Fertility Preservation in Childhood Cancer: Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment

Marsida Hutka; Prashant Kadam; Dorien Van Saen; Natalie Z M Homer; Jaime Onofre; W Hamish B Wallace; Lee B Smith; Jan-Bernd Stukenborg; Ellen Goossens; Rod T Mitchell

doi:10.3390/cancers12102830

Fertility Preservation in Childhood Cancer: Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment

Marsida Hutka, Prashant Kadam, Dorien Van Saen, Natalie Z M Homer, Jaime Onofre, W Hamish B Wallace, Lee B Smith, Jan-Bernd Stukenborg, Ellen Goossens, Rod T Mitchell

Basic (bio-) Medical Sciences

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Abstract

Survivors of childhood cancer are at risk for long-term treatment-induced health sequelae, including gonadotoxicity and iatrogenic infertility. At present, for prepubertal boys there are no viable clinical options to preserve future reproductive potential. We investigated the effect of a pubertal induction regimen with gonadotrophins on prepubertal human testis xenograft development. Human testis tissue was obtained from patients with cancer and non-malignant haematological disorders (n = 6; aged 1–14 years) who underwent testis tissue cryopreservation for fertility preservation. Fresh and frozen-thawed testis fragments were transplanted subcutaneously or intratesticularly into immunocompromised mice. Graft-bearing mice received injections of vehicle or exogenous gonadotrophins, human chorionic gonadotrophin (hCG, 20 IU), and follicle-stimulating hormone (FSH, 12.5 IU) three times a week for 12 weeks. The gross morphology of vehicle and gonadotrophin-exposed grafts was similar for both transplantation sites. Exposure of prepubertal human testis tissue xenografts to exogenous gonadotrophins resulted in limited endocrine function of grafts, as demonstrated by the occasional expression of the steroidogenic cholesterol side-chain cleavage enzyme (CYP11A1). Plasma testosterone concentrations (0.13 vs. 0.25 ng/mL; p = 0.594) and seminal vesicle weights (10.02 vs. 13.93 mg; p = 0.431) in gonadotrophin-exposed recipient mice were comparable to vehicle-exposed controls. Regardless of the transplantation site and treatment, initiation and maintenance of androgen receptor (AR) expression were observed in Sertoli cells, indicating commitment towards a more differentiated status. However, neither exogenous gonadotrophins (in castrated host mice) nor endogenous testosterone (in intact host mice) were sufficient to repress the expression of markers associated with immature Sertoli cells, such as anti-Müllerian hormone (AMH) and Ki67, or to induce the redistribution of junctional proteins (connexin, CX43; claudin 11, CLDN11) to areas adjacent to the basement membrane. Spermatogonia did not progress developmentally but remained the most advanced germ cell type in testis xenografts. Overall, these findings demonstrate that exogenous gonadotrophins promote partial activation and maturation of the somatic environment in prepubertal testis xenografts. However, alternative hormone regimens or additional factors for pubertal induction are required to complete the functional maturation of the spermatogonial stem cell (SSC) niche.

Original language	English
Article number	2830
Pages (from-to)	1-21
Number of pages	21
Journal	Cancers
Volume	12
Issue number	10
DOIs	https://doi.org/10.3390/cancers12102830
Publication status	Published - Oct 2020

Bibliographical note

Funding Information:
Funding: M.H., J.O., and E.G. were supported by the ITN Marie Skłodowska-Curie programme “Growsperm” (EU-FP7-PEOPLE-2013-ITN 603568). D.V.S is a postdoctoral fellow of the Fund for Scientific Research Flanders. J-B.S. was supported by the Swedish Childhood Cancer Foundation. R.T.M. was supported by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) and a UKRI Future Leaders Fellowship (MR/S017151/1). Children with Cancer UK (15-0198) supported tissue collection. These studies were carried out at the MRC Centre for Reproductive Health, funded by the Grant MR/N022556/1. Part of this work was undertaken in the Vrije Universiteit Brussel.

