New Insights of OLFM2 and OLFM4 in Gut-Liver Axis and Their Potential Involvement in Nonalcoholic Fatty Liver Disease

Olfactomedins (OLFMs) are a family of glycoproteins that play a relevant role in embryonic development and in some pathological processes. Although OLFM2 is involved in the regulation of the energy metabolism and OLFM4 is an important player in inflammation, innate immunity and cancer, the role of OLFMs in NAFLD-related intestinal dysbiosis remains unknown. In this study, we analysed the hepatic mRNA expression of OLFM2 and the jejunal expression of OLFM4 in a well-established cohort of women with morbid obesity (MO), classified according to their hepatic histology into normal liver (n = 27), simple steatosis (n = 26) and nonalcoholic steatohepatitis (NASH, n = 16). Our results showed that OLFM2 hepatic mRNA was higher in NASH, in advanced degrees of steatosis and in the presence of lobular inflammation. Additionally, we obtained positive correlations between hepatic OLFM2 and glucose, cholesterol, trimethylamine N-oxide and deoxycholic acid levels and hepatic fatty acid synthase, and negative associations with weight and jejunal Toll-like receptors (TLR4) and TLR5 expression. Regarding jejunal OLFM4, we observed positive correlations with circulating interleukin (IL)-8, IL-10, IL-17 and jejunal TLR9. In conclusion, OLFM2 in the liver seems to play a relevant role in NAFLD progression, while OLFM4 in the jejunum could be involved in gut dysbiosis-related inflammatory events.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is characterized by ectopic fat accumulation in hepatic tissue in the absence of secondary causes of steatosis, such as alcohol intake and/or viral infection [1,2]. NAFLD is a general term used for a variety of liver conditions that comprises many stages from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH), this last one defined by the presence of hepatic inflammation, ballooning and/or

Baseline Characteristics of Subjects
The clinical and biochemical measurements of the cohort formed by women with MO (BMI > 40 kg/m 2 ) classified according to the hepatic histology as normal liver (NL, n = 27), SS (n = 26) and NASH (n = 16), are shown in Table 1. The participants did not present significant differences between groups in terms of weight, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), homeostatic model assessment method-insulin resistance (HOMA1-IR), insulin, glycosylated haemoglobin (HbA1c), cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), aspartate-aminotransferase (AST), alanine-aminotransferase (ALT) and gamma-glutamyltransferase (GGT). However, in this analysis, we found higher levels of glucose and alkaline phosphatase (ALP) in the SS group than in the NL group, increased levels of triglycerides in the NASH cohort compared to NL women and increased levels of ALP in SS subjects in comparison to NASH patients.

Evaluation of the Relative mRNA Abundance of OLFM2 and OLFM4 According to Hepatic Histology
To achieve the aim of this study, that was to explore the role of OLFMs in NAFLD progression, we evaluated the OLFM2 mRNA expression in liver samples in a cohort of women with MO. Moreover, given that the importance of the gut-liver axis in NAFLD progression has been reported, we also analysed the jejunal OLFM4 mRNA expression.
First, when we analysed OLFM2 and OLFM4 relative mRNA expressions between NL and NAFLD in hepatic and jejunal samples, respectively, we only reported significant differences between groups in the case of OLFM2 (p = 0.005). Then, we subclassified the patients according to the hepatic histopathological grades into NL, SS and NASH. In this sense we found a higher expression of hepatic OLFM2 in SS and NASH women compared to the NL cohort ( Figure 1A). Later, when the subjects were divided according to the presence of NASH, an increase in OLFM2 relative expression in the NASH group was observed in comparison with the non-NASH group, as shown in Figure 1B. However, regarding OLFM4, we did not report significant differences between groups ( Figure 1C,D). sense we found a higher expression of hepatic OLFM2 in SS and NASH women compared to the NL cohort ( Figure 1A). Later, when the subjects were divided according to the presence of NASH, an increase in OLFM2 relative expression in the NASH group was observed in comparison with the non-NASH group, as shown in Figure 1B. However, regarding OLFM4, we did not report significant differences between groups ( Figure 1C,D).

Figure 1.
Differential relative mRNA abundance of OLFM2 in hepatic tissue between women with MO (A) classified as NL, SS and NASH and (B) classified according to the presence or absence of NASH. Differential relative mRNA abundance of OLFM4 in jejunal tissue between women with MO (C) classified as NL, SS and NASH and (D) classified according to the presence or absence of NASH. OLFM, olfactomedin; NL, normal liver; SS, simple steatosis; NASH, nonalcoholic steatohepatitis; A.U arbitrary units. Differences between groups were calculated using Mann-Whitney test and p < 0.05 was considered statistically significant.

