Alterations of Sphingolipid Metabolism in Different Types of Polycystic Ovary Syndrome

The roles of sphingolipids in polycystic ovary syndrome (PCOS) are still unknown. This study aimed to investigate the sphingolipid characteristics for different types of PCOS using liquid chromatography-mass spectrometry (LC-MS). A total of 107 women with PCOS and 37 healthy women as normal controls were studied. PCOS patients were further classified into non-obesity with insulin resistance (IR) (NOIR), obesity with IR (OIR), and non-obesity and non-IR (NIR) subgroups. A total of 87 serum sphingolipids, including 9 sphingosines, 3 sphinganines, 1 sphingosine-1-phosphate (S1P), 19 ceramides (Cers), 1 ceramide-1-phosphate, 44 sphingomyelins (SMs), 4 hexosylceramides, and 6 lactosylceramides (LacCers) were analyzed using an improved sphingolipidomic approach based on LC-MS. Notable elevations in the levels of S1P, Cer, and SM were observed in PCOS patients when compared with healthy women, and SM species with long saturated acyl chains showed potential as novel biomarkers of PCOS. In addition, the level of LacCer was only elevated in NIR, and there was almost no change in NOIR and OIR. This study is the first to report the comprehensive sphingolipidomic profiling of different subgroups of PCOS with or without IR or obesity and suggests that serum sphingolipids might be useful as diagnostic biomarkers for different types of PCOS.


Results
Baseline characteristics. A total of 107 PCOS patients and 37 healthy women were enrolled. Thirty-four (31.8%) of the 107 PCOS patients presented with a body mass index (BMI) < 25 kg/m 2 and homeostatic model assessment of insulin resistance (HOMA-IR) ≥ 2.14 (the non-obesity with IR (NOIR) group), 41 patients (38.3%) had BMI ≥ 25 kg/m 2 and HOMA-IR ≥ 2.14 kg/m 2 (the obesity with IR (OIR) group), and 32 patients (29.9%) had BMI < 25 kg/m 2 and HOMA-IR < 2.14 kg/m 2 (the non-obesity and non-IR (NIR) group). The demographic, endocrine, and glycolipid metabolic features of controls and patients with different types of PCOS are described in Table 1. There were no significant differences in age for any of the groups. PCOS patients in the OIR group had a significantly greater BMI, waist-to-hip ratio (WHR), and HOMA-IR; higher levels of total cholesterol (CHOL), triglycerides (TG), and low-density lipoprotein (LDL); and lower levels of high-density lipoprotein (HDL) compared to controls. Except for BMI and HDL, these characteristics were also significantly increased in the NOIR group compared with the controls. However, PCOS in the NIR group only presented with obviously increased luteinizing hormone (LH) to follicle-stimulating hormone (FSH) ratio, LH level, and total testosterone (TT) level compared with controls. Overall, these changes in PCOS patients were clinically related to PCOS type and complemented the metabolomics analysis.
Identification of serum sphingolipids. The sphingolipids in human serum were comprehensively profiled using an established sphingolipidomic approach 23 . A total of 87 sphingolipids were identified and quantified in the serum samples, including 9 sphingosines, 3 sphinganines (Sas), S1P, 19 Cers, 1 ceramide-1-phosphate (Cer1P), 44 sphingomyelins (SMs), 4 hexosylceramides (HexCers), and 6 lactosylceramides (LacCers). The MS and MS/MS data of identified sphingolipids and the multiple reaction monitoring (MRM) transitions used to monitor each sphingolipid are listed in Table 2. The MRM chromatograms of all sphingolipids in human serum are shown in Fig. 1. Fig. 2a, the total sphingolipid content in PCOS patients showed a significant increase (15.9% increase) when compared with healthy women. Among the sphingolipids, three subclasses demonstrated remarkable elevations in PCOS patients, including S1P (10.8% increase), Cer (12.2% increase), and SM (16.3% increase). Sa was the only subclass showing a decrease in PCOS patients. Multivariate analysis was subsequently carried out to discriminate between PCOS patients and healthy women. As shown in Fig. 2b, the three-dimensional (3D) orthogonal partial least squares discriminant analysis (OPLS-DA) score plot demonstrated a clear separation between the PCOS group and the healthy group (R 2 X = 0.523, R 2 Y = 0.697, Q 2 = 0.533). Sphingolipids with a variable importance plot (VIP) value > 1.5 were regarded