Fatty acid composition of the follicular fluid of normal weight, overweight and obese women undergoing assisted reproductive treatment: a descriptive cross-sectional study

Fatty acids (FAs) are important compounds in the micro-environment of the ovarian follicle. Besides their role as cellular energy source, they have important biological functions in cell membrane biogenesis [1, 2] and signaling [3]. In addition, they act as precursors for steroids and prostaglandins, which are essential for normal reproductive function [4]. In the follicular fluid (FF), FAs are present in an esterified form [triglycerides (TG), cholesterol esters (CHE) and phospholipids (PL)] or as non-esterified FAs (NEFAs), mainly bound to albumin [5]. Maternal diet can have a profound effect on the FA composition of the FF, which may subsequently affect the FA composition of the cumulus cells and the oocyte [6‐8]. Numerous animal studies, mostly in cattle, sheep and rodents, have demonstrated the effect of diet-induced FA changes on ovarian physiology in general and on the quality of the oocyte more specifically [6, 8‐10]. Dietary induced FA changes or obesogenic diets during oocyte maturation or early embryo development affect oocyte and embryo quality [11, 12] and these effects may even have consequences for the offspring’s health [13, 14]. However, results of these studies are not always in agreement and difficult to compare as not only the quantity of the FA offered, but also the physiological status, the type of FA (ratios of omega-3, -6 or −9 FAs, number of double bonds, carbon chain length) and the duration of treatment may determine the final FA concentrations in the FF [3]. Besides dietary effects, body condition and metabolic status may also impact on the profile of FAs in the FF [15]. Metabolic disorders such as obesity and type II diabetes, but also a negative energy balance, are associated with upregulated lipolysis, leading to elevated NEFA concentrations in the serum [16], that are reflected in the FF of the dominant follicle [15, 17] and could therefore directly affect oocyte quality and metabolism. It has been shown that TG and NEFA concentrations are increased in the FF of obese women subjected to ovum pick-up [18], while such elevated NEFA and/or TG concentrations were negatively associated with human cumulus-oocyte-complex morphology [19] and affected both murine oocyte maturation [18] and bovine embryo quality [20, 21]. Other studies investigating the influence of serum or FF FAs on fertility in general and oocyte quality more specifically, are difficult to compare as experimental designs, number and treatment of patients included and the relation between specific FAs studied and fertility outcome parameters often differ [19, 22, 23]. Additional to the heterogeneity in study design, most research on FAs has focused on the total fat fraction, rather than investigating FA differences in different lipid fraction. This is important though, because the cellular response to FAs possibly depends on the lipid fraction the FA belongs to. For this reason, knowledge on the specific FA composition of each lipid fraction in human FF is essential. In this study, we hypothesized that the FF FA composition may be altered by the woman’s metabolic body condition (as determined by her body mass index, BMI), being a well-documented risk factor for fertility failure [24]. Therefore, we aimed (1) to determine the quantity, the distribution and the relative abundance of FAs in different lipid fractions (PL, CHE, TG and NEFA) in the FF of the dominant follicle of normal weight, overweight and obese women, undergoing assisted reproductive treatment (ART) and (2) to define how this FA distribution is affected by BMI in these women. To our knowledge, this is the first time that a detailed description of FA concentrations and abundance in different lipid fractions in the FF of normal weight, overweight and obese women, undergoing ART is provided. This study further discusses potential implications of BMI-related FF FA changes on oocyte and embryo quality.

The aim of this study was to provide insight in the FA profile, specified per lipid fraction, in the FF of the pre-ovulatory follicle from normal weight, overweight and obese women, undergoing ART. To our knowledge, this is the first time that FAs in human FF lipid fractions are described in such detail. Our results showed that approximately 42% of the total FA concentration was esterified in the PL fraction and 34% in the CHE fraction, whereas only 10% were present in both the TG and NEFA fraction. Interestingly, only TG (trend) and NEFA concentrations were affected by BMI. Even though FA concentrations in the NEFA fraction were well below those of the other fractions, they showed the most BMI-related variability.

