
==== Front
Clin Exp Reprod Med
Clin Exp Reprod Med
CERM
Clinical and Experimental Reproductive Medicine
2233-8233
2233-8241
Korean Society for Reproductive Medicine

38525520
10.5653/cerm.2023.06569
cerm-2023-06569
Review Article
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
http://orcid.org/0000-0003-1169-6843
Bashiri Zahra 1 2 3
Hosseini Seyed Jamal 4 5
Salem Maryam 6
http://orcid.org/0000-0003-2661-0117
Koruji Morteza 2 7
1 Endometrium and Endometriosis Research Center, Hamadan University of Medical Sciences, Hamadan, Iran
2 Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
3 Omid Fertility and Infertility Clinic, Hamedan, Iran
4 4Biomedical Engineering Department, Amirkabir University of Technology, Tehran, Iran
5 Department of Pharmaceutical Biomaterials and Medical Biomaterials Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
6 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
7 Stem 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
9 2024
25 3 2024
51 3 171180
3 10 2023
16 11 2023
14 12 2023
© 2024. THE KOREAN SOCIETY FOR REPRODUCTIVE MEDICINE
2024
https://creativecommons.org/licenses/by-nc/4.0/ 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.
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.

Fertility preservation
In vitro and in vivo spermatogenesis
Sperm production
Transplantation
==== Body
pmcIntroduction

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].

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.

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.