as the important variables contributing to the 3D OPLS-DA model. As a result, 7 sphingolipids, including 6 SMs (SM (d40:0), SM (d38:0), SM (d18:1/22:0), SM (d18:1/24:0), SM (d41:0), and SM (d18:1/23:0)) and 1 Cer (Cer (d18:0/24:0)) were selected as potential biomarkers (Fig. 2c). Differences in serum sphingolipidomes between different subgroups of PCOS and healthy women. The PCOS group was classified into three subgroups (OIR, NOIR, NIR) as described above for further comparison with the healthy group. As shown in Fig. 3, the levels of total sphingolipids and of each sphingolipid subclass in all three subgroups of PCOS followed similar trends as the overall PCOS group. Notably, OIR showed the most obvious elevation (23.83%) in the total sphingolipid level when compared to the healthy group. All three subgroups of PCOS demonstrated significant increases in the level of SM (OIR > NOIR > NIR), while only NOIR and OIR showed significant increases in the level of Cer (OIR > NOIR), and only OIR showed significant increases in the level of S1P. The level of Sa was decreased in all three subgroups of PCOS, in which only OIR demonstrated a significant change. Unexpectedly, the level of LacCer was only elevated in NIR (28.72%), and there was almost no change in NOIR and OIR.

Differences in serum sphingolipidomes between PCOS patients and healthy women. As shown in
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Discussion
In this study, the serum sphingolipids in healthy controls and PCOS patients were comprehensively investigated using the improved LC-MS-based sphingolipidomic approach 23 . As a result, 87 sphingolipids were identified and quantified in each sample. Notable elevations in the levels of S1P, Cer, and SM were observed in PCOS patients, and these made the largest contribution to the significant increase in the total sphingolipid content in PCOS patients. Our results are in accordance with a previous study that suggested that PCOS is associated with increased TG and SM and decreased phosphatidylethanolamines and lysophosphatidylcholines in patient plasma 35 . The multivariate analysis between PCOS patients and healthy women also suggested that SM was the most dominant subclass of sphingolipid involved in the pathogenesis of PCOS because 6 out of 7 potential markers were SMs. Notably, all potential markers, including the one Cer marker, have saturated acyl chains. A previous report showed that the increase in SM species with long saturated acyl chains (18:0, 20:0, 22:0, and 24:0) was closely correlated with the development of metabolic syndrome associated with lipid metabolism, obesity, and IR 36 . Therefore, we suggest that SM species with long saturated acyl chains might serve as novel biomarkers of PCOS, and further studies should be carried out to confirm this.
In addition to PCOS, other metabolic diseases such as obesity and IR have also been shown to be associated with lipid alterations 37 . Therefore, we further divided the PCOS patients into three subgroups (NIR, NOIR, OIR) according to their HOMA-IR and BMI values. NIR represented the group without IR or obesity, NOIR represented the group with IR only, and OIR represented the group with both IR and obesity. The levels of total sphingolipids and of each subclass in all three subgroups of PCOS followed similar trends as for all PCOS patients combined, while the subgroups of PCOS with IR or obesity exhibited greater changes than their corresponding subgroups without IR or obesity. As mentioned above, an increase in SM species with long saturated acyl chains was closely associated with lipid metabolism, obesity, and IR. Therefore, the presence of IR or both IR and obesity contributed to the increase in the level of SM in the order of OIR > NOIR > NIR. In addition, plasma Cer was reported to be elevated in obese subjects with type 2 diabetes mellitus, and increased levels of plasma Cer might also be a marker of IR 38 . However, in our study the occurrence of PCOS seems to have no direct relationship with Cer, and only NOIR and OIR showed significant increases in the level of Cer (OIR > NOIR). Furthermore, S1P was also reported to be elevated in the plasma of obese subjects and to correlate with IR 39 , and this is the likely explanation for why only OIR demonstrated a significant increase in the level of S1P in our study.