Evaluation of the Relative mRNA Abundance of OLFM2 and OLFM4 according to the Severity of Steatosis
To deepen the knowledge of the link between OLFMs and NAFLD, first we wanted to focus on the hepatic lipid content degree, so we classified the cohort into different grades of steatosis. In this sense, we reported an enhanced expression of hepatic OLFM2 mRNA in moderate and severe stages of steatosis in comparison to those subjects without steatosis ( Figure 2A); while we did not find significant differences when OLFM4 was analysed, as graphically represented in Figure 2B. OLFM, olfactomedin; NL, normal liver; SS, simple steatosis; NASH, nonalcoholic steatohepatitis; A.U arbitrary units. Differences between groups were calculated using Mann-Whitney test and p < 0.05 was considered statistically significant.

Evaluation of the Relative mRNA Abundance of OLFM2 and OLFM4 According to the Severity of Steatosis
To deepen the knowledge of the link between OLFMs and NAFLD, first we wanted to focus on the hepatic lipid content degree, so we classified the cohort into different grades of steatosis. In this sense, we reported an enhanced expression of hepatic OLFM2 mRNA in moderate and severe stages of steatosis in comparison to those subjects without steatosis ( Figure 2A); while we did not find significant differences when OLFM4 was analysed, as graphically represented in Figure 2B. Later, we wanted to focus the analysis on the main parameters of the advanced stage

Evaluation of the Relative mRNA Abundance of OLFM2 and OLFM4 According to NASH-Related Parameters
Later, we wanted to focus the analysis on the main parameters of the advanced stage of NAFLD, such as inflammation and hepatocellular ballooning; we therefore evaluated their link with OLFM2 and OLFM4. Concerning portal inflammation, we did not observe differential expressions of OLFM2 in the liver and OLFM4 in the jejunum between groups ( Figure 3A,B). Regarding lobular inflammation, we found an increase in OLFM2 hepatic expression in the cohort presenting this type of inflammation ( Figure 3C), but we did not find differences with regard to OLFM4 jejunal expression ( Figure 3D). Moreover, nonsignificant differences in OLFM2 and OLFM4 were reported when the cohort was classified based on the presence of hepatocyte ballooning ( Figure 3E,F).  A.U arbitrary units. Differences between groups were calculated using Mann-Whitney test. p < 0.05 was considered statistically significant.

Correlations of Relative mRNA Abundance of Hepatic OLFM2 and Jejunal OLFM4, with Clinical and Biochemical-Related Parameters
To broaden the study of OLFM2, we analysed their correlations with different parameters related to glucose and lipid metabolism, gut microbiota and inflammation in a cohort of women with MO. First, we observed positive correlations between hepatic OLFM2 mRNA expression and glucose, cholesterol, trimethylamine N-oxide (TMAO), deoxycholic acid (DCA) levels and fatty acid synthase (FAS) mRNA expression in the liver, as shown in Figure 4A-E, respectively. We also found a negative association between hepatic OLFM2 mRNA expression and weight, jejunal TLR4 and jejunal TLR5 expression, as . Differential relative mRNA abundance of hepatic OLFM2 between, (A) absence or presence of portal inflammation, (C) lobular inflammation absence or presence and (E) ballooning absence or presence. Differential relative mRNA abundance of jejunal OLFM4 between, (B) absence or presence of portal inflammation, (D) lobular inflammation absence or presence and (F) ballooning absence or presence. OLFM, olfactomedin. A.U arbitrary units. Differences between groups were calculated using Mann-Whitney test. p < 0.05 was considered statistically significant.

Correlations of Relative mRNA Abundance of Hepatic OLFM2 and Jejunal OLFM4, with Clinical and Biochemical-Related Parameters
To broaden the study of OLFM2, we analysed their correlations with different parameters related to glucose and lipid metabolism, gut microbiota and inflammation in a cohort of women with MO. First, we observed positive correlations between hepatic OLFM2 mRNA expression and glucose, cholesterol, trimethylamine N-oxide (TMAO), deoxycholic acid (DCA) levels and fatty acid synthase (FAS) mRNA expression in the liver, as shown in Figure 4A-E, respectively. We also found a negative association between hepatic OLFM2 mRNA expression and weight, jejunal TLR4 and jejunal TLR5 expression, as was graphically represented in Figure 4F On the other hand, when we focused on the OLFM4 in the jejunum, we observed positive correlations between OLFM4 and circulating interleukin (IL)-8, IL-10, IL-17 levels and jejunal TLR9 expression, as shown in Figure 5A-D, respectively. On the other hand, when we focused on the OLFM4 in the jejunum, we observed positive correlations between OLFM4 and circulating interleukin (IL)-8, IL-10, IL-17 levels and jejunal TLR9 expression, as shown in Figure 5A-D, respectively.

