In vivo and in vitro sperm production: An overview of the challenges and advances in male fertility restoration

Experimental approaches for preservation of male fertility

Article information

Clin Exp Reprod Med. 2024;51(3):171-180

Publication date (electronic) : 2024 March 25

doi : https://doi.org/10.5653/cerm.2023.06569

Zahra Bashiri^,¹^,²^,³

, Seyed Jamal Hosseini ⁴^,⁵, Maryam Salem ⁶, Morteza Koruji^,²^,⁷

¹Endometrium and Endometriosis Research Center, Hamadan University of Medical Sciences, Hamadan, Iran

²Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

³Omid Fertility and Infertility Clinic, Hamedan, Iran

⁴4Biomedical Engineering Department, Amirkabir University of Technology, Tehran, Iran

⁵Department of Pharmaceutical Biomaterials and Medical Biomaterials Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

⁶Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

⁷Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran

Corresponding author: Zahra Bashiri Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran 1449614535, Iran Fax: +98-21-88622689 E-mail: zbashiri88@gmail.com

Co-corresponding author: Morteza Koruji Stem Cell and Regenerative Medicine Research Center and Department of Anatomy, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran 1449614535, Iran Fax: +98-21-88622689 E-mail: koruji.m@iums.ac.ir

Received 2023 October 3; Revised 2023 November 16; Accepted 2023 December 14.

Abstract

Male infertility can be caused by genetic anomalies, endocrine disorders, inflammation, and exposure to toxic chemicals or gonadotoxic treatments. Therefore, several recent studies have concentrated on the preservation and restoration of fertility to enhance the quality of life for affected individuals. It is currently recommended to biobank the tissue extracted from testicular biopsies to provide a later source of spermatogonial stem cells (SSCs). Another successful approach has been the in vitro production of haploid male germ cells. The capacity of SSCs to transform into sperm, as in testicular tissue transplantation, SSC therapy, and in vitro or ex vivo spermatogenesis, makes them ideal candidates for in vivo fertility restoration. The transplantation of SSCs or testicular tissue to regenerate spermatogenesis and create embryos has been achieved in nonhuman mammal species. Although the outcomes of human trials have yet to be released, this method may soon be approved for clinical use in humans. Furthermore, regenerative medicine techniques that develop tissue or cells on organic or synthetic scaffolds enriched with bioactive molecules have also gained traction. All of these methods are now in different stages of experimentation and clinical trials. However, thanks to rigorous studies on the safety and effectiveness of SSC-based reproductive treatments, some of these techniques may be clinically available in upcoming decades.

Keywords: Fertility preservation; In vitro and in vivo spermatogenesis; Sperm production; Transplantation

Introduction

In recent decades, spermatogonial stem cell (SSC)-based approaches to overcoming infertility caused by gonadotoxic therapy have become an important topic of investigation. The increasing survival rates of childhood cancer have drawn attention to the effects of gonadotoxic treatments on future fertility [1]. Unfortunately, sperm cryopreservation is not an ideal option for prepubertal boys who have not yet started to produce sperm. However, prespermatogonia, or SSCs that are responsible for initiating spermatogenesis at puberty, exist in prepubertal testicular tissue (TT); thus, cryopreservation of TT containing SSCs can preserve their reproductive potential [2]. Several medical centers worldwide currently use this technique and offer patients the option of freezing testicular biopsies before administering gonadotoxic therapy [3]. In addition, some centers admit patients who suffer from genetic or developmental disorders associated with prepubertal germ cell loss [4]. Currently, two major experimental protocols to restore fertility are being investigated: (1) SSC or TT transplantation and (2) SSC or TT culture [5].

The hypothetical purpose of preserving TT from biopsies is to allow for tissue autotransplantation in adulthood after the disease period. Maintaining interactions between the germ cells and their supporting somatic cells enables SSCs to regain differentiation within their natural niche [6]. Subsequently, these preserved SSCs or tissue fragments can be engrafted on a three-dimensional (3D) substrate by TT engineering. These 3D-culture systems provide a suitable microenvironment for cell attachment and specific growth factors for testicular regeneration [7]. The emergence of advanced bioengineered systems has offered new hope for maintaining male fertility through the development of functional male germ cells. Although SSC-based therapies provide an opportunity to restore fertility [8], technical and ethical barriers have limited the ability to complete spermatogenesis, and more efforts are required to establish a reliable culture system for clinical use.

Although some studies have focused on in vitro spermatogenesis resulting in mature gametes, none have found a sufficiently effective technique for differentiating human SSCs into functional sperm [9]. Despite the promising results obtained in recent years, further research is required to develop a therapeutic tool that will provide prepubertal boys and men with azoospermia the chance of fertility. This brief review highlights the next steps required to transform experimental approaches into clinical practice and emphasizes the current achievements and future challenges of fertility preservation in prepubertal boys and patients with azoospermia.

TT transplantation

TT transplantation involves the implantation of TT into various body sites, such as the testis, scrotum, and ectopic tissues [10]. One potential benefit of TT transplantation is the re-introduction of SSCs into the patient’s natural extracellular matrices. After TT transplantation, spermatogenesis can be induced through the systemic regulation of hormones, nutrition, and oxygen supply. Revascularization is also promoted in the TT grafts, which in turn generates mature sperm. The successful transplantation of TT, with subsequent offspring following intracytoplasmic sperm injection, was first reported in mice by Shinohara et al. [11] and Honaramooz et al. [12] in 2002, then in rat models [13], and later in higher mammals such as pigs, monkeys, and macaques [2,14-17].

The other option for sustaining fertility is transplantation of TT into experimental animals. The SSCs differentiate into sperm via the TT implanted in animal models, and then those cells are returned to the patient. However, no authentic cases of completed spermatogenesis using immature human TT xenografts have been reported [18]. This procedure is not yet authorized in clinical settings due to the substantial risk that germ cells can be contaminated by unidentified host tissue viruses such as retroviruses, as well as the endocrine differences between donor and recipient [12,19,20].