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.
==== Refs
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 24753928
2 Fayomi AP Peters K Sukhwani M Valli-Pulaski H Shetty G Meistrich ML Autologous grafting of cryopreserved prepubertal rhesus testis produces sperm and offspring Science 2019 363 1314 9 30898927
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 35403667
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 35130799
5 Yokonishi T Ogawa T Cryopreservation of testis tissues and in vitro spermatogenesis Reprod Med Biol 2016 15 21 8 26709347
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 31640294
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 35360062
9 Smart E Lopes F Rice S Nagy B Anderson RA Mitchell RT Chemotherapy drugs cyclophosphamide, cisplatin and doxorubicin induce germ cell loss in an in vitro model of the prepubertal testis Sci Rep 2018 8 1773 29379115
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 32111477
11 Shinohara T Inoue K Ogonuki N Kanatsu-Shinohara M Miki H Nakata K Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination Hum Reprod 2002 17 3039 45 12456600
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 12181567
13 Shinohara T Kato M Takehashi M Lee J Chuma S Nakatsuji N Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination Proc Natl Acad Sci U S A 2006 103 13624 8 16945902
14 Nakai M Kaneko H Somfai T Maedomari N Ozawa M Noguchi J Production of viable piglets for the first time using sperm derived from ectopic testicular xenografts Reproduction 2010 139 331 5 20015869
15 Kaneko H Kikuchi K Nakai M Somfai T Noguchi J Tanihara F Generation of live piglets for the first time using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice PLoS One 2013 8 e70989 23923039
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 12606381
17 Liu Z Nie YH Zhang CC Cai YJ Wang Y Lu HP Generation of macaques with sperm derived from juvenile monkey testicular xenografts Cell Res 2016 26 139 42 26369429
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 18664476
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 17916184
20 Goossens E Van Saen D Tournaye H Spermatogonial stem cell preservation and transplantation: from research to clinic Hum Reprod 2013 28 897 907 23427228
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 25593971
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 28070774
23 Brinster RL Zimmermann JW Spermatogenesis following male germ-cell transplantation Proc Natl Acad Sci U S A 1994 91 11298 302 7972053
24 Honaramooz A Behboodi E Megee SO Overton SA Galantino-Homer H Echelard Y Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats Biol Reprod 2003 69 1260 4 12801978
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 28174013
26 Hermann BP Sukhwani M Winkler F Pascarella JN Peters KA Sheng Y Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm Cell Stem Cell 2012 11 715 26 23122294
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 30123997
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 25209022
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 22016413
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 18042634
31 Eslahi N Shakeri-Zadeh A Ashtari K Pirhajati-Mahabadi V Tohidi Moghadam T Shabani R In vitro cytotoxicity of folate-silica-gold nanorods on mouse acute lymphoblastic leukemia and spermatogonial cells Cell J 2019 21 14 26 30507084
32 Shabani R Ashjari M Ashtari K Izadyar F Behnam B Khoei S 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 29849458
33 Shams A Shabani R Asgari H Karimi M Najafi M Asghari-Jafarabadi M 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 35264013
34 Ashtari B Shams A Esmaeilzadeh N Tanbakooei S Koruji M Moghadam MJ Separating mouse malignant cell line (EL4) from neonate spermatogonial stem cells utilizing microfluidic device in vitro Stem Cell Res Ther 2020 11 191 32448280
35 Picton HM Wyns C Anderson RA Goossens E Jahnukainen K Kliesch S A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys Hum Reprod 2015 30 2463 75 26358785
36 Samplaski MK Deault-Bonin M Lo KC Genetic and epigenetic changes after spermatogonial stem cell culture and transplantation EJIFCC 2014 25 27 41 27683455
37 Singh D Paduch DA Schlegel PN Orwig KE Mielnik A Bolyakov A 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 28369535
38 Gassei K Orwig KE Experimental methods to preserve male fertility and treat male factor infertility Fertil Steril 2016 105 256 66 26746133
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 23484114
41 Ishikura Y Ohta H Sato T Murase Y Yabuta Y Kojima Y In vitro reconstitution of the whole male germ-cell development from mouse pluripotent stem cells Cell Stem Cell 2021 28 2167 79 34496297
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 21820164
43 Easley CA 4th Phillips BT McGuire MM Barringer JM Valli H Hermann BP Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells Cell Rep 2012 2 440 6 22921399
44 Sasaki K Yokobayashi S Nakamura T Okamoto I Yabuta Y Kurimoto K Robust in vitro induction of human germ cell fate from pluripotent stem cells Cell Stem Cell 2015 17 178 94 26189426
45 Cyranoski D Mouse eggs made from skin cells in a dish Nature 2016 538 301 27762380
46 Irie N Weinberger L Tang WW Kobayashi T Viukov S Manor YS SOX17 is a critical specifier of human primordial germ cell fate Cell 2015 160 253 68 25543152
47 Dong G Shang Z Liu L Liu C Ge Y Wang Q Retinoic acid combined with spermatogonial stem cell conditions facilitate the generation of mouse germ-like cells Biosci Rep 2017 37 BSR20170637 28314787
48 Khampang S Cho IK Punyawai K Gill B Langmo JN Nath S Blastocyst development after fertilization with in vitro spermatids derived from nonhuman primate embryonic stem cells F S Sci 2021 2 365 75 34970648
49 Shlush E Maghen L Swanson S Kenigsberg S Moskovtsev S Barretto T In vitro generation of Sertoli-like and haploid spermatid-like cells from human umbilical cord perivascular cells Stem Cell Res Ther 2017 8 37 28202061
50 Smith JF Yango P Altman E Choudhry S Poelzl A Zamah AM Testicular niche required for human spermatogonial stem cell expansion Stem Cells Transl Med 2014 3 1043 54 25038247
51 Maghen L Shlush E Gat I Filice M Barretto T Jarvi K 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 Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells Nat Commun 2018 9 2620 29976922
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 28126234
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 29076267
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 28884412
56 Hassan AI Alam SS Evaluation of mesenchymal stem cells in treatment of infertility in male rats Stem Cell Res Ther 2014 5 131 25422144
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 26025454
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 27586027
59 Nayernia K Lee JH Drusenheimer N Nolte J Wulf G Dressel R Derivation of male germ cells from bone marrow stem cells Lab Invest 2006 86 654 63 16652109
60 Laurent LC Ulitsky I Slavin I Tran H Schork A Morey R 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 21211785
61 Hussein SM Batada NN Vuoristo S Ching RW Autio R Narva E Copy number variation and selection during reprogramming to pluripotency Nature 2011 471 58 62 21368824