Discussion
This study is important as it is the first to report an interesting association between OLFMs and NAFLD in hepatic and jejunal samples of women with MO. It is already known that more in-depth studies of the molecular mechanisms involved in NAFLD are necessary to find new therapeutic targets. In this sense, given that OLFMs are strongly linked to cellular processes [16,17] and have been related to some disorders, such as obesity and hepatocarcinoma (HCC), among others [20,[31][32][33][34], we wanted to evaluate the possible roles of OLFM2 and OLFM4 in NAFLD pathogenesis.
Regarding our first finding, when patients were classified according to their hepatic histopathology, OLFM2 mRNA expression in the liver increased when hepatic health worsened. Moreover, a significant increase in OLFM2 expression in the presence of NASH was observed when the cohort was classified into non-NASH and NASH groups. These facts seem to suggest that OLFM2 is involved in NAFLD progression, especially in NASH. Although OLFM2 mRNA abundance has been found in liver tissues [18], its expression has not been previously evaluated, and this protein has not been related to NAFLD. Therefore, OLFM2 in liver tissues seems to play a role in NAFLD progression in subjects with obesity, but further studies are needed in this field.
Then, when we evaluated hepatic OLFM2 and jejunal OLFM4 based on the steatosis stage of the disease, we found a significant increase in hepatic OLFM2 in patients with moderate and severe steatosis grades. Later, when we analysed the role of these OLFMs in accordance with steatohepatitis parameters, such as lobular and portal inflammation

