Figure 1. Histological sample of a functional primate ovary. (A) This ovary shows follicles in several phases of development, from primordial and primary stages (B) up to antral follicles, with the cavity full of follicular fluid (*)(1).
The quality of gametes is a critical factor that influences the chances to achieve a successful fertilization, embryo development and pregnancy outcomes, both in vitro and in vivo (2, 3).
The female gamete, the oocyte, undergoes a process of growth in size, as well as nuclear and cytoplasmic maturation. This is evidenced by the follicle progressive transformation through several developmental stages. These stages are: primordial, primary, secondary, pre‐antral, antral (small or large) and preovulatory follicles (Figure 1) or Graafian follicles (named after the Dutch anatomist Regnier de Graaf). Theca and granulosa cells from secondary follicles secrete a fluid that accumulates in the antral cavity or “antrum”, which eventually surrounds the oocyte (2). Such fluid is the product of blood filtration through the theca and additional secretions from both the theca and the granulosa layers (2, 4, 5) and is called follicular fluid (FF). Cumulus cells (CCs) surrounding the oocyte nurture it with essential compounds obtained from the FF. The communication between mural granulosa cells and cumulus cells is achieved through secretions of extracellular vesicles to the FF (2). The oocyte completely depends on the CCs during its maturation and it is in continuous contact with the FF. Therefore, variations in FF composition may affect oocyte development directly or indirectly (Table 1), and the subsequent impaired oocyte maturation will influence future reproductive outcomes (4).
The FF is mainly composed of steroid hormones, proteins, electrolytes, reactive oxygen species (ROS), antioxidants, metabolites, growth factors and fatty acids (2-4). The actual components or their concentrations vary through the different developmental stages under hormonal regulation (6). Furthermore, it can also be affected by pathologies or environmental conditions such as nutrition, stress or contaminants (7).
A balanced FF composition through the follicle development cycle is critical to produce good-quality oocytes (2). During ovulation, FF is released to the oviduct along with the oocyte, and it plays a role in the modulation of sperm function, since it seems to influence sperm capacitation and, as a consequence, acrosome reaction (2). Taking into account the importance of FF in different processes of natural reproduction, it seems logical to consider possible uses in assisted reproduction.
FF collection and analysis in assisted reproduction practice
Composition of the FF is directly linked to oocyte quality. The concentration of different components is commonly used in assisted reproduction as a parameter to distinguish the best oocytes (2, 4, 8). This knowledge may allow for optimisation of ART outcomes while avoiding embryo overproduction (8). Because FF is easily obtained during oocyte collection, it is a ready and available source for “–omics” analysis, with the aim of identifying oocyte quality indicators (5, 9).
The first challenge to use FF as a source of oocyte quality biomarkers in ART is the implantation of a suitable collection method during daily practice (5, 8, 10). FF should be collected separately for every single follicle with the use of culture medium for needle flushing between samples (5). During ovum pick-up a buffered solution may be used for flushing and then mixed with the collected FF. In order to obtain a known dilution of FF components, the concentrations of flushing liquid must be standardized and the volume used should be measured (10). However, in order to fulfil such requirements, multiple vaginal punctures may be needed, which increases both the risk of bleeding and the patient’s discomfort (5).
An additional hurdle is encountered when trying to estimate oocyte quality during FF studies, since nuclear maturity needs to be assessed soon after oocyte recovery (5). Nevertheless, this is currently performed just for those cycles involving ICSI, as opposed to IVF cycles, in which oocyte maturity is not assessed until the following day. In order to reliably select quality biomarkers for FF, it is mandatory to identify the maturation status of the oocyte from the same follicle; if the examination of the nucleus is delayed, part of the maturation process may be actually taking place during in vitro culture, which may bias the end result (5).
The technical difficulties previously mentioned could be solved with relative ease through certain changes in the daily routine of assisted reproduction practice. However, although interest in identifying reliable biomarkers indicative of gamete quality or future embryo development has been increasing in the last two decades, analysis of FF still presents additional limitations (8). Therefore, FF is currently not being used yet in the daily routine practice in human assisted reproduction (10). The research conducted so far includes proteomics and metabolomics analysis (5, 9). The latter can be subdivided into two categories: one focused on the study of a single or a pair of compounds and the other seeking a more complete analysis encompassing several FF metabolites (5, 8, 10). The studied molecules comprise all the different types of compounds that constitute FF, briefly mentioned in the introduction (2-4).
Proteomics analysis has brought to light certain proteins previously unknown to be present in the FF. Also, differences have been appreciated in protein expression between healthy women and ART patients, and between ART patients undergoing different protocols. From these results some potential biomarkers of oocyte quality have been identified (9); however, proteomics studies tend to evaluate FF differences between individuals with different ART outcomes, rather than comparing FF from different follicles to assess oocyte quality (9).
Because the analysis of a single or a pair of FF compounds often shows controversial results when comparing different authors (5), the latest trend leans towards the analysis of extended FF metabolomics. This type of study comprises the identification and quantification of low molecular weight end-products in the FF (10). Among the available techniques, the most widely used one suitable as an approach for metabolomics studies is mass spectrometry (MS) (11), which may also be combined along with other techniques (5). The results of these analyses provide information on events downstream of gene expression and any effect on the metabolism of that particular follicle and, therefore, of its development (5, 10). Although gene expression could be more directly studied through its transcripts by means of microarrays and high-fidelity RNA amplification techniques, such methods would require the lysis of the oocyte, thus rendering it useless to improve oocyte selection (5). A suitable alternative is studying cumulus cells transcripts, since the metabolic support provided by these cells is indispensable for oocyte growth (4, 12). In this case, cell lysis would not pose a problem.
