Acylcarnitine esters profiling of serum and follicular fluid in patients undergoing in vitro fertilization

Normal oocyte maturation, fertility and embryo development is closely associated with energy metabolism [1‐4]. The prominent role of fatty acids in energy supply to acquire developmental competence of oocyte and early embryos has been established [5‐8]. However, growing body of evidences suggests, that non-esterified fatty acid supply in excess of its metabolic utilization results in fatty acid accumulation that may compromise oocyte maturation and developmental capacity of early embryo [9‐11]. Interestingly, when free fatty acid composition of serum and/or follicular fluid (FF) was analyzed it became apparent that not isolated individual fatty acids, but rather physiologically relevant ratios and /or combinations of fatty acids cause significant dysregulation of cellular processes [8].

Free fatty acids are metabolised via beta-oxidation that is mediated by L-carnitine. L-carnitine is present in free and esterified forms in tissues and body fluids. It has multiple metabolic functions including transport of long-chain fatty acids into the mitochondria for beta-oxidation, transfer of short- and medium-chain acyl groups from the peroxisome to mitochondria, regulation of intracellular acyl-CoA/free CoA ratio and export of toxic acyl residues from the mitochondria [12‐17]. Accumulation of acylcarnitines, (AC)s therefore is regarded as indicative of mitochondrial dysfunction and impaired cellular fatty acid metabolism [18]. The importance of L-carnitine in improving oocyte quality and reproductive performance has been demonstrated in animal and human studies [19‐24].

On the basis of these observations it was intriguing to investigate further the relationship between carnitine profile and reproductive potential. In women who wants to conceive a child we can only analyze the serum carnitine profile and no FF samplaes are available, therefore patients receiving IVF can be an observational model. Also testing whether L-carnitine or individual carnitine esters alone or in combination can serve as potential biomarkers of pregnancy outcome.

The present study was conducted to determine the patterns of free carnitine (FC) and AC esters in serum and FF in women undergoing IVF. Attempts were also made to assess the properties of blood-follicular barrier by quantifying simultaneously the short-, medium- and long-chain ACs in the two distinct fluid compartments. In addition, composition of carnitine pools was related to indices of reproductive potential such as number of oocytes and that of viable embryos.

The present study provided evidences that serum carnitine profile of patients undergoing IVF is comparable to that of healthy women. FC and major AC esters can be detected in the FF, and the serum to FF ratio of individual carnitine compounds is inversely related to the carbon chain length of carnitine esters. Moreover, markers of reproductive potential (number of oocytes and embryos) appeared to be associated with reduction of FC and some individual ACs both in serum and FF.

In recent years considerable efforts have been made to identify potential biomarkers in FF to predict IVF outcome. FF serves as dynamic, physiological environment of maturing oocytes and embryos, therefore, it is assumed to reflect metabolic changes that occur during maturation. It has been shown to contain hormones, growth factors, reactive oxygen species, cytokines, apoptotic factors and several metabolic intermediates [27, 28]. Targeted analysis of specific biomarkers or certain combination of biomarkers has revealed important correlation with oocyte quality and/or related embryo.

The recent introduction of metabolomic and proteomic profiling of FF demonstrated that combined use of a panel of biomarkers as opposed to a single biomarker proved to be a reliable estimate of pregnancy outcome [29‐32].

Our present study was prompted by the observations that L-carnitine-mediated β-oxidation of fatty acids is an essential energy source for oocyte and embryo development [5‐8]. In this regard Dunning et al. reported that inhibition of carnitine palmitoyl transferase I (CPT I), the enzyme that catalyzes the initial step of β-oxidation, with etomoxir impaired subsequent embryo development. On the other hand, upregulation of β-oxidation during oocyte maturation by L-carnitine increased oocyte developmental competence as manifested by the increased rate of cleavage to 2-cell stage [21]. It was also demonstrated by the same group that L-carnitine supplementation during in vitro 3D follicle culture significantly increased β-oxidation and markedly improved both fertilization rate and blastocyst development without altering survival, growth or differentiation of follicles [22]. The beneficial effect of L-carnitine on reproductive performance has been well- documented, it has been claimed, however, that in addition to its essential role in β-oxidation l-carnitine has the capacity to protect against oxidative stress, inflammation and apoptosis [20, 23, 24, 33]. L-carnitine-related improvement of insulin resistance and glucose utilization may also contribute to the better IVF outcome [34]. However several animal studies investigated the impact of L-carnitine supplementation on oocyte quality and preimplantation embryo development [22‐24, 35‐37], very limited data are accessible for the carnitine levels of human ovarian follicular samples [38, 39] and no reports are available on their acylcarnitine profiles. In their study Montjean et al. could not observe correlations between the total carnitine content of the follicular fluid and either the circulating estradiol content of the serum or the outcome of the IVF attempt. Robker et al. investigated the L-carnitine concentrations in follicular fluid samples from women who were administered human chorionic gonadotropin. It is not known whether the L-carnitine level is regulated during the menstrual cycle by gonadotropin [38]. In our study in an attempt to further explore the relationship between IVF parameters and the composition of endogenous carnitine pool we determined the concentrations of FC and 20 major AC esters in serum and FF samples obtained from patients receiving IVF. Importantly, all individual ACs measured could be detected in FF, their concentrations, however, proved to be dependent on maternal serum concentrations and on the carbon chain length of acyl groups. These findings are consistent with the notion that FC and AC esters cross the blood-follicular barrier but the passage through this barrier is reduced as molecular weight and lipophilicity increases with the increasing carbon chain length. Moreover, serum and FF carnitine profiling revealed marked reduction in total carnitine, FC and AC levels in IVF patients with oocyte number of >9 and/or with embryo number of >6 as compared to those with the respective values of <9 and/or <6. Based on these results we suggest that the L-carnitine/AC pathway is upregulated and the actual carnitine pool is depleted in patients with better reproductive potential.

Mitochondrial transport of long-chain fatty acids for β-oxidation is achieved via the carnitine circle. This requires the consecutive action of CPT I, carnitine-AC translocase, and CPT II [40, 41]. The coordinated increase in the activity of these enzymes may result in enhanced transfer of activated long-chain fatty acids across the mitochondrial membranes and in an increase of substrate availability for β-oxidation to meet the greater energy requirements of IVF patients with better developmental performance. It is conceivable that this accelerated process consumes excess carnitine and deprives carnitine pools.

While our study underlines the importance of carnitine-mediated β-oxidation in reproduction it is to be considered that women with disrupted carnitine cycle can conceive and proceed to successful pregnancy. Indeed, there are case reports of patients with adult-onset CPT II deficiency who become pregnant and went on to deliver preterm or full-term newborns [42‐45]. In order to identify IVF patients at risk of functional L-carnitine deficiency further studies are to be conducted to measure simultaneously carnitine compounds and markers of free fatty acid metabolism.

Carnitine profiling of women undergoing IVF provided suggestive evidences that L-carnitine metabolism is accelerated and the developmental competence of oocytes and early embryos can be optimalized by giving supplemental L-carnitine.