Discussion
This study is important as it is the first to report an interesting association between OLFMs and NAFLD in hepatic and jejunal samples of women with MO. It is already known that more in-depth studies of the molecular mechanisms involved in NAFLD are necessary to find new therapeutic targets. In this sense, given that OLFMs are strongly linked to cellular processes [16,17] and have been related to some disorders, such as obesity and hepatocarcinoma (HCC), among others [20,[31][32][33][34], we wanted to evaluate the possible roles of OLFM2 and OLFM4 in NAFLD pathogenesis.
Regarding our first finding, when patients were classified according to their hepatic histopathology, OLFM2 mRNA expression in the liver increased when hepatic health worsened. Moreover, a significant increase in OLFM2 expression in the presence of NASH was observed when the cohort was classified into non-NASH and NASH groups. These facts seem to suggest that OLFM2 is involved in NAFLD progression, especially in NASH. Although OLFM2 mRNA abundance has been found in liver tissues [18], its expression has not been previously evaluated, and this protein has not been related to NAFLD. Therefore, OLFM2 in liver tissues seems to play a role in NAFLD progression in subjects with obesity, but further studies are needed in this field.
Then, when we evaluated hepatic OLFM2 and jejunal OLFM4 based on the steatosis stage of the disease, we found a significant increase in hepatic OLFM2 in patients with moderate and severe steatosis grades. Later, when we analysed the role of these OLFMs in accordance with steatohepatitis parameters, such as lobular and portal inflammation and hepatocyte ballooning, we found an increase in hepatic OLFM2 expression in patients with lobular inflammation. These findings indicate that OLFM2 increases as the disease progresses, since it is greatly increased in NASH, which is usually characterized by hepatic inflammation and elevated lipid content [3,35]. As we have previously mentioned, this is a novel finding; thus, no previous reports exist in this regard, not even reports on the expression of this protein in the liver. More studies are necessary to corroborate this evidence.
Unfortunately, although OLFM4 seems to play relevant roles as an anti-inflammatory molecule in Helicobacter pylori infection [31] and gastric and intestinal disorders [33,34], which could suggest that OLFM4 is involved in NAFLD pathogenesis through microbiota changes and the gut-liver axis, in our study, the mRNA expression of this protein in jejunal samples was not significantly different between the hepatic histopathological groups. In the literature there are no other studies in this area, but probably in those patients with MO who present an intestinal dysbiosis associated with obesity, there is an activation of inflammasomes and also of the immune system [36,37] that gives rise to an inflammatory microenvironment capable of masking the possible relationship that could have expression in the jejunum of OLFM4 with liver histology. Therefore, to further investigate this relationship, additional studies should be performed in subjects of normal weight.
To deepen the knowledge of OLFMs in regard to NAFLD, we analysed several correlations. Positive associations between hepatic OLFM2 mRNA and hepatic FAS mRNA expression and cholesterol and glucose levels, which are parameters closely related to NAFLD, were found [38,39]. When there is an excess of carbohydrates in the diet, glucose can be stored as lipids in the liver, and FAS, a lipogenic enzyme, synthesizes triglycerides from free fatty acids; therefore, its action enhances hepatic steatosis, the principal characteristic of NAFLD [39,40]. In addition, another study reported increased FAS mRNA expression and high glucose levels in people with NAFLD [41], so our current results are consistent with previously mentioned publications showing dysregulation of hepatic lipogenesis in NAFLD [42,43]. All this information, as well as our findings of positive correlations of OLFM2 in the liver with glucose and cholesterol levels and hepatic FAS, corroborates our previous hypothesis that hepatic expression of OLFM2 increases as NAFLD progresses, when at the same time, the metabolic imbalance also worsens [44].
Additionally, we found a negative significant correlation between hepatic OLFM2 and weight. This association is difficult to explain, given that González-García et al. observed that a global lack of OLFM2 induces weight loss [20]. This contradiction could be explained by the fact that our cohort presented a degree of severe obesity (BMI between 40 and 46 Kg/m 2 ), and perhaps some correlations may have been skewed due to such excessive BMI.
On the other hand, we also observed a positive correlation of hepatic OLFM2 with DCA and TMAO levels. TMAO is a gut microbiota-dependent metabolite, and some publications [42,45,46] reported increased TMAO levels in patients with NAFLD. The same authors suggested that TMAO could contribute to the development and severity of this disease due to its role in glucose regulation, cholesterol homeostasis and lipid absorption [45,47]. An increase in DCA levels, which are involved in the metabolism of ingested lipids, was also seen in NAFLD patients [48]. Additionally, Grzych et al. showed high levels of DCA in the presence of NASH [49]. Therefore, these positive associations of hepatic OLFM2 with DCA and TMAO levels remain consistent with previous results, reinforcing the hypothesis that OLFM2 may play an important role in the progression of NAFLD.
Later, we wanted to analyse whether OLFM2 in the liver is associated with jejunal TLRs, since these are involved in gut-liver axis crosstalk that can influence NAFLD appearance and progression [50]. In this regard, although we previously observed that hepatic OLFM2 mRNA expression is enhanced when the disease becomes severe, we observed a negative correlation between jejunal TLR4 and 5 with hepatic OLFM2 mRNA expression. This is a contradictory result given that TLRs are known to be involved in NAFLD progression [50], but this result may be because women in our cohort followed a very low-calorie diet for 3 weeks before bariatric surgery [51]. In this sense, Macedo Rogero et al. demonstrated that omega-3 polyunsaturated fatty acids, usually included in the very low-calorie diet [52], attenuate the activation of the TLR4 signalling pathway, exerting an anti-inflammatory effect [53] and inhibiting lipid accumulation [54]. Concerning TLR5, this receptor has been shown to not necessarily be altered by diet [55]. Otherwise, TLR5 seems to play a key role in liver protection against intestinal dysbiosis-induced NAFLD in mice [56], preventing gut inflammation-related disorders [57]. Moreover, an increase in TLR5 seems to contribute to liver regeneration events [58]. Hence, in this case, the negative correlation of TLR5 with OLFM2 mRNA expression in the liver makes sense given that OLFM2 increases as NAFLD worsens, while TLR5 in the gut could be decreased due to the high inflammation pattern induced by intestinal dysbiosis and liver damage. Similarly, mouse models without TLR5 expression were used to present obesity, hepatic steatosis and insulin resistance, the main characteristics of our study cohort [59].
Focusing on OLFM4, as stated above, its relative mRNA expression in the jejunum did not present significant differences between NAFLD groups, but we did observe positive associations with IL-8, IL-10 and IL-17 levels and jejunal TLR9 expression. In this sense, circulating IL-8 and IL-17 are defined as proinflammatory cytokines that present enhanced circulating levels in NAFLD [60][61][62]. Therefore, in this work, OLFM4 seems to be related to a proinflammatory state, despite previously being linked to an anti-inflammatory role in other tissues [31,63]. Hence, we cannot definitively conclude that OLFM4 in the jejunum is involved in gut dysbiosis-related inflammation because our patients presented low-grade chronic inflammation due to obesity [64], but it would be interesting to evaluate OLFM4 jejunal abundance in lean subjects.
IL-10 is a well-defined anti-inflammatory cytokine [65] with protective effects in liver injury [66], but high levels of circulating IL-10 have sometimes been reported in subjects with obesity [67]. Given that IL-10 attenuates the secretion of proinflammatory cytokines, a continuous increase in IL-10 levels could be explained by the competition of IL-10 with proinflammatory cytokines in an attempt to balance the inflammatory state of these patients [67]. Moreover, IL-10 exhibits feedback regulation to inhibit proinflammatory cytokine production [68]. These findings could explain the positive correlation between OLFM4 mRNA in the jejunum, which was previously correlated with proinflammatory cytokines, and IL-10 circulating levels, since this anti-inflammatory cytokine seems to counteract the low-grade chronic inflammatory pattern of our MO patients.
TLR9 is a receptor that is closely linked to metabolism and inflammatory events [69], and it has been suggested it plays a relevant role in NAFLD pathogenesis [50]. Additionally, it was clinically demonstrated that TLR9 is a key driver of NASH [69,70]. Hence, the positive association of TLR9 with OLFM4 jejunal expression also makes sense, as it is consistent with its correlation with proinflammatory cytokines. This finding reinforces the possible inflammatory role of OLFM4 in the jejunum, but more studies are needed.
In summary, we have carried out a novel study showing that the hepatic expression of OLFM2 seems to increase as NAFLD progresses, and this protein seems to be associated with NASH and with a severe steatosis pattern in NAFLD patients. Additionally, OLFM4 was suggested to be more related to inflammation that occurs due to intestinal dysbiosis, usually linked to NAFLD. In this sense, we are pioneers in demonstrating a relationship between OLFM2 and NAFLD and between OLFM4 and inflammation related to gut dysbiosis. However, there are some limitations to this study; for example, in the literature, there is little or no experience in this field. In addition, this is a cross-sectional study, so a causal relationship cannot be confirmed. Lastly, a cohort of patients made up of only MO women was used, so these results cannot be extrapolated to other sexes or other groups of people with obesity, overweight or normal weight. Hence, future studies with a larger cohort and assessing the role of these proteins in lean subjects with NAFLD, are needed to understand the specific role of these OLFMs in the pathogenesis of NAFLD and their relationship with the gut-liver axis. Moreover, to analyse OLFMs expression in in vivo and in vitro models of liver lipid overload would also be necessary. In addition, it could also be interesting to evaluate the OLFM4 expression in other sections of the gastrointestinal tract to elucidate its involvement in gut inflammation and its relationship to NAFLD.

