Human ovarian follicular fluid (FF) contains high density lipoprotein (HDL) as the predominant lipoprotein with only traces of other lipoproteins [¹, ²]. The importance of HDL in mammalian female reproduction is demonstrated by scavenger receptor class B group I, knockout (SR-BI KO) female mouse-generated embryos which arrest due to aberrant HDL metabolism [³–⁶]. In clinical studies, we have demonstrated negative correlations between FF HDL cholesterol and ApoAI levels with embryo fragmentation during in vitro fertilization (IVF) [⁷, ⁸]. These observations prompted us to further examine particular FF HDL components and examine their association with embryo morphology parameters during IVF. Since HDL is the major FF lipoprotein, we hypothesized that HDL is also the major carrier of lipid-soluble vitamins and micronutrients in FF. Our objective was to measure the lipid-soluble vitamin and micronutrient content of FF and to examine their associations with embryo morphology parameters in women undergoing IVF.

Oocytes were retrieved from the sampled ovarian follicle in 83 of the 87 study participants undergoing IVF. FF volume collected from two participants was insufficient for any analysis therefore 81 participants with harvested oocytes and sufficient FF volume for analysis comprised the final study sample. Among all study oocytes 83.9% (n = 68) were arrested in Metaphase-II and 66.2% (n = 45) of these mature oocytes were normally fertilized (2PN) by either conventional IVF or intracytoplasmic sperm injection (ICSI). Sample size varied for certain analytes due to the limited volume of FF collected during this pilot study. The complete demographic, treatment and outcome variables for the study sample are described in Table 1. Table 2 presents the distributions of the absolute levels of all study analytes in FF as well as the correlation of each variable with FF-HDL cholesterol, EFS and ECN adjusted for day of transfer.

Except for β-carotene and γ-tocopherol, all FF fat-soluble vitamins and micronutrients are significantly positively correlated with FF HDL cholesterol concentration. With this expansion of the dataset from our prior report (8), FF total HDL cholesterol remains negatively correlated with EFS (r = −0.47, P = 0.003). FF concentrations of α-tocopherol (r = −0.41, P = 0.009), δ-tocopherol (r = −0.38, P = 0.017) and β-cryptoxanthine (r = −0.42, P = 0.008) are also negatively correlated with EFS. While other FF micronutrients also correlate negatively with EFS, these do not approach statistical significance (P > 0.10). No meaningful correlations are detected between ECN, adjusted for day of embryo transfer, and FF analytes (P > 0.15).

Table 3 presents the ordinal logistic regression model for EFS in which a 0.1 µmol/L increase in FF β-cryptoxanthin concentration is associated with a statistically significant (P = 0.010) decrease of 75% in the cumulative odds (odds ratio = 0.25) for an increased EFS, conditional on FF γ-tocopherol. FF γ-tocopherol is itself significantly associated with a 66% decrease in the cumulative odds (odds ratio = 0.34) for an increased EFS (P = 0.035) for each one 1.0 µmol/L increase in concentration. There are no significant predictors of ECN.

In summary, we extend our initial observations [⁷, ⁸] with additional evidence in support of a role for the HDL particle in the developmental potential of the human oocyte during early embryogenesis. We have now demonstrated that multiple components of FF HDL particles including cholesterol, ApoAI, and the micronutrients, ß-cryptoxanthine and γ-tocopherol, represent unique clinical predictors of embryo fragmentation during IVF. From these results it is unclear whether the HDL particles themselves or co-linear HDL particle components such as ß-crytoxanthin and α-tocopherol are responsible for the protective effect against embryo fragmentation. It is clear that the strong correlations between HDL and lipophilic micronutrient levels in FF prompt all future studies to consider adjusting for associated covariates during statistical analyses. While previous studies have not demonstrated significant clinical effects associated with carotenoids, retinoids, or tocopherols, it is not clear that either study analyzed the relationship with embryo morphology parameters [¹², ¹³]. Consistent with Schweigert et al. we observe similarly reduced levels of micronutrients in FF compared to plasma [¹³]. Our observations suggest that numerous lipophilic components of HDL particles including micronutrients may be influencing the membrane and cytoplasmic properties of the oocyte with downstream effects on embryo fragmentation occurring during in vitro cytokinesis.

There are several notable limitations to this study: (1) we cannot make any conclusions regarding a role for sperm factors even though we considered total motile sperm count in our multivariate analyses; and (2) these findings are simply associations and do not necessarily imply causal effect. We acknowledge that more detailed human and mammalian studies need to be performed in order to assess causality.

To what extent the constitution of the FF HDL particle is a reflection of intra-follicular metabolic processing versus extra-follicular modeling is not known. Our understand of FF HDL metabolism during folliculogenesis and oocyte development is limited and further complicated by the multiple proteins and lipids that interact with and contribute to the biological roles of HDL particles [⁸]. Given the relative predominant existence of HDL particles in FF during folliculogenesis, it remains to be determined if these lipophilic micronutrients are influencing oocyte development independent of intra-follicular cholesterol and phospholipid transport mechanisms [⁸]. Our findings raise interesting questions regarding the biological mechanisms that are directing apparent effects of HDL-associated micronutrients on oocyte development and competence. Furthermore, the negative correlations between multiple FF HDL components and embryo fragmentation provide reasons to consider HDL particle composition as a useful biomarker of embryo fragmentation during IVF.

Presented in part at the American Society of Reproductive Medicine Annual Meeting in San Francisco, November 2008.

Capsule

Follicular fluid lipid-soluble vitamins and micronutrients are correlated with HDL and may explain the observed association between HDL and embryo morphology parameters during IVF.

We acknowledge the assistance of Julia Sandler, Giulia Conti, and Natasha Narayan and the support of the physicians and staff of the UCSF Center for Reproductive Health, San Francisco, CA.

Funding This work was not supported by any grant funding. Institutional discretionary research funds available to Drs. Browne and Fujimoto were used to support this work.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.