In the current study, we found that hypertrophy of LF was associated with quantitative and qualitative lipid changes and demonstrated total lipid accumulation in the HLF group. Specific PCs, such as PC(O-16:0_16:0) + H + and PC(O-18:1_16:0) + H+, were remarkably increased in the HLF group, evident from the MALDI-IMS analysis. Of note, the expression of PC(O-16:0_16:0) + H + and PC(O-18:1_16:0) + H + in HLF was highly uniform; MALDI-IMS proved their accumulation, visually. We also demonstrated that PC(26:0) + H+, PC(25:0) + H+, and PC(23:0) + H + showed a reproducible tendency to be elevated in the HLF group.
The distribution of the levels of PC(O-16:0_16:0) + H + and PC(O-18:1_16:0) + H + were highly variable in the HLF group. However, some cases showed low levels of both PC(O-16:0_16:0) + H + and PC(O-18:1_16:0) + H+ (Fig. 8). Interestingly, the subjects with low PC(O-16:0_16:0) + H + and PC(O-18:1_16:0) + H + levels were less likely to show LF thickness compared with the other subjects. A possible explanation for this fact might be that PC(O-16:0_16:0) + H + and PC(O-18:1_16:0) + H + increase proportionally to LF thickness.
PC(26:0) + H + was the main phospholipid found to be increased in the HLF group and correlated with LF thickness. Several risk factors such as T2DM, hypertension, obesity, and smoking habits have been identified as contributors to the pathogenesis of LSCS.17–20 T2DM played an essential role in the induction of HLF in several east and west Asian populations.20 Previous research has reported that T2DM induces adipose tissue signaling and increases the rate of de novo PC biosynthesis.21 Similarly, we demonstrated that in the HLF group, the median values of the area ratio of saturated PC(26:0) + H + in patients with T2DM tended to increase in comparison to those without T2DM (11.2 vs. 8.8, respectively) (Fig. 7A). These results suggest that increased PCs may not only correlate with T2DM, but it may also be a novel risk factor for LSCS. Therefore, inhibiting increases in PC(26:0) + H + levels would be beneficial in preventing LSCS.
Zhou et al. described that LPA concentration in the cerebrospinal fluid of patients with HLF was higher than those in subjects with NHLF, and LPA induced hypertrophy of LF via LPA receptor1 to Akt signal pathway.22 However, their study did not show the lipid distributions in HLF. In that respect, our study shows the importance of evaluating the lipids profiles of HLF. In this study, there were several reasons that LPAs were not identified. First, the modified Bligh-Dyer method mainly extracted neutral lipids, suggesting that acid lipids like LPAs were not determined. Second, LPAs have been unstable and quickly break down to other lipids at room temperature. Then, harvested LF at room temperature may allow LPAs to turn down for other lipids.
The previous report described that lysophosphatidylcholines (LPCs), hydrolyzed from PCs via phospholipase C, increases growth factors contributing to thickness for LF. We also identified enriched LPC(17:0) + H, which may be possible for deriving from PC(17:0_18:1) + H (supplemental table). In HLF, we identified enriched OAHFAs (supplemental Fig. 4) leading to age-related macular degeneration with yellow corneal rings23. Interestingly, HLF also appears as yellow in gross (supplemental Fig. 1), originated from lipofuscin. Lipofuscin formed by oxidation of unsaturated fatty acid exerts autofluorescence, and the fluorescent particle generates fluorescent light of 500 to 650 nm wavelength. Therefore, it might be possible that yellow ligament appearance derived from OAHFAs.
We acknowledge that there are limitations to this study. First, we included LF from the LDH of younger patients in the NHLF group, leading to a significant difference in the mean patient age between the two groups. Older patients with LDH frequently present hypertrophied ligamentum flavum simultaneously. Therefore, we could not include older patients in the NHLF group and cannot deny the possibility that aging increased the PC levels. Second, the LF samples were left at room temperature during a surgical operation, and several lipids are unstable under such conditions. Therefore, their lipid compositions might have changed due to endogenous enzymes. However, our results are potentially significant in that specific PCs were increased compared to the other lipids in the HLF group and correlated with LF thickness.
In conclusions, phosphatidylcholines (PCs), ceramides (Cers), O-acyl-ω-hydroxy fatty acids (OAHFAs), and triglyceride (TGs) were increased in HLF. Those findings would provide an insight into LSCS pathogenesis that can be exploited to design an alternative treatment strategy in the future.