DOI: 10.

1159/000507197
Received: 10/28/2019
Accepted: 3/7/2020
Published(online): 3/13/2020
---------------
The effect of diabetes mellitus on corneal endothelial cells and central corneal thickness
: A case-control study
Papadakou P. Chatziralli I. Papathanassiou M. Lambadiari V. Siganos C.S. Theodossiadis P.
Kozobolis V.
---------------
ISSN: 0030-3747 (Print), eISSN: 1423-0259 (Online)
https://www.karger.com/ORE
Ophthalmic Research

t
---------------

ip
Disclaimer:

cr
Accepted, unedited article not yet assigned to an issue. The statements, opinions and data contained in this
us
publication are solely those of the individual authors and contributors and not of the publisher and the
editor(s). The publisher and the editor(s) disclaim responsibility for any injury to persons or property
resulting from any ideas, methods, instructions or products referred to in the content.
an

Copyright:
m

All rights reserved. No part of this publication may be translated into other languages, reproduced or
utilized in any form or by any means, electronic or mechanical, including photocopying, recording,
ed

microcopying, or by any information storage and retrieval system, without permission in writing from the
publisher.
© 2020 S. Karger AG, Basel
pt

---------------
ce
Ac

137.132.123.69 - 3/17/2020 6:31:15 PM

National Univ. of Singapore
Downloaded by:
 1

The effect of diabetes mellitus on corneal endothelial cells and central corneal

thickness: A case-control study

Running title: Corneal changes in diabetes

Authors:

Panagiota Papadakou1*, Irini Chatziralli1*, Miltiadis Papathanassiou1, Vaia

Lambadiari2, Charalambos S. Siganos3, Panagiotis Theodossiadis1, Vassilios

t
Kozobolis4

ip
cr
*
us
Panagiota Papadakou and Irini Chatziralli have equal contribution to the manuscript.
an
m

Affiliation:
1
2nd Department of Ophthalmology, National and Kapodistrian University of Athens,
ed

Athens, Greece
pt

2 nd
ce

2 Department of Internal Medicine, Research Institute and Diabetes Center,

National and Kapodistrian University of Athens, Athens, Greece

Ac

3
Ophthalmology Department, University of Crete, Heraklion, Crete, Greece
4
Eye Institute of Thrace, Democritus University of Thrace, Alexandroupolis, Greece

Corresponding author and reprints requests:

Irini Chatziralli

Attikon University Hospital,

1, Rimini street, Haidari, 12462, Athens, Greece

Tel: 00306973046326; Email: eirchat@yahoo.gr

Keywords: corneal endothelium; diabetic retinopathy; endothelial cell density

137.132.123.69 - 3/17/2020 6:31:15 PM
National Univ. of Singapore
Downloaded by:
 2

Abstract

Purpose: To evaluate the characteristics of corneal endothelial cells and central corneal

thickness (CCT) in patients with diabetes mellitus (DM), to compare them with those

of healthy subjects (controls) and to determine potential factors, affecting the corneal

parameters in patients with DM.

Methods: Participants in this study were 72 patients with DM and 88 healthy controls.

Diabetic patients were further classified into 4 groups depending on severity of diabetic

retinopathy (no retinopathy, mild, moderate, severe non-proliferative diabetic

t
retinopathy and proliferative diabetic retinopathy). All participants underwent non-

ip
cr
contact specular microscopy to evaluate corneal endothelium parameters and CCT,
us
while factors affecting endothelial cell density and CCT in patients with DM were also
an
analyzed.
m

Results: Patients with DM presented significantly decreased endothelial cell density

compared to controls (2297.9±311.3 and 2518.3±243.7 cells/mm2 respectively,

ed

p<0.001), while the two groups did not differ significantly in any other measured
pt
ce

corneal parameter. In the diabetic group, the multivariate analysis showed a significant

association between decreased endothelial cell density and increased HbA1c (p<0.001),
Ac

longer DM duration (p=0.003) and more severe diabetic retinopathy status (p=0.008).

