The Effect of a 6 Week Dietary Intervention on the Triglyceride and Total Lipid Concentrations in the Follicular Fluid of Women Undergoing In-Vitro Fertilisation, and the Impact on Oocyte Competence, Fertilisation and Embryo Development

BACKGROUND: Diet and nutrition have been acknowledged as being important factors in affecting birth outcomes for many years (Ho et al. 2016). For couples undergoing fertility treatment, despite many technological advances, live birth rates have improved very little in the last 20 years. Whereas age is has the largest impact on the success of a woman’s treatment, there is a growing body of evidence that preconception health and lifestyle choices can have a major effect on treatment outcomes (Panth et al. 2018). Hence, several groups have turned their attention to improving the quality of gametes entering the laboratory, by interventions in the periconceptual period. Observational cohort studies carried out by Vujkovic et al. (2010), and Karayianis et al. (2017) suggested a link between preconception diet and reproductive outcomes, with a positive correlation between adherence to the Mediterranean diet (high in vegetable oils, fish, vegetables and legumes and low in carbohydrate rich snacks) and chances of success with in vitro fertilisation (IVF). Furthermore, in a prospective observational study, Hammiche et al. (2011) correlated increased dietary intake of polyunsaturated fatty acids EPA and DHA with improved embryo morphology. Fatty acids are a major source of energy for the oocyte and early embryo (Sturmey et al. 2009) and are required for incorporation into the rapidly increasing cell membrane of the developing embryo (Carnegie Institution 2013).

Prior to PREPARE, only two RCTs have attempted to describe the effect of dietary supplements on reproductive outcomes in sub-fertile women. Both Agrawal et al. (2012) and Westphal et.al (2006) showed improved pregnancy rates in the groups taking the supplement in question; however the studies were limited by the small number of participants included. Despite the limited evidence, there is now a multi-million-pound nutraceutical industry producing products that claim to improve a couple’s chance of conceiving. The lack of evidence from robust RCTs regarding the advice given about optimal lifestyle choices for those suffering with subfertility, was highlighted in a Cochrane review by Anderson et al. (2010) and points to a current gap in the knowledge around this topic. One proposed reason for the lack of prospective RCTs in this area would be difficulty in monitoring adherence to specific dietary interventions over a prolonged period of time (Kermack et al. 2014). However, animal studies have demonstrated that even short dietary manipulations, around the time of implantation, can have an effect on parameters such as oocyte competence, blastocyst formation and implantation rate (Fleming et al. 2011; Schelbach et al. 2011; Minge et al. 2008).

The hypothesis for PREPARE was that a diet rich in Omega-3 fatty acids and Vitamin D would be beneficial to the developing embryo. The single centre study included 111 couples undergoing their first or second cycle of IVF, in which the female was aged between 18-41 and had a BMI between 20 and 32 kg/m², and the male was to provide a fresh semen sample for use in treatment. Further inclusion criteria were an anti-műllerian hormone levels above 7pmol/l or an antral follicle count greater than 10. Exclusion criteria were previously diagnosed diabetes, prescribed medication (apart from simple pain killers), taking herbal remedies or eating oily fish more than once a week. Both male and female participants in the trial group received a six-week supply of olive oil for cooking, olive oil-based spread and daily drinks supplemented with Vitamin D (10 µg), DHA (1 g) and EPA (1 g). The control group received sunflower seed oil for cooking, sunflower oil based spread and daily supplement drinks excluding Vitamin D, DHA and EPA (Kermack et al. 2014). Pertinent to the study in question here, blood samples were taken from female patients prior to randomisation. On the day of oocyte retrieval, following ovarian gonadotropin stimulation, further blood samples were collected, along with follicular fluid. This study intends to examine any differences in the TG and total lipid content within the follicular fluid between the intervention and control group. A meta-analysis review performed by Musa-Veloso et al. (2010) concluded that DHA and EPA dose dependently reduce serum concentrations of TGs. There is growing evidence in the literature that a high fat diet causes lipotoxic responses in the intra-ovarian environment and can lead to poorer IVF outcomes (Wu et al. 2010). As TGs provide cells with NEFAs, intracellular overexposure to NEFAs may result from increased local availability of TGs, leading to inefficient β-oxidation and generation of cytotoxic lipid peroxides. Conversion of NEFAs to energy via β-oxidation protects cells from lipotoxic effects such as mitochondrial dysfunction (Gervais et al. 2015; Wu et al. 2010). Energy supply from mitochondria is considered the most critical factor with respect to oocyte fertilisation competence and embryo developmental competence (Van Blerkom 2011). Excess circulatory lipids may be taken up by lean organs such as the ovaries and there is some evidence to suggest that women presenting with high follicular fluid NEFA levels, produce lower quality oocytes during IVF treatment (Jungheim et al. 2011). Gervais et al. (2015) demonstrated that follicular fluid concentrations of TGs were strongly correlated with testosterone (T) levels (in gonadotrophin stimulated women undergoing IVF) and, in turn, increased stimulated T levels were associated with a decrease in the number of oocytes fertilised. The hypothesis for this study is that a dietary intervention, involving supplementation with EPA and DHA, will reduce TG and total lipid concentrations in follicular fluid and, in turn reduce inefficient β-oxidation of fatty acids, potentially leading to oocytes of improved competence being available for IVF. AIMS: The aim of this project is to investigate whether a pre-conceptional dietary intervention, based on the Mediterranean diet, affects the triglyceride and total lipid content in the follicular fluid and whether there is any correlation with oocyte competence, fertilisation rates and embryo development. This will be done through analysis of samples taken during a double blinded randomised controlled study (RCT), in which the trial group took dietary supplements with increased levels of the Omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and the control group were given a placebo. This project will investigate a proposed secondary outcome from the NIHR BRC study PREAPARE (PREconception dietary supplements in Assisted Reproduction), described by Kermack et al. (2014). The author of this proposal was directly involved in refining the PREPARE study protocol and collecting samples of follicular fluid, cumulus cells and semen from participating couples. The primary endpoint of the original RCT was the time taken for cleaved embryos in the study to develop from the 2-cell to 3-cell stage. Secondary outcomes include the analysis of further morphokenetic markers, the concentration of nutritional markers in blood, follicular fluid an endometrial fluid, changes in immune cell populations within the endometrium and changes in semen parameters. Originally the project proposal was to analyse the triglyceride (TG) content only in the follicular fluid of both the trial and control groups. However, since submitting, further findings from PREPARE have been published. The group demonstrated that adherence to the 6-week dietary intervention altered the concentrations of specific fatty acids within the serum and follicular fluid (Kermack et al. 2018). Also, Gervais et al. (2015) have previously demonstrated that both TG and total lipid concentrations in follicular fluid are associated with increased testosterone levels and, in turn, impaired fertility outcomes. Total lipid concentration is the sum of TG and non-esterified fatty acid (NEFA) concentrations. For these reasons, the proposal for this study has altered in that we intend to analyse both the TG and total lipid content of the follicular fluid collected from both the trial and control groups.