To date, numerous attempts to induce the maturation of human TT in vivo have been associated with only limited proliferation of SSCs. After the transplantation of TT, hypoxia and ischemic stress lead to tissue necrosis or activation of the apoptotic pathway [21], and ischemia-reperfusion may damage the SSCs’ niche as a result. Recent studies have succeeded in revascularizing testicular grafts by encapsulating the tissue or by applying molecular supplements such as angiogenic agents and antioxidants. These functionalized grafts have shown better outcomes [22].

Overall, the TT grafting technique has led to successful spermatogenesis in a range of animal models, but is still not an efficient clinical practice model because of the possibility of cancer cells spreading. Therefore, research aimed at improving the efficacy of tissue transplantation is still ongoing, and future studies must consider the significant variables affecting the survival rate of transplanted TT.

SSC transplantation

Since they can proliferate and differentiate, SSCs can restore fertility after being injected into the rete testis and ductuli afferents. A mouse model was used to evaluate SSC autotransplantation for the first time in 1994 [23], and promising results have been reported in other species since then [24-26]. Many studies have confirmed SSC migration to recipient seminiferous tubules and the formation of small colonies in those tubules after the transplantation of human SSCs into mouse testis [27,28]. However, the differentiation of autotransplanted SSCs into sperm has not been successful in humans. In autotransplantation, there is an inherent risk of reinfecting the patient with cancer cells and reintroducing the disease [10].

Attempts have been made with cell transplantation to exclude cancer cells from the testicular cell suspension by using fluorescence-activated cell sorting, magnetic-activated cell sorting [29,30], smart nanoparticles [31-33], and microfluidic devices [34]. Despite the current advancements, however, more reliable diagnostic techniques are required. Furthermore, because there are few SSCs in the testis, sufficient quantities must be created by in vitro proliferation for a successful treatment. The two major limitations in grafting efficiency include the low rate of cell proliferation in vitro and the absence of a standardized procedure with a high success rate [35].

Furthermore, the appearance of normal spermatogenesis after transplantation does not necessarily indicate normal functionality of the SSCs. These offspring may exhibit abnormal DNA methylation and low reproduction rates [36], which are probably due to the problems and inefficiencies of the SSC transplantation technique. The blood-testis barrier (BTB) can also be another major barrier in SSC transplantation. Singh et al. [37] investigated the high levels of glial cell-derived neurotrophic factor (GDNF) produced by immature Sertoli cells that resulted in increased SSC proliferation and significantly larger colonies in immature mice testes without the BTB. investigated the high levels of glial cell-derived neurotrophic factor (GDNF) produced by immature Sertoli cells that resulted in increased SSC proliferation and significantly larger colonies in immature mice testes without the BTB.

To summarize, removing cancer cells from testicular cell suspensions using specific culture conditions for the proliferation of SSCs and addressing the safety issues related to potential cell modification in the culture are concerns that should be addressed before clinical use [25].

In-laboratory sperm production using stem cells

To take advantage of assisted reproductive technologies, an infertile person must produce at least a few functional gametes. However, germ cells are not fully available in some azoospermia people, such as those with Sertoli-cell-only syndrome. Therefore, researchers have investigated the process of multipotent/pluripotent stem cell differentiation to produce functional sperm in vitro [38,39]. These studies have shown significant potential in animal models, but differences between human and other animal germ cells have prevented their widespread use in humans [40]. Several new studies are planned or currently underway to use stem cell therapy to treat male infertility [39,41]. Previous studies have reported that embryonic stem cells (ESCs) and induced pluripotent stem cells can be differentiated into germ cells in rodents, monkeys, and humans [42-44]. In studies by Hayashi et al. [42] and Cyranoski [45], sperm-like cells generated from mouse ESCs in a step-by-step process were injected into oocytes to produce offspring. Recently, two research groups produced spermatozoon-like cells from human ESCs, which were employed to treat azoospermic males [44,46]. Irie et al. [46] and Sasaki et al. [44] differentiated human ESCs into primordial germ cells (PGCs) with a gene expression pattern similar to nascent PGCs. Dong et al. [47] differentiated mouse ESCs into male germ cells using retinoic acid and placed them in special culture conditions to induce spermatogonial cell differentiation. After 6 days, differentiation of the cells was confirmed by evaluation of the acrosin gene [47]. In 2021, nonhuman primate ESCs were differentiated into spermatid-like cells by Khampang et al. [48] for the first time. Pronucleus formation was observed after microinjection of the spermatid-like cells into rhesus macaque mature oocytes. After artificial activation, they observed embryonic divisions, from the one-cell zygote stage to expanded blastocysts [48].

Mesenchymal stem cells (MSCs) are adult stem cells with the potential to enhance the efficiency of fertility restoration methods like SSC or TT transplantation and maintain fertility [48-58]. In 2006, Nayernia et al. [59] first reported that MSCs could differentiate into germ cells and express pre-meiotic germ cell markers. Shlush et al. [49] treated MSCs with retinoic acid, GDNF, putrescine, and leukemia inhibitory factor in a cell culture in vitro study. After 3 weeks, large flat cells and small round cells showed a morphology similar to Sertoli cells and germ cells. A xenotransplantation assay showed haploid cells with a flagellum-like structure that expressed meiotic markers and markers associated with spermatid cells [49]. However, these stem cell-based investigations have yet to document the production of morphological sperm. Since studies using transplantation or offspring production in humans cannot be confirmed for obvious ethical reasons, a different approach is required to verify the potential of human SSCs. It is also worth noting that a thorough examination for chromosomal abnormalities and epigenetic changes should be made to ensure that stem-cell-derived cells have normal genomes [60,61].