Subjects
The institutional review board (Institut Investigació Sanitària Pere Virgili (IISPV) CEIm; 23c/2015; 11 May 2015) approved this research. Informed written consent was obtained from all participants. The cohort was formed of 69 Caucasian women with MO (BMI > 40 kg/m 2 ). Liver and jejunum biopsies were collected during a planned laparoscopic bariatric surgery and liver samples were indicated for clinical diagnosis. The exclusion criteria were as follows: (1) an intake of ethanol higher than 10 g/day or other toxins; (2) patients who had infectious disease or neoplastic disease or an acute or chronic hepatic disease different from NAFLD; (3) menopausal women or women using contraceptives in order to avoid the interference of hormones which can cause biases in glucose and lipid metabolism as well as in cytokine determinations; (4) patients treated with fibrates because this treatment can interfere with the metabolism of some microbiota-derived metabolites studied in this work.

Sample Size
The work was mainly focused on defining the specific role of OLFM2 in hepatic tissue and OLFM4 in jejunum samples in MO patients with or without NAFLD. To achieve our objective, sample sizes were calculated using a GRANMO calculator accepting an alpha risk of 0.05 and a beta risk of less than 0.2 in a bilateral contrast; 25 subjects in the first group (NL: control group) and 50 in the second (NAFLD: SS and NASH) are needed to detect as statistically significant the difference between two proportions, which for group 1 is expected to be of 0.33 and group 2 of 0.67. The ARCSINUS approach was used.