No previous report has discussed the relation between Sa and PCOS. However, because Sa is upstream of Cer in the metabolism pathway of sphingolipids, we speculate that the decrease we observed in Sa might be the  www.nature.com/scientificreports www.nature.com/scientificreports/ result of the upregulation of Cer synthases leading to the accumulation of Cer and further downstream products. However, further studies are needed to confirm this hypothesis. It is interesting that the level of LacCer was only elevated in NIR, while there was almost no change in NOIR and OIR. This implies that the level of LacCer was significantly increased in PCOS patients who did not suffer from IR and obesity. However, previous studies indicated that there is a negative correlation between gangliosides (complex glycosphingolipids) and insulin responsiveness 40 , and it was observed that inhibition of glucosylceramide synthase activity reversed IR in several rodent models of obesity 41 . Therefore, in theory the level of LacCer  www.nature.com/scientificreports www.nature.com/scientificreports/ would be expected to be increased in PCOS patients with IR. However, up to now there have been no studies to investigate the direct interaction of LacCer on insulin responsiveness and the relationship between glucosylceramide synthase activity and IR in humans. Thus the results of the subgroup analysis further suggest that PCOS patients have unique sphingolipid biomarkers that are not caused only by obesity and IR.
In the multivariate statistical analysis of sphingolipid alterations among the three subgroups of PCOS, it was found that the discrimination between the three subgroups of PCOS was unsatisfactory, especially between NOIR and OIR. Only a single sphingolipid showed a significant difference between NOIR and OIR, and this might be explained by the close relationship between IR and obesity, which often cause similar alterations in sphingolipid metabolism. The differentiation between NIR and OIR was comparatively better. These results suggested that alterations in sphingolipid metabolism might play a role in the pathogenesis of PCOS and that sphingolipids might be useful as diagnostic biomarkers for different types of PCOS.
This study represents the first report of a comprehensive sphingolipidomic profiling of PCOS and different subgroups of PCOS with or without IR and/or obesity. The results shed light on the diagnosis and pathogenesis of PCOS. A limitation of the study is that we only detected some of the sphingolipid characteristics for different types of PCOS patients, and the mechanism of alterations of sphingolipid metabolism in PCOS needs further investigation in both human subjects and animal models.

Materials and Methods
Study Participants. PCOS patients aged 18 to 40 years were recruited between January 2014 and May 2015 from the Department of Traditional Chinese Medicine of the First Affiliated Hospital of Guangzhou Medical University. The first woman was recruited on January 2, 2014, and the last one was on May 28, 2015. Agematched healthy female subjects were selected from community volunteers. PCOS was diagnosed as having two of the following three Rotterdam criteria 42 : (1) oligomenorrhea/amenorrhea; (2) more than 12 follicles ≤9 mm in diameter or ovarian volume >10 ml on pelvic scanning; and (3) clinical or biochemical hyperandrogenism. Oligomenorrhea is defined as an intermenstrual interval >35 days or <8 menstrual bleedings in the past year. Amenorrhea is defined as an intermenstrual interval >90 days. Biochemical hyperandrogenism is defined as a TT level ≥ 0.6 ng/ml 43 , and clinical hyperandrogenism is defined as a Ferriman-Gallwey score ≥ 5 44 . Women were excluded if they had other endocrine disorders such as hyperprolactinemia, type I or type II diabetes mellitus, non-classic congenital adrenal hyperplasia, suspected Cushing's syndrome, androgen-secreting tumors, thyroid diseases, or drug-induced androgen excess. Women who had received any hormonal treatments, Chinese herbal prescriptions, or acupuncture treatments in the past 3 months were also excluded from the study. All controls had regular menstrual cycles and normal hormone levels.  www.nature.com/scientificreports www.nature.com/scientificreports/ Affiliated Hospital of Guangzhou Medical University, and serum FSH, LH, estradiol (E2), TT, prolactin, TG, CHOL, LDL, HDL, fasting plasma glucose, and fasting insulin were measured. The intra-assay and inter-assay coefficients of variation were less than 5%. HOMA-IR was calculated to assess changes in insulin sensitivity 40 . FSH, LH, and TT levels were measured with a Beckman-Coulter Unicel DXi800 automatic chemiluminescence analyzer (Beckman Coulter, Brea, USA), and TG, CHOL, LDL, and HDL levels were measured on a Beckman-Coulter AU5800 automatic biochemical analyzer. Fasting insulin was analyzed using the Modular E170 ® automatic electrochemiluminescent analyzer (Roche Diagnostics, Mannheim, Germany). Fasting plasma glucose was measured on a Beckman Coulter LX20 automatic biochemical analyzer. All assays were performed based on the instructions of manufacturers and with reagents and materials provide by the manufacturers.