Funding Information:
M.H., J.O., and E.G. were supported by the ITN Marie Sk?odowska-Curie programme ?Growsperm? (EU-FP7-PEOPLE-2013-ITN 603568). D.V.S is a postdoctoral fellow of the Fund for Scientific Research Flanders. J-B.S. was supported by the Swedish Childhood Cancer Foundation. R.T.M. was supported by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) and a UKRI Future Leaders Fellowship (MR/S017151/1). Children with Cancer UK (15-0198) supported tissue collection. These studies were carried out at the MRC Centre for Reproductive Health, funded by the Grant MR/N022556/1. Part of this work was undertaken in the Vrije Universiteit Brussel.

Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.

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Hutka, M., Kadam, P., Van Saen, D., Homer, N. Z. M., Onofre, J., Wallace, W. H. B., Smith, L. B., Stukenborg, J-B., Goossens, E., & Mitchell, R. T. (2020). Fertility Preservation in Childhood Cancer: Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment. Cancers, 12(10), 1-21. [2830]. https://doi.org/10.3390/cancers12102830

Hutka, Marsida ; Kadam, Prashant ; Van Saen, Dorien ; Homer, Natalie Z M ; Onofre, Jaime ; Wallace, W Hamish B ; Smith, Lee B ; Stukenborg, Jan-Bernd ; Goossens, Ellen ; Mitchell, Rod T. / Fertility Preservation in Childhood Cancer : Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment. In: Cancers. 2020 ; Vol. 12, No. 10. pp. 1-21.

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title = "Fertility Preservation in Childhood Cancer: Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment",

abstract = "Survivors of childhood cancer are at risk for long-term treatment-induced health sequelae, including gonadotoxicity and iatrogenic infertility. At present, for prepubertal boys there are no viable clinical options to preserve future reproductive potential. We investigated the effect of a pubertal induction regimen with gonadotrophins on prepubertal human testis xenograft development. Human testis tissue was obtained from patients with cancer and non-malignant haematological disorders (n = 6; aged 1–14 years) who underwent testis tissue cryopreservation for fertility preservation. Fresh and frozen-thawed testis fragments were transplanted subcutaneously or intratesticularly into immunocompromised mice. Graft-bearing mice received injections of vehicle or exogenous gonadotrophins, human chorionic gonadotrophin (hCG, 20 IU), and follicle-stimulating hormone (FSH, 12.5 IU) three times a week for 12 weeks. The gross morphology of vehicle and gonadotrophin-exposed grafts was similar for both transplantation sites. Exposure of prepubertal human testis tissue xenografts to exogenous gonadotrophins resulted in limited endocrine function of grafts, as demonstrated by the occasional expression of the steroidogenic cholesterol side-chain cleavage enzyme (CYP11A1). Plasma testosterone concentrations (0.13 vs. 0.25 ng/mL; p = 0.594) and seminal vesicle weights (10.02 vs. 13.93 mg; p = 0.431) in gonadotrophin-exposed recipient mice were comparable to vehicle-exposed controls. Regardless of the transplantation site and treatment, initiation and maintenance of androgen receptor (AR) expression were observed in Sertoli cells, indicating commitment towards a more differentiated status. However, neither exogenous gonadotrophins (in castrated host mice) nor endogenous testosterone (in intact host mice) were sufficient to repress the expression of markers associated with immature Sertoli cells, such as anti-M{\"u}llerian hormone (AMH) and Ki67, or to induce the redistribution of junctional proteins (connexin, CX43; claudin 11, CLDN11) to areas adjacent to the basement membrane. Spermatogonia did not progress developmentally but remained the most advanced germ cell type in testis xenografts. Overall, these findings demonstrate that exogenous gonadotrophins promote partial activation and maturation of the somatic environment in prepubertal testis xenografts. However, alternative hormone regimens or additional factors for pubertal induction are required to complete the functional maturation of the spermatogonial stem cell (SSC) niche.",

author = "Marsida Hutka and Prashant Kadam and {Van Saen}, Dorien and Homer, {Natalie Z M} and Jaime Onofre and Wallace, {W Hamish B} and Smith, {Lee B} and Jan-Bernd Stukenborg and Ellen Goossens and Mitchell, {Rod T}",