In vitro maturation of TTs or SSCs

Since SSC implantation into cultured testicular fragments is difficult and demands a high level of proficiency [62], an alternative approach could be the differentiation of SSCs into sperm via cell or TT culture (Figure 1) [10].

Figure 1.

In vitro maturation of testicular tissue or spermatogonial stem cells. Testis fragments can be cultured in dynamic or static systems. In the dynamic system, tissues are cultured with a continuous flow of fresh culture medium. In the static system, the tissues are cultured at the gas-liquid interface or in a hanging drop system, which requires constant changes in the environment. Isolated testicular cells can also be cultured in two-dimensional (2D) or three-dimensional (3D) culture systems. In 2D culture systems, testicular cells are seeded on a flat 2D culture surface, with or without co-culturing with other types of cells. In a 3D culture system, cells are engrafted into a 3D environment that allows for cell-cell or paracrine interactions. The 3D cell culture systems include porous, nanofiber, hydrogel scaffold, and organoid systems.

1. TT culture

TT cultures have been used for the study of mammalian spermatogenesis because the tubules and interstitial tissue preserve their spatial integrity. The earliest laboratory-based report of spermatogenesis using rabbit TT was published in 1920; however, most of the testicular cells rapidly degenerated [63]. The first research to successfully produce functional mouse sperm in the laboratory was not documented until 2011 [64]. To restore human fertility, haploid spermatids were injected into the oocytes of patients with azoospermia in 1999 [65], which ultimately led to the birth of healthy offspring. Subsequent studies provided possible treatments for spermatogenesis disorders using TT cultures with additional supplements to cure without genetic manipulation. One such experiment was conducted by Sato et al. [66] in 2012. When stem cell factor and colony stimulating factor-1 supplements were added to immature mouse testes cultured on agarose gel, spermatogenesis increased significantly and resulted in the production of long spermatids, flagellated sperm, and live offspring after microinjection. Although supplements are a critical factor for SSC differentiation, they are insufficient on their own to generate mature human sperm in vitro. According to some studies, gonadotropins can induce SSCs to differentiate into primary spermatocytes when they are added to a culture medium containing vitamins [67,68]. Furthermore, recent studies have developed dynamic culture systems in which TT is exposed to a continuous and controlled flow of fresh culture medium [69,70]. Komeya et al. [71] reported the successful 6-month maintenance of mouse spermatogenesis using a microfluidic system. They also achieved healthy offspring following microinjection of the sperm and spermatids derived from the cultured testis [71]. In another study, testicular fragments of immature mice cultivated on agarose gel showed a lower rate of spermatogenesis than tissue produced in a perfusion mini-bioreactor, indicating that the dynamic culture system could better simulate the physiological environment of the testis [72]. Yuan et al. [73] demonstrated that self-renewing SSCs and the organization of mature seminiferous epithelium from in vitro organogenesis of the fetal gonadal ridge of human testicular in vitro-derived spermatids (from spermatogonia) could fertilize oocytes and support subsequent blastocyst formation. Although in vitro spermatogenesis using laboratory organotypic cultures preserves the 3D structure and spatial arrangement of TT, this method still faces a range of challenges. These include the need for a large volume of tissue and the ultimate loss of significant portions of that tissue, as well as the inability to genetically modify the candidate cells [74].

2. SSC culture

To overcome some of the constraints of tissue culture and minimize cell mortality caused by the scarcity of nutrition and oxygen, two-dimensional (2D) culture systems were developed for SSCs. In addition, researchers examined the addition of growth factors or the co-culturing of germ cells and feeder cells (such as Sertoli cells, Vero cells, and mouse fibroblast cells) to promote spermatogenesis [75,76]. Since 2D culture systems could not create cell-cell interactions or the exchange of nutrients and gases for stem cell differentiation, the use of 3D substrates, while maintaining normal cell morphology, was proposed [77,78].

In recent years, a wide range of synthetic polymers (synthetic carbon [79,80], polycaprolactone [81], poly-L-lactic acid [82,83], polyvinyl alcohol [84], polyamide [85], and glycolic acid [83]) and natural polymers (alginate [86,87], gelatin [88], methyl cellulose [89], collagen [90,91], fibroin [92,93], chitosan [94], Matrigel [95-97], and agarose [75,98]) have been used to fabricate scaffolds for the purpose of improving spermatogenesis. Most synthetic scaffolds were found unsuitable for SSC differentiation, whereas natural biomaterials demonstrated superior performance. In research published in 2012, mature mouse sperm were produced on a soft agar culture system (SACS) [99]. In another recent study, the completion of human spermatogenesis was observed on agarose gel plus a laminin supplement in the presence of Sertoli cells after being cultured for 74 days [100]. Analysis showed that the laminin-enhanced 3D matrix supported all physiological activities of the SSCs, including survival and proliferation, and led to the differentiation of spermatogonial cells into morphological sperm. Despite this success in spermatogenesis, the method failed to retrieve live sperm from the culture system.

In addition to the type of biomaterial, the scaffold synthesis approach could be important in the process of cell differentiation [101]. Artificial testes have been designed using various scaffolding techniques (fibrous [81-83], porous [92,102], hydrogel [89,103], and 3D printed [104-107]). Nanofibrous scaffolds could not support spermatogenesis through the final stages due to their inability to simulate the topography of TT. Over the past decade, studies have shown that extracellular matrix (ECM)-based systems of decellularized TT in the form of testicular organoids [108-116], hydrogels [116,117], sponges [102], 3D systems containing ECM [111,112], and 2D and 3D immersion culture systems [114] lead to better survival and accumulation of the SSCs for proliferation and differentiation. However, none of these studies revealed evidence of complete spermatogenesis. In our previous studies, ECM solution was used as the ioink for fabrication of a hydrogel-printed scaffold following TT decellularization with a hypertonic solution. Mouse sperm with tail-like structures that were easily separated from the surface of semi-tubular structures were identified 3 weeks after the cultivation of testicular cells [106,107]. This method can be applied to regenerate TT and restore fertility in human studies. Investigations into spermatogenesis currently focus on the secretions derived from lab-grown cell cultures, including the role of the exosomes synthesized by Sertoli cells in the survival [118,119] and differentiation of SSCs [120]. Another study also showed that epididymosomes increased the proliferation of SSCs in a decellularized TT-derived 3D system [121]. Multiple studies have reported the use of a cell-derived ECM made of a decellularized matrix to stimulate differentiation in a variety of stem cells [122-125]. Therefore, it is recommended that somatic cells from the ECM produced with decellularized TT be used to evaluate the differentiation of SSCs in the future.

