Search for Olfactory Cells Within the Fetal Ethmoid Epithelium

Overview

The recent rhinological functional approach known as "Evo-Devo" leads us to consider the nose not as a single organ, but as the construction of a specifically olfactory organ within which functional respiratory structures have developed over the course of species evolution in the same way they develop during human embryogenesis. In adult humans, the olfactory structures are limited to two narrow regions situated at the upper part of the nasal passages known as olfactory clefts. Nevertheless, phylogenetic evidence indicates that these olfactory clefts are vestiges of an olfactory organ that once occupied the entire ethmoid bone in the front of the skull base. The hypothesis of this study posits that, during the embryological development of the ethmoid bone, it would initially be covered with olfactory mucosa, which would gradually diminish as the lateral masses of the ethmoid formed, eventually persisting exclusively within the olfactory clefts of the ethmoid by the conclusion of the first trimester (end of organogenesis). Besides their fundamental interest, the results of this study could help understand the still unknown pathogenesis of nasal polyposis, a chronic anosmic disease with a prevalence of 2 to 4% in the French and European population. This study requires a series of ethmoidal samples from human fetuses at various ages between 7 and 15 weeks of gestational age. Specific immunohistological labeling techniques under standard optical microscopy will be used to search for and quantify olfactory nerve cells within the fetal ethmoidal mucosa.

This research project is based on the hypothesis that olfactory epithelium is present within the mucosa of the lateral ethmoid masses, similar to the olfactory fissures at the beginning of the fetal organogenesis period, gradually disappearing before the end of the first trimester in the lateral ethmoid masses as they form. This study aims to be descriptive, with the objective of identifying this fetal olfactory epithelium and assessing its distribution changes within the ethmoidal mucosa as fetal age progresses. The chosen method for conducting this study relies on histological analysis using a standard optical microscope of fetal ethmoids (whole bodies or only the cephalic extremity depending on sample size) to preserve their architecture. Microscopic analysis will be coupled with immunohistochemistry or immunofluorescence techniques to characterize the cells of interest: primary olfactory neurons. Sample collection will be prospective, with the recruitment of fetuses spread over the course of the study, spanning 18 to 24 months. The anticipated minimum sample size is 20 cases. Intermediate analyses are planned in case of non-contribution of samples or if there are a sufficient number of samples to meet the primary and secondary objectives. The samples are fetal products obtained from either medical or surgical induced abortion procedures, with a gestational age of a minimum of 7 weeks amenorrhea and up to 14 weeks + 6 days. Sample recruitment is planned to take place within the Orthogeny Service of the University Hospital Maternity in Nancy, integrated into existing service protocols (medical or surgical abortions), and after clear and fair information and obtaining written informed consent from the patients by the investigators. The relevant healthcare teams are informed of the ongoing research. The impact of the research on patient care should be minimized as much as possible to avoid disrupting the patients' process and the medical procedure being carried out. No treatment protocols should be modified or disturbed by this protocol or, if necessary, by the on-site investigators. In accordance with the 2021 update of the Good Practice Recommendations provided by the French National Authority for Health (HAS) and validated by the French National College of Gynecology and Obstetrics (CNGOF), every abortion procedure includes a prior medical consultation held within 5 days following a woman's request. This consultation precedes the procedure by a few days and aims to provide information about the procedure itself to the patients and address the clinical and paraclinical medical prerequisites necessary for its successful execution. During this time, there is no intervention by the investigators to respect the proper implementation of the medical procedure, which requires a crucial information period during which interference is preferable to be avoided. Patient information about the conducted research and obtaining written consent is planned to occur upon the patients' admission to the Outpatient Unit of Gynecology and Obstetrics. This phase, in the form of a brief oral interview following their admission by the medical and paramedical teams, is the responsibility of the investigators. It is documented in a paper document that complies with legal establishment directives, produced in duplicate, with one copy retained by the investigators for the DRCI (Clinical Research and Innovation Department), and the second retained by the patient. The date of the last menstrual period is recorded by the investigators after the interview. This is the only personal or medical data collected from hospitalized patients. Once the procedure is completed (medical or surgical) according to the medical protocol established by the practitioners in the Orthogeny Service, the expulsion or aspiration products are collected and immediately placed in a sealed biopathological collection container while still fresh. Each sample is marked with an inclusion number: COEF#N° and the gestational age in weeks calculated from the date of the last menstrual period. These data are systematically indexed in an Excel® spreadsheet on a secure computer in the hospital establishment. The sample is promptly transported by the investigators to the Fetal Pathology Laboratory of the University Hospital Maternity in Nancy for rapid freezing at -80°C. A preliminary examination of the products under a binocular microscope is performed to isolate fetal material of interest: whole bodies, complete or fragmented cephalic extremities, including the intact facial mass. The conditioning of each sample is planned to be standardized. After the recruitment of 10 cases, frozen samples are subjected to a cryosection procedure using a specific Cryocut® microtome. This step allows the creation of serial sections with a thickness of approximately 5 micrometers, mounted on glass slides. An initial extemporaneous examination phase with standard HES staining enables the precise identification of the ethmoid block and subsequently the creation of a series of coronal sections. At this stage of development, skeletal structures are not ossified, avoiding a decalcification phase. Each produced slide is marked with a "Research Number," computer-assigned and automatically given by the histological analysis software used by the Laboratory: Ariane SX®. These numbers are also indexed electronically in relation to the sample data (COEF#N° and gestational age). The series of slides thus created are treated with immunohistochemistry or immunofluorescence techniques. The planned immunohistological markers include: Synaptophysin (SP), Neurospecific Enolase (NSE), Neuronal Cell Adhesion Molecule (NCAM), and Oligodendrocyte Transcription Factor (OLIG2). These markers are known monoclonal antibodies referenced in the literature for identifying mature and immature primary olfactory neurons. After labeling the slides, they are passed through a photographic scanner to generate a digital file containing all the images obtained for each sample. Each file is titled according to the indexing number of each sample: COEF#N°. The recorded image files bear the corresponding slide " Research " number. Given the purely descriptive nature of this study, descriptive statistics are planned. After analysis, each series of slides is physically stored at 4°C in the Fetal Pathology Laboratory under the responsibility of the investigators. At the end of the study, these slides will be grouped for preservation within a dedicated collection at the Lorraine Biological Resources Center.