Vitrification preserves proliferation capacity in human spermatogonia

Poels, Jonathan; Langendonckt, Anne Van; Many, Marie‐Christine; Wese, François-Xavier; Wyns, Christine

doi:10.1093/humrep/des455

Cited by 120 publications

(113 citation statements)

References 39 publications

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“…However, vitrification represents an innovative alternative strategy for pre-pubertal testicular tissue cryopreservation. Indeed, it has been suggested that vitrification may be an effective method to cryopreserve testicular tissue and maintain its ability to initiate (Poels et al, 2013) or even complete (Abrishami et al, 2010;Kaneko et al, 2013;Yokonishi et al, 2014) spermatogenesis after thawing. Vitrification is a physical phenomenon that allows, by an ultrafast cooling velocity, the transformation of a liquid into an amorphous glassy solid (Fahy et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

“…In mice, some promising recent results have shown good preservation of vitrified/warmed pre-pubertal testicular tissue after allografting (Baert et al, 2012) or short term in vitro organ culture (Curaba et al, 2011b;Gouk et al, 2011). However, CSF and vitrification protocols resulted in similar SSCs survival rates, suggesting that multifactorial parameters other than the cryopreservation procedure may be implicated (Poels et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa

et al. 2015

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SUMMARYTesticular tissue cryopreservation offers the hope of preserved future fertility to pre-pubertal boys with cancer before exposition to gonadotoxic treatments. The objective of this study was to compare controlled slow freezing (CSF) with five vitrification techniques for cryopreservation of murine pre-pubertal testicular tissue and to evaluate the best protocol that could provide a successful completion of spermatogenesis after in vitro maturation. Testicular tissue from 24 mice at 6.5 days post-partum (dpp) was used to compare several vitrification protocols with one another, as well as with a CSF protocol. Toxicity test using additional 12 mice was performed for all cryopreservation solutions. Fresh tissue (FT) from six mice was used as a control. Once the optimal vitrification protocol was selected [the modified solid surface vitrification No. 1 (mSSV 1 )], testes from 18 mice were cultured in vitro for 30 days with (i) fresh, (ii) slow-frozen/thawed and (iii) vitrified/warmed tissues. Testes from six mice at 36.5 dpp were used as controls. At day 30 of in vitro culture, germ cells of the seminiferous tubules showed a high ability to proliferate and elongated spermatids were observed after both freezing techniques, confirming the successful completion of in vitro spermatogenesis. However, after mSSV 1 , the morphological alterations and the percentage of pyknotic seminiferous tubules were lower than CSF (4.67 AE 0.53 vs. 10.1 AE 1.12 and 22.7 AE 2.83% vs. 37.3 AE 4.24% respectively). Moreover, the number of flagellated spermatozoa produced per mg of tissue was higher for mSSV 1 than for CSF (35 AE 3 vs. 9 AE 4 cells), with amounts of secreted testosterone during the culture close to those of FT. The mSSV 1 protocol resulted in success rates better than CSF in maintaining testicular tissue structure, tubular morphology and tissue functions not solely for immediate frozen/thawed tissues but also after a long-term in vitro culture.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa

et al. 2015

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“…In addition, cryopreservation of cellular aggregates or tissues may allow for the isolation and culture of SSCs from frozen-thawed tissues in order to mimic conditions that will occur in oncology patients. 35,36 To date, immature testicular pieces have been cryopreserved by slow-freezing and vitrification in both mice 37 and humans 38,39 and have been hetero-grafted beneath the tunica albuginea of a busulfantreated recipient testis. These studies demonstrate that spermatogonia can survive in xenogeneic recipients after cryopreservation and therefore result in an offspring after sperm microinsemination.…”

