Identifying Meibomian Gland Dysfunction Biomarkers in A Cohort Of Patients Affected By DM Type II Part Ⅱ

3. Results 

One hundred twenty-three patients with type II DM with symptoms of MGD attended the Department of Ophthalmology at A. Fiorini Hospital in Terracina (Latina, Italy). Of these, 69 patients had a recent history of cataract surgery, four had thyroid disease, 22 were heavy smokers, three were taking anti-androgen therapy, and seven menopause hormonal replacement therapy; therefore, 18 female patients (14.6%) met the inclusion criteria and were included in the study. Demographics and clinical signs of the studied subjects are summarised in Table 2. 

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Seventy percent of patients had a Schirmer test I value < 10 mm/5 min and 94.1% of patients had a Schirmer test II <10 mm/5 min and 47.1% of patients had a value lower than 4 mm/5 min. TFBUT was lower than 5 s in 94.4% of patients. The median value of the ML score was 3 and vBs was 2. OSDI score was higher than 20 in 53.1%; the median value was 28.5. Fifty-six percent of patients were classified as normal weight according to their BMI percentile and 44% were overweight.

3.1. Blood Biomarkers 

Mean glycemia values were 124.56 ± 16.65 mg/dL (min 88 max 152), and Hb1Ac of 6.5% ± 0.69 (min 5.7%, max 8.3%). Seventy-five percent of patients had a value of Hb1Ac ≤ 6.5%. Mean triglycerides levels were: 145.5 ± 67.9 mg/dL (min 70 max 319); mean total cholesterol was 165 ± 32.59 mg/dL (min 117 max 245); mean HDL value was 45.5 ± 9.15 mg/dL (min 32 max 60); mean LDL was 90.3 ± 20.3 mg/dL (min 54 max 129); mean testosterone level was 1.0 ±1.7 nmol/L (min 0.13 max 5.14); mean DHEA-S value was 122.37 ± 110.52 µg/dL (min 40.5 max 343); mean ASD value was 1.6 ± 0.93 ng/dL (min 0.35 max 3.29); mean cortisol value was 11.54 ± 3.46 µg/dL (min 6.8 max 17.9). 

DHEA-S showed a negative correlation with age (r = −0.88; p = 0.02) and with the vBS (r = −0.78; p = 0.02). Cortisol and ASD were positively correlated with MG length (r = 0.51; p = 0.04 and r = 0.75; p = 0.02), while triglycerides had a negative correlation (r = −0.51; p = 0.02). Scatter plots of the significantly correlated variables are reported in Figure 4. No significant correlation was found for the other parameters analyzed.

3.2. Meibography Analysis 

An average of 17.12 ± 2.83 MGs were identified in each eyelid analyzed (min 13 – max 18). The mean total MG length was 1023.94 ± 213.07 mm/mm2 (min 653 max 1384.) The MG lengths had a negative association with the loss of MG area (r = −0.51; p = 0.02). The mean MG width was 75.88 ± 15.53 mm/mm2 and was negatively correlated to Schirmer test II and TFBUT (r = −0.5; p = 0.02; r = −0.56; p = 0.01). The MG area was 172.69 ± 31.29 mm2 and was also negatively correlated to Schirmer test II (r= −0.49; p = 0.04;). 

Fifty percent of patients had a meiboscore value of 2 (loss of 26–50% of MGs) and the other 50% had a meiboscore of 1 (loss of ≤25%). Both meiboscore and loss of MG area were positively associated with age (r = 0.55; p = 0.01 and r = 0.47; p = 0.04). There was a statistically significant difference between the loss of MG area and age groups as determined by one-way ANOVA (F = 5.61, p =0.015). 

A Tukey post hoc test revealed that there was a statistically significant difference between the older group and patients younger than 60 years (p = 0.01) and the group of patients between 61 and 79 years (p = 0.04). However, there were no differences between 61–79 years (p = 0.75). The mean Loss of MG area was 26.29 ± 11.62% (min 5–max 47). Loss of MG area was higher in the older group (≥80 years; four patients mean value 39.8 ± 6.61%) in comparison to a group of patients of 61–79 years (seven patients, mean value 24.23 ± 11.87%) and ≤ 60 years (seven patients, mean value 20.64 ± 7.47%); see Figure 5. There was a high agreement between observers in calculating MG length, width, area, and meiboscore (Intraclass correlation coefficient (ICC), respectively, of 0.98; 0.88, 0.96, and 0.94).

