Trehalose and Dry Eye Disease: A Comprehensive Systematic Review of Randomized Controlled Trials

Citation: Ballesteros-Sánchez, A.; Martinez-Perez, C.; Alvarez- Peregrina, C.; Sánchez-Tena, M.Á.; De-Hita-Cantalejo, C.; Sánchez- González, M.C.; Sánchez-González, J.-M. Trehalose and Dry Eye Disease: A Comprehensive Systematic Review of Randomized Controlled Trials. J. Clin. Med. 2023,12, 7301. https:// doi.org/10.3390/jcm12237301 Academic Editor: Bárbara Burgos-Blasco Received: 27 September 2023 Revised: 22 November 2023 Accepted: 23 November 2023 Published: 25 November 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Journal of Clinical Medicine Review Trehalose and Dry Eye Disease: A Comprehensive Systematic Review of Randomized Controlled Trials Antonio Ballesteros-Sánchez 1,2 , Clara Martinez-Perez 3, Cristina Alvarez-Peregrina 4, Miguel Ángel Sánchez-Tena 4, Concepción De-Hita-Cantalejo 1, María Carmen Sánchez-González 1 and José-María Sánchez-González 1,* 1Department of Physics of Condensed Matter, Optics Area, University of Seville, 41004 Seville, Spain; [email protected] (M.C.S.-G.) 2Department of Ophthalmology, Clínica Novovisión, 30008 Murcia, Spain 3ISEC LISBOA (Instituto Superior de Educação e Ciências), 1750-142 Lisbon, Portugal; [email protected] 4Optometry and Vision Department, Faculty of Optics and Optometry, Complutense University of Madrid, 28037 Madrid, Spain *Correspondence: [email protected] Abstract: The purpose of the research project was to extensively review the efficacy and safety of a trehalose tear-substitute treatment in cases of dry eye disease (DED). A systematic review that included only full-length randomized controlled studies (RCTs) reporting the effects of trehalose tear-substitute treatment in three databases, PubMed, Scopus and Web of Science, was performed according to the PRISMA statement. The search period included papers published before 8 August 2023. The Cochrane risk-of-bias tool was used to analyze the quality of the studies selected. A total of 10 RCTs were included in this systematic review. Trehalose tear-substitute treatments achieved a higher improvement than did control group interventions in all reported variables. The mean differences between both groups were in favor of trehalose, and were as follows: ocular surface disease index (OSDI) questionnaire score of − 8.5 ± 7 points, tear film breakup time (TBUT) of 1.9 ± 1 s, tear film thickness (TFT) of 0.25 ± 0.1 µ m, tear meniscus height (TMH) of 0.02 ± 0.02 mm, Schirmer test (ST) of 0.8 ± 1.4 mm, corneal fluorescein staining (CFS) of − 0.7 ± 0.1 points and visual acuity (VA) of 0.3 ± 2.1 letters. No adverse events after trehalose tear-substitute treatments were reported. Trehalose tear substitutes are a safe and effective treatment for DED. Therefore, trehalose tear substitutes should be recommended for patients with dry eye disease. In addition, there is specific evidence to support its use in the preoperative cataract surgery period. Keywords: trehalose; hyaluronic acid; tear substitutes; dry eye disease 1. Introduction Dry eye disease (DED) is a chronic, multifactorial and inflammatory disease of the ocular surface that affects up to 30% of adults over the age of 50 and is more frequent in women; additionally, its prevalence increases with age [ 1 , 2 ]. DED is characterized by a reduced production or excessive evaporation of tears, which leads to symptoms such as dryness, irritation, burning, and blurred vision [ 3 ]. DED can have a significant impact on the quality of life of affected individuals and is a leading cause of visits to eye care professionals worldwide [4,5]. The management of DED involves a range of treatments aimed at relieving symptoms, improving tear film stability, and reducing inflammation [ 6 , 7 ]. According to the management and therapy subcommittee of the Tear Film and Ocular Surface (TFOS) Dry Eye Workshop (DEWS) II, tear substitutes constitute the first-step therapy for DED [ 6 ]. However, this treatment has limitations, and many patients continue to experience symptoms despite treatment [ 6 , 8 ]. Therefore, new tear-substitute formulations are under research. Trehalose, J. Clin. Med. 2023,12, 7301. https://doi.org/10.3390/jcm12237301 https://www.mdpi.com/journal/jcm J. Clin. Med. 2023,12, 7301 2 of 13 a