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In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels
BACKGROUND: In vivo studies involving molecular markers of the follicle wall associated with follicular fluid (FF) milieu are crucial for a better understanding of follicle dynamics. The inability to obtain in vivo samples of antral follicle wall (granulosa and theca cells) without jeopardizing ovar...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6064614/ https://www.ncbi.nlm.nih.gov/pubmed/30055625 http://dx.doi.org/10.1186/s12958-018-0380-8 |
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author | Ishak, G. M. Bashir, S. T. Dutra, G. A. Gastal, G. D. A. Gastal, M. O. Cavinder, C. A. Feugang, J. M. Gastal, E. L. |
author_facet | Ishak, G. M. Bashir, S. T. Dutra, G. A. Gastal, G. D. A. Gastal, M. O. Cavinder, C. A. Feugang, J. M. Gastal, E. L. |
author_sort | Ishak, G. M. |
collection | PubMed |
description | BACKGROUND: In vivo studies involving molecular markers of the follicle wall associated with follicular fluid (FF) milieu are crucial for a better understanding of follicle dynamics. The inability to obtain in vivo samples of antral follicle wall (granulosa and theca cells) without jeopardizing ovarian function has restricted advancement in knowledge of folliculogenesis in several species. The purpose of this study in mares was to develop and validate a novel, minimally invasive in vivo technique for simultaneous collection of follicle wall biopsy (FWB) and FF samples, and repeated collection from the same individual, during different stages of antral follicle development. We hypothesized that the in vivo FWB technique provides samples that maintain the normal histological tissue structure of the follicle wall layers, offers sufficient material for various cellular and molecular techniques, and allows simultaneous retrieval of FF. METHODS: In Experiment 1 (ex vivo), each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from 10-, 20-, and 30-mm follicles were repeatedly and simultaneously obtained through transvaginal ultrasound-guided technique. RESULTS: In Experiment 1, the thickness of granulosa, theca interna, and theca externa layers was not influenced (P > 0.05) by the harvesting techniques. In Experiment 2, the overall recovery rates of FWB and FF samples were 97 and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size. The expression of luteinizing hormone receptor (LHR) was mostly confined in the theca interna layer, with the estradiol-related receptor alpha (ERRα) in the granulosa and theca interna layers. The 30-mm follicle group had greater (P < 0.05) LHR expression in the theca interna and ERRα in the granulosa layer compared to the other groups. The overall expression of LHR and ERRα, and the intrafollicular estradiol were higher (P < 0.05 – P < 0.0001) in the 30-mm follicle group. CONCLUSION: The in vivo technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function, and has the potential to be translated to other species, including humans. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12958-018-0380-8) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6064614 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-60646142018-08-01 In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels Ishak, G. M. Bashir, S. T. Dutra, G. A. Gastal, G. D. A. Gastal, M. O. Cavinder, C. A. Feugang, J. M. Gastal, E. L. Reprod Biol Endocrinol Methodology BACKGROUND: In vivo studies involving molecular markers of the follicle wall associated with follicular fluid (FF) milieu are crucial for a better understanding of follicle dynamics. The inability to obtain in vivo samples of antral follicle wall (granulosa and theca cells) without jeopardizing ovarian function has restricted advancement in knowledge of folliculogenesis in several species. The purpose of this study in mares was to develop and validate a novel, minimally invasive in vivo technique for simultaneous collection of follicle wall biopsy (FWB) and FF samples, and repeated collection from the same individual, during different stages of antral follicle development. We hypothesized that the in vivo FWB technique provides samples that maintain the normal histological tissue structure of the follicle wall layers, offers sufficient material for various cellular and molecular techniques, and allows simultaneous retrieval of FF. METHODS: In Experiment 1 (ex vivo), each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from 10-, 20-, and 30-mm follicles were repeatedly and simultaneously obtained through transvaginal ultrasound-guided technique. RESULTS: In Experiment 1, the thickness of granulosa, theca interna, and theca externa layers was not influenced (P > 0.05) by the harvesting techniques. In Experiment 2, the overall recovery rates of FWB and FF samples were 97 and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size. The expression of luteinizing hormone receptor (LHR) was mostly confined in the theca interna layer, with the estradiol-related receptor alpha (ERRα) in the granulosa and theca interna layers. The 30-mm follicle group had greater (P < 0.05) LHR expression in the theca interna and ERRα in the granulosa layer compared to the other groups. The overall expression of LHR and ERRα, and the intrafollicular estradiol were higher (P < 0.05 – P < 0.0001) in the 30-mm follicle group. CONCLUSION: The in vivo technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function, and has the potential to be translated to other species, including humans. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12958-018-0380-8) contains supplementary material, which is available to authorized users. BioMed Central 2018-07-28 /pmc/articles/PMC6064614/ /pubmed/30055625 http://dx.doi.org/10.1186/s12958-018-0380-8 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Ishak, G. M. Bashir, S. T. Dutra, G. A. Gastal, G. D. A. Gastal, M. O. Cavinder, C. A. Feugang, J. M. Gastal, E. L. In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels |
title | In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels |
title_full | In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels |
title_fullStr | In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels |
title_full_unstemmed | In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels |
title_short | In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels |
title_sort | in vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6064614/ https://www.ncbi.nlm.nih.gov/pubmed/30055625 http://dx.doi.org/10.1186/s12958-018-0380-8 |
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