FF and fertility-related pathologies
Several researchers have reported differences in FF composition or concentration in women suffering from certain pathologies (9, 13-15). Consequently, some research groups have aimed to deeper study FF biomarkers related to these patients, such as women with polycystic ovary syndrome (PCOS) or obesity (13-15).
For instance, glucose is collected and metabolised by CCs from FF, which provide the resulting pyruvate to the oocyte (14). However, high glucose levels in FF are negatively related to oocyte quality and may be linked to the cause of overweight or obese women lower pregnancy and live-birth rates (14). This is further supported by the fact that these fertility problems are usually overcome when donor oocytes are used, suggesting that oocyte quality may be the main issue. Furthermore, obese women have also been found to present higher concentrations of insulin, glucagon, GLP-1, C-peptide or leptin. These compounds have functions related to glucose, energy homeostasis and regulation of fat stores, which suggests that oocyte quality problems observed in patients with high body mass index (BMI) are likely related to glucose and/or lipid energy source metabolic pathways (13, 14). However, the exact reason for this decrease in oocyte quality in women with high BMI is still unclear. Further study of metabolic dynamics in FF associated to these pathways may help to find the specific causes and subsequent treatment.
PCOS is a highly variable pathology and relatively common in women and frequently associated to fertility problems. Significant differences have been found regarding the FF of PCOS patients compared to normally fertile women. PCOS patients are likely to present increased levels of glycoprotein, acetate and cholesterol, as well as significantly decreased values of lactic acid, glutamine, pyruvate and alanine (13). There are multiple ways these differences may be related to their lower fertility. As an example, lactate and pyruvate are products involved in the glycolysis pathway; normal levels would indicate normal energy production and consumption. However, low levels are suggestive of abnormal oocyte metabolism, which has been linked to reduced oocyte quality (16, 17). It has also been hypothesised that the insulin resistance associated with PCOS, along with elevated LH levels, increases androgen production and compromises the role of insulin in glucose metabolism (15).
FF as a source for in vitro gamete culture and freezing media supplements
The uses of FF are not limited to the study of its components and their concentrations (18-20). Assisted reproduction techniques aim to mimic the in vivo environment as much as possible, but some molecules present in the natural environment of gamete development or fertilization are too difficult or too complex to replicate (mRNAs, extracellular vesicles, etc.). While some groups are wary of the use of biological materials such as FF due to its full compositions and dynamics being unclear, others had been researching the use of FF as a supplement for oocyte culture (18-20).
In vitro maturation (IVM) of oocytes is an alternative practice offered in IVF centres for fertility preservation or in cases where the collection of immature oocytes is the safest or only choice available. This is the case for some women suffering from PCOS, poor ovarian reserve or repeated IVF failures (18). FF can be used as a complement for IVM to increase the efficiency of oocyte maturation (19). Madkour et al (2018) reported a new IVM protocol using heterologous follicular fluid (HFF) and supernatant of cumulus-granulosa cells (CGCs), which showed increased rates of oocyte development up to blastocyst when compared to the use of autologous FF or the in vivo condition of specifics patients (19).
A different alternative is the partial use of FF resources. Small extracellular vesicles (EVs) rich in mRNA can be added to oocyte IVM media (until the embryo reaches the 4-cell stage) to reduce the differences in mRNA expression profiles between in vitro and in vivo environments (2). This supplementation has been proved to cause changes in blastocyst formation rates and in the transcription levels of genes related to embryonic metabolism and development (2).
The vitrification of oocytes using the corona radiata and autologous follicular fluid (AFF) instead of vitrification solution has been previously reported (20). In the study, clinical outcomes obtained were similar to those in the control group (using regular vitrification solution), comparing oocyte survival rate after thawing, blastocyst formation and pregnancy rates between both groups (20).
Follicular fluid constituents and their concentrations are closely linked to the development and maturation of oocytes (2-4). FF is a by-product of oocyte retrieval, and the standardization of a protocol for FF collection during oocyte retrieval for its subsequent analysis is a doable option (8). Still, performing extensive profiling of FF is, at present, time-consuming, expensive and dependent on the availability of specialised equipment and highly trained professionals (5). Nonetheless, integrating metabolomics or proteomics technologies with assisted reproduction practice would greatly benefit the study of several pathologies. Simultaneously, it would provide useful information for oocyte grading through biomarkers, consequently helping raise embryo quality (8).
So far, a large part of the research has focused on finding individual biomarkers in FF (5). However, focusing on a wider spectrum of FF metabolites has the potential to provide more information about FF compounds, their roles and their relationships to fertility parameters (8). It is also important to carry out these studies in healthy individuals, avoiding comparisons just between ART patients with and without a certain pathology or successful versus unsuccessful ART outcomes. This would help to better define and standardize the references for “normal” parameters in FF (5).
Advances in the study of FF composition and dynamics would help improve the treatments of several fertility-related pathologies, oocyte selection, in vitro maturation and cryopreservation protocols (2, 8, 14, 19, 20). Therefore, the vast potential these fields present despite their novelty should be further explored, not only by validating the findings of small sample size studies, but also by expanding the research scope to encompass the true complexity of metabolomics and proteomics (10).
Even though assisted reproduction practice does have a significant percentage of successful outcomes, it is still lower than desired. Detailed knowledge of FF dynamics and its connections to oocyte development and overall reproductive health of the patient would help to provide customized treatments and raise the efficiency of oocyte selection and culture, consequently helping to improve the overall quality of such treatments.
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