note = "Funding Information: Funding: M.H., J.O., and E.G. were supported by the ITN Marie Sk{\l}odowska-Curie programme “Growsperm” (EU-FP7-PEOPLE-2013-ITN 603568). D.V.S is a postdoctoral fellow of the Fund for Scientific Research Flanders. J-B.S. was supported by the Swedish Childhood Cancer Foundation. R.T.M. was supported by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) and a UKRI Future Leaders Fellowship (MR/S017151/1). Children with Cancer UK (15-0198) supported tissue collection. These studies were carried out at the MRC Centre for Reproductive Health, funded by the Grant MR/N022556/1. Part of this work was undertaken in the Vrije Universiteit Brussel. Funding Information: M.H., J.O., and E.G. were supported by the ITN Marie Sk?odowska-Curie programme ?Growsperm? (EU-FP7-PEOPLE-2013-ITN 603568). D.V.S is a postdoctoral fellow of the Fund for Scientific Research Flanders. J-B.S. was supported by the Swedish Childhood Cancer Foundation. R.T.M. was supported by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) and a UKRI Future Leaders Fellowship (MR/S017151/1). Children with Cancer UK (15-0198) supported tissue collection. These studies were carried out at the MRC Centre for Reproductive Health, funded by the Grant MR/N022556/1. Part of this work was undertaken in the Vrije Universiteit Brussel. Publisher Copyright: {\textcopyright} 2020 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.",

year = "2020",

month = oct,

doi = "10.3390/cancers12102830",

language = "English",

volume = "12",

pages = "1--21",

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Fertility Preservation in Childhood Cancer : Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment. / Hutka, Marsida; Kadam, Prashant ; Van Saen, Dorien; Homer, Natalie Z M; Onofre, Jaime; Wallace, W Hamish B; Smith, Lee B; Stukenborg, Jan-Bernd; Goossens, Ellen; Mitchell, Rod T.

In: Cancers, Vol. 12, No. 10, 2830, 10.2020, p. 1-21.

Research output: Contribution to journal › Article › peer-review

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T1 - Fertility Preservation in Childhood Cancer

T2 - Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment

AU - Hutka, Marsida

AU - Kadam, Prashant

AU - Van Saen, Dorien

AU - Homer, Natalie Z M

AU - Onofre, Jaime

AU - Wallace, W Hamish B

AU - Smith, Lee B

AU - Stukenborg, Jan-Bernd

AU - Goossens, Ellen

AU - Mitchell, Rod T

N1 - Funding Information: Funding: M.H., J.O., and E.G. were supported by the ITN Marie Skłodowska-Curie programme “Growsperm” (EU-FP7-PEOPLE-2013-ITN 603568). D.V.S is a postdoctoral fellow of the Fund for Scientific Research Flanders. J-B.S. was supported by the Swedish Childhood Cancer Foundation. R.T.M. was supported by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) and a UKRI Future Leaders Fellowship (MR/S017151/1). Children with Cancer UK (15-0198) supported tissue collection. These studies were carried out at the MRC Centre for Reproductive Health, funded by the Grant MR/N022556/1. Part of this work was undertaken in the Vrije Universiteit Brussel. Funding Information: M.H., J.O., and E.G. were supported by the ITN Marie Sk?odowska-Curie programme ?Growsperm? (EU-FP7-PEOPLE-2013-ITN 603568). D.V.S is a postdoctoral fellow of the Fund for Scientific Research Flanders. J-B.S. was supported by the Swedish Childhood Cancer Foundation. R.T.M. was supported by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) and a UKRI Future Leaders Fellowship (MR/S017151/1). Children with Cancer UK (15-0198) supported tissue collection. These studies were carried out at the MRC Centre for Reproductive Health, funded by the Grant MR/N022556/1. Part of this work was undertaken in the Vrije Universiteit Brussel. Publisher Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.