Conclusion: Diabetes mellitus seems to affect corneal endothelium, since endothelial

cell density was decreased in the diabetic group while duration of disease, HbA1c levels

and severity of retinopathy were significantly associated with changes in endothelial

cell density and should be taken into account.

137.132.123.69 - 3/17/2020 6:31:15 PM
National Univ. of Singapore
Downloaded by:
 3

Introduction

Diabetes mellitus (DM) is a systemic metabolic disease, affecting over 400

million people worldwide and it is expected to affect 642 million by 2040 [1]. It is

commonly associated with the development of long-term macrovascular and

microvascular complications and can affect almost all tissues of the human body,

including the eye [2]. Diabetic retinopathy (DR) is one of the most common

complications of DM and is the leading cause of blindness among adults under 45 years

old in the industrialized world [3,4]. Besides DR, patients with DM are prone to

t
developing corneal endothelial damage, such as endothelial defects, punctate epithelial

ip
cr
keratopathy, recurrent corneal erosions and persistent epithelial defects [5,6]. Indeed,
us
although diabetic keratopathy (DK) is often subclinical, it may occur in up to 70% of
an
patients with DM [7].
m

Previous studies have also shown that corneal endothelial cells in patients with

DM have morphological abnormalities, including decrease in endothelial cell density

ed

(ECD) and hexagonality, increase in polymegathism, pleomorphism and central corneal

pt
ce

thickness (CCT) [8-12]. These findings could be attributed to the chronic metabolic

alterations at the cellular level due to hyperglycemia, affecting primarily the single layer
Ac

of the coherent endothelial cells [13], while the damaged corneal endothelial function

can disturb the balance of stromal hydration, leading to corneal edema, changes in

corneal transparency and reduced corneal sensitivity in patients with DM [12].

Although morphological changes in corneal endothelium and CCT have been

previously described in patients with DM, there is no consistency between studies. In

addition, most of the studies had small sample size and lacked control group.

Considering the above, the purpose of this study was to evaluate the characteristics of

corneal endothelial cells and the CCT in patients with DM and to compare them with
137.132.123.69 - 3/17/2020 6:31:15 PM
National Univ. of Singapore
Downloaded by:
 4

those of healthy subjects (controls). Moreover, this study aims to determine potential

factors, affecting the corneal parameters in patients with DM.

Methods

Participants in this study were 72 patients with DM, who were recruited at 2nd

Department of Ophthalmology, University of Athens, Greece between May and August

2019. The diagnosis of DM was based on medical history and all subjects were on oral

or parenteral antidiabetic medication. In addition, 88 subjects without DM were

recruited at the same period and served as controls. One eye was randomly chosen per

t
ip
patient, so as to avoid bias due to the intercorrelation of values between the eyes of the

cr
same patient. Eyes with previous ocular surgery or trauma, any corneal disease, mature
us
cataract, retinal diseases other than DR, intraocular inflammation, contact lens use,
an

glaucoma and pseudoexfoliation were excluded from the study.

m

The demographic characteristics (age, gender), the medical history and

ed

comorbidities (hypertension, hyperlipidemia) of all participants were recorded. Serum

pt

glycosylated hemoglobin (HbA1c) was measured in all patients with DM to evaluate

ce

their glycemic status. All patients underwent complete ophthalmological examination,

Ac

including best-corrected visual acuity measurement, intraocular pressure (IOP)

measurement, slit-lamp examination and dilated fundoscopy. Patients with DM were

classified based on the severity of DR, as follows: without any DR, mild non-

proliferative DR (NPDR), moderate NPDR, severe NPDR, and proliferative DR (PDR).

Furthermore, all participants underwent corneal specular microscopy, using

TOMEY EM-3000 by the same examiner (PP). Patient’s chin was placed on the chin

rest and his/her forehead rested on the special head area. The patient was asked to fixate

his/her eye for a few seconds on the red light coming from inside the device until the
137.132.123.69 - 3/17/2020 6:31:15 PM

instrument automatically took a clear image of the corneal endothelium and measured
National Univ. of Singapore
Downloaded by:
 5

the CCT at the same time. Corneal ECD (cells ⁄mm2), the mean cell area (μm2) and

variation in size of endothelial cells (CV) as an index of the extent of variation in cell

area (polymegathism), the percentage of hexagonal cells as an index of variation in the

cell shape (pleomorphism), and CCT were analyzed.