Total NEFA concentrations in the FF were elevated in obese women, which is confirmed by earlier work [18]. However, it is in contrast with our previous findings [17] and with the data of Robker et al.[35], who could not show a BMI-related difference in FF total NEFA concentrations. The difference between these data and those of our previous study, could partially be explained by a large variation in NEFA concentrations, the use of a different (colorimetric versus gas chromatographic) analysis methods and the inequality of the number of patients within each BMI group in our previous study [17]. Remarkably, our data also showed that even though absolute NEFA concentrations presented with a great BMI-related variability, relative concentrations only showed minor differences, suggesting that increasing BMI did not cause a shift in the relative abundance of the different FAs. Interestingly, Jungheim et al.[19] found that women with elevated levels of FF NEFAs displayed poorer cumulus-oocyte complex (COC) morphology. They differentiated between FF palmitic, stearic, oleic and linoleic acid in the NEFA fraction, but no correlations with BMI were found. Our data showed that 87% (20/23) of all FAs in the NEFA fraction, including palmitic, stearic, oleic and linoleic acid, correlated with BMI, which suggests that BMI causes changes in the FF NEFA concentrations that could directly affect COC function and quality. This is substantiated by the fact that we previously showed that bovine oocyte exposure, during the final stage of maturation, to elevated NEFA concentrations (oleic, palmitic and/or stearic acid) was detrimental for the oocyte’s developmental capacity [15]. It also altered gene expression patterns and energy/amino acid metabolism in blastocysts from oocytes exposed during the last 24 h of maturation [20]. Aardema et al.[36] showed that oocytes actively take up and metabolize NEFAs out of their environment (mitochondrial β-oxidation for the purpose of ATP production) and that this may influence lipid storage in lipid droplets within the oocyte, depending on the type and amount of FA offered. More specifically, oocytes exposed to palmitic and stearic acid presented with less intracellular fat storage and a hampered oocyte developmental competence. This effect could be counteracted by the addition of oleic acid to the treatment [36]. Such a protective effect of the mono-unsaturated FA oleic acid has also been proposed by Cnop et al. [37] in pancreatic islet cells, where oleic acid was described to reduce palmitate induced lipotoxicity, possibly by promoting triglyceride formation (cytoprotective mechanism). Our recent research investigated the mechanisms behind this lipotoxicity and showed that the degree of mitochondrial FA beta-oxidation has a strong impact on the development of NEFA exposed bovine oocytes and on the quality of the resulting embryos [21]. Besides the oocyte, the somatic cells of the follicle might also be influenced by the composition of FAs in the FF. For example, palmitic acid has been shown to inhibit in vitro bovine granulosa and theca cell proliferation and to alter steroid production by inducing apoptosis [38, 39]. Arachidonic acid, on the other hand, protects human granulosa cells from palmitic and stearic acid-induced apoptosis [40]. The effects of poly-unsaturated FAs are less uniform as for example linoleic acid (18:2 n-6) hampers and linolenic acid (18:3 n-3) stimulates bovine nuclear oocyte maturation [6, 8, 41, 42]. Interestingly, linoleic acid was present in the FF as one of the most predominant FAs. Marei et al. [41] showed that exposure of bovine cumulus-oocyte complexes to elevated linoleic acid concentrations during final maturation impaired oocyte maturation and decreased oocyte developmental competence. This may imply that elevated levels of linoleic acid could have harmful effects on fertility outcome in women. Under normal circumstances, linoleic acid concentrations are decreased in the FF of large follicles, compared to smaller follicles [43]. This suggest that linoleic acid can play an important role in the regulation of oocyte maturation, with decreasing levels allowing progression of oocyte maturation.

In all the previously discussed in vitro studies, FAs are added to the medium as non-esterified FAs. It remains, however, unclear if the same FAs, but esterified to a different fat fraction might elicit a differential effect. For example, a FA derived from the hydrolysis of TGs, contained in for example very low-density lipoproteins, by lipoprotein lipase [44], could potentially exert a different effect at the level of the cell, compared to the same FA in the NEFA fraction.

Besides the total NEFA concentrations, also total TG concentrations tended to be increased in overweight women. Interestingly, when mouse oocytes were matured in medium supplemented with NEFA/TG rich human FF, a dramatic decrease in oocyte maturation to metaphase 2, an increased oocyte lipid content and an upregulation of genes related to endoplasmic reticulum stress could be observed [18]. This suggests a detrimental effect of elevated TG and/or NEFA concentrations on oocyte quality. It is however not clear whether this is caused by elevated TG or NEFA concentrations or both or to what particular compound in the FF this effect can be attributed. Remarkably, 18:2 n-6 and 22:6 n-3 in the TG fraction were elevated in the FF of overweight, but not obese women, compared to normal weight women. A potential reason for this could be that in a first coping mechanism excess FAs, present in overweight individuals, are stored in lipid droplets [45]. However, in obese women, much of the circulating FAs originate from abdominal fat adipocytes [45], which are rich in saturated and mono-unsaturated FAs [46, 47]. High levels of the saturated palmitic and stearic acid have been shown to reduce lipid storage in maturing bovine oocytes [36]. This is in agreement with our data, showing that obese women presented with elevated levels of many FA in the NEFA fraction, rather than in the TG fraction for potential storage in lipid droplets.

We have previously shown that a strong correlation exists between serum and FF metabolites, indicating that the serum composition influences the FF composition [17]. However, these changes were not BMI-related, indicating that factors, other than serum composition have a large impact on FF composition. Furthermore, the potential impact of differential FAs in the FF on oocyte developmental competence depends on the actual presence and the ratios of FAs within the FF. Therefore, the primary focus of our study was to describe FF FAs, rather than to investigate their relation to the serum composition. We furthermore did not have any knowledge on diet or fasting before sampling, even though it has been well described that diet can change the FF FA composition and can thereby affect oocyte developmental competence and subsequent embryo quality [48].

Another remark is that obese women required a higher dosage of gonadotropins to reach the same stimulatory effect on follicular development, compared to normal weight women. However, it has also been described that gonadotropin releasing hormone analogues can alter serum lipoprotein levels and can increase insulin resistance [49], potentially deteriorating the insulin resistant state in many obese women. Because of ethical restraints in proposing suboptimal gonadotropin concentration administration, we were unable to account for this potential confounding factor in our study.

This descriptive study reports on the FA concentrations in the PL, CHE, TG and NEFA fraction in the FF of normal weight, overweight and obese women, undergoing ART. Our study highlights that most FAs in the FF belong to the PL and CHE fat fraction, but that NEFAs presented with the greatest BMI-related variability, with most individual FA concentrations increased in obese women. These differences may affect oocyte quality and subsequent embryo development, possibly by acting directly on oocyte metabolism.