Conclusions

Spermatogenic arrest and the absence of haploid male germ cells are causes of infertility in men. Since infertility secondary to cancer treatments is rising, new methods to preserve and differentiate male germ cells are needed. Researchers have offered new hope in the treatment of these patients by using the transplantation of SSCs and tissue pieces or the cell suspension-derived laboratory sperm. Sperm have been successfully produced on ECM-derived 3D printing scaffolds and SACS, which may be a step towards the creation of artificial testes.

Although fertility restoration strategies have achieved promising results in animal models, these methods are currently not suitable for the human clinical setting due to the complexity of human spermatogenesis and the lack of sufficient human tissue. In addition, more research is required to confirm that these fertility-protection strategies are safe. The simplicity of in vitro cultures and the achievements obtained thus far imply that TT transplantation can be a secure and effective treatment for fertility preservation. However, it is important to optimize this method by purifying the suspensions and removing lingering cancer cells, as well as increasing the number of SSCs in vitro before transplantation. Despite the innovations in design and fabrication technology, customization of testicular scaffolds is still a critical issue and should be further investigated to confirm its therapeutic relevance. There is reason to hope that reproductive technology will soon advance through the design of new and efficient systems that benefit humans.

Notes

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Author contributions

Conceptualization: ZB, SJH. Data curation: ZB. Project administration: ZB. Visualization: ZB. Writing-original draft: ZB, MS. Writing-review & editing: SJH, MK.

References

1. Van Saen D. In search of the most efficient fertility preservation strategy for prepubertal boys. Facts Views Vis Obgyn 2013;5:45–58.

2. Fayomi AP, Peters K, Sukhwani M, Valli-Pulaski H, Shetty G, Meistrich ML, et al. Autologous grafting of cryopreserved prepubertal rhesus testis produces sperm and offspring. Science 2019;363:1314–9.

3. Tran KT, Valli-Pulaski H, Colvin A, Orwig KE. Male fertility preservation and restoration strategies for patients undergoing gonadotoxic therapies. Biol Reprod 2022;107:382–405.

4. Ozcan MC, Snegovskikh V, Adamson GD. Oocyte and embryo cryopreservation before gonadotoxic treatments: principles of safe ovarian stimulation, a systematic review. Womens Health (Lond) 2022;18:17455065221074886.

5. Yokonishi T, Ogawa T. Cryopreservation of testis tissues and in vitro spermatogenesis. Reprod Med Biol 2016;15:21–8.

6. Ntemou E, Alexandri C, Lybaert P, Goossens E, Demeestere I. Oncofertility: pharmacological protection and immature testicular tissue (ITT)-based strategies for prepubertal and adolescent male cancer patients. Int J Mol Sci 2019;20:5223.

7. Bhaskar R, Gupta MK, Han SS. Tissue engineering approaches for the in vitro production of spermatids to treat male infertility: a review. Eur Polym J 2022;174:111318.

8. Delgouffe E, Braye A, Goossens E. Testicular tissue banking for fertility preservation in young boys: which patients should be included? Front Endocrinol (Lausanne) 2022;13:854186.

9. Smart E, Lopes F, Rice S, Nagy B, Anderson RA, Mitchell RT, et al. Chemotherapy drugs cyclophosphamide, cisplatin and doxorubicin induce germ cell loss in an in vitro model of the prepubertal testis. Sci Rep 2018;8:1773.

10. Pelzman DL, Orwig KE, Hwang K. Progress in translational reproductive science: testicular tissue transplantation and in vitro spermatogenesis. Fertil Steril 2020;113:500–9.

11. Shinohara T, Inoue K, Ogonuki N, Kanatsu-Shinohara M, Miki H, Nakata K, et al. Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination. Hum Reprod 2002;17:3039–45.

12. Honaramooz A, Snedaker A, Boiani M, Scholer H, Dobrinski I, Schlatt S. Sperm from neonatal mammalian testes grafted in mice. Nature 2002;418:778–81.

13. Shinohara T, Kato M, Takehashi M, Lee J, Chuma S, Nakatsuji N, et al. Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination. Proc Natl Acad Sci U S A 2006;103:13624–8.

14. Nakai M, Kaneko H, Somfai T, Maedomari N, Ozawa M, Noguchi J, et al. Production of viable piglets for the first time using sperm derived from ectopic testicular xenografts. Reproduction 2010;139:331–5.

15. Kaneko H, Kikuchi K, Nakai M, Somfai T, Noguchi J, Tanihara F, et al. Generation of live piglets for the first time using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice. PLoS One 2013;8e70989.

16. Schlatt S, Honaramooz A, Boiani M, Scholer HR, Dobrinski I. Progeny from sperm obtained after ectopic grafting of neonatal mouse testes. Biol Reprod 2003;68:2331–5.

17. Liu Z, Nie YH, Zhang CC, Cai YJ, Wang Y, Lu HP, et al. Generation of macaques with sperm derived from juvenile monkey testicular xenografts. Cell Res 2016;26:139–42.

18. Wyns C, Van Langendonckt A, Wese FX, Donnez J, Curaba M. Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue. Hum Reprod 2008;23:2402–14.