Regression analysis was performed between MG parameters obtained by meibography and serum biomarkers. Results are reported in Table 3.

Regression analysis using a pairwise selection method identified that age had a significant impact on the loss of MG area (F = 5.4; p = 0.02). There was a 19.1% difference between the mean MG loss area of patients older than 80 years and patients younger than 60 years. The difference was statistically significant (IC 95% 4.2–34.1; p = 0.01). Similarly, the 15.5% difference between the loss of MG area of patients 61–79 years old and patients older than 80 years was statistically significant (IC95% 0.67–30.45; p = 0.04). In contrast, no statistically significant value was identified for all other analyzed variables.

4. Discussion and Conclusions 

The purpose of this study was to identify any serum biomarkers in patients affected by type II DM and MGD. Cortisol and ASD were associated with an increase in MG tortuosity and a high level of triglycerides was associated with a reduction of MG length. Moreover, a decay of DHEAS was correlated to a negative modification of ocular surface staining using lissamine green (vBs score), and MG width was negatively correlated with Schirmer test II and TFBUT.

4.1. Role of Age and Diabetes 

Previous studies have demonstrated how aging is a key factor for the development of MGD [18–20] deriving from MG atrophy [21] and a decrease in meibocyte differentiation [19]. Hashemi et al. [20] reported an MGD prevalence of 71.2% in patients older than 60 years. Furthermore, the prevalence gradually increased from 64.4% in the group of 60–64 years to 82.4% in patients older than 80 years [19]. 

Our paper reported a similar finding in that the loss of MG area was more common in those more than 80 years old. In this group of patients, there was a mean loss of MG area equal to 40% compared to a mean of 20% in the younger group. This reduction of MG area can lead to a reduction of meibomian in the tear film and lead to an increase in evaporative DES. However, data obtained from meibography alone cannot discriminate MGD from non-MGD changes as a percentage of MG atrophy is normal amongst older people; thus, morphological and functional MG tests are necessary to determine the presence of MGD. The association between type II DM and MGD has already been reported in the literature [6,22–28]. 

A prospective randomized controlled trial conducted to investigate MG and tear film function in type 2 DM patients found eyelid margin abnormalities to be significantly higher and the number of expressible glands to be significantly lower compared to a control group. Tao Yu et al. [22,26] showed that 57.6% of people in the DM group had MG dropout, while it was 33% in the control group. In addition, they described modifications of MGs, such as enlargement of acinar units, irregular shape with acinus, and reduction in density of acinar units. The authors theorized that there may be a certain degree of occlusion of the MG ductal in patients with type 2 DM. 

The obstruction of the MG is ductal leads to accumulation of meibum, thus causing the cystic dilatation and the altered morphology of the acini, and further causing the atrophy of the acini and decrease in acinar density. These findings supported our result of MG loss and modification of ocular surface staining as a reduction of MG acini function. We also found that MG width was negatively correlated to TFBUT and Schirmer test II value. 

This means that this MG width can predict the reduction of the function of MGs. We hypothesized that if the MG body accumulates meibomian and it is not able to express it through orifices, it can lead to progressive MG atrophy. This condition is reflected by ocular surface parameters that influenced negatively both the TFBUT and the Schirmer test. Fan Fang et al. [23] investigated the relationship between MGD and levels of HbA1c in patients with DM type II and reported that HbA1c ≥ 7% is likely to result in MG dysfunctions, especially related to lipid layer thickness and MGs percentage loss. 

We performed a similar analysis dividing patients according to their level of HbA1c, but we did not identify any differences among the variables analyzed. This fact could be related to our small sample but also a population of diabetics with good glycemic compensation and low fluctuation of HbA1c. Only five patients had Hba1c values higher than 7%, so a comparison between these groups cannot provide useful information. It would be interesting, to understand how much age and diabetes affect the MG change, to study a large sample over the years including non-diabetic patients and compare meibography changes according to glycemia value.

4.2. Hormones 

Role The role of androgens in MG function has been already elucidated [29–31]. The MGs are sebaceous glands, and androgens are well known to regulate the development, differentiation, and lipid production of sebaceous glands throughout the body [29,32]. Sebaceous gland activity and secretion decrease with age, and this aging-associated dysfunction has been correlated with both atrophy of acinar cells and a reduction in serum androgen levels [33]. Sullivan et al. have demonstrated that aging in men and women goes together with a significant increase in lower eyelid erythema, telangiectasia, keratinization, irregular posterior margins, orifice metaplasia, and opaque secretions [34]. 

