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

Abstract

BackgroundIn 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.MethodsIn 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.ResultsIn 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.ConclusionThe 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 materialThe online version of this article (10.1186/s12958-018-0380-8) contains supplementary material, which is available to authorized users.

Transition to the ovulatory season in mares: An investigation of antral follicle receptor gene expression in vivo

“…This study compares the expression pattern of receptors and proteins related to proliferation (EGFR and Ki‐67), hormones (ERRα and LHR), angiogenesis (VEGFR), apoptosis (Bax and Bcl‐2), and intrafollicular concentration of estradiol among different stages of in vivo antral follicle development and between the SAN and SOV seasons using the same mares. Follicle wall layers (i.e., granulosa, theca interna, and theca externa) and FF samples were obtained using the antral FWB technique recently developed in our laboratory (Ishak et al, ). The main findings of the present study were (a) 30–34‐mm follicles had greater EGFR, Ki‐67, VEGFR, and LHR expression in specific follicle wall layers during the SOV season than the SAN season; (b) a greater overall expression of EGFR in the whole follicle wall was found in the SOV season than in the SAN season in all follicle classes; (c) the expression pattern of the VEGFR at follicle selection (20–24‐mm class) was similar between the two seasons; however, VEGFR expression increased during the SOV season and decreased during the SAN season when follicles grew to 30 mm; and (d) Bax/Bcl‐2 ratio revealed early apoptosis signaling in the granulosa layer of 30‐mm growing follicles during the SAN season.…”

“…In this regard, slaughterhouse and ovariectomized materials have been used previously to study antral folliculogenesis in different species (Brown et al, ; Ellenberger et al, ; King et al, ; McNatty et al, ; Mishra et al, ; Muller et al, ; Oberhaus et al, ; Scarlet et al, ; Sirois & Doré, ; Watson & Al‐Zi'abi, ; Watson et al, ). Therefore, the application of antral FWB technique with simultaneous FF sampling (Ishak et al, ) is a promising tool for advancing knowledge of folliculogenesis and when drug treatments and/or pathologies may affect its course.…”

“…Hence, there is a need for in vivo studies regarding the expression of different hormone and growth factor receptors and proteins in the antral follicle wall layers (i.e., granulosa, theca interna, and theca externa), and its relationships with intrafollicular fluid (FF) milieu during different follicle developmental stages. The recent advent of the follicle wall biopsy (FWB) technique for in vivo ovarian studies in mares (Ishak et al, ) has provided an innovative approach that has the potential to advance knowledge of different stages of antral folliculogenesis.…”

A Nano-Platform for in situ Investigations of Ovarian Follicles

“…developed in our lab [29] was used to simultaneously harvest FWB and FF from PBS-and liposome-injected follicles (7 small and 7 large) and one non-injected (control) follicle. This Oberkochen, Germany).…”

“…Following intrafollicular injections, the minimally invasive in vivo Follicle Wall Biopsy (FWB) technique is a recently improved methodology for assisted reproduction allowing for simultaneous sampling of the follicle environment (FF, granulosa, theca interna and externa cells) for ex vivo examinations. Most importantly, the FWB technique is performed on living mares without affecting their fertility, ovarian function, or the FF composition, therefore providing an excellent cocktail for biomarkers research and clinical purposes [28,29]. Its combination with the encapsulated liposomes for intrafollicular deliveries can be highly effective for a more in-depth understanding of the molecular mechanisms governing follicle growth and oocyte maturation.…”