Is vitrification with microinjection of single seminiferous tubules an efficient cryopreservation approach for limited testicular tissue?
Testicular tissue from 10 patients with normal spermatogenesis were assigned to a fresh control group or one of the following cryopreservation procedures: uncontrolled slow freezing (USF) using either 1.5 or 2.1 M DMSO combined with sucrose and vitrification with or without single seminiferous tubules microinjection.
Single seminiferous tubules microinjected with cryoprotective agents (CPA) enhanced the penetration of CPA compared with CPA-treated testicular tissue fragments. Microinjection of seminiferous tubules (VLP) maintained tubule structural integrity and germ cell numbers, and reduced spermatogonial apoptosis after cryopreservation compared with vitrification without microinjection (apoptosis rate: VLP versus vitrification without microinjection, P = 0.047; VLP versus USF, P= 0.049). Freezing of single seminiferous tubules using 0.25-ml straws and traditional sperm freezing methods protected sperm retrieval and recovery rates, and the progressive motility index.
Vitrification of single seminiferous tubule with microinjection of low CPA concentration is an effective approach to testicular cryopreservation.
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- Minipuberty in Klinefelter syndrome: Current status and future directions.American Journal of Medical Genetics Part C: Seminars in Medical Genetics. 2020; 184: 320-326https://doi.org/10.1002/ajmg.c.31794
- DMSO inhibits human platelet activation through cyclooxygenase-1 inhibition. A novel agent for drug eluting stents?.Biochem. Biophys. Res. Commun. 2010; 391: 1629-1633https://doi.org/10.1016/j.bbrc.2009.12.102
- What is the best cryopreservation protocol for human testicular tissue banking?.Hum. Reprod. 2013; 28: 1816-1826https://doi.org/10.1093/humrep/det100
- Oocyte vitrification for fertility preservation in women with endometriosis: an observational study.Fertil. Steril. 2020; 113: 836-844https://doi.org/10.1016/j.fertnstert.2019.11.017
- Insights on cryoprotectant toxicity from gene expression profiling of endothelial cells exposed to ethylene glycol.Cryobiology. 2015; 71: 405-412https://doi.org/10.1016/j.cryobiol.2015.10.142
- Cryopreservation of prepubertal mouse testicular tissue by vitrification.Fertil. Steril. 2011; 95 (e1221): 1229-1234https://doi.org/10.1016/j.fertnstert.2010.04.062
- Male infertility in cancer patients: Review of the literature.Int. J. Urol. 2010; 17: 327-331https://doi.org/10.1111/j.1442-2042.2010.02484.x
- Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa.Andrology. 2015; 3: 611-625https://doi.org/10.1111/andr.12042
- Testicular biopsy in prepubertal boys: a worthwhile minor surgical procedure?.Nat. Rev. Urol. 2016; 13: 141-150https://doi.org/10.1038/nrurol.2015.312
- Autologous grafting of cryopreserved prepubertal rhesus testis produces sperm and offspring.Science. 2019; 363: 1314-1319https://doi.org/10.1126/science.aav2914
- Cryopreservation of hematopoietic and non-hematopoietic stem cells.Transfus Apher. Sci. 2006; 34: 309-315https://doi.org/10.1016/j.transci.2005.11.012
- Vitrification of Rhesus Macaque Mesenchymal Stem Cells and the Effects on Global Gene Expression.Stem. Cells Int. 2017; 20173893691https://doi.org/10.1155/2017/3893691
- Does prepubertal testicular tissue vitrification influence spermatogonial stem cells (SSCs) viability?.J. Assist. Reprod. Genet. 2013; 30: 1271-1277https://doi.org/10.1007/s10815-013-0050-x
- An experimental protocol for fertility preservation in prepubertal boys recently diagnosed with cancer: a report of acceptability and safety.Hum. Reprod. 2010; 25: 37-41https://doi.org/10.1093/humrep/dep371
- Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm.Cell. Stem. Cell. 2012; 11: 715-726https://doi.org/10.1016/j.stem.2012.07.017
- Association analysis identifies new risk loci for non-obstructive azoospermia in Chinese men.Nat. Commun. 2014; 5: 3857https://doi.org/10.1038/ncomms4857
- Cryopreservation of Human Stem Cells for Clinical Application: A Review.Transfus Med. Hemother. 2011; 38: 107-123https://doi.org/10.1159/000326623
- Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success.Reprod. Biomed. Online. 2003; 6: 191-200https://doi.org/10.1016/s1472-6483(10)61710-5
- Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose.Reproduction. 2008; 136: 167-173https://doi.org/10.1530/REP-07-0463
- Long-term storage of tissues by cryopreservation: critical issues.Biomaterials. 1996; 17: 243-256https://doi.org/10.1016/0142-9612(96)85562-1
- Genetics of male infertility.Nat. Rev. Urol. 2018; 15: 369-384https://doi.org/10.1038/s41585-018-0003-3
- A novel micro-straw for cryopreservation of small number of human spermatozoon.Asian J. Androl. 2017; 19: 326-329https://doi.org/10.4103/1008-682X.173452
- Sertoli cells from non-obstructive azoospermia and obstructive azoospermia patients show distinct morphology, Raman spectrum and biochemical phenotype.Hum. Reprod. 2013; 28: 1863-1873https://doi.org/10.1093/humrep/det068
