Title: Progress in translational reproductive science: testicular tissue transplantation and in vitro spermatogenesis
Abstract: Since the birth of the first child conceived via in vitro fertilization 40 years ago, fertility treatments and assisted reproductive technology have allowed many couples to reach their reproductive goals. As of yet, no fertility options are available for men who cannot produce functional sperm, but many experimental therapies have demonstrated promising results in animal models. Both autologous (stem cell transplantation, de novo morphogenesis, and testicular tissue grafting) and outside-the-body (xenografting and in vitro spermatogenesis) approaches exist for restoring sperm production in infertile animals with varying degrees of success. Once safety profiles are established and an ideal patient population is chosen, some of these techniques may be ready for human experimentation in the near future, with likely clinical implementation within the next decade. Since the birth of the first child conceived via in vitro fertilization 40 years ago, fertility treatments and assisted reproductive technology have allowed many couples to reach their reproductive goals. As of yet, no fertility options are available for men who cannot produce functional sperm, but many experimental therapies have demonstrated promising results in animal models. Both autologous (stem cell transplantation, de novo morphogenesis, and testicular tissue grafting) and outside-the-body (xenografting and in vitro spermatogenesis) approaches exist for restoring sperm production in infertile animals with varying degrees of success. Once safety profiles are established and an ideal patient population is chosen, some of these techniques may be ready for human experimentation in the near future, with likely clinical implementation within the next decade. Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/58846-29751 Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/users/16110-fertility-and-sterility/posts/58846-29751 In 1978, Louise Brown, the world's first baby conceived by in vitro fertilization was born (1Steptoe P.C. Edwards R.G. Birth after the reimplantation of a human embryo.Lancet. 1978; 312: 366Abstract Google Scholar), beginning the era of assisted reproduction. In the ensuing 40 years, nearly 8 million children were conceived and born using assisted reproductive technologies (ART) (2Niederberger C. Pellicer A. Cohen J. Gardner D.K. Palermo G.D. O'Neill C.L. et al.Forty years of IVF.Fertil Steril. 2018; 110: 185-324.e5Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). For many men with azoospermia or oligospermia, ART remains a viable option to produce offspring when sperm can be found. However, for those men who cannot produce mature sperm due to congenital disorders, acquired diseases, or gonadotoxic therapies, no fertility options currently exist. One of the more active areas of infertility research is discovery and refinement of new techniques that may someday allow these men to produce offspring. Although none of these techniques has yet reached the clinical stage, the results in larger mammals have been promising. This review will detail experimental stem cell and testicular tissue-based approaches for infertility and describe what further work needs to be done to bring these techniques into the clinical realm. Spermatogonial stem cells (SSCs), the most primitive type of spermatogonia, are found on the basement membrane of seminiferous tubules and characterized by their ability to both self-renew and differentiate. Male fertility requires proper functioning of SSCs to maintain continuous spermatogenesis throughout the life span (3De Rooij D.G. The nature and dynamics of spermatogonial stem cells.Development. 