PY - 2020/10

Y1 - 2020/10

N2 - Survivors of childhood cancer are at risk for long-term treatment-induced health sequelae, including gonadotoxicity and iatrogenic infertility. At present, for prepubertal boys there are no viable clinical options to preserve future reproductive potential. We investigated the effect of a pubertal induction regimen with gonadotrophins on prepubertal human testis xenograft development. Human testis tissue was obtained from patients with cancer and non-malignant haematological disorders (n = 6; aged 1–14 years) who underwent testis tissue cryopreservation for fertility preservation. Fresh and frozen-thawed testis fragments were transplanted subcutaneously or intratesticularly into immunocompromised mice. Graft-bearing mice received injections of vehicle or exogenous gonadotrophins, human chorionic gonadotrophin (hCG, 20 IU), and follicle-stimulating hormone (FSH, 12.5 IU) three times a week for 12 weeks. The gross morphology of vehicle and gonadotrophin-exposed grafts was similar for both transplantation sites. Exposure of prepubertal human testis tissue xenografts to exogenous gonadotrophins resulted in limited endocrine function of grafts, as demonstrated by the occasional expression of the steroidogenic cholesterol side-chain cleavage enzyme (CYP11A1). Plasma testosterone concentrations (0.13 vs. 0.25 ng/mL; p = 0.594) and seminal vesicle weights (10.02 vs. 13.93 mg; p = 0.431) in gonadotrophin-exposed recipient mice were comparable to vehicle-exposed controls. Regardless of the transplantation site and treatment, initiation and maintenance of androgen receptor (AR) expression were observed in Sertoli cells, indicating commitment towards a more differentiated status. However, neither exogenous gonadotrophins (in castrated host mice) nor endogenous testosterone (in intact host mice) were sufficient to repress the expression of markers associated with immature Sertoli cells, such as anti-Müllerian hormone (AMH) and Ki67, or to induce the redistribution of junctional proteins (connexin, CX43; claudin 11, CLDN11) to areas adjacent to the basement membrane. Spermatogonia did not progress developmentally but remained the most advanced germ cell type in testis xenografts. Overall, these findings demonstrate that exogenous gonadotrophins promote partial activation and maturation of the somatic environment in prepubertal testis xenografts. However, alternative hormone regimens or additional factors for pubertal induction are required to complete the functional maturation of the spermatogonial stem cell (SSC) niche.

AB - Survivors of childhood cancer are at risk for long-term treatment-induced health sequelae, including gonadotoxicity and iatrogenic infertility. At present, for prepubertal boys there are no viable clinical options to preserve future reproductive potential. We investigated the effect of a pubertal induction regimen with gonadotrophins on prepubertal human testis xenograft development. Human testis tissue was obtained from patients with cancer and non-malignant haematological disorders (n = 6; aged 1–14 years) who underwent testis tissue cryopreservation for fertility preservation. Fresh and frozen-thawed testis fragments were transplanted subcutaneously or intratesticularly into immunocompromised mice. Graft-bearing mice received injections of vehicle or exogenous gonadotrophins, human chorionic gonadotrophin (hCG, 20 IU), and follicle-stimulating hormone (FSH, 12.5 IU) three times a week for 12 weeks. The gross morphology of vehicle and gonadotrophin-exposed grafts was similar for both transplantation sites. Exposure of prepubertal human testis tissue xenografts to exogenous gonadotrophins resulted in limited endocrine function of grafts, as demonstrated by the occasional expression of the steroidogenic cholesterol side-chain cleavage enzyme (CYP11A1). Plasma testosterone concentrations (0.13 vs. 0.25 ng/mL; p = 0.594) and seminal vesicle weights (10.02 vs. 13.93 mg; p = 0.431) in gonadotrophin-exposed recipient mice were comparable to vehicle-exposed controls. Regardless of the transplantation site and treatment, initiation and maintenance of androgen receptor (AR) expression were observed in Sertoli cells, indicating commitment towards a more differentiated status. However, neither exogenous gonadotrophins (in castrated host mice) nor endogenous testosterone (in intact host mice) were sufficient to repress the expression of markers associated with immature Sertoli cells, such as anti-Müllerian hormone (AMH) and Ki67, or to induce the redistribution of junctional proteins (connexin, CX43; claudin 11, CLDN11) to areas adjacent to the basement membrane. Spermatogonia did not progress developmentally but remained the most advanced germ cell type in testis xenografts. Overall, these findings demonstrate that exogenous gonadotrophins promote partial activation and maturation of the somatic environment in prepubertal testis xenografts. However, alternative hormone regimens or additional factors for pubertal induction are required to complete the functional maturation of the spermatogonial stem cell (SSC) niche.

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Fertility Preservation in Childhood Cancer: Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment

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