Improved preservation of ovarian tissue morphology that is compatible with antigen detection using a fixative mixture of formalin and acetic acid

Abstract

STUDY QUESTION Can ovarian tissue morphology be better preserved whilst enabling histological molecular analyses following fixation with a novel fixative, neutral buffered formalin (NBF) with 5% acetic acid (referred to hereafter as Form-Acetic)? SUMMARY ANSWER Fixation with Form-Acetic improved ovarian tissue histology compared to NBF in multiple species while still enabling histological molecular analyses. WHAT IS KNOWN ALREADY NBF fixation results in tissue shrinkage in various tissue types including the ovary. Components of ovarian tissue, notably follicles, are particularly susceptible to NBF-induced morphological alterations and can lead to data misrepresentation. Bouin’s solution (which contains 5% acetic acid) better preserves tissue architecture compared to NBF but is limited for immunohistochemical analyses. STUDY DESIGN, SIZE, DURATION A comparison of routinely used fixatives, NBF and Bouin’s, and a new fixative, Form-Acetic was carried out. Ovarian tissue was used from three different species: human (n = 5 patients), sheep (n = 3; 6 ovaries; 3 animals per condition) and mouse (n = 14 mice; 3 ovaries from 3 different animals per condition). PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian tissue from humans (aged 13 weeks to 32 years), sheep (reproductively young i.e. 3–6 months) and mice (10 weeks old) were obtained and fixed in 2 ml NBF, Bouin’s or Form-Acetic for 4, 8, and 24 h at room temperature. Tissues were embedded and sectioned. Five-micron sections were stained with haemotoxylin and eosin (H&E) and the percentage of artefact (clear space as a result of shrinkage) between ovarian structures was calculated. Additional histological staining using Periodic acid-Schiff and Masson’s trichrome were performed on 8 and 24 h NBF, Bouin’s and Form-Acetic fixed samples to assess the compatibility of the new fixative with stains. On ovarian tissue fixed for both 8 and 24 h in NBF and Form-Acetic, immunohistochemistry (IHC) studies to detect FOXO3a, FoxL2, collagen IV, laminin and anti-Müllerian hormone (AMH) proteins were performed in addition to the terminal deoxynucleotidyl transferase nick end labelling (TUNEL) assay to determine the compatibility of Form-Acetic fixation with types of histological molecular analyses. MAIN RESULTS AND THE ROLE OF CHANCE Fixation in Form-Acetic improved ovarian tissue morphology compared to NBF from all three species and either slightly improved or was comparable to Bouin’s for human, mouse and sheep tissues. Form-Acetic was compatible with H&E, Periodic acid-Schiff and Masson’s trichrome staining and all proteins (FOXO3a, FoxL2, collagen IV and laminin and AMH) could be detected via IHC. Furthermore, Form-Acetic, unlike NBF, enabled antigen recognition for most of the proteins tested without the need for antigen retrieval. Form-Acetic also enabled the detection of damaged DNA via the TUNEL assay using fluorescence. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION In this study, IHC analysis was performed on a select number of protein types in ovarian tissue thus encouraging further studies to confirm the use of Form-Acetic in enabling the detection of a wider range of protein forms in addition to other tissue types. WIDER IMPLICATIONS OF THE FINDINGS The simplicity in preparation of Form-Acetic and its superior preservative properties whilst enabling forms of histological molecular analyses make it a highly valuable tool for studying ovarian tissue. We, therefore, recommend that Form-Acetic replaces currently used fixatives and encourage others to introduce it into their research workflow. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Oxford Medical Research Council Doctoral Training Programme (Oxford MRC-DTP) grant awarded to B.D.B. (Grant no. MR/N013468/1), the Fondation Hoffmann supporting R.A. and the Petroleum Technology Development Fund (PTDF) awarded to B.V.A.

3D Bioprinted Spheroidal Droplets for Engineering the Heterocellular Coupling between Cardiomyocytes and Cardiac Fibroblasts

“…DNA fragmentation in the cells within the 3D bioprinted cardiac spheroidal droplets was determined using Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection (Invitrogen™, Thermo Fisher Scientific, USA). After 7 days of culture, 3D spheroidal droplets were fixed in 4% paraformaldehyde for 15 min and then cut in half exposing the core of the scaffold followed by a staining procedure with picolyl azide Alexa Fluor™488 visualizing the DNA degradation according to the manufacturer’s protocol [ 65 ]. The nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA).…”

Analysing culture methods of frozen human ovarian tissue to improve follicle survival

“…While morphology is a widely used and generally accepted measure of follicle health and survival (McLaughlin et al 2014, Talevi et al 2018, Pampanini et al 2019, Walker et al 2019, Lopes et al 2020, there are some limitations in using morphological parameters alone to evaluate follicle health. Namely, fixatives such as neutral buffered formalin cause tissuedependant shrinkage, which we have observed can obscure the morphology of human ovarian follicles in particular, and may result in misinterpretation of data (Adeniran et al 2021). In the present study, tissue was fixed in Bouin's solution, which prevents tissue shrinkage and better preserves morphology, however, this fixative is incompatible with molecular analysis such as immunohistochemistry.…”

Fixation in Form-Acetic allows hyaluronic acid detection in mouse ovaries

“…Bouin’s solution reduces the shrinkage artefact but is not ideal for immuno-labelling because of its coagulative properties. In 2021, we developed a new fixative, Form-Acetic ( Adeniran et al . 2021 ), which is composed of NBF supplemented with 5% acetic acid and allows exquisite preservation of the tissue while enabling a range of histological molecular analyses ( Adeniran et al .…”

“…In 2021, we developed a new fixative, Form-Acetic ( Adeniran et al . 2021 ), which is composed of NBF supplemented with 5% acetic acid and allows exquisite preservation of the tissue while enabling a range of histological molecular analyses ( Adeniran et al . 2021 ).…”

“…However, although aldehyde-based fixatives (e.g. NBF) have been used to detect HA ( Lo et al 2019 ), detection of HA was not assessed after fixation with Form-Acetic ( Adeniran et al 2021 ). Consequently, it was important to verify whether Form-Acetic facilitated the detection of HA in the ovary.…”