Follicular fluid metabolome: a better alternative than serum metabolome for new insights on reproductive health?

Untargeted metabolome is increasingly used to identify potential biomarkers related to reproductive health outcomes, using high resolution mass spectrometry technology and expanding metabolome database. Both C18 hydrophobic chromatography column with negative electrospray ionization and hydrophilic interaction liquid chromatography (HILIC) column with positive electrospray ionization are used in liquid chromatography high resolution mass spectrometry to enhance the yield of metabolites. Although serum or plasma metabolome is more commonly examined, the biospecimens can include urine, amniotic fluid, breast milk, and innovatively, follicular fluid in ovarian follicles upon ovum retrieval for patients undergoing in vitro fertilization (IVF). In this issue of Fertility and Sterility, Hood et al. (1Hood R.B. Liang D. Tan Y. Ford J. Souter I. Jones D.P. et al.Characterizing the follicular fluid metabolome: quantifying the correlation across follicles and differences with the serum metabolome.Fertil Steril. 2022; 118: 970-979Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar) presented high correlation (overall 0.99, feature-specific controlling for subject variability 0.74–0.85, subject-specific 0.88–0.91, including both C18 and HILIC columns albeit analyzed separately) between follicular fluid metabolites measured in the first, second, and third follicles collected from woman undergoing IVF treatment (n = 26, 23, 18 for first-second, first-third, second-third follicle comparisons, respectively). In contrast, the correlation between first follicle and serum metabolome was moderate to weak (overall 0.45–0.63, feature-specific 0.35–0.37, subject-specific 0.42–0.59) in 125 women. The overlapping features between follicular fluid and serum (n = 3,970 from C18 negative and 2,352 from HILIC positive) accounted for 37%–51% of all features (with >20% detection frequency, same hereafter) in the C18 negative, and similarly 26%–42% of all features in the HILIC positive columns. Further refining the features for a 1:1 match between the follicular fluid and serum, after removing potential 1:n match and selecting features with the closest retention time, reduced the overlapping features to 1,928 from C18 negative (18%–25% of all features) and 1,149 from HILIC positive (13%–21% of all features) columns. Of these features, only 27 and 36, respectively, were level-1 metabolites that were already validated for the molecular structure using a reference standard. It is possible that follicular fluid represents a new biospecimen for metabolome, and its metabolites are related to fertility and reproductive health outcomes, particularly in women under IVF treatment. Follicular fluid has 2 major origins: from blood supply that passes through the blood-follicle barrier (BFB), and from secretions by the cells within the follicle, particularly the granulosa cells (2Basuino L. Silveira Jr., C.F. Human follicular fluid and effects on reproduction.JBRA assisted reproduction. 2016; 20: 38-40Crossref PubMed Scopus (38) Google Scholar). The BFB consists of the tight junction between endothelial cells of the blood vessels surrounding the follicles, subendothelial basement membrane, the thecal interstitial cells, the follicular basement membrane, and the membrana granulosa cells (3Siu M.K. Cheng C.Y. The blood-follicle barrier (BFB) in disease and in ovarian function.Adv Exp Med Biol. 2012; 763: 186-192Crossref PubMed Scopus (52) Google Scholar). The BFB, acting as a “molecular sieve,” is found to be more permeable to proteins <100 KDa. Although small molecular metabolites tend to cross the blood-tissue barriers, the metabolomic difference between follicular fluid and serum has not been examined before this study. Because of the moderate to weak correlation in the metabolome between the 2 matrices in this study, along with less than a quarter of overlapping features with a 1:1 matching, the role of another source of follicular fluid appears to be important. Follicular fluid metabolome may provide additional insights into the metabolic exchange between the granulosa cells and ovum as well as shed light on the potential mechanisms underlying infertility and adverse reproductive outcomes (4Brinca A.T. Ramalhinho A.C. Sousa Â. Oliani A.H. Breitenfeld L. Passarinha L.A. et al.Follicular fluid: a powerful tool for the understanding and diagnosis of polycystic ovary syndrome.Biomedicines. 2022; 10Crossref PubMed Scopus (11) Google Scholar). Oxidative stress has been associated with ovarian aging, and follicular fluid provides antioxidant defense system to prevent the generation of reactive oxygen species and scavenge existing free radicals. Free fatty acids and lipid profiles in the follicular fluid may be relate to oxidative stress and polycystic ovary syndrome, a major cause of female infertility. Glucose and derivatives are critical energy sources in follicular fluid to the developing oocyte. Abnormal levels of steroid acids, hormones, and other metabolites in follicular fluid may also be related to pathologic outcomes in fertility and reproductive health. The article by Hood et al. (1Hood R.B. Liang D. Tan Y. Ford J. Souter I. Jones D.P. et al.Characterizing the follicular fluid metabolome: quantifying the correlation across follicles and differences with the serum metabolome.Fertil Steril. 2022; 118: 970-979Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar) has also raised few questions in the interpretation when pondering about the future use of follicular fluid as a biospecimen. First, the median raw feature-specific correlation was below <0.1, whereas, the median adjusted correlation was between 0.35 and 0.37. Year of sample collection was the single factor that had the largest influence on the adjusted correlation. More work is needed to examine the role of sample collection procedures on follicular metabolites. Second, the subject-specific correlation between serum and follicular fluid was largely similar between different infertility diagnosis: male, female, or unknown, suggesting the strength of the correlation was not affected by the infertility etiology. More research is needed to understand the difference in follicular fluid metabolome between women with and without infertility. Previous research of follicular fluid proteomics has additionally included healthy ovum donors to identify proteins related to infertility (5Zamah A.M. Hassis M.E. Albertolle M.E. Williams K.E. Proteomic analysis of human follicular fluid from fertile women.Clin Proteomics. 2015; 12: 5Crossref PubMed Scopus (90) Google Scholar). A similar approach can be used in follicular fluid metabolome if the blood metabolome is substantially different from follicular fluid. Third, microbiome as a source of follicular fluid metabolites was hinted in the paper, and more research is warranted to examine the potential implications of the microbiome metabolic pathway. Fourth, the high correlation between the first, second, and third follicles for follicular fluid metabolome during a given IVF cycle may still call for the answers to the variation in follicular metabolome by age, obesity, insulin resistance, lifestyle, infection, and dietary patterns. Fifth, the vast number of features discovered by the untargeted metabolome has not been validated with a reference standard, and this limitation may reduce the impact of mechanistic as well as human studies that aim to identify etiologic or therapeutic targets. It could be that both targeted and untargeted approaches may be needed to provide a more comprehensive metabolomic profile. In summary, although follicular fluid metabolome may be potentially useful in reproductive health research, with clinical implications, substantially more work needs to be planned to uncover the natural variation of metabolome, the source of metabolites, its role in disease etiology, and the validated identity of more metabolites. Characterizing the follicular fluid metabolome: quantifying the correlation across follicles and differences with the serum metabolomeFertility and SterilityVol. 118Issue 5PreviewTo compare the variability in metabolomes between the serum and follicular fluid, as well as across 3 dominant follicles. Full-Text PDF