2017; 144: 3022-3030Crossref PubMed Scopus (68) Google Scholar). Within the basal compartment of the seminiferous tubules exist the population of spermatogonia, which can be divided into type A intermediate and B subpopulations. In primates, undifferentiated type A spermatogonia, which contain all SSCs, are further subdivided into Adark (Ad) and Apale (Ap) subtypes (4Oatley J.M. Brinster R.L. The germline stem cell niche unit in mammalian testes.Physiol Rev. 2012; 92: 577-595Crossref PubMed Scopus (261) Google Scholar, 5Fayomi A.P. Orwig K.E. Spermatogonial stem cells and spermatogenesis in mice, monkeys and men.Stem Cell Res. 2018; 29: 207-214Crossref PubMed Scopus (33) Google Scholar). For many years, it was believed that Ad and Ap were two morphologically and histologically distinct populations of cells serving different roles—reserve stem cells (Ad) and active stem cells (Ap) that self-renew and differentiate to maintain steady state spermatogenesis in the adult testis (6Clermont Y. Leblond C.P. Differentiation and renewal of spermatogonia in the monkey, Macacus rhesus.Am J Anat. 1959; 104: 237-273Crossref PubMed Google Scholar, 7Ehmcke J. Simorangkir D.R. Schlatt S. Identification of the starting point for spermatogenesis and characterization of the testicular stem cell in adult male rhesus monkeys.Hum Reprod. 2005; 20: 1185-1193Crossref PubMed Scopus (0) Google Scholar). However, recent work has suggested that at least some Ad and Ap cells are instead from the same population but at different stages of the cell cycle (8Hermann B.P. Sukhwani M. Hansel M.C. Orwig K.E. Spermatogonial stem cells in higher primates: are there differences from those in rodents?.Reproduction. 2010; 139: 479-493Crossref PubMed Scopus (105) Google Scholar, 9Von Kopylow K. Staege H. Spiess A.-N. Schulze W. Will H. Primig M. et al.Differential marker protein expression specifies rarefaction zone-containing human Adark spermatogonia.Reproduction. 2012; 143: 45-57Crossref PubMed Scopus (44) Google Scholar). Differentiation and renewal of SSCs within the seminiferous tubules are regulated by the surrounding stem cell niche (Fig. 1), a dynamic microenvironment consisting of Sertoli cells and the testicular interstitial cells that produce molecular signals to mediate SSC function (10Chen S.-R. Liu Y.-X. Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling.Reproduction. 2015; 149: R159-R167Crossref PubMed Scopus (91) Google Scholar, 11Heinrich A, DeFalco T. Essential roles of interstitial cells in testicular development and function. Andrology. Published online August 24, 2019. https://doi.org/10.1111/andr.12703Google Scholar, 12Hess R.A. Cooke P.S. Hofmann M.-C. Murphy K.M. Mechanistic insights into the regulation of the spermatogonial stem cell niche.Cell Cycle. 2006; 5: 1164-1170Crossref PubMed Google Scholar, 13Chen C. Ouyang W. Grigura V. Zhou Q. Carnes K. Lim H. et al.ERM is required for transcriptional control of the spermatogonial stem cell niche.Nature. 2005; 436: 1030-1034Crossref PubMed Scopus (224) Google Scholar). Due to the relative rarity of SSCs within the testis and lack of definitive SSC markers, identifying a precise anatomic location of the niche has been difficult (14Mäkelä J.-A. Hobbs R.M. Molecular regulation of spermatogonial stem cell renewal and differentiation.Reproduction. 2019; 158: 169-187Crossref PubMed Scopus (1) Google Scholar, 15Jones D.L. Wagers A.J. No place like home: anatomy and function of the stem cell niche.Nat Rev Mol Cell Biol. 2008; 9: 11-21Crossref PubMed Scopus (464) Google Scholar). However, previous mouse studies have suggested that distribution of SSCs along the basement membrane of seminiferous tubules may be skewed toward regions adjacent to vasculature and the surrounding interstitium (16Chiarini-Garcia H. Hornick J.R. Griswold M.D. Russell L.D. Distribution of type A spermatogonia in the mouse is not random.Biol Reprod. 2001; 65: 1179-1185Crossref PubMed Google Scholar, 17Chiarini-Garcia H. Raymer A.M. Russell L.D. Non-random distribution of spermatogonia in rats: evidence of niches in the seminiferous tubules.Reproduction. 2003; 126: 669-680Crossref PubMed Google Scholar, 18Yoshida S. Sukeno M. Nabeshima Y.-I. A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis.Science. 