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Reproductive biology and endocrinology : RB&E2018; 16(1); 71; doi: 10.1186/s12958-018-0380-8

In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels.

Abstract: 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. Conclusions: 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.
Publication Date: 2018-07-28 PubMed ID: 30055625PubMed Central: PMC6064614DOI: 10.1186/s12958-018-0380-8Google Scholar: Lookup
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  • Journal Article

Summary

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This research presents a new, minimally invasive technique to assess ovarian function by collecting samples from the antral follicle wall in mares. These samples provide enough material for various cellular and molecular studies, including analyses of specific molecular markers.

Research Purpose and Hypothesis

  • The aim of this research was to devise and test a new method for safely collecting samples from the follicle wall and the follicle fluid (FF) within mares. The goal was to collect these samples in a way that did not jeopardize the animal’s ability to function normally.
  • The researchers hypothesized that this method would enable them to collect samples which retained the regular structure of the follicle wall layers and would offer adequate material for a variety of cellular and molecular procedures. They also believed this method would allow for the simultaneous retrieval of FF.

Methods and Experiments

  • The researchers conducted two experiments, one in vivo and the other ex vivo. For the ex vivo experiment, they sampled each follicle using two techniques: biopsy forceps and a scalpel blade (control) to compare their effects.
  • In the in vivo experiment, they used a transvaginal ultrasound-guided technique to simultaneously and repeatedly obtain FWB and FF samples from various sizes of follicles (10mm, 20mm, and 30mm). This way, they could compare the sample results for the success rate, thickness of the layers, and specific gene expressions.

Results

  • The first experiment showed that the thickness of the granulosa, theca interna, and theca externa layers was not affected by the sampling methods used.
  • For the second experiment, the overall recovery rates were excellent, with 97% for FWB samples and 100% for FF. The success rates, however, varied according to the size of the follicle, regarding obtaining samples with all layers of the follicle wall.
  • The expression of the luteinizing hormone receptor (LHR) was predominantly found in the theca interna layer, whereas the estradiol-related receptor alpha (ERRα) was found in the granulosa and theca interna layers.
  • Larger follicles exhibited a higher expression of LHR and ERRα, and higher levels of intrafollicular estradiol.

Conclusions

  • The newly developed in vivo technique for collecting samples proved to be successful as it can be repeatedly and simultaneously used to collect sufficient FWB and FF samples without disrupting the ovarian function.
  • By shedding more light on folliculogenesis process, this research could potentially contribute to the study of ovarian function not only in mares but potentially in other species, including humans.

Cite This Article

APA
Ishak GM, Bashir ST, Dutra GA, Gastal GDA, Gastal MO, Cavinder CA, Feugang JM, Gastal EL. (2018). In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels. Reprod Biol Endocrinol, 16(1), 71. https://doi.org/10.1186/s12958-018-0380-8

Publication

ISSN: 1477-7827
NlmUniqueID: 101153627
Country: England
Language: English
Volume: 16
Issue: 1
Pages: 71
PII: 71

Researcher Affiliations

Ishak, G M
  • Department of Animal Science, Food and Nutrition, Southern Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA.
  • Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq.
Bashir, S T
  • Department of Animal Science, Food and Nutrition, Southern Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA.
Dutra, G A
  • Department of Animal Science, Food and Nutrition, Southern Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA.
Gastal, G D A
  • Department of Animal Science, Food and Nutrition, Southern Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA.
Gastal, M O
  • Department of Animal Science, Food and Nutrition, Southern Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA.
Cavinder, C A
  • Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA.
Feugang, J M
  • Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA.
Gastal, E L
  • Department of Animal Science, Food and Nutrition, Southern Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA. egastal@siu.edu.

MeSH Terms

  • Animals
  • Biomarkers / metabolism
  • Biopsy / instrumentation
  • Biopsy / methods
  • Biopsy / veterinary
  • Female
  • Follicular Fluid / metabolism
  • Horses
  • Immunohistochemistry
  • Ovarian Follicle / surgery
  • Ovary / pathology
  • Ovary / physiopathology
  • Ovary / surgery

Grant Funding

  • P20 GM103476 / NIGMS NIH HHS
  • 8-6402-3-018 / USDA-ARS Biophotonics Initiative
  • PPGMV-UFRRJ#88881.133485/2016-01 / CAPES (Coordination for the Improvement of Higher Education Personnel)
  • 246741/ 2012-0 / CNPq (The National Council for Scientific and Technological Development)

Conflict of Interest Statement

ETHICS APPROVAL AND CONSENT TO PARTICIPATE: All experimental procedures were performed according to the United States Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training (grants.nih.gov/grants/olaw/references/phspol.htm). In Experiment 1, the research protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Mississippi State University, Mississippi State, USA; in Experiment 2, the research protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Southern Illinois University, Carbondale, IL, USA. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Citations

This article has been cited 4 times.
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  2. Dutra GA, Ishak GM, Pechanova O, Pechan T, Peterson DG, Jacob JCF, Willard ST, Ryan PL, Gastal EL, Feugang JM. Seasonal variation in equine follicular fluid proteome. Reprod Biol Endocrinol 2019 Mar 6;17(1):29.
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  3. Feugang JM, Gad A, Menjivar NG, Ishak GM, Gebremedhn S, Gastal MO, Dlamini NH, Prochazka R, Gastal EL, Tesfaye D. Seasonal influence on miRNA expression dynamics of extracellular vesicles in equine follicular fluid. J Anim Sci Biotechnol 2024 Oct 9;15(1):137.
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  4. Bashir ST, Baerwald AR, Gastal MO, Pierson RA, Gastal EL. Dominant follicle growth patterns and associated endocrine dynamics in anovulatory and ovulatory waves in women. Reprod Fertil 2023 May 1;4(2).
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