natural disaccharide consisting of two glucose molecules, has been shown to have protective properties against various stressors and is known to stabilize proteins and membranes, prevent denaturation and inhibit oxidative damage [ 9 ]. Trehalose has been found to have several potential benefits for the management of DED [ 10 ]. Firstly, trehalose can stabilize the lipid layer of the tear film, which is essential for maintaining tear-film stability and preventing evaporation [ 11 , 12 ]. Secondly, trehalose can reduce tear-film osmolarity, which is often elevated in DED and contributes to ocular surface damage [13]. Thirdly, trehalose has been shown to enhance the survival of corneal epithelial cells, which are essential for maintaining the integrity and function of the ocular surface [ 13 , 14 ]. Lastly, trehalose has anti-inflammatory properties and can modulate the expression of genes involved in the regulation of tear secretion and ocular surface homeostasis [14,15]. To date, some published studies have evaluated the effects of trehalose tear substitutes in DED. However, to the best of our knowledge, no systematic reviews have explored the available literature regarding the benefits of trehalose tear-substitute treatments. Therefore, the objective of this systematic review is to evaluate the efficacy and safety of trehalose tear substitutes in the management of DED based on the available randomized controlled trials (RCTs). Through this review, we aim to provide a comprehensive overview of the current evidence on trehalose, enabling evidence-based decision making and guiding future research directions. 2. Materials and Methods 2.1. Data Sources and Search Strategy This systematic review was performed according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) method [ 16 , 17 ]. We identified 136 articles published before 8 August 2023 through the following databases: PubMed, Scopus and Web of Science. The data search strategy with Boolean operators was as follows: (trehalose OR trehalose solution) AND (artificial tears OR tear substitutes OR eye drops) AND (dry eye disease OR dry eye syndrome OR DED OR dysfunctional tear syndrome OR keratoconjunctivitis sicca). These keywords were selected by consensus among the researchers. The references of the retrieved articles were reviewed to identify other related studies that might meet the inclusion criteria. 2.2. Study Selection All of the 136 articles identified through the search strategy were considered and analyzed. Duplicate studies were removed by DistillerSR software (Version 2.35, DistillerSR Inc., Ottawa, ON, Canada) [ 18 ]. The remaining studies underwent additional screening stages, which included title screening, abstract screening and full-text screening. Studies unrelated to the topic were excluded from the review during title and abstract screenings. In full-text screening, studies that did not include the instillation of trehalose eye drops were also excluded from the review. The studies were reviewed by two researchers (ABS and JMSG), who selected them according to the inclusion and exclusion criteria. In cases of discrepancies between the reviewers, a third author was consulted to resolve the disagreement. The inclusion criteria were as follows: human studies, full-length original articles, and RCTs. The exclusion criteria excluded non-English-language publications and unindexed journals. There were no restrictions set as to the country in which the study was performed, the follow-up period, the sample size, or the results of the study. 2.3. Quality Assessment and Data Extraction The data from each study were collected and summarized independently in tables designed by two researchers (ABS and JMSG). The following information was obtained from each article: (1) author and date of publication (year), (2) study design, (3) mean follow-up duration for all patients in the whole procedure (expressed in hours or months), (4) number of patients, (5) mean age of the patients (expressed in years), (6) patient sex J. Clin. Med. 2023,12, 7301 3 of 13 (male/female), (7) number of eyes involved, (8) study group intervention, (9) control group intervention, (10) adverse events and (11) conflicts of interest. Regarding the results of the studies, the following data were collected according to