2007; 317: 1722-1726Crossref PubMed Scopus (330) Google Scholar), suggesting that blood-borne compounds, vascular endothelial cells, and paracrine products of various interstitial cell types (including Leydig cells, peritubular cells, and macrophages) are necessary for normal SSC activity (11Heinrich A, DeFalco T. Essential roles of interstitial cells in testicular development and function. Andrology. Published online August 24, 2019. https://doi.org/10.1111/andr.12703Google Scholar). A multitude of conditions and their associated treatments may compromise SSC function or the stem cell niche, subsequently resulting in subfertility or infertility. Although oncology patients are the best-known example, many patient populations including those treated with immunosuppressive agents and transgender individuals may benefit from fertility preservation (FP) (19Johnson E.K. Finlayson C. Rowell E.E. Gosiengfiao Y. Pavone M.E. Lockart B. et al.Fertility preservation for pediatric patients: current state and future possibilities.J Urol. 2017; 198: 186-194Crossref PubMed Scopus (33) Google Scholar). With improving cancer survival rates and a corresponding increase in survivorship into reproductive age, FP has become an essential component of oncologic treatment (20Oktay K. Harvey B.E. Partridge A.H. Quinn G.P. Reinecke J. Taylor H.S. et al.Fertility preservation in patients with cancer: ASCO clinical practice guideline update.J Clin Oncol. 2018; 36: 1994-2001Crossref PubMed Scopus (187) Google Scholar). Rapidly dividing cells such as spermatogenic cells in the testis are sensitive to the cytotoxic effects of radiation and certain chemotherapies (most notably alkylating or platinum-based agents); accordingly, many cancer treatment regimens can result in impaired fertility (21Meistrich M.L. Effects of chemotherapy and radiotherapy on spermatogenesis in humans.Fertil Steril. 2013; 100: 1180-1186Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Among survivors of childhood cancers, 48% reported infertility, and 52% had a sperm count <15 million/mL in long-term follow-up observation (22Green D.M. Liu W. Kutteh W.H. Ke R.W. Shelton K.C. Sklar C.A. et al.Cumulative alkylating agent exposure and semen parameters in adult survivors of childhood cancer: a report from the St Jude Lifetime Cohort Study.Lancet Oncol. 2014; 15: 1215-1223Abstract Full Text Full Text PDF PubMed Google Scholar, 23Wasilewski-Masker K. Seidel K.D. Leisenring W. Mertens A.C. Shnorhavorian M. Ritenour C.W. et al.Male infertility in long-term survivors of pediatric cancer: a report from the childhood cancer survivor study.J Cancer Surviv. 2014; 8: 437-447Crossref PubMed Scopus (57) Google Scholar). For these reasons, the American Society of Clinical Oncology, American Academy of Pediatrics, and American Society for Reproductive Medicine all recommend pretreatment FP counseling in patients facing gonadotoxic therapies (20Oktay K. Harvey B.E. Partridge A.H. Quinn G.P. Reinecke J. Taylor H.S. et al.Fertility preservation in patients with cancer: ASCO clinical practice guideline update.J Clin Oncol. 2018; 36: 1994-2001Crossref PubMed Scopus (187) Google Scholar, 24Ethics Committee of the American Society for Reproductive MedicineFertility preservation and reproduction in patients facing gonadotoxic therapies: an Ethics Committee opinion.Fertil Steril. 2018; 110: 380-386Abstract Full Text Full Text PDF PubMed Google Scholar, 25Fallat M.E. Hutter J. American Academy of Pediatrics Committee on Bioethics, American Academy of Pediatrics Section on Hematology/Oncology, American Academy of Pediatrics Section on Surgery. Preservation of fertility in pediatric and adolescent patients with cancer.Pediatrics. 2008; 121: e1461-e1469Crossref PubMed Scopus (0) Google Scholar). Despite this high incidence of treatment-induced infertility, only 29% of male cancer patients reported discussing FP with their oncologist, and 11% went on to bank sperm (26Grover N.S. Deal A.M. Wood W.A. Mersereau J.E. Young men with cancer experience low referral rates for fertility counseling and sperm banking.J Oncol Pract. 