Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system

Alison Ting, R. R. Yeoman, J. R. Campos, M. S. Lawson, S. F. Mullen, G. M. Fahy, Mary Zelinski

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Study Questio: NWhat are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? Summary Answer: The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. What Is Known Already: For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers.STUDY Design: , SIZE, DURATIONNon-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). Participants/Materials, Setting, Methods: Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm3) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 24/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A4), estradiol (E2)] production were analyzed weekly. Main Results and the Role of Chance: Dense stroma and intact pre-antral follicles were observed using VS containing 27% glycerol, 27% EG and 0.8% polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12%) and tissues vitrified with VEG (52 ± 2%) showed similar survival rates (all data: mean ± SEM; P > 0.05). For both groups, the initial follicle diameter was similar and increased (P <0.05) by Week 3, but diameters in vitrified follicles were smaller (P <0.05) by Week 6 (566 ± 27 μm) than those of the fresh follicles (757 ± 26 μm). Antrum formation rates were lower (P <0.05) for vitrified (37 ± 6%) relative to fresh (64 ± 8%) follicles. There was no significant change in levels in culture media of E2, P4 and A4 between fresh and VEG groups at any time point during culture.LIMITATIONS, REASONS FOR CAUTIONOnly in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues. Wider Implications of the Findings: This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined. Study Funding/Competing Interest: (S)National Institutes of Health (NIH) Oncofertility Consortium UL1 RR024926 (1RL1-HD058293, HD058295, PL1 EB008542), the Eunice Kennedy Shriver NICHD/NIH (U54 HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.

Original languageEnglish (US)
Pages (from-to)1267-1279
Number of pages13
JournalHuman Reproduction
Volume28
Issue number5
DOIs
StatePublished - May 2013

Fingerprint

Vitrification
Macaca
Glycerol
Ethylene Glycol
Polymers
Primates
Antral
National Institutes of Health (U.S.)
Live Birth
Bromodeoxyuridine
Dimethyl Sulfoxide
Fertility
Glia Maturation Factor
Neoplasms
Cryoprotective Agents
National Institute of Child Health and Human Development (U.S.)
Heterotopic Transplantation
Tissue Transplantation
Androstenedione
Autologous Transplantation

Keywords

  • cryopreservation
  • follicle culture
  • ovary
  • primate
  • vitrification

ASJC Scopus subject areas

  • Rehabilitation
  • Obstetrics and Gynecology
  • Reproductive Medicine

Cite this

Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system. / Ting, Alison; Yeoman, R. R.; Campos, J. R.; Lawson, M. S.; Mullen, S. F.; Fahy, G. M.; Zelinski, Mary.

In: Human Reproduction, Vol. 28, No. 5, 05.2013, p. 1267-1279.

Research output: Contribution to journalArticle

Ting, Alison ; Yeoman, R. R. ; Campos, J. R. ; Lawson, M. S. ; Mullen, S. F. ; Fahy, G. M. ; Zelinski, Mary. / Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system. In: Human Reproduction. 2013 ; Vol. 28, No. 5. pp. 1267-1279.
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abstract = "Study Questio: NWhat are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? Summary Answer: The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. What Is Known Already: For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers.STUDY Design: , SIZE, DURATIONNon-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). Participants/Materials, Setting, Methods: Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm3) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 24/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A4), estradiol (E2)] production were analyzed weekly. Main Results and the Role of Chance: Dense stroma and intact pre-antral follicles were observed using VS containing 27{\%} glycerol, 27{\%} EG and 0.8{\%} polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12{\%}) and tissues vitrified with VEG (52 ± 2{\%}) showed similar survival rates (all data: mean ± SEM; P > 0.05). For both groups, the initial follicle diameter was similar and increased (P <0.05) by Week 3, but diameters in vitrified follicles were smaller (P <0.05) by Week 6 (566 ± 27 μm) than those of the fresh follicles (757 ± 26 μm). Antrum formation rates were lower (P <0.05) for vitrified (37 ± 6{\%}) relative to fresh (64 ± 8{\%}) follicles. There was no significant change in levels in culture media of E2, P4 and A4 between fresh and VEG groups at any time point during culture.LIMITATIONS, REASONS FOR CAUTIONOnly in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues. Wider Implications of the Findings: This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined. Study Funding/Competing Interest: (S)National Institutes of Health (NIH) Oncofertility Consortium UL1 RR024926 (1RL1-HD058293, HD058295, PL1 EB008542), the Eunice Kennedy Shriver NICHD/NIH (U54 HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.",
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year = "2013",
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T1 - Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system

AU - Ting, Alison

AU - Yeoman, R. R.

AU - Campos, J. R.

AU - Lawson, M. S.

AU - Mullen, S. F.

AU - Fahy, G. M.

AU - Zelinski, Mary

PY - 2013/5

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N2 - Study Questio: NWhat are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? Summary Answer: The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. What Is Known Already: For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers.STUDY Design: , SIZE, DURATIONNon-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). Participants/Materials, Setting, Methods: Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm3) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 24/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A4), estradiol (E2)] production were analyzed weekly. Main Results and the Role of Chance: Dense stroma and intact pre-antral follicles were observed using VS containing 27% glycerol, 27% EG and 0.8% polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12%) and tissues vitrified with VEG (52 ± 2%) showed similar survival rates (all data: mean ± SEM; P > 0.05). For both groups, the initial follicle diameter was similar and increased (P <0.05) by Week 3, but diameters in vitrified follicles were smaller (P <0.05) by Week 6 (566 ± 27 μm) than those of the fresh follicles (757 ± 26 μm). Antrum formation rates were lower (P <0.05) for vitrified (37 ± 6%) relative to fresh (64 ± 8%) follicles. There was no significant change in levels in culture media of E2, P4 and A4 between fresh and VEG groups at any time point during culture.LIMITATIONS, REASONS FOR CAUTIONOnly in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues. Wider Implications of the Findings: This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined. Study Funding/Competing Interest: (S)National Institutes of Health (NIH) Oncofertility Consortium UL1 RR024926 (1RL1-HD058293, HD058295, PL1 EB008542), the Eunice Kennedy Shriver NICHD/NIH (U54 HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.

AB - Study Questio: NWhat are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? Summary Answer: The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. What Is Known Already: For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers.STUDY Design: , SIZE, DURATIONNon-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). Participants/Materials, Setting, Methods: Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm3) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 24/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A4), estradiol (E2)] production were analyzed weekly. Main Results and the Role of Chance: Dense stroma and intact pre-antral follicles were observed using VS containing 27% glycerol, 27% EG and 0.8% polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12%) and tissues vitrified with VEG (52 ± 2%) showed similar survival rates (all data: mean ± SEM; P > 0.05). For both groups, the initial follicle diameter was similar and increased (P <0.05) by Week 3, but diameters in vitrified follicles were smaller (P <0.05) by Week 6 (566 ± 27 μm) than those of the fresh follicles (757 ± 26 μm). Antrum formation rates were lower (P <0.05) for vitrified (37 ± 6%) relative to fresh (64 ± 8%) follicles. There was no significant change in levels in culture media of E2, P4 and A4 between fresh and VEG groups at any time point during culture.LIMITATIONS, REASONS FOR CAUTIONOnly in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues. Wider Implications of the Findings: This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined. Study Funding/Competing Interest: (S)National Institutes of Health (NIH) Oncofertility Consortium UL1 RR024926 (1RL1-HD058293, HD058295, PL1 EB008542), the Eunice Kennedy Shriver NICHD/NIH (U54 HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.

KW - cryopreservation

KW - follicle culture

KW - ovary

KW - primate

KW - vitrification

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