Improved preservation of ovarian tissue morphology that is compatible with antigen detection using a fixative mixture of formalin and acetic acid
Authors: Adeniran B V , Bjarkadottir B D , Appeltant R , Lane S , Williams S A 
Published: March 2021

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.