2016; 12: 465-471Crossref PubMed Google Scholar). Given the gonadotoxic nature of many nononcologic immunotherapies (such as hydroxyurea or sirolimus) and hormone/surgical treatments for gender dysphoria, FP should be considered in these patient populations as well (27Vakeesan B. Weidman D.R. Maloney A.M. Allen L. Lorenzo A.J. Gupta A.A. Fertility preservation in pediatric subspecialties: a pilot needs assessment beyond oncology.J Pediatr. 2018; 194: 253-256Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 28Mattawanon N. Spencer J.B. Schirmer D.A. Tangpricha V. Fertility preservation options in transgender people: a review.Rev Endocr Metab Disord. 2018; 19: 231-242Crossref PubMed Scopus (15) Google Scholar, 29Ethics Committee of the American Society for Reproductive MedicineAccess to fertility services by transgender persons: an Ethics Committee opinion.Fertil Steril. 2015; 104: 1111-1115Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Options for FP depend on a patient's sexual maturity level. The established and preferred method of preserving fertility in postpubertal males is cryopreservation of ejaculated semen obtained via masturbation, which is possible in approximately 90% of patients seeking FP (30Menon S. Rives N. Mousset-Siméon N. Sibert L. Vannier J.P. Mazurier S. et al.Fertility preservation in adolescent males: experience over 22 years at Rouen University Hospital.Hum Reprod. 2009; 24: 37-44Crossref PubMed Scopus (0) Google Scholar). Institutional protocols vary, but typically two or three samples are collected, either in-office or at home, with 48-hour abstinence periods between collections (31Abram McBride J. Lipshultz L.I. Male fertility preservation.Curr Urol Rep. 2018; 19: 49Crossref PubMed Scopus (6) Google Scholar). Cryopreserved sperm can then remain frozen and usable for years, with multiple live births reported more than 20 years after initial collection (32Feldschuh J. Brassel J. Durso N. Levine A. Successful sperm storage for 28 years.Fertil Steril. 2005; 84: 1017Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), and one live birth from a sample that had been frozen for 40 years (33Szell A.Z. Bierbaum R.C. Hazelrigg W.B. Chetkowski R.J. Live births from frozen human semen stored for 40 years.J Assist Reprod Genet. 2013; 30: 743-744Crossref PubMed Scopus (2) Google Scholar). A large meta-analysis of 801 patients showed a 49% live-birth success rate for those men who chose to use cryopreserved sperm (34Ferrari S. Paffoni A. Filippi F. Busnelli A. Vegetti W. Somigliana E. Sperm cryopreservation and reproductive outcome in male cancer patients: a systematic review.Reprod Biomed Online. 2016; 33: 29-38Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Long-term storage of sperm additionally does not adversely affect live-birth rates, with similar rates noted at 5, 10, and 15 years of cryopreservation (35Huang C. Lei L. Wu H.-L. Gan R.-X. Yuan X.-B. Fan L.-Q. et al.Long-term cryostorage of semen in a human sperm bank does not affect clinical outcomes.Fertil Steril. 2019; 112: 663-669.e1Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). While collection of a semen sample via masturbation is optimal, some men are either too unwell or unwilling to produce a sample in this manner. Men with anorgasmia or anejaculation secondary to neurologic or other medical disorders, pelvic or retroperitoneal surgery, or psychogenic causes may benefit from other means of sperm retrieval. Both electroejaculation and penile vibratory stimulation are relatively noninvasive procedures by which semen can be collected in this patient population (36Mehta A. Sigman M. Management of the dry ejaculate: a systematic review of aspermia and retrograde ejaculation.Fertil Steril. 2015; 104: 1074-1081Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 37Meng X. Fan L. Wang T. Wang S. Wang Z. Liu J. Electroejaculation combined with assisted reproductive technology in psychogenic anejaculation patients refractory to penile vibratory stimulation.Transl Androl Urol. 2018; 7: S17-S22Crossref PubMed Scopus (5) Google Scholar). Surgical testicular sperm extraction is reserved for azoospermic patients or men who fail less invasive therapies (38Halpern J.A. Hill R. Brannigan R.E. Guideline based approach to male fertility preservation.Urol Oncol. 