Comparisons among the two groups were performed, using t-test or Mann-

Whitney-Wilcoxon test for continuous variables and Chi-square test or Fisher’s exact

test for categorical variables. Multivariate regression analysis was also done to

determine factors associated with ECD and CCT in patients with DM. Statistical

t
significance was set to p<0.05. Statistical analysis was performed using SPSS 22.0

ip
cr
statistical software (IBM, SPSS Inc, Chicago, IL).
us
an
Results
m

Table 1 shows the demographic and clinical characteristics of our study sample.

The mean age of patients and controls was 67.1±10.7 years and 67.6±11.1 years
ed

respectively. In diabetic group, 28 out of 72 patients were male (38.9%) and 44 (61.1%)
pt
ce

female. In the control group, 37 out of 88 subjects (42%) were male and 51 (58%)

female. There was no statistically significant difference between patients with DM and
Ac

controls regarding age (p=0.747) and gender (p=0.686). However, 58.3% of patients

with DM and 31.8% of controls had hypertension (p<0.001), while 43.1% of patients

with DM and 17% of controls had hyperlipidaemia (p<0.001). The mean HbA1c in

patients with DM was 7.2±1.6%. Intraocular pressure did not differ significantly

between the two groups (13.5±3.7 mmHg for the diabetic group vs. 14.2±3.1 for the

control group, p=0.195).

The analysis of corneal parameters showed that patients with DM presented

significantly decreased ECD compared to controls (2297.9±311.3 and 2518.3±243.7

137.132.123.69 - 3/17/2020 6:31:15 PM
National Univ. of Singapore
Downloaded by:
 6

cells/mm2 for diabetic and control group respectively, p<0.001), while the two groups

did not differ significantly in any other measured corneal parameter i.e., mean cell size

area (p=0.084), CV (p=0.339) and hexagonality (p=0.059). The mean CCT in patients

with DM was 543.2±38.7 μm and was higher compared to that of the control group

(531.9±37.2 μm) at a borderline level (p=0.062), although the difference did not reach

statistical significance.

Table 2 shows the results of the multivariate analysis, assessing factors

potentially associated with ECD and CCT in the diabetic group. Specifically, the CCT

t
was not associated with any of the examined variables. Regarding ECD, there was a

ip
cr
significant association between decreased ECD and increased HbA1c (p<0.001), longer
us
DM duration (p=0.003) and more severe DR (p=0.008).
an

Discussion
m

Our study showed that patients with DM had decreased ECD compared to
ed

normal subjects of the same age and gender, while endothelial cells hexagonality, size
pt

area and CV, as well as CCT did not differ between patients with DM and controls.
ce

Previous studies have also studied corneal parameters in patients with DM. Our
Ac

findings regarding ECD decrease were in accordance with the majority of previous

studies. Specifically, Inoue et al found a 4.1% reduction in ECD in patients with DM

compared to controls in line with other authors, who also reported a significant

reduction in ECD in diabetic patients [9-11, 14-20]. However, there is no absolute

consensus in the literature. Storr-Paulsen et al mentioned that type 2 DM has no impact

on ECD or morphology in patients with good glycemic status, while they found that

higher HbA1c was associated with lower ECD, an observation which was also

confirmed in our study [21].

137.132.123.69 - 3/17/2020 6:31:15 PM
National Univ. of Singapore
Downloaded by:
 7

An interesting finding of our study was that ECD decrease was significantly

associated with longer DM duration, higher HbA1c levels and more severe DR.

Nevertheless, other studies were not consistent with our results. Inoue et al found that

none of the systemic or ocular factors was significantly correlated with the ECD [11],

while several authors have demonstrated that only duration of DM may play an

important role in corneal morphological abnormalities [10,14].

