimental designs, while promising and often based on niche concepts, face long-term failure due to the absence of a dynamic platform that simulates the microvascular 3D physiology of the seminiferous tubules. This lack of dynamic conditions leads to shortcomings in physical elements such as vascular flow, shear stress, O2 gradient, and equal distribution of nutrients.

Addressing these limitations requires a novel biomedical transdisciplinary approach that recapitulates the embryologic development of male germ stem/progenitor cells within the testicular compartments and provides effective cell-to-cell and cell-to-ECM crosstalk under appropriate physical conditions.

The authors propose that bioengineered organoids supported by smart bio-printed tubules and microfluidic organ-on-a-chip systems represent promising future tools. These platforms offer the potential for efficient, precise, and personalized systems that allow autologous pluripotent stem cells to undergo the spermatogenetic cycle. Combining smart bioengineered materials with amassed cell-sourced organoids mimicking embryologic nest topography is considered promising. The development of such systems, devoid of animal sources or matrices, is an imperative future trend, requiring interdisciplinary collaboration in fields like biophysics, biochemistry, and molecular biology to establish a unified and stable technical system.

The article also mentions Very Small Embryonic-Like Cells (VSELs) as potential alternative sources for infertility studies. These cells are reported to be adult testicular primitive primordial germ cells or putative PSCs.

In conclusion, while current in vitro spermatogenesis techniques have shown progress, particularly in modeling the testicular niche, the critical challenges lie in achieving sufficient yield and functionality of germ cells. The next destination for this field involves developing dynamic 3D bioengineered systems, such as organoids, bio-printed structures, and organ-on-a-chip platforms, that more accurately mimic the complex physical, chemical, and cellular environment of the in vivo testis, with the ultimate goal of providing a viable therapeutic option for infertile males, including those with complete testicular aplasia.

Recent Publications

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