2020; 28: 31-35Crossref Scopus (2) Google Scholar, 39Berookhim B.M. Mulhall J.P. Outcomes of operative sperm retrieval strategies for fertility preservation among males scheduled to undergo cancer treatment.Fertil Steril. 2014; 101: 805-811Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The therapeutic principles of FP in adolescent boys are similar to those in adults, with some additional considerations. Many younger pubertal patients are sexually naïve, which can lead to difficult discussions between patient, parents, and providers (40Moss J.L. Choi A.W. Fitzgerald Keeter M.K. Brannigan R.E. Male adolescent fertility preservation.Fertil Steril. 2016; 105: 267-273Abstract Full Text Full Text PDF PubMed Google Scholar). Accordingly, conversations both with the patient and parents, individually and together, are necessary to broach this topic successfully (41Leonard M. Hammelef K. Smith G.D. Fertility considerations, counseling, and semen cryopreservation for males prior to the initiation of cancer therapy.Clin J Oncol Nurs. 2004; 8 (145): 127-131Crossref PubMed Google Scholar). Ethical issues specific to adolescents, such as parental versus patient decisional capacity, may also arise, necessitating possible consultation with a medical ethicist (42Burns K.C. Hoefgen H. Strine A. Dasgupta R. Fertility preservation options in pediatric and adolescent patients with cancer: fertility options in pediatric patients.Cancer. 2018; 124: 1867-1876Crossref PubMed Scopus (0) Google Scholar). However, once a decision is made to proceed with FP, success rates are high, with up to 65% of patients aged 11–13 years and 80% of patients aged 14–17 years able to successfully cryopreserve sperm (43DiNofia A.M. Wang X. Yannekis G. Ogle S. Hobbie W.L. Carlson C.A. et al.Analysis of semen parameters in a young cohort of cancer patients.Pediatr Blood Cancer. 2017; 64: 381-386Crossref PubMed Scopus (17) Google Scholar). For adolescent patients who are unable or unwilling to produce a semen sample, testicular sperm extraction or testicular tissue biopsy are viable FP options. In prepubertal patients who have not yet begun spermatogenesis, only experimental options exist for cryopreservation of testicular tissue obtained by testicular biopsy or orchiectomy. Although successful restoration of fertility after prepubertal testicular tissue cryopreservation has not yet been demonstrated in humans, several centers across the world have collected and cryopreserved biopsied testicular tissues (44Braye A. Tournaye H. Goossens E. Setting up a cryopreservation programme for immature testicular tissue: lessons learned after more than 15 years of experience.Clin Med Insights Reprod Health. 2019; 13: 1-8Crossref Google Scholar, 45Picton H.M. Wyns C. Anderson R.A. 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: 2463-2475Crossref PubMed Scopus (119) Google Scholar, 46Valli-Pulaski H. Peters K.A. Gassei K. Steimer S.R. Sukhwani M. Hermann B.P. et al.Testicular tissue cryopreservation: 8 years of experience from a coordinated network of academic centers.Hum Reprod. 2019; 34: 966-977Crossref PubMed Scopus (6) Google Scholar, 47Keros V. Hultenby K. Borgström B. Fridström M. Jahnukainen K. Hovatta O. Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment.Hum Reprod. 2007; 22: 1384-1395Crossref PubMed Scopus (198) Google Scholar, 48Ginsberg J.P. Li Y. Carlson C.A. Gracia C.R. Hobbie W.L. Miller V.A. et al.Testicular tissue cryopreservation in prepubertal male children: an analysis of parental decision-making.Pediatr Blood Cancer. 2014; 61: 1673-1678Crossref PubMed Scopus (42) Google Scholar, 49Ho W.L.C. Bourne H. Gook D. Clarke G. Kemertzis M. Stern K. et al.A short report on current fertility preservation strategies for boys.Clin Endocrinol. 2017; 87: 279-285Crossref PubMed Scopus (13) Google Scholar, 50Heckmann L. Langenstroth-Röwer D. Pock T. Wistuba J. Stukenborg J.-B. Zitzmann M. et al.A diagnostic germ cell score for immature testicular tissue at risk of germ cell loss.Hum Reprod. 