Furthermore, our study demonstrated no significant difference in the other

measured corneal parameters, such as cell size area, CV and hexagonality, between

t
patients with DM and controls. These findings were in agreement with the results of

ip
cr
previous studies by Inoue et al, Storr-Paulsen et al and Sudhir et al [11,21,22], and in
us
disagreement with those of Lee et al and Choo et al [10,23].
an
As far as CCT is concerned, there was no statistically significant difference
m

between patients with DM and controls in our study, in line with several authors

[8,11,22,23], although most of the so far studies have controversial results, showing
ed

increase of CCT in diabetic patients [9,10,12-20]. We also did not find any correlation
pt

between CCT and patients’ characteristics i.e., DM duration, HbA1c levels, disease
ce

severity, as it was found for ECD, which was in line with Toygar et al, who also
Ac

suggested that retinal disease severity does not seem to have an effect on CCT [24].

The discrepancy between our results and those of other studies and generally

the variation of results in previous studies could be attributed to the variability in the

studied populations, having different characteristics regarding age, glycemic status, DR

severity, DM duration and comorbidities. In addition, the different methods used to

examine corneal endothelium may lead to differentiation of results.

It is, however, worthy to note that based on our results and on those of the

literature, DM seems to have an impact on corneal endothelium, either in ECD and cells
137.132.123.69 - 3/17/2020 6:31:15 PM
National Univ. of Singapore
Downloaded by:
 8

morphology or in CCT, although it has not been defined which alteration precede. The

mechanism, which may induce morphological changes in diabetic cornea, involves

mainly the polyol (sorbitol-aldose reductase) pathway. In this pathway, hyperglycaemia

leads to increased activity of the aldose reductase, causing sorbitol build-up in the

corneal epithelial and endothelial cells. Since sorbitol acts as osmotic agent, its

accumulation results in the swelling of endothelial cells and therefore in increased CCT.

Another possible explanation for corneal changes in patients with DM pertains to the

fact that DM reduces Na+-K+-ATPase activity of the corneal endothelium, leading to

t
morphological and permeability changes in diabetic cornea, and to corneal

ip
cr
decompensation in advanced stages. Moreover, endothelial pump function was proven
us
to be affected by decreased ATP production as a result of slowing down of the Krebs
an
cycle in diabetic cornea [21,25].
m

Potential limitation of our study may be the fact that measurements have not

been performed at the same time for each patient, thus affecting the CCT. In addition,
ed

hypertension and hyperlipidemia rates differ between patients with DM and controls,
pt
ce

which may affect our results. However, strengths of the study were the case-control

design and the fact that all measurements have been done by one examiner.
Ac

In conclusion, our study demonstrated that corneal endothelium in patients with

DM seems to be affected, since ECD was found to be decreased in patients with DM

compared to controls, while duration of DM, HbA1c levels and DR severity were

significantly associated with changes in ECD and should be taken into account. The

clinical relevance of our results suggests that this difference in corneal endothelium

between patients with DM and controls may infer higher susceptibility to surgical stress

and delayed healing following cataract surgery in diabetic patients and surgeons should

inform patients for prognosis.

137.132.123.69 - 3/17/2020 6:31:15 PM
National Univ. of Singapore
Downloaded by:
 9

Acknowledgement: None

Statement of Ethics: The study was in accordance with the Tenets of Helsinki

Declaration and was approved by the institutional review board of our hospital. Inform

consent was obtained from all participants before entering the study.

Disclosure statement: The authors declare no conflict of interest.

Funding sources: None

Authors’ contributions: Panagiota Papadakou conceived the idea, designed the study

and collected data. Irini Chatziralli conceived the idea, designed the study, collected

t
data, performed the statistical analysis and drafted the manuscript. Miltiadis

ip
cr
Papathanassiou, Vaia Lambadiari, Charalambos Siganos, Panagiotis Theodossiadis and
us
Vassilios Kozobolis collected data. All authors have read and approved the current
an
version of the manuscript.
m
ed
pt
ce
Ac

137.132.123.69 - 3/17/2020 6:31:15 PM

National Univ. of Singapore
Downloaded by:
 10

References

1. International Diabetes Federation Diabetes Atlas 8th Edition, Brussels, Belgium

http://www.diabetesatlas.org/ , last accessed 29/09/2019.