19. Hou M, Andersson M, Zheng C, Sundblad A, Soder O, Jahnukainen K. Immunomagnetic separation of normal rat testicular cells from Roser’s T-cell leukaemia cells is ineffective. Int J Androl 2009;32:66–73.

20. Goossens E, Van Saen D, Tournaye H. Spermatogonial stem cell preservation and transplantation: from research to clinic. Hum Reprod 2013;28:897–907.

21. Poels J, Abou-Ghannam G, Herman S, Van Langendonckt A, Wese FX, Wyns C. In search of better spermatogonial preservation by supplementation of cryopreserved human immature testicular tissue xenografts with N-acetylcysteine and testosterone. Front Surg 2014;1:47.

22. Vermeulen M, Poels J, de Michele F, des Rieux A, Wyns C. Restoring fertility with cryopreserved prepubertal testicular tissue: perspectives with hydrogel encapsulation, nanotechnology, and bioengineered scaffolds. Ann Biomed Eng 2017;45:1770–81.

23. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994;91:11298–302.

24. Honaramooz A, Behboodi E, Megee SO, Overton SA, Galantino-Homer H, Echelard Y, et al. Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats. Biol Reprod 2003;69:1260–4.

25. Giudice MG, de Michele F, Poels J, Vermeulen M, Wyns C. Update on fertility restoration from prepubertal spermatogonial stem cells: how far are we from clinical practice? Stem Cell Res 2017;21:171–7.

26. Hermann BP, Sukhwani M, Winkler F, Pascarella JN, Peters KA, Sheng Y, et al. Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm. Cell Stem Cell 2012;11:715–26.

27. Mohaqiq M, Movahedin M, Mazaheri Z, Amirjannati N. Successful human spermatogonial stem cells homing in recipient mouse testis after in vitro transplantation and organ culture. Cell J 2019;20:513–20.

28. Mirzapour T, Movahedin M, Koruji M, Nowroozi MR. Xenotransplantation assessment: morphometric study of human spermatogonial stem cells in recipient mouse testes. Andrologia 2015;47:626–33.

29. Hermann BP, Sukhwani M, Salati J, Sheng Y, Chu T, Orwig KE. Separating spermatogonia from cancer cells in contaminated prepubertal primate testis cell suspensions. Hum Reprod 2011;26:3222–31.

30. Hou M, Andersson M, Zheng C, Sundblad A, Soder O, Jahnukainen K. Decontamination of leukemic cells and enrichment of germ cells from testicular samples from rats with Roser’s T-cell leukemia by flow cytometric sorting. Reproduction 2007;134:767–79.

31. Eslahi N, Shakeri-Zadeh A, Ashtari K, Pirhajati-Mahabadi V, Tohidi Moghadam T, Shabani R, et al. In vitro cytotoxicity of folate-silica-gold nanorods on mouse acute lymphoblastic leukemia and spermatogonial cells. Cell J 2019;21:14–26.

32. Shabani R, Ashjari M, Ashtari K, Izadyar F, Behnam B, Khoei S, et al. Elimination of mouse tumor cells from neonate spermatogonial cells utilizing cisplatin-entrapped folic acid-conjugated poly(lactic-co-glycolic acid) nanoparticles in vitro. Int J Nanomedicine 2018;13:2943–54.

33. Shams A, Shabani R, Asgari H, Karimi M, Najafi M, Asghari-Jafarabadi M, et al. In vitro elimination of EL4 cancer cells from spermatogonia stem cells by miRNA-143- and 206-loaded folic acid-conjugated PLGA nanoparticles. Nanomedicine (Lond) 2022;17:531–45.

34. Ashtari B, Shams A, Esmaeilzadeh N, Tanbakooei S, Koruji M, Moghadam MJ, et al. Separating mouse malignant cell line (EL4) from neonate spermatogonial stem cells utilizing microfluidic device in vitro. Stem Cell Res Ther 2020;11:191.

35. Picton HM, Wyns C, Anderson RA, Goossens E, Jahnukainen K, Kliesch S, et al. A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys. Hum Reprod 2015;30:2463–75.

36. Samplaski MK, Deault-Bonin M, Lo KC. Genetic and epigenetic changes after spermatogonial stem cell culture and transplantation. EJIFCC 2014;25:27–41.

37. Singh D, Paduch DA, Schlegel PN, Orwig KE, Mielnik A, Bolyakov A, et al. The production of glial cell line-derived neurotrophic factor by human Sertoli cells is substantially reduced in Sertoli cell-only testes. Hum Reprod 2017;32:1108–17.

38. Gassei K, Orwig KE. Experimental methods to preserve male fertility and treat male factor infertility. Fertil Steril 2016;105:256–66.

39. Ferguson W. Sperm stem cells restore male fertility. New Sci 2012;216:10.

40. Martin LA, Seandel M. Propagation of adult SSCs: from mouse to human. Biomed Res Int 2013;2013:384734.

41. Ishikura Y, Ohta H, Sato T, Murase Y, Yabuta Y, Kojima Y, et al. In vitro reconstitution of the whole male germ-cell development from mouse pluripotent stem cells. Cell Stem Cell 2021;28:2167–79.

42. Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 2011;146:519–32.

43. Easley CA 4th, Phillips BT, McGuire MM, Barringer JM, Valli H, Hermann BP, et al. Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells. Cell Rep 2012;2:440–6.

44. Sasaki K, Yokobayashi S, Nakamura T, Okamoto I, Yabuta Y, Kurimoto K, et al. Robust in vitro induction of human germ cell fate from pluripotent stem cells. Cell Stem Cell 2015;17:178–94.

45. Cyranoski D. Mouse eggs made from skin cells in a dish. Nature 2016;538:301.

46. Irie N, Weinberger L, Tang WW, Kobayashi T, Viukov S, Manor YS, et al. SOX17 is a critical specifier of human primordial germ cell fate. Cell 2015;160:253–68.

47. Dong G, Shang Z, Liu L, Liu C, Ge Y, Wang Q, et al. Retinoic acid combined with spermatogonial stem cell conditions facilitate the generation of mouse germ-like cells. Biosci Rep 2017;37:BSR20170637.

48. Khampang S, Cho IK, Punyawai K, Gill B, Langmo JN, Nath S, et al. Blastocyst development after fertilization with in vitro spermatids derived from nonhuman primate embryonic stem cells. F S Sci 2021;2:365–75.

49. Shlush E, Maghen L, Swanson S, Kenigsberg S, Moskovtsev S, Barretto T, et al. In vitro generation of Sertoli-like and haploid spermatid-like cells from human umbilical cord perivascular cells. Stem Cell Res Ther 2017;8:37.

50. Smith JF, Yango P, Altman E, Choudhry S, Poelzl A, Zamah AM, et al. Testicular niche required for human spermatogonial stem cell expansion. Stem Cells Transl Med 2014;3:1043–54.

51. Maghen L, Shlush E, Gat I, Filice M, Barretto T, Jarvi K, et al. Human umbilical perivascular cells: a novel source of MSCs to support testicular niche regeneration. Reproduction 2016;153:85–95.

52. Guo R, Ye X, Yang J, Zhou Z, Tian C, Wang H, et al. Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells. Nat Commun 2018;9:2620.

53. Guadix JA, Zugaza JL, Galvez-Martin P. Characteristics, applications and prospects of mesenchymal stem cells in cell therapy. Med Clin (Barc) 2017;148:408–14.

54. Samsonraj RM, Raghunath M, Nurcombe V, Hui JH, van Wijnen AJ, Cool SM. Concise review: multifaceted characterization of human mesenchymal stem cells for use in regenerative medicine. Stem Cells Transl Med 2017;6:2173–85.

55. Fazeli Z, Abedindo A, Omrani MD, Ghaderian SM. Mesenchymal stem cells (MSCs) therapy for recovery of fertility: a systematic review. Stem Cell Rev Rep 2018;14:1–12.

56. Hassan AI, Alam SS. Evaluation of mesenchymal stem cells in treatment of infertility in male rats. Stem Cell Res Ther 2014;5:131.

57. Hsiao CH, Ji AT, Chang CC, Cheng CJ, Lee LM, Ho JH. Local injection of mesenchymal stem cells protects testicular torsion-induced germ cell injury. Stem Cell Res Ther 2015;6:113.

58. Zhang ZY, Xing XY, Ju GQ, Zhong L, Sun J. Mesenchymal stem cells from human umbilical cord ameliorate testicular dysfunction in a male rat hypogonadism model. Asian J Androl 2017;19:543–7.

59. Nayernia K, Lee JH, Drusenheimer N, Nolte J, Wulf G, Dressel R, et al. Derivation of male germ cells from bone marrow stem cells. Lab Invest 2006;86:654–63.

60. Laurent LC, Ulitsky I, Slavin I, Tran H, Schork A, Morey R, et al. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell 2011;8:106–18.

61. Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R, Narva E, et al. Copy number variation and selection during reprogramming to pluripotency. Nature 2011;471:58–62.

62. Takashima S, Shinohara T. Culture and transplantation of spermatogonial stem cells. Stem Cell Res 2018;29:46–55.

63. Martinovitch PN. Development in vitro of the mammalian gonad. Nature 1937;139:413.

64. Sato T, Katagiri K, Gohbara A, Inoue K, Ogonuki N, Ogura A, et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature 2011;471:504–7.

65. Tesarik J, Bahceci M, Ozcan C, Greco E, Mendoza C. Restoration of fertility by in-vitro spermatogenesis. Lancet 1999;353:555–6.

66. Sato T, Yokonishi T, Komeya M, Katagiri K, Kubota Y, Matoba S, et al. Testis tissue explantation cures spermatogenic failure in c-Kit ligand mutant mice. Proc Natl Acad Sci U S A 2012;109:16934–8.

67. Steinberger E, Steinberger A, Perloff WH. Initiation of spermatogenesis in vitro. Endocrinology 1964;74:788–92.

68. Boitani C, Politi MG, Menna T. Spermatogonial cell proliferation in organ culture of immature rat testis. Biol Reprod 1993;48:761–7.

69. Kanbar M, de Michele F, Poels J, Van Loo S, Giudice MG, Gilet T, et al. Microfluidic and static organotypic culture systems to support ex vivo spermatogenesis from prepubertal porcine testicular tissue: a comparative study. Front Physiol 2022;13:884122.

70. AbuMadighem A, Shuchat S, Lunenfeld E, Yossifon G, Huleihel M. Testis on a chip-a microfluidic three-dimensional culture system for the development of spermatogenesis in-vitro. Biofabrication 2022;14:035004.

71. Komeya M, Kimura H, Nakamura H, Yokonishi T, Sato T, Kojima K, et al. Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device. Sci Rep 2016;6:21472.

72. Amirkhani Z, Movahedin M, Baheiraei N, Ghiaseddin A. Mini bioreactor can support in vitro spermatogenesis of mouse testicular tissue. Cell J 2022;24:277–84.

73. Yuan Y, Li L, Cheng Q, Diao F, Zeng Q, Yang X, et al. In vitro testicular organogenesis from human fetal gonads produces fertilization-competent spermatids. Cell Res 2020;30:244–55.

74. von Kopylow K, Schulze W, Salzbrunn A, Schaks M, Schafer E, Roth B, et al. Dynamics, ultrastructure and gene expression of human in vitro organized testis cells from testicular sperm extraction biopsies. Mol Hum Reprod 2018;24:123–34.

75. Huleihel M, Nourashrafeddin S, Plant TM. Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta). Asian J Androl 2015;17:972–80.

76. Voigt AL, Thiageswaran S, de Lima E Martins Lara N, Dobrinski I. Metabolic requirements for spermatogonial stem cell establishment and maintenance in vivo and in vitro. Int J Mol Sci 2021;22:1998.

