A methodological and functional proteomic approach of human follicular fluid en route for oocyte quality evaluation☆
Graphical abstract
Introduction
The human ovarian follicular fluid (HFF) prevalently results from granulosa and theca cell secretion and from capillary diffusion [1], [2], [3], [4], [5]. During folliculogenesis, the blood-follicle barrier becomes more permeable to plasma molecular components and the follicular fluid (FF) acquires a consistent similarity to the serum [1], [5]. Reflecting granulosa and theca metabolic activity as well as changing in follicle permeability, FF biochemical composition may reveal not only the stage of follicular development but also the general functional state of the follicle itself and the health condition of the generating organism. Moreover, variations in concentration of follicular fluid components can also affect the oocyte quality [6], [7]. Actually, the FF is the microenvironment in which the oocyte develops and undergoes maturation, and it has been reasonably thought, and to some extent proven, to affect oocyte quality, fertilization and, maybe, embryo development [8].
Being aspirated with female gametes during oocyte recovery in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) procedures, follicular fluid represents an abundant and easily-available biological material for follicle and oocyte quality investigations, without affecting the collected precious oocytes. As a matter of fact, being a probable source of biomarkers for oocyte/embryo quality and competence evaluation, the biomolecular characterization of HFF has attracted considerable attention. Actually, as a consequence of legal and ethical problems related to the over production of embryos and to their morphological/genetic selection, the oocyte and embryo quality assessment has become one of the major aims in reproductive biomedicine. At present, oocyte and embryo selection is prevalently attained using morphological criteria [9], [10], [11], [12], [13], [14]. Nevertheless, the morphologic assessment is generally considered controversial and unsatisfactory, and, due to cumulus and corona cell presence, it can only be properly applied in ICSI procedure where the sperm injection occurs in “naked” oocytes [12], [15]. Despite the biomedical urging of its functional characterization, FF protein composition as well as its role in follicular growth and oocyte maturation still remain to be clarified. Follicular fluid, like plasma, is actually characterized by a vast protein complexity and a very broad dynamic range of protein abundances that hinder its analysis. In the last years a number of proteomics attempts to determine the HFF protein composition have been performed but several of them show consistent sample treatment and/or a number of limitations in protein recovery, resolution, visualization and identification, as well as in protein isoform detection [4], [6], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28]. In order to obtain an overall proteomic snapshot of the fluid minimizing sample manipulation and experimental artifacts and increasing the amount of detectable protein isoforms and species, we tried to set up a proper solubilization and resolution method of HFF for 2-DE analysis. In spite of all its technical limits and the existence of alternative successful experimental methodologies developed to overcome such limits, 2-DE is in fact still retained, according to its high potential in protein resolution and in simultaneous visualization of hundreds of proteins, a powerful tool in investigations of complex protein mixtures, also towards biomarker discovery [29], [30].
Here we proposed and compared two different protocols for sample preparation and three different modalities for sample loading in the first dimension. No prefractionation process was performed, nor depletion of the most-abundant proteins was applied. Even if the less-abundant protein detection may be affected by overlooking proteins, such as albumin or α-macroglobulin, we opted for the most direct analytical-way with the minimum of sample treatment and, consequently, with a bona fide reduction in the occurrence of methodological artifacts. Moreover, some of the most abundant serum proteins are retained to play specific roles in follicle and oocyte development and maturation. Consequently, their removal may limit functional characterization of the FF.
Using a denaturation/solubilization process by heating samples in a SDS/DTE solution and then performing protein precipitation and anodic cup-loading, very high quality 2-D gels were obtained. This allowed to properly select and excise protein spots to be analyzed by mass spectrometry. Numerous protein spots were thus unambiguously identified and, worthy of note, also some spots localizing at low molecular weight, that were excluded by the list of identified proteins in previously FF 2-D investigations, were successfully identified. Afterwards, to functionally correlate the identified proteins, a pathway analysis was performed by the MetaCore program and the built paths suggested the presence in mature follicles of a fine and tight control in enzymatic proteolysis that induces, regulates and/or inhibits inflammatory reaction, wounding response, coagulation cascade, and extracellular matrix (ECM) degradation/remodeling. According to scientific literature, the biochemical activities of identified proteins described, as expected, a functional environment characteristic of the ending phase of follicle maturation and ovulation. Based on the identified proteins and on the designation of functional hubs in the most significant network, albumin and alpha-1-antitrypsin were immunostained in FF and in serum from the same patients to evaluate correlation existing between the serum and the follicular pattern of these proteins.
Section snippets
Follicular fluid and serum collection
Patients undergoing controlled ovarian hyperstimulation for in vitro fertilization were recruited for the study at the Centre for Diagnosis and Treatment of Couple Sterility, Institute of Obstetrics and Gynaecology, “Le Scotte” General Hospital in Siena. Human follicular fluid samples were obtained from 6 normo-ovulatory young women, 25–32 years old, and used for methodological analysis, anti-albumin Western blot, and Mass Spectrometry (Supplementary Data: Table S1). From the same patients, on
Results and Discussion
In a global context in which couples affected by infertility are astonishingly increasing, the demand for infertility medical care is becoming an urgent topic in clinical medicine. In order to ensure the highest possible number of couples with fertility problems to adhere to assisted reproduction programs, an improvement of molecular-medicine methodologies currently applied in in vitro fertilization is absolutely required. Actually, towards an increase of pregnancy rates with positive outcome,
Concluding remarks
Morphological and microscopic applied criteria to grade the most competent oocyte/embryo are subjective and inadequately related to successful pregnancy rate. In the last decade, the application of proteomics high throughput methodologies to human reproductive fluid analysis has delineated a novel biochemical functional profile of molecular processes that characterize and may affect follicular development and maturation, and, as a consequence, the oocyte/embryo competence and viability, and
Acknowledgments
This work was supported by the FIRB project “Italian Human ProteomeNet” (BRN07BMCT_013-MIUR) to Luca Bini.
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This article is part of a Special Issue entitled: From Genome to Proteome: Open Innovations.