Liver Pathology
Liver samples were classified by the method described elsewhere [71,72], using haematoxylin and eosin and Masson's trichrome stains. They were then scored by an experienced hepatopathologist. According to their hepatic histopathology, women with MO were classified into NL (n = 27) and NAFLD (n = 42). Patients with NAFLD were subclassified SS (micro/macrovesicular steatosis without inflammation or fibrosis, n = 26), and NASH (Brunt grades 1-2, n = 16). None of the patients with NASH in our cohort presented fibrosis.

Biochemical Analyses
Physical, anthropometric, and biochemical evaluation were performed on all the studied cohort. Blood samples were extracted through a BD Vacutainer ® system by specialized nurses, after overnight fasting and before bariatric surgery. Venous blood samples were obtained in tubes with or without ethylenediaminetetraacetic acid, which were separated in plasma and serum aliquots by centrifugation (3500 rpm, 4 • C, 15 min). Conventional automated analyser was used to analyse biochemical parameters. IR was estimated using homeostatic model assessment for IR (HOMA1-IR). Cytokines, such as interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-17, TNF-α and adiponectin, were determined using multiplex sandwich immunoassays and the MILLIPLEX MAP Human Adipokine Magnetic Bead Panel 1 (HADK1MAG-61K, Millipore, Billerica, MA, USA), the MILLIPLEX MAP Human High-Sensitivity T Cell Panel (HSTCMAG28SK, Millipore, Billerica, MA, USA), and the Bio-Plex 200 instrument, according to the manufacturer's instructions. Absolute quantification of circulating bile acids, choline, trimethylamine (TMA), trimethylamine N-oxide (TMAO), betaine and short-chain fatty acids were analysed by liquid chromatography coupled to triple-quadrupole-mass spectrometry (LC-QqQ). All these analyses were assessed at the Center for Omic Sciences (Rovira i Virgili University-Eurecat).

mRNA Expression in Liver and Jejunum
Hepatic and jejunal samples were collected during bariatric surgery and conserved in tubes with RNAlater (Qiagen, Hilden, Germany) at 4 • C. Samples were then processed and stored at −80 • C. RNeasy mini kit (Qiagen, Barcelona, Spain) was used to extract total RNA from the liver and the jejunum. Reverse transcription to cDNA was performed with the High-Capacity RNA-to-cDNA Kit (Applied Biosystems, Madrid, Spain). Realtime quantitative PCR was carried out with the TaqMan Assay predesigned by Applied Biosystems for the detection of OLMF2 (Hs01017934_m1) mRNA in the liver and OLFM4 (Hs00197437_m1) mRNA in the jejunum; we also evaluated the mRNA of some hepatic lipid metabolism-related genes such as sterol-regulatory-element-binding protein 1c (SREBP1c) (Hs01088691_m1), liver X receptor alpha (LXRα) (Hs00173195_m1), fatty acid synthase (FAS) (Hs00188012_m1); and TLRs in jejunum (TLR2 (Hs02621280_s1), TLR4 (Hs00152939_m1), TLR5 (Hs05021301_s1), TLR9 (Hs00370913_s1)). The expression of each gene was calculated standardized to the mRNA expression of 18S RNA (Fn04646250_s1) for hepatic genes, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hs02786624_g1) for jejunal genes, after they were normalized using the control group (NL) as a reference. All reactions were duplicated in 96-well plates using the QuantStudio™ 7 Pro Real-Time PCR System (Applied Biosystem, Foster City, CA, USA).

Statistical Analysis
The data were analysed using the SPSS/PC+ for Windows statistical package (version 27.0; SPSS, Chicago, IL, USA). The distribution of variables was obtained using the Kolmogorov-Smirnov test. All results were expressed as the median and the interquartile range (25th-75th). The different comparative analyses were assessed using Mann-Whitney U test to compare groups. The coefficient of correlation (rho) between variables was calculated using Spearman's method. p-values < 0.05 were statistically significant. Graphics were elaborated using GraphPad Prism software (version 7.0; GraphPad, San Diego, CA, USA).

Conclusions
In conclusion, the first novel finding of this study is that OLFM2 expression in the liver seems to play a relevant role in NAFLD progression in women presenting MO. Secondly, our results suggested that jejunal OLFM4 expression could be involved in gut dysbiosisrelated inflammatory events in women with MO. In any case, more research in this field is necessary to confirm these hypotheses.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Conflicts of Interest:
The authors declare no conflict of interest.