77. Galdon G, Atala A, Sadri-Ardekani H. In vitro spermatogenesis: how far from clinical application? Curr Urol Rep 2016;17:49.

78. Wu X, Su J, Wei J, Jiang N, Ge X. Recent advances in three-dimensional stem cell culture systems and applications. Stem Cells Int 2021;2021:9477332.

79. Rafeeqi T, Kaul G. Carbon nanotubes as a scaffold for spermatogonial cell maintenance. J Biomed Nanotechnol 2010;6:710–7.

80. Pan F, Chi L, Schlatt S. Effects of nanostructures and mouse embryonic stem cells on in vitro morphogenesis of rat testicular cords. PLoS One 2013;8e60054.

81. Talebi A, Sadighi Gilani MA, Koruji M, Ai J, Rezaie MJ, Navid S, et al. Colonization of mouse spermatogonial cells in modified soft agar culture system utilizing nanofibrous scaffold: a new approach. Galen Med J 2019;8e1319.

82. Eslahi N, Hadjighassem MR, Joghataei MT, Mirzapour T, Bakhtiyari M, Shakeri M, et al. The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture. Int J Nanomedicine 2013;8:4563–76.

83. Lee JH, Oh JH, Lee JH, Kim MR, Min CK. Evaluation of in vitro spermatogenesis using poly(D,L-lactic-co-glycolic acid) (PLGA)-based microporous biodegradable scaffolds. J Tissue Eng Regen Med 2011;5:130–7.

84. Ziloochi Kashani M, Bagher Z, Asgari HR, Najafi M, Koruji M, Mehraein F. Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold. Syst Biol Reprod Med 2020;66:202–15.

85. Shakeri M, Kohram H, Shahverdi A, Shahneh AZ, Tavakolifar F, Pirouz M, et al. Behavior of mouse spermatogonial stem-like cells on an electrospun nanofibrillar matrix. J Assist Reprod Genet 2013;30:325–32.

86. Lee DR, Kaproth MT, Parks JE. In vitro production of haploid germ cells from fresh or frozen-thawed testicular cells of neonatal bulls. Biol Reprod 2001;65:873–8.

87. Lee DR, Kim KS, Yang YH, Oh HS, Lee SH, Chung TG, et al. Isolation of male germ stem cell-like cells from testicular tissue of non-obstructive azoospermic patients and differentiation into haploid male germ cells in vitro. Hum Reprod 2006;21:471–6.

88. Vardiani M, Gholipourmalekabadi M, Ghaffari Novin M, Koruji M, Ghasemi Hamidabadi H, Salimi M, et al. Three-dimensional electrospun gelatin scaffold coseeded with embryonic stem cells and Sertoli cells: a promising substrate for in vitro coculture system. J Cell Biochem 2019;120:12508–18.

89. Stukenborg JB, Schlatt S, Simoni M, Yeung CH, Elhija MA, Luetjens CM, et al. New horizons for in vitro spermatogenesis?: an update on novel three-dimensional culture systems as tools for meiotic and post-meiotic differentiation of testicular germ cells. Mol Hum Reprod 2009;15:521–9.

90. Lee JH, Kim HJ, Kim H, Lee SJ, Gye MC. In vitro spermatogenesis by three-dimensional culture of rat testicular cells in collagen gel matrix. Biomaterials 2006;27:2845–53.

91. Zhang J, Hatakeyama J, Eto K, Abe S. Reconstruction of a seminiferous tubule-like structure in a 3 dimensional culture system of re-aggregated mouse neonatal testicular cells within a collagen matrix. Gen Comp Endocrinol 2014;205:121–32.

92. Bashiri Z, Moghaddaszadeh A, Falak R, Khadivi F, Afzali A, Abbasi M, et al. Generation of haploid spermatids on silk fibroin-alginate-laminin-based porous 3D scaffolds. Macromol Biosci 2023;23e2200574.

93. Narimanpour Z, Bojnordi MN, Hamidabadi HG. Spermatogenic differentiation of spermatogonial stem cells on three-dimensional silk nanofiber scaffold. Middle East Fertil Soc J 2022;27:15.

94. Perrard MH, Sereni N, Schluth-Bolard C, Blondet A, d′Estaing SG, Plotton I, et al. Complete human and rat ex vivo spermatogenesis from fresh or frozen testicular tissue. Biol Reprod 2016;95:89.

95. Sun M, Yuan Q, Niu M, Wang H, Wen L, Yao C, et al. Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system. Cell Death Differ 2018;25:749–66.

96. Zhang X, Wang L, Zhang X, Ren L, Shi W, Tian Y, et al. The use of knockout serum replacement (KSR) in three dimensional rat testicular cells co-culture model: an improved male reproductive toxicity testing system. Food Chem Toxicol 2017;106(Pt A):487–95.

97. Legendre A, Froment P, Desmots S, Lecomte A, Habert R, Lemazurier E. An engineered 3D blood-testis barrier model for the assessment of reproductive toxicity potential. Biomaterials 2010;31:4492–505.

98. Stukenborg JB, Wistuba J, Luetjens CM, Elhija MA, Huleihel M, Lunenfeld E, et al. Coculture of spermatogonia with somatic cells in a novel three-dimensional soft-agar-culture-system. J Androl 2008;29:312–29.

99. Abu Elhija M, Lunenfeld E, Schlatt S, Huleihel M. Differentiation of murine male germ cells to spermatozoa in a soft agar culture system. Asian J Androl 2012;14:285–93.

100. Jabari A, Gholami K, Khadivi F, Koruji M, Amidi F, Gilani MA, et al. In vitro complete differentiation of human spermatogonial stem cells to morphologic spermatozoa using a hybrid hydrogel of agarose and laminin. Int J Biol Macromol 2023;235:123801.