Section: Introductionmentioning

confidence: 99%

The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture

Eslahi¹,

Hadjighassem²,

Joghataei³

et al. 2013

IJN

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Introduction A 3D-nanofiber scaffold acts in a similar way to the extracellular matrix (ECM)/basement membrane that enhances the proliferation and self-renewal of stem cells. The goal of the present study was to investigate the effects of a poly L-lactic acid (PLLA) nanofiber scaffold on frozen-thawed neonate mouse spermatogonial stem cells (SSCs) and testis tissues. Methods The isolated spermatogonial cells were divided into six culture groups: (1) fresh spermatogonial cells, (2) fresh spermatogonial cells seeded onto PLLA, (3) frozen-thawed spermatogonial cells, (4) frozen-thawed spermatogonial cells seeded onto PLLA, (5) spermatogonial cells obtained from frozen-thawed testis tissue, and (6) spermatogonial cells obtained from frozen-thawed testis tissue seeded onto PLLA. Spermatogonial cells and testis fragments were cryopreserved and cultured for 3 weeks. Cluster assay was performed during the culture. The presence of spermatogonial cells in the culture was determined by a reverse transcriptase polymerase chain reaction for spermatogonial markers ( Oct4, GFRα-1, PLZF, Mvh(VASA), Itgα6 , and Itgβ1 ), as well as the ultrastructural study of cell clusters and SSCs transplantation to a recipient azoospermic mouse. The significance of the data was analyzed using the repeated measures and analysis of variance. Results The findings indicated that the spermatogonial cells seeded on PLLA significantly increased in vitro spermatogonial cell cluster formations in comparison with the control groups (culture of SSCs not seeded on PLLA) ( P ≤0.001). The viability rate for the frozen cells after thawing was 63.00% ± 3.56%. This number decreased significantly (40.00% ± 0.82%) in spermatogonial cells obtained from the frozen-thawed testis tissue. Both groups, however, showed in vitro cluster formation. Although the expression of spermatogonial markers was maintained after 3 weeks of culture, there was a significant downregulation for some spermatogonial genes in the experimental groups compared with those of the control groups. Furthermore, transplantation assay and transmission electron microscopy studies suggested the presence of SSCs among the cultured cells. Conclusion Although PLLA can increase the in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells, it may also cause them to differentiate during cultivation. The study therefore has implications for SSCs proliferation and germ cell differentiation in vitro.

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“…Other authors have analyzed the histology and morphology of testicular tubules stained with hematoxylin-eosin (Baert et al, 2012;Poels et al, 2013). The main limitation of this method is that it cannot definitively evaluate the impact of the procedure on the function of cryopreserved testicular tissue (Kvist et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

“…Several animal (Curaba et al, 2011;Gouk et al, 2011;Baert et al, 2012;Poels et al, 2012;Liu et al, 2013) and human (Curaba et al, 2011;Baert et al, 2013;Poels et al, 2013;Sá et al, 2012) vitrification in the cryopreservation of prepubertal testicular tissue with encouraging results. These studies have shown that the preservation of testicular cells and tissue through vitrification has been at least as effective as traditional slow freezing protocols (Curaba et al, 2011;Baert et al, 2012;Sá et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

A comparison between a new vitrification protocol and the slow freezing method in the cryopreservation of prepubertal testicular tissue

Radaelli¹,

Almodin²,

Minguetti-Câmara³

et al. 2017

JBRA Assisted Reproduction

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Objective: This study aimed to compare a new vitrification protocol with reduced cryoprotectant exposure to the slow freezing method in the cryopreservation of prepubertal rat testicular tissue.Methods: Five sexually immature male Wistar rats were submitted to bilateral orchiectomy. Tissue samples from each testicle were fragmented into small pieces and randomly assigned to three groups: Group A, fresh tissue (control); Group B, slow programmable freezing (SPF); and Group C (vitrification). Frozen/thawed, vitrified/warmed, and fresh testicular tissue were histologically compared. A pathologist blinded to the procedures assessed the morphology (cell differentiation, nuclei, and epithelium) of 10 seminiferous tubules from each testicle (100 tubules per Group).Results: Sertoli and spermatogonial stem cells were easily differentiated, and the nucleoli were easily viewed in the tubules assessed in all three groups. Small alterations in tissue architecture were observed in the control group as a result of tissue handling. Moderate alterations of the epithelium with the formation of small gaps and cell detachment from the basement membrane were observed in 28% of the frozen and 9% of the vitrified tubules. Condensed nuclei involving a small proportion of cells were observed in six and three tubules of the frozen and vitrified group, respectively. Despite the alterations, 97% of the frozen and 99% of the vitrified tubules were considered well preserved.Conclusions: The findings indicate that the vitrification protocol tested in this study adequately preserved the morphological integrity of prepubertal testicular tissue in a rat model. Further studies are required to confirm testicular tissue function after grafting.

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Vitrification preserves proliferation capacity in human spermatogonia

Abstract: The present study was supported by a grant from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie N° 7. 4.572.09.F). The authors declare that there is no conflict of interest.

Cited by 120 publications

References 39 publications

Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa

Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa

The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture

A comparison between a new vitrification protocol and the slow freezing method in the cryopreservation of prepubertal testicular tissue

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