Moreover, the assumption of anti-androgen medications leads to MGD, altered lipid profiles in MG secretions, decreased tear film stability, and evaporative dry eye [35]. Our population reflected these studies; DHEAS decreased with age, and it was associated with a modification of ocular surface staining (vBS). The regression analysis between DHEAS and age showed that age predicts DHEAS level well (F = 17.9; p < 0.001), suggesting that 52% of the variation was predicted by age. 

The decrease in the production of DHEAS with age is considered to play an important role in IL-6-mediated pro-inflammatory effects in humans [36]. DHEAS and ASD concentration-dependently inhibited IL-6 production from peripheral blood mononuclear cells but their levels significantly decreased with age and this fact leads to the rise in IL-6 production during the process of aging [36]. This finding could be a significant cofactor for the manifestation of inflammatory and age-related diseases such as MGD. 

Higher tear levels of interleukin (IL)-1β, IL-6, chemokine IL-8, IL-10, IFN-γ, and tumor necrosis factor-α, TNF-α have been found in dry eye patients [10]; further studies are needed to define if IL-6 in MGD patients can be a good biomarker of this pathology. A high level of cortisol was associated with a higher level of IL-6 and the severity of inflammation was reported to be associated with a low DHEAS/cortisol ratio [37]. Interestingly, we highlighted also that both cortisol and ASD were positively associated with MG length; this result could mean that these hormones are biomarkers of MG length and that they can have a role in the follow-up of MGD patients. However, only ASD showed a significant result in the regression analysis. ASD predicts a significant effect on the MG length (F = 4.72; p < 0.045), suggesting that 22% of the variation is predicted by this factor. This means that it can be considered as a serum biomarker of MGs modification even if a large sample needs to be studied to confirm this theory.

4.3. Blood Lipid Levels BMI and Role 

A systematic metanalysis reported a strong positive correlation between dyslipidemia and MGD and suggested performing prospective studies to demonstrate a temporal relationship with MGD preceding dyslipidemia [38]. In our study, we do not identify this association because our population had an overall normal value of total cholesterol and only one patient had a value higher than 200 mg/mL, so the sample was not representative of this condition. Interestingly, a high level of triglycerides was related to a reduction of MG length. Butovich et al. [39] described a new condition that they termed High Triglycerides/Low Waxes (HTLW) syndrome. 

They observed severely decreased pools of normal meibomian lipids such as wax esters and cholesteryl esters in meibum and tears, and a 20× to 30× rise in the triglyceride fraction over the norm without any change in the routine blood lipid panel test. Our association between high triglyceride level and reduction of MG length is quite innovative and we can assume that it is the first morphology change in meibomian shape that reflects the modification in the meibomian secret. A change in the meibomian component may modify its viscosity and cause the pressure to express meibomian from orifices to increase. Tao Yu et al. [26] identified an increase in resistance to the outflow of meibomian in patients with type 2 DM due to some MG orifice obstruction. 

We believe that there are at least two factors to justify these changes. From one side, there is an increase in meibomian viscosity, and from the other side, MG orifice obstruction is due to keratinization related to the pro-inflammatory process. This increase in pressure in the lumen of MGs could be an explanation for the increasing MG width, and this anatomical change can lead MGs to become atrophic. BMI percentile was found to be a predictor of MG tortuosity and atrophy in pediatric patients [40] and also a risk for MGD in the adult population [41]. 

In our research, BMI was not associated with any parameter analyzed. Other colleagues [38] reported similar findings. In our population, BMI distribution was not representative because patients were classified only as normal and overweight without any patients in the obese percentile. This study has several limitations; the sample we analyzed is small and was obtained from a single medical center. The incorporation of elderly subjects may also cloud the relationship between blood sample lipid abnormalities and MGD. 

In addition, our sample was representative only of female patients. In our center, male patients complaining of MGD symptoms were very few and many of them were taking anti-androgen medication or they were heavy smokers. Both were exclusion criteria for our study. To provide a correct representation of the population, the sample would need to be increased including also male patients. All data were obtained from a single visit and meibography was performed at only one point in time without prospective longitudinal studies, so it is difficult to ascertain whether MG changes in morphology were associated with DM status and/or change in hormone levels. In addition, the absence of control groups cannot allow to state any differences between MGD and non-MGD patients. 