Sample collection and processing. All of the blood samples were collected by staff in the First Affiliated Hospital of Guangzhou Medical University. The blood samples were 5 ml and were centrifuged for 15 minutes at 3,000 rpm within 30 minutes after the blood samples were drawn. The separated serum was stored at −80 °C and was delivered to the State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, within 3 months of being collected.
Sample preparation and sphingolipidomic assays. Extraction of sphingolipids. Serum sphingolipids were extracted using an established method 23 . Briefly, 20 μl of serum was transferred into a borosilicate glass tube, and 10 μl of internal standards (2.5 μM each) and 0.75 ml of extraction solvent [methanol (MeOH): chloroform, 2:1, v/v] were added. The contents were dispersed in an ultrasonicator at room temperature for 30 seconds, followed by incubation at 48 °C for 12 hours. After cooling, 75 μl of potassium hydroxide in MeOH (1 M) was added and incubated with shaking at 37 °C for 2 hours, after which 3 μl acetic acid was added to neutralize the extract. After centrifugation, the supernatant was stored in a 4 ml bottle, and the residue was re-extracted twice. Finally, the extract was dried by nitrogen and re-dissolved in 150 μl of MeOH for liquid chromatography-mass spectrometry (LC-MS) detection. The quality control (QC) sample was pooled with equal quantities of samples from the different groups for the sphingolipid analysis.  Table 2.
Data analysis. An in-house sphingolipid database has been established in our laboratory based on the Agilent MassHunter Personal Compound Database and Library software and information from the LIPID MAPS Lipidomics Gateway. Using this customized sphingolipid database and the "find-by-formula" option in the MassHunter Qualitative Analysis Software (version B.06.00), potential sphingolipids were identified based on a comparison of accurate mass, abundance of the isotopes, and isotope spacing with the calculated theoretical masses and abundances. The structures of potential identified sphingolipids were further determined according to the characterized fragments obtained from high-resolution MS/MS data.
The raw data for quantitative analysis were processed with Agilent MassHunter Quantitative Analysis B.06.00 software. The resulting data were first transferred into a Microsoft Excel-type spreadsheet and then imported into the SIMCA-P + 14.0 software (Umetrics, Umea, Sweden) for multivariate statistical analysis. Principal component analysis (PCA) was used to visualize general clustering among the different groups. 3D OPLS-DA was carried out to identify differences in sphingolipid expression between the different groups based on their VIP values. Variables that were significantly changed among different samples were selected as potential biomarkers based on VIP > 1.5 and then validated using t-test analysis.
Analysis of variance (ANOVA) or Kruskal-Wallis tests were performed to examine the differences in the clinical characteristics among the different groups using the SPSS version 21.0 software (SPSS Inc., Chicago, IL, USA). Values are shown as the mean and 95% confidence interval, and a value of p < 0.05 was considered statistically significant.
Ethical approval. All  www.nature.com/scientificreports www.nature.com/scientificreports/ Informed consent. Informed consent was obtained from all individual participants included in the study.

Conclusion
In conclusion, remarkable elevations in the levels of S1P, Cer, and SM were observed in PCOS patients compared to healthy women. Notably, SM (d40:0), SM (d38:0), SM (d18:1/22:0), SM (d18:1/24:0), SM (d41:0) and Cer (d18:0/24:0) showed the most significant alterations, which showed that SM species with long saturated acyl chains were the best candidates to serve as novel biomarkers of PCOS. Our results are in accordance with previous studies suggesting that PCOS is associated with increased SM and that SM species with long saturated acyl chains are closely correlated with the development of metabolic syndrome, obesity, and IR. In all three subgroups of PCOS (NIR, NOIR, OIR), the alteration of sphingolipid levels followed a similar trend as the overall PCOS group except for LacCer, which was only elevated in NIR. Our results suggest that serum sphingolipids might be useful as diagnostic biomarkers for different subgroups of PCOS.