62 Takashima S Shinohara T Culture and transplantation of spermatogonial stem cells Stem Cell Res 2018 29 46 55 29587218
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 In vitro production of functional sperm in cultured neonatal mouse testes Nature 2011 471 504 7 21430778
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 Testis tissue explantation cures spermatogenic failure in c-Kit ligand mutant mice Proc Natl Acad Sci U S A 2012 109 16934 8 22984182
67 Steinberger E Steinberger A Perloff WH Initiation of spermatogenesis in vitro Endocrinology 1964 74 788 92 14168569
68 Boitani C Politi MG Menna T Spermatogonial cell proliferation in organ culture of immature rat testis Biol Reprod 1993 48 761 7 8485240
69 Kanbar M de Michele F Poels J Van Loo S Giudice MG Gilet T Microfluidic and static organotypic culture systems to support ex vivo spermatogenesis from prepubertal porcine testicular tissue: a comparative study Front Physiol 2022 13 884122 35721544
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 Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device Sci Rep 2016 6 21472 26892171
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 35717571
73 Yuan Y Li L Cheng Q Diao F Zeng Q Yang X In vitro testicular organogenesis from human fetal gonads produces fertilization-competent spermatids Cell Res 2020 30 244 55 32086476
74 von Kopylow K Schulze W Salzbrunn A Schaks M Schafer E Roth B Dynamics, ultrastructure and gene expression of human in vitro organized testis cells from testicular sperm extraction biopsies Mol Hum Reprod 2018 24 123 34 29304256
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 26067870
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 33670439
77 Galdon G Atala A Sadri-Ardekani H In vitro spermatogenesis: how far from clinical application? Curr Urol Rep 2016 17 49 27107595
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 34671401
79 Rafeeqi T Kaul G Carbon nanotubes as a scaffold for spermatogonial cell maintenance J Biomed Nanotechnol 2010 6 710 7 21361137
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 8 e60054 23555881
81 Talebi A Sadighi Gilani MA Koruji M Ai J Rezaie MJ Navid S Colonization of mouse spermatogonial cells in modified soft agar culture system utilizing nanofibrous scaffold: a new approach Galen Med J 2019 8 e1319 34466493
82 Eslahi N Hadjighassem MR Joghataei MT Mirzapour T Bakhtiyari M Shakeri M The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture Int J Nanomedicine 2013 8 4563 76 24348035
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 20603864
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 32138551
85 Shakeri M Kohram H Shahverdi A Shahneh AZ Tavakolifar F Pirouz M Behavior of mouse spermatogonial stem-like cells on an electrospun nanofibrillar matrix J Assist Reprod Genet 2013 30 325 32 23274510
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 11514353
87 Lee DR Kim KS Yang YH Oh HS Lee SH Chung TG 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 16210386
88 Vardiani M Gholipourmalekabadi M Ghaffari Novin M Koruji M Ghasemi Hamidabadi H Salimi M 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 30977186
89 Stukenborg JB Schlatt S Simoni M Yeung CH Elhija MA Luetjens CM 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 19561342
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 16430959
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 24717811
92 Bashiri Z Moghaddaszadeh A Falak R Khadivi F Afzali A Abbasi M Generation of haploid spermatids on silk fibroin-alginate-laminin-based porous 3D scaffolds Macromol Biosci 2023 23 e2200574 37116215
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 Complete human and rat ex vivo spermatogenesis from fresh or frozen testicular tissue Biol Reprod 2016 95 89 27580986
95 Sun M Yuan Q Niu M Wang H Wen L Yao C Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system Cell Death Differ 2018 25 749 66 29305586
96 Zhang X Wang L Zhang X Ren L Shi W Tian Y 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 28478099
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 20206992
98 Stukenborg JB Wistuba J Luetjens CM Elhija MA Huleihel M Lunenfeld E Coculture of spermatogonia with somatic cells in a novel three-dimensional soft-agar-culture-system J Androl 2008 29 312 29 18046051
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 22057383
100 Jabari A Gholami K Khadivi F Koruji M Amidi F Gilani MA 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 36842740
101 Bashiri Z Gholipourmalekabadi M Khadivi F Salem M Afzali A Cham TC In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility Cell Tissue Res 2023 394 393 421 37721632
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 30666997
103 Cham TC Chen X Honaramooz A Current progress, challenges, and future prospects of testis organoids† Biol Reprod 2021 104 942 61 33550399
104 Baert Y Dvorakova-Hortova K Margaryan H Goossens E Mouse in vitro spermatogenesis on alginate-based 3D bioprinted scaffolds Biofabrication 2019 11 035011 30921781
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 35560010
106 Bashiri Z Zahiri M Allahyari H Esmaeilzade B Proliferation of human spermatogonial stem cells on optimized PCL/gelatin nanofibrous scaffolds Andrologia 2022 54 e14380 35083770
107 Bashiri Z Amiri I Gholipourmalekabadi M Falak R Asgari H Maki CB Artificial testis: a testicular tissue extracellular matrix as a potential bio-ink for 3D printing Biomater Sci 2021 9 3465 84 33949391
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 29281008
109 Bredenoord AL Clevers H Knoblich JA Human tissues in a dish: the research and ethical implications of organoid technology Science 2017 355 eaaf9414 28104841
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 23759307
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 28017656
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 28339648
113 Sakib S Uchida A Valenzuela-Leon P Yu Y Valli-Pulaski H Orwig K Formation of organotypic testicular organoids in microwell culture† Biol Reprod 2019 100 1648 60 30927418
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 32492667
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 34359871
116 Vermeulen M Del Vento F Kanbar M Pyr Dit Ruys S Vertommen D Poels J Generation of organized porcine testicular organoids in solubilized hydrogels from decellularized extracellular matrix Int J Mol Sci 2019 20 5476 31684200
117 Yang Y Lin Q Zhou C Li Q Li Z Cao Z 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 32509769
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 34371438
119 Gao H Cao H Li Z Li L Guo Y Chen Y Exosome-derived small RNAs in mouse Sertoli cells inhibit spermatogonial apoptosis Theriogenology 2023 200 155 67 36806925
120 Li Q Li H Liang J Mei J Cao Z Zhang L 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 33608983
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 36220369
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 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 31916699
124 Zhang W Yang J Zhu Y Sun X Guo W Liu X 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 31218457
125 Kanninen LK Porola P Niklander J Malinen MM Corlu A Guguen-Guillouzo C Hepatic differentiation of human pluripotent stem cells on human liver progenitor HepaRG-derived acellular matrix Exp Cell Res 2016 341 207 17 26854693