Overall, this study provides a starting point for further research. We believe that the identification of clinical biomarkers is extremely useful for treating these patients, especially in those centers where meibography is not available. Cortisol, DHEAS, ASD, and triglyceride levels showed interesting associations in our diabetic population. Regarding meibography in patients with type 2 DM, MG width enlargement seems to be the primary movement and its early detection can lead to an improvement in patient treatment. 

We cannot state that early treatment can halt the progression of the disease and reduce the loss of MG area but it would be interesting to study the evolution of this parameter in longitudinal studies. Even if this study has several limitations, it is the first study to have reported some associations between MG parameters obtained by meibography and serum biomarkers. Future studies, perhaps including meibum lipid analysis and tear cytokine levels, may also further elucidate the connection between these parameters, MG architecture, and function.

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References 

1 Tiffany, J.M. Physiological Functions of the Meibomian Glands. Prog Retinal Eye Res. 1995, 14, 47–74. [CrossRef] 

2. Rolando, M.; Merayo-Lloves, J. Management Strategies for Evaporative Dry Eye Disease and Future Perspective. Curr. Eye Res. 2022, 47, 813–823. [CrossRef] [PubMed] 

3. Sullivan, D.A.; Sullivan, B.D.; Evans, J.E.; Schirra, F.; Yamagami, H.; Liu, M.; Richards, S.M.; Suzuki, T.; Schaumberg, D.A.; Sullivan, R.M.; et al. Androgen Deficiency, Meibomian Gland Dysfunction, and Evaporative Dry Eye. Ann. N. Y. Acad. Sci. 2002, 966, 211–222. [CrossRef] 

4. Bu, J.; Wu, Y.; Cai, X.; Jiang, N.; Jeyalatha, M.V.; Yu, J.; He, X.; He, H.; Guo, Y.; Zhang, M.; et al. Hyperlipidemia induces meibomian gland dysfunction. Ocul. Surf. 2019, 17, 777–786. [CrossRef] [PubMed] 

5. Ding, J.; Liu, Y.; A Sullivan, D. Effects of Insulin and High Glucose on Human Meibomian Gland Epithelial Cells. Investig. Opthalmology Vis. Sci. 2015, 56, 7814–7820. [CrossRef] [PubMed] 

6. Yoo, T.K.; Oh, E. Diabetes mellitus is associated with dry eye syndrome: A meta-analysis. Int. Ophthalmol. 2019, 39, 2611–2620. [CrossRef] 

7. Botion, L.M.; Green, A. Long-term regulation of lipolysis and hormone-sensitive lipase by insulin and glucose. Diabetes 1999, 48, 1691–1697. [CrossRef] 

8. Wei, Y.; Asbell, P.A. The Core Mechanism of Dry Eye Disease Is Inflammation. Eye Contact Lens: Sci. Clin. Pr. 2014, 40, 248–256. [CrossRef] 

9. Kang, M.H.; Kim, M.K.; Lee, H.J.; Lee, H.I.; Wee, W.R.; Lee, J.H. Interleukin-17 in Various Ocular Surface Inflammatory Diseases. J. Korean Med Sci. 2011, 26, 938–944. [CrossRef] 

10. Acera, A.; Rocha, G.; Vecino, E.; Lema, I.; Durán, J.A. Inflammatory Markers in the Tears of Patients with Ocular Surface Disease. Ophthalmic Res. 2008, 40, 315–321. [CrossRef] 

11. Schiffman, R.M.; Christianson, M.D.; Jacobsen, G.; Hirsch, J.D.; Reis, B.L. Reliability and Validity of the Ocular Surface Disease Index. Arch. Ophthalmol. 2000, 118, 615–621. [CrossRef] [PubMed] 

12. Yamaguchi, M.; Kutsuna, M.; Uno, T.; Zheng, X.; Kodama, T.; Ohashi, Y. Marx Line: Fluorescein Staining Line on the Inner Lid as Indicator of Meibomian Gland Function. Am. J. Ophthalmol. 2006, 141, 669–669.e8. [CrossRef] [PubMed] 

13. van Bijsterveld, O.P. Diagnostic Tests in the Sicca Syndrome. Arch. Ophthalmol. 1969, 82, 10–14. [CrossRef] [PubMed] 14. Yanoff, M.; Duker, J.S. (Eds.) Ophthalmology: ExpertConsult.Com, 4th ed.; Elsevier Saunders: Philadelphia, PA, USA, 2014; ISBN 978-1-4557-3984-4. 

15. Pult, H.; Riede-Pult, B. Comparison of subjective grading and objective assessment in meibography. Contact Lens Anterior Eye 2013, 36, 22–27. [CrossRef] [PubMed] 

16. Müller, R.T.; Abedi, F.; Cruzat, A.; Witkin, D.; Baniasadi, N.; Cavalcanti, B.; Jamali, A.; Chodosh, J.; Dana, R.; Pavan-Langston, D.; et al. Degeneration and Regeneration of Subbasal Corneal Nerves after Infectious Keratitis. Ophthalmology 2015, 122, 2200–2209. [CrossRef] 

17. Setu, A.K.; Horstmann, J.; Schmidt, S.; Stern, M.E.; Steven, P. Deep learning-based automatic meibomian gland segmentation and morphology assessment in infrared meibography. Sci. Rep. 2021, 11, 7649. [CrossRef] 

18. Den, S.; Shimizu, K.; Ikeda, T.; Tsubota, K.; Shimmura, S.; Shimazaki, J. Association Between Meibomian Gland Changes and Aging, Sex, or Tear Function. Cornea 2006, 25, 651–655. [CrossRef]

19. Nien, C.J.; Massei, S.; Lin, G.; Nabavi, C.; Tao, J.; Brown, D.J.; Paugh, J.R.; Jester, J.V. Effects of age and dysfunction on human meibomian glands. JAMA Ophthalmol 2011, 129, 462–469. [CrossRef] 

20. Hashemi, H.; Asharlous, A.; Aghamirsalim, M.; Yekta, A.; Pourmatin, R.; Sajjadi, M.; Pakbin, M.; Asadollahi, M.; Khabazkhoob, M. Meibomian gland dysfunction in the geriatric population: Tehran geriatric eye study. Int. Ophthalmol. 2021, 41, 2539–2546. [CrossRef] 

21. Hykin, P.G.; Bron, A.J. Age-Related Morphological Changes in Lid Margin and Meibomian Gland Anatomy. Cornea 1992, 11, 334–342. [CrossRef] 

22. Yu, T.; Shi, W.-Y.; Song, A.-P.; Gao, Y.; Dang, G.-F.; Ding, G. Changes of meibomian glands in patients with type 2 diabetes mellitus. Int. J. Ophthalmol. 2016, 9, 1740–1744. [CrossRef] [PubMed] 

23. Fan, F.; Li, X.; Li, K.; Jia, Z. To Find Out the Relationship Between Levels of Glycosylated Hemoglobin with Meibomian Gland Dysfunction in Patients with Type 2 Diabetes. Ther. Clin. Risk Manag. 2021, 17, 797–807. [CrossRef] 

24. Abu, E.K.; O Ofori, A.; Boadi-Kusi, S.B.; Ocansey, S.; Yankah, R.K.; Kyei, S.; Awuku, A.Y. Dry eye disease and meibomian gland dysfunction among a clinical sample of type 2 diabetes patients in Ghana. Afr. Heal. Sci. 2022, 22, 293–302. [CrossRef] [PubMed] 

25. Lin, X.; Xu, B.; Zheng, Y.; Coursey, T.G.; Zhao, Y.; Li, J.; Fu, Y.; Chen, X.; Zhao, Y.-E. Meibomian Gland Dysfunction in Type 2 Diabetic Patients. J. Ophthalmol. 2017, 2017, 3047867. [CrossRef] [PubMed] 

26. Yu, T.; Han, X.-G.; Gao, Y.; Song, A.-P.; Dang, G.-F. Morphological and cytological changes of meibomian glands in patients with type 2 diabetes mellitus. Int. J. Ophthalmol. 2019, 12, 1415–1419. [CrossRef] [PubMed] 

27. Wu, H.; Fang, X.; Luo, S.; Shang, X.; Xie, Z.; Dong, N.; Xiao, X.; Lin, Z.; Liu, Z. Meibomian Glands and Tear Film Findings in Type 2 Diabetic Patients: A Cross-Sectional Study. Front. Med. 2022, 9, 762493. [CrossRef] 

28. Arunachalam, C.; Shamsheer, R. A clinical study of meibomian gland dysfunction in patients with diabetes. Middle East Afr. J. Ophthalmol. 2015, 22, 462–466. [CrossRef] 

29. A Sullivan, D.; Sullivan, B.D.; Ullman, M.D.; Rocha, E.; Krenzer, K.L.; Cermak, J.M.; Toda, I.; Doane, M.G.; E Evans, J.; A Wickham, L. Androgen influence on the meibomian gland. Investig. Opthalmology Vis. Sci. 2000, 41, 3732–3742.