101. Bashiri Z, Gholipourmalekabadi M, Khadivi F, Salem M, Afzali A, Cham TC, et al. In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility. Cell Tissue Res 2023;394:393–421.

102. Rezaei Topraggaleh T, Rezazadeh Valojerdi M, Montazeri L, Baharvand H. A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids. Biomater Sci 2019;7:1422–36.

103. Cham TC, Chen X, Honaramooz A. Current progress, challenges, and future prospects of testis organoids†. Biol Reprod 2021;104:942–61.

104. Baert Y, Dvorakova-Hortova K, Margaryan H, Goossens E. Mouse in vitro spermatogenesis on alginate-based 3D bioprinted scaffolds. Biofabrication 2019;11:035011.

105. Robinson M, Bedford E, Witherspoon L, Willerth SM, Flannigan R. Using clinically derived human tissue to 3-dimensionally bioprint personalized testicular tubules for in vitro culturing: first report. F S Sci 2022;3:130–9.

106. Bashiri Z, Zahiri M, Allahyari H, Esmaeilzade B. Proliferation of human spermatogonial stem cells on optimized PCL/gelatin nanofibrous scaffolds. Andrologia 2022;54e14380.

107. Bashiri Z, Amiri I, Gholipourmalekabadi M, Falak R, Asgari H, Maki CB, et al. Artificial testis: a testicular tissue extracellular matrix as a potential bio-ink for 3D printing. Biomater Sci 2021;9:3465–84.

108. Alves-Lopes JP, Stukenborg JB. Testicular organoids: a new model to study the testicular microenvironment in vitro? Hum Reprod Update 2018;24:176–91.

109. Bredenoord AL, Clevers H, Knoblich JA. Human tissues in a dish: the research and ethical implications of organoid technology. Science 2017;355eaaf9414.

110. Yokonishi T, Sato T, Katagiri K, Komeya M, Kubota Y, Ogawa T. In vitro reconstruction of mouse seminiferous tubules supporting germ cell differentiation. Biol Reprod 2013;89:15.

111. Baert Y, De Kock J, Alves-Lopes JP, Soder O, Stukenborg JB, Goossens E. Primary human testicular cells self-organize into organoids with testicular properties. Stem Cell Reports 2017;8:30–8.

112. Pendergraft SS, Sadri-Ardekani H, Atala A, Bishop CE. Three-dimensional testicular organoid: a novel tool for the study of human spermatogenesis and gonadotoxicity in vitro. Biol Reprod 2017;96:720–32.

113. Sakib S, Uchida A, Valenzuela-Leon P, Yu Y, Valli-Pulaski H, Orwig K, et al. Formation of organotypic testicular organoids in microwell culture†. Biol Reprod 2019;100:1648–60.

114. Edmonds ME, Woodruff TK. Testicular organoid formation is a property of immature somatic cells, which self-assemble and exhibit long-term hormone-responsive endocrine function. Biofabrication 2020;12:045002.

115. Cham TC, Ibtisham F, Fayaz MA, Honaramooz A. Generation of a highly biomimetic organoid, including vasculature, resembling the native immature testis tissue. Cells 2021;10:1696.

116. Vermeulen M, Del Vento F, Kanbar M, Pyr Dit Ruys S, Vertommen D, Poels J, et al. Generation of organized porcine testicular organoids in solubilized hydrogels from decellularized extracellular matrix. Int J Mol Sci 2019;20:5476.

117. Yang Y, Lin Q, Zhou C, Li Q, Li Z, Cao Z, et al. A testis-derived hydrogel as an efficient feeder-free culture platform to promote mouse spermatogonial stem cell proliferation and differentiation. Front Cell Dev Biol 2020;8:250.

118. Salek F, Baharara J, Shahrokhabadi KN, Amini E. The guardians of germ cells: sertoli-derived exosomes against electromagnetic field-induced oxidative stress in mouse spermatogonial stem cells. Theriogenology 2021;173:112–22.

119. Gao H, Cao H, Li Z, Li L, Guo Y, Chen Y, et al. Exosome-derived small RNAs in mouse Sertoli cells inhibit spermatogonial apoptosis. Theriogenology 2023;200:155–67.

120. Li Q, Li H, Liang J, Mei J, Cao Z, Zhang L, et al. Sertoli cell-derived exosomal microRNA-486-5p regulates differentiation of spermatogonial stem cell through PTEN in mice. J Cell Mol Med 2021;25:3950–62.

121. Rahbar M, Asadpour R, Azami M, Mazaheri Z, Hamali H. Improving the process of spermatogenesis in azoospermic mice using spermatogonial stem cells co-cultured with epididymosomes in three-dimensional culture system. Life Sci 2022;310:121057.

122. Yang L, Jiang Z, Zhou L, Zhao K, Ma X, Cheng G. Hydrophilic cell-derived extracellular matrix as a niche to promote adhesion and differentiation of neural progenitor cells. RSC Adv 2017;7:45587–94.

123. Silva JC, Carvalho MS, Udangawa RN, Moura CS, Cabral JM, da Silva CL, et al. Extracellular matrix decorated polycaprolactone scaffolds for improved mesenchymal stem/stromal cell osteogenesis towards a patient-tailored bone tissue engineering approach. J Biomed Mater Res B Appl Biomater 2020;108:2153–66.

124. Zhang W, Yang J, Zhu Y, Sun X, Guo W, Liu X, et al. Extracellular matrix derived by human umbilical cord-deposited mesenchymal stem cells accelerates chondrocyte proliferation and differentiation potential in vitro. Cell Tissue Bank 2019;20:351–65.

125. Kanninen LK, Porola P, Niklander J, Malinen MM, Corlu A, Guguen-Guillouzo C, et al. Hepatic differentiation of human pluripotent stem cells on human liver progenitor HepaRG-derived acellular matrix. Exp Cell Res 2016;341:207–17.

Article information Continued

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.