2. Pearce I, Simó R, Lövestam-Adrian M, Wong DT, Evans M. Association between

diabetic eye disease and other complications of diabetes: Implications for care. A

systematic review. Diabetes Obes Metab 2019;21:467-478.

3. Yau JW, Rogers SL, Kawasaki R, Lamoureux EL, Kowalski JW, Bek T, et al.

Global prevalence and major risk factors of diabetic retinopathy. Diabetes Care

t
ip
2012;35:556-564.

cr
4. Antonetti DA, Klein R, Gardner TW. Diabetic retinopathy. N Engl J Med
us
2012;366:1227-1239.
an

5. Schultz RO, Matsuda M, Yee RW, Edelhauser HF, Schultz KJ. Corneal
m

endothelial changes in type I and type II diabetes mellitus. Am J Ophthalmol

ed

1984;98:401-410.
pt

6. Schultz RO, Van Horn DL, Peters MA, Klewin KM, Schutten WH. Diabetic
ce

keratopathy. Trans Am Ophthalmol Soc 1981;79:180-199.

Ac

7. Didenko TN, Smoliakova GP, Sorokin EL, Egorov VV. Clinical and pathogenetic

features of neurotrophic corneal disorders in diabetes. Vestn Oftalmol

1999;115:7-11.

8. Siribunkum J, Kosrirukvongs P, Singalavanija A. Corneal abnormalities in

diabetes. J Med Assoc Thai 2001;84:1075-1083.

9. Shenoy R, Khandekar R, Bialasiewicz A, Al Muniri A. Corneal endothelium in

patients with diabetes mellitus: a historical cohort study. Eur J Ophthalmol

2009;19:369-375.
137.132.123.69 - 3/17/2020 6:31:15 PM
National Univ. of Singapore
Downloaded by:
 11

10. Lee JS, Oum BS, Choi HY, Lee JE, Cho BM. Differences in corneal thickness and

corneal endothelium related to duration in diabetes. Eye 2006;20:315-318.

11. Inoue K, Kato S, Inoue Y, Amano S, Oshika T. The corneal endothelium and

thickness in type II diabetes mellitus. Jpn J Ophthalmol 2002;46:65-69.

12. Roszkowska AM, Tringali CG, Colosi P, Squeri CA, Ferreri G. Corneal

endothelium evaluation in type I and type II diabetes mellitus. Ophthalmologica

1999;213:258-261.

13. Morikubo S, Takamura Y, Kubo E, Tsuzuki S, Akagi Y. Corneal changes after

t
small-incision cataract surgery in patients with diabetes mellitus. Arch

ip
cr
Ophthalmol 2004;122:966-969.
us
14. Islam QU, Mehboob MA, Amin ZA. Comparison of corneal morphological
an
characteristics between diabetic and non diabetic population. Pak J Med Sci
m

2017;33:1307-1311.

15. Briggs S, Osuagwu UL, AlHarthi EM. Manifestations of type 2 diabetes in corneal
ed

endothelial cell density, corneal thickness and intraocular pressure. J Biomed Res
pt
ce

2015;30.

16. Leelawongtawun W, Surakiatchanukul B, Kampitak K, Leelawongtawun R.

Ac

Study of Corneal Endothelial Cells Related to Duration in Diabetes. J Med Assoc

Thai 2016;99:S182-188.

17. Urban B, Raczyńska D, Bakunowicz-Łazarczyk A, Raczyńska K, Krętowska M.

Evaluation of corneal endothelium in children and adolescents with type 1

diabetes mellitus. Mediators Inflamm 2013;2013:913754.

18. Galgauskas S, Laurinavičiūtė G, Norvydaitė D, Stech S, Ašoklis R. Changes in

choroidal thickness and corneal parameters in diabetic eyes. European Journal of

Ophthalmology 2016;26:163-167.
137.132.123.69 - 3/17/2020 6:31:15 PM
National Univ. of Singapore
Downloaded by:
 12

19. Calvo-Maroto AM, Pérez-Cambrodí RJ, Esteve-Taboada JJ, García-Lázaro S,

Cerviño AN. Corneal backscatter in insulin-dependent and non-insulin-dependent

diabetes mellitus patients: a pilot study. Arquivos Brasileiros de Oftalmologia

2017;80:148-153.