2018; 33: 636-645Crossref PubMed Scopus (8) Google Scholar, 51Radford J. Restoration of fertility after treatment for cancer.Horm Res. 2003; 59: 21-23Crossref PubMed Scopus (0) Google Scholar) with the expectation that experimental stem cell or testicular tissue-based technologies will make this possible in the future. The remainder of this review will discuss the translational approaches to this clinical problem (Fig. 2). The concept of sperm cryopreservation dates back more than 200 years, when Italian scientist Lazzaro Spallanzani noted in 1776 that sperm became motionless when cooled in snow (52Anger J.T. Gilbert B.R. Goldstein M. Cryopreservation of sperm: indications, methods and results.J Urol. 2003; 170: 1079-1084Crossref PubMed Scopus (0) Google Scholar). Further research in the mid-20th century considerably advanced the field when cryoprotectants such as glycerol were studied and applied to livestock artificial insemination techniques. Successful cryopreservation relies on freezing to temperatures that arrest cellular metabolism, but these temperatures also lead to increased intracellular reactive oxygen species, organelle damage, and caspase-mediated apoptosis, which decrease sperm fertility potential (53Thomson L.K. Fleming S.D. Aitken R.J. De Iuliis G.N. Zieschang J.-A. Clark A.M. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis.Hum Reprod. 2009; 24: 2061-2070Crossref PubMed Scopus (222) Google Scholar). Other studies have demonstrated statistically significant declines in post-thaw sperm morphology (54O'Connell M. McClure N. Lewis S.E.M. The effects of cryopreservation on sperm morphology, motility and mitochondrial function.Hum Reprod. 2002; 17: 704-709Crossref PubMed Google Scholar, 55Ozkavukcu S. Erdemli E. Isik A. Oztuna D. Karahuseyinoglu S. Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa.J Assist Reprod Genet. 2008; 25: 403-411Crossref PubMed Scopus (0) Google Scholar), motility (56MacKenna A. Crosby J. Huidobro C. Correa E. Duque G. Semen quality before cryopreservation and after thawing in 543 patients with testicular cancer.JBRA Assist Reprod. 2017; 21: 31-34Crossref PubMed Scopus (6) Google Scholar), and DNA integrity (57Raad G. Lteif L. Lahoud R. Azoury J. Azoury J. Tanios J. et al.Cryopreservation media differentially affect sperm motility, morphology and DNA integrity.Andrology. 2018; 6: 836-845Crossref PubMed Scopus (0) Google Scholar, 58Lusignan M.F. Li X. Herrero B. Delbes G. Chan P.T.K. Effects of different cryopreservation methods on DNA integrity and sperm chromatin quality in men.Andrology. 2018; 6: 829-835Crossref PubMed Scopus (0) Google Scholar). Accordingly, it is a critical research goal to discover optimal cryopreservation protocols to maximize successful sperm retrieval and function. There has been sparse research comparing the post-thaw effects of different cryoprotective agents in humans. Although newer agents have been studied and shown some promise (59O'Neill H.C. Nikoloska M. Ho H. Doshi A. Maalouf W. Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage.J Assist Reprod Genet. 2019; 36: 1713-1720Crossref PubMed Scopus (0) Google Scholar), dimethyl sulfoxide remains the standard cryoprotectant in many American and European FP centers (45Picton H.M. Wyns C. Anderson R.A. 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: 2463-2475Crossref PubMed Scopus (119) Google Scholar, 46Valli-Pulaski H. Peters K.A. Gassei K. Steimer S.R. Sukhwani M. Hermann B.P. et al.Testicular tissue cryopreservation: 8 years of experience from a coordinated network of academic centers.Hum Reprod. 2019; 34: 966-977Crossref PubMed Scopus (6) Google Scholar) for prepubertal boys. Slow freezing and vitrification protocols have also been compared. Vitrification uses differing cryoprotectant concentrations and ultrarapid cooling to prevent ice crystal formation. A recent meta-analysis comparing these two protocols (60Li Y.-X. Zhou L. Lv M.-Q. Ge P. Liu Y.-C. Zhou D.-X. Vitrification and conventional freezing methods in sperm cryopreservation: a systematic review and meta-analysis.Eur J Obstet Gynecol Reprod Biol. 2019; 233: 84-92Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) showed improved post-thaw parameters in the vitrification cohort. Other studies have corroborated this finding (59O'Neill H.C. Nikoloska M. Ho H. Doshi A. Maalouf W. Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage.J Assist Reprod Genet. 2019; 36: 1713-1720Crossref PubMed Scopus (0) Google Scholar, 61Le M.T. Nguyen T.T.T. Nguyen T.T. Nguyen V.T. Nguyen T.T.A. Nguyen V.Q.H. et al.Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects.Eur J Obstet Gynecol Reprod Biol. 2019; 234: 14-20Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 62Karthikeyan M. Arakkal D. Mangalaraj A.M. Kamath M.S. Comparison of conventional slow freeze versus permeable cryoprotectant-free vitrification of abnormal semen sample: a randomized controlled trial.J Hum Reprod Sci. 2019; 12: 150-155Crossref PubMed Scopus (3) Google Scholar), and the first live-birth after intracytoplasmic sperm injection (ICSI) with vitrified sperm has just been reported (63Spis E. Bushkovskaia A. Isachenko E. Todorov P. Sanchez R. Skopets V. et al.Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: three live births.Cryobiology. 2019; 90: 100-102Crossref PubMed Scopus (3) Google Scholar). However, only two small trials have examined the feasibility of vitrification for immature testicular tissue, limiting its clinical applicability in this setting (64Curaba M. Poels J. van Langendonckt A. Donnez J. Wyns C. Can prepubertal human testicular tissue be cryopreserved by vitrification?.Fertil Steril. 2011; 95: 2123.e9-2123.e12Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 65Poels J. Van Langendonckt A. Many M.-C. Wese F.-X. Wyns C. Vitrification preserves proliferation capacity in human spermatogonia.Hum Reprod. 2013; 28: 578-589Crossref PubMed Scopus (62) Google Scholar). These studies have suggested that a lower concentration of cryoprotectant in vitrification protocols may decrease organelle and membrane damage, and subsequently improve sperm survivability. Two strategies exist for the long-term storage of SSCs: cryopreservation of testicular tissue (TT) or testicular cell suspensions. Cryopreservation of TT fragments using vitrification or slow-freeze protocols relies on permeable cryoprotectants, which as previously mentioned may cause cellular damage. Additionally, usage of macroscopic tissue samples, as in TT, introduces heat and mass transfer during freezing, which leads to nonuniform cooling rates and subsequent changes in cell-cell interactions (66Karlsson J.O. Toner M. Long-term storage of tissues by cryopreservation: critical issues.Biomaterials. 1996; 17: 243-256Crossref PubMed Scopus (332) Google Scholar). These biophysical transfer phenomena are substantially reduced when using microscopic SSC suspensions. One major advantage to TT, however, is preservation of the SSC niche architecture, which may improve post-thaw SSC viability and function (67Kanbar M. de Michele F. Wyns C. Cryostorage of testicular tissue and retransplantation of spermatogonial stem cells in the infertile male.Best Pract Res Clin Endocrinol Metab. 2019; 33: 103-115Crossref PubMed Scopus (3) Google Scholar). Preservation of testicular cell suspensions has been less commonly studied in humans (68Brook P.F. Radford J.A. Shalet S.M. Joyce A.D. Gosden R.G. Isolation of germ cells from human testicular tissue for low temperature storage and autotransplantation.Fertil Steril. 2001; 75: 269-274Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 69Pacchiarotti J. Ramos T. Howerton K. Greilach S. Zaragoza K. Olmstead M. et al.Developing a clinical-grade cryopreservation protocol for human testicular tissue and cells.Biomed Res Int. 2013; 2013: 930962Crossref PubMed Scopus (0) Google Scholar, 70Unni S. Kasiviswanathan S. D'Souza S. Khavale S. Mukherjee S. Patwardhan S. et al.Efficient cryopreservation of testicular tissue: effect of age, sample state, and concentration of cryoprotectant.Fertil Steril. 2012; 97: 200-208.e1Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 71Sá R. Cremades N. Malheiro I. Sousa M. Cryopreservation of human testicular diploid germ cell suspensions.Andrologia. 2012; 44: 366-372Crossref PubMed Scopus (0) Google Scholar), and only one group (72Yango P. Altman E. S