- A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells.Exp. Cell. Res. 1972; 71: 345-355https://doi.org/10.1016/0014-4827(72)90303-5
- Influence of temperature, serum, and gonadotropin supplementation in short- and long-term organotypic culture of human immature testicular tissue.Fertil. Steril. 2018; 110 (e1043): 1045-1057https://doi.org/10.1016/j.fertnstert.2018.07.018
- Effects of chemotherapy and radiotherapy on spermatogenesis in humans.Fertil. Steril. 2013; 100: 1180-1186https://doi.org/10.1016/j.fertnstert.2013.08.010
- Optimizing methods for human testicular tissue cryopreservation and spermatogonial stem cell isolation.J. Cell. Biochem. 2019; 120: 613-621https://doi.org/10.1002/jcb.27419
- ROS are required for mouse spermatogonial stem cell self-renewal.Cell. Stem. Cell. 2013; 12: 774-786https://doi.org/10.1016/j.stem.2013.04.001
- Spermatogonial stem cell autotransplantation and germline genomic editing: a future cure for spermatogenic failure and prevention of transmission of genomic diseases.Hum. Reprod. Update. 2016; 22: 561-573https://doi.org/10.1093/humupd/dmw017
- Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation.Hum. Reprod. Update. 2016; 22: 744-761https://doi.org/10.1093/humupd/dmw029
- Propagation of human spermatogonial stem cells in vitro.JAMA. 2009; 302: 2127-2134https://doi.org/10.1001/jama.2009.1689
- In vitro production of functional sperm in cultured neonatal mouse testes.Nature. 2011; 471: 504-507https://doi.org/10.1038/nature09850
- In vitro sperm production from mouse spermatogonial stem cell lines using an organ culture method.Nat. Protoc. 2013; 8: 2098-2104https://doi.org/10.1038/nprot.2013.138
- Validation-verification of a highly effective, practical human testicular tissue in vitro culture-cryopreservation procedure aimed to optimize pre-freeze and post-thaw motility.J. Assist. Reprod. Genet. 2016; 33: 519-528https://doi.org/10.1007/s10815-016-0659-7
- Testicular stem cells for fertility preservation: preclinical studies on male germ cell transplantation and testicular grafting.Pediatr. Blood Cancer. 2009; 53: 274-280https://doi.org/10.1002/pbc.22002
- Successful delivery derived from cryopreserved rare human spermatozoa with novel cryopiece.Andrology. 2017; 5: 832-837https://doi.org/10.1111/andr.12380
- Up-regulation of heme oxygenase-1 expression modulates reactive oxygen species level during the cryopreservation of human seminiferous tubules.Fertil. Steril. 2014; 102 (e974): 974-980https://doi.org/10.1016/j.fertnstert.2014.07.736
- Fibroblast growth factor-5 promotes spermatogonial stem cell proliferation via ERK and AKT activation.Stem. Cell. Res. Ther. 2019; 10: 40https://doi.org/10.1186/s13287-019-1139-7
- NODAL secreted by male germ cells regulates the proliferation and function of human Sertoli cells from obstructive azoospermia and nonobstructive azoospermia patients.Asian J. Androl. 2015; 17: 996-1005https://doi.org/10.4103/1008-682X.159722
- Assessment of freezing procedures for rat immature testicular tissue.Theriogenology. 2011; 76: 981-990https://doi.org/10.1016/j.theriogenology.2011.04.025
- Vitrification media: toxicity, permeability, and dielectric properties.Cryobiology. 2002; 44: 24-37https://doi.org/10.1016/S0011-2240(02)00002-0
- Options for fertility preservation in prepubertal boys.Hum. Reprod. Update. 2010; 16: 312-328https://doi.org/10.1093/humupd/dmp054
- Measurement of essential physical properties of vitrification solutions.Theriogenology. 2007; 67: 81-89https://doi.org/10.1016/j.theriogenology.2006.09.029
- Offspring production with sperm grown in vitro from cryopreserved testis tissues.Nat. Commun. 2014; 5: 4320https://doi.org/10.1038/ncomms5320
- In vitro testicular organogenesis from human fetal gonads produces fertilization-competent spermatids.Cell Research. 2020; 30: 244-255https://doi.org/10.1038/s41422-020-0283-z
- Ice nucleation and antinucleation in nature.Cryobiology. 2000; 41: 257-279https://doi.org/10.1006/cryo.2000.2289
- Single-cell analysis of developing and azoospermia human testicles reveals central role of Sertoli cells.Nat. Commun. 2020; 11: 5683https://doi.org/10.1038/s41467-020-19414-4
Dr Li Zheng is Professor of Department of Andrology at Shanghai General Hospital. His main research interests are fertility preservation and molecular mechanism of male infertility. With over 30 years’ experience in reproductive biology, he developed new carriers for cryopreservation of sperm and evaluated its safety, and established a rare sperm cryopreservation protocol and applied it in clinical practice which gave birth to healthy offspring.
Vitrification using 1.05-M DMSO and 1.35-M ethylene glycol for 10 min and 2.1-M DMSO and 2.7-M ethylene glycol for 3 min with microinjection was an efficient alternative to uncontrolled slow freezing for human testicular tissue. This protocol will provide an unprecedented cryopreservation solution for limited human testicular tissue.
Published online: July 03, 2021
Accepted: June 28, 2021
Received in revised form: June 8, 2021
Received: March 30, 2021Declaration: The authors report no financial or commercial conflicts of interest
© 2021 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.