20. Módis L Jr1, Szalai E, Kertész K, Kemény-Beke A, Kettesy B, et al. Evaluation

of the corneal endothelium in patients with diabetes mellitus type I and II. Histol

Histopathol 2010;25:1531-7.

21. Storr-Paulsen A, Singh A, Jeppesen H, Norregaard JC, Thulesen J. Corneal

t
endothelial morphology and central thickness in patients with type II diabetes

ip
cr
mellitus. Acta Ophthalmol 2014;92:158-160.
us
22. Sudhir RR, Raman R, Sharma T. Changes in the corneal endothelial cell density
an
and morphology in patients with type 2 diabetes mellitus: a population-based
m

study, Sankara Nethralaya Diabetic Retinopathy and Molecular Genetics Study

(SN-DREAMS, Report 23). Cornea 2012;31:1119-1122.

ed

23. Choo M, Prakash K, Samsudin A, Soong T, Ramli N, Kadir A. Corneal changes

pt
ce

in type II diabetes mellitus in Malaysia. Int J Ophthalmol 2010;3:234-236.

24. Toygar O, Sizmaz S, Pelit A, Toygar B, Yabaş Kiziloğlu Ö, et al. Central corneal
Ac

thickness in type II diabetes mellitus: is it related to the severity of diabetic

retinopathy? Turk J Med Sci 2015;45:651-654.

25. El-Agamy A, Alsubaie S. Corneal endothelium and central corneal thickness

changes in type 2 diabetes mellitus. Clin Ophthalmol 2017;11:481-486.

137.132.123.69 - 3/17/2020 6:31:15 PM
National Univ. of Singapore
Downloaded by:
 Table 1. Demographic and clinical characteristics of the study sample.

Patients with diabetes Controls (n=88) p value

mellitus (n=72)
Age (mean±SD, years) 67.1±10.7 67.6±11.1 0.747
Gender (n, %) 0.686
Male 28 (38.9%) 37 (42%)
Female 44 (61.1%) 51 (58%)
Hypertension (n, %) 42 (58.3%) 28 (31.8%) <0.001
Hyperlipidaemia (n, %) 31 (43.1%) 15 (17.0%) <0.001
HbA1c (mean±SD, %) 7.2±1.6 -
Duration of diabetes mellitus (mean±SD, years) 12.3±5.9 -
Endothelial cell density (mean±SD, cells/mm2) 2297.9±311.3 2518.3±243.7 <0.001
Endothelial cell size area (mean±SD, μm2) 415.6±61.3 401.2±43.2 0.084
Cell size coefficient of variation (mean±SD) 0.43±0.06 0.44±0.07 0.339
Hexagonality (mean±SD, %) 46.3±8.2 48.8±8.3 0.059

t
ip
Central Corneal Thickness (mean±SD, μm) 543.2±38.7 531.9±37.2 0.062
Classification of DR (n, %) -

cr
No DR 23 (31.9%)
us
Mild non-proliferative DR 29 (40.3%)
Moderate non-proliferative DR 15 (20.8%)
an
Severe non-proliferative DR 2 (2.8%)
Proliferative DR 3 (4.2%)
Intraocular pressure ( mean±SD, mmHg) 13.5±3.7 14.2±3.1 0.195
m
ed
pt
ce
Ac

137.132.123.69 - 3/17/2020 6:31:15 PM

National Univ. of Singapore
Downloaded by:
 Table 2. Results of the multivariate analysis of factors associated with endothelial cell
density.

Variable Category/Increment Coefficient (95%CI) p value

HbA1c levels <7.5% vs. ≥7.5% +10.4 (+5.5 to +14.1) <0.001
Diabetes mellitus duration 10 years increase -6.5 (-9.9 to -1.8) 0.003
Severity of diabetic retinopathy One level increase -5.3 (-8.7 to -3.1) 0.008

t
ip
cr
us
an
m
ed
pt
ce
Ac

137.132.123.69 - 3/17/2020 6:31:15 PM

National Univ. of Singapore
Downloaded by: