Germ cell quantification in human fetal and prepubertal testis tissues: a comparison of current methodologies
Methods to quantify germ cell number in human immature testicular tissues are essential to evaluate the impact of chemotherapy exposures and to optimise cryopreservation protocols used in fertility preservation for prepubertal boys. Established quantification methods rely on the presence of round tu...
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| Format: | Article |
| Language: | English |
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Bioscientifica
2025-02-01
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| Series: | Reproduction and Fertility |
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| Online Access: | https://raf.bioscientifica.com/view/journals/raf/6/1/RAF-24-0116.xml |
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| author | Emma Kearney David Greenald Gabriele Matilionyte Sheila Lane Melissa D Tharmalingam Jill Davies Jan-Bernd Stukenborg Grace Forsyth Rod T Mitchell |
| author_facet | Emma Kearney David Greenald Gabriele Matilionyte Sheila Lane Melissa D Tharmalingam Jill Davies Jan-Bernd Stukenborg Grace Forsyth Rod T Mitchell |
| author_sort | Emma Kearney |
| collection | DOAJ |
| description | Methods to quantify germ cell number in human immature testicular tissues are essential to evaluate the impact of chemotherapy exposures and to optimise cryopreservation protocols used in fertility preservation for prepubertal boys. Established quantification methods rely on the presence of round tubules within the tissue. However, round tubular cross sections are limited in human prepubertal testicular tissues, especially when using in vitro culture. We aimed to assess whether an alternative method of germ cell quantification would provide similar results to recently established methods, without the requirement for round tubules. Human testicular samples included fetal tissue (exposed in vitro to cisplatin, carboplatin or control) or prepubertal tissue (fresh, cryopreserved, fresh in vitro cultured or cryopreserved in vitro cultured). Immunofluorescence assessed AP2γ (gonocytes) and MAGE-A4 ((pre)spermatogonia) expression. Germ cells were quantified by tubular germ cell density (Method 1), which was compared to methods that require round tubules, including spermatogonial number per round tubular cross section (S/T) (Method 2), fertility index (Method 3) and round tubular germ cell density (Method 4). A correlation analysis between methods was performed. Method 1 is strongly and significantly correlated with Method 2 (r = 0.838, P < 0.0001; r = 0.833, P < 0.0001), Method 3 (r = 0.752, P < 0.001; r = 0.802, P < 0.0001) and Method 4 (r = 0.863, P < 0.0001; r = 0.914, P < 0.0001) for fetal and prepubertal tissues, respectively. Given that Method 1 assess tubules irrespective of shape, it may increase the total number of germ cells available for quantification, validating its use for quantification of human testicular tissue samples where the amount of tissue or presence of round tubules is limited. |
| format | Article |
| id | doaj-art-8da0bae6cb574b21bae111e4b3d6f706 |
| institution | Kabale University |
| issn | 2633-8386 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Bioscientifica |
| record_format | Article |
| series | Reproduction and Fertility |
| spelling | doaj-art-8da0bae6cb574b21bae111e4b3d6f7062025-02-10T16:58:39ZengBioscientificaReproduction and Fertility2633-83862025-02-016110.1530/RAF-24-01161Germ cell quantification in human fetal and prepubertal testis tissues: a comparison of current methodologiesEmma Kearney0David Greenald1Gabriele Matilionyte2Sheila Lane3Melissa D Tharmalingam4Jill Davies5Jan-Bernd Stukenborg6Grace Forsyth7Rod T Mitchell8Centre for Reproductive Health, Institute of Regeneration and Repair, University of Edinburgh, Edinburgh, United KingdomCentre for Reproductive Health, Institute of Regeneration and Repair, University of Edinburgh, Edinburgh, United KingdomCentre for Reproductive Health, Institute of Regeneration and Repair, University of Edinburgh, Edinburgh, United KingdomDepartment of Paediatric Oncology and Haematology, Children’s Hospital Oxford, Oxford University Hospitals NHS Foundation Trust, Oxford, United KingdomCentre for Reproductive Health, Institute of Regeneration and Repair, University of Edinburgh, Edinburgh, United KingdomOxford Cell and Tissue Biobank, Children’s Hospital Oxford, Oxford University Hospitals NHS Foundation Trust, Oxford, United KingdomNORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women’s and Children’s Health, Karolinska Institutet and Karolinska University Hospital, Solna, SwedenCentre for Reproductive Health, Institute of Regeneration and Repair, University of Edinburgh, Edinburgh, United KingdomCentre for Reproductive Health, Institute of Regeneration and Repair, University of Edinburgh, Edinburgh, United KingdomMethods to quantify germ cell number in human immature testicular tissues are essential to evaluate the impact of chemotherapy exposures and to optimise cryopreservation protocols used in fertility preservation for prepubertal boys. Established quantification methods rely on the presence of round tubules within the tissue. However, round tubular cross sections are limited in human prepubertal testicular tissues, especially when using in vitro culture. We aimed to assess whether an alternative method of germ cell quantification would provide similar results to recently established methods, without the requirement for round tubules. Human testicular samples included fetal tissue (exposed in vitro to cisplatin, carboplatin or control) or prepubertal tissue (fresh, cryopreserved, fresh in vitro cultured or cryopreserved in vitro cultured). Immunofluorescence assessed AP2γ (gonocytes) and MAGE-A4 ((pre)spermatogonia) expression. Germ cells were quantified by tubular germ cell density (Method 1), which was compared to methods that require round tubules, including spermatogonial number per round tubular cross section (S/T) (Method 2), fertility index (Method 3) and round tubular germ cell density (Method 4). A correlation analysis between methods was performed. Method 1 is strongly and significantly correlated with Method 2 (r = 0.838, P < 0.0001; r = 0.833, P < 0.0001), Method 3 (r = 0.752, P < 0.001; r = 0.802, P < 0.0001) and Method 4 (r = 0.863, P < 0.0001; r = 0.914, P < 0.0001) for fetal and prepubertal tissues, respectively. Given that Method 1 assess tubules irrespective of shape, it may increase the total number of germ cells available for quantification, validating its use for quantification of human testicular tissue samples where the amount of tissue or presence of round tubules is limited.https://raf.bioscientifica.com/view/journals/raf/6/1/RAF-24-0116.xmlhumantestiscisplatincarboplatinprepubertalfetalfertilitygerm cellsgonocytesspermatogoniacell quantification |
| spellingShingle | Emma Kearney David Greenald Gabriele Matilionyte Sheila Lane Melissa D Tharmalingam Jill Davies Jan-Bernd Stukenborg Grace Forsyth Rod T Mitchell Germ cell quantification in human fetal and prepubertal testis tissues: a comparison of current methodologies Reproduction and Fertility human testis cisplatin carboplatin prepubertal fetal fertility germ cells gonocytes spermatogonia cell quantification |
| title | Germ cell quantification in human fetal and prepubertal testis tissues: a comparison of current methodologies |
| title_full | Germ cell quantification in human fetal and prepubertal testis tissues: a comparison of current methodologies |
| title_fullStr | Germ cell quantification in human fetal and prepubertal testis tissues: a comparison of current methodologies |
| title_full_unstemmed | Germ cell quantification in human fetal and prepubertal testis tissues: a comparison of current methodologies |
| title_short | Germ cell quantification in human fetal and prepubertal testis tissues: a comparison of current methodologies |
| title_sort | germ cell quantification in human fetal and prepubertal testis tissues a comparison of current methodologies |
| topic | human testis cisplatin carboplatin prepubertal fetal fertility germ cells gonocytes spermatogonia cell quantification |
| url | https://raf.bioscientifica.com/view/journals/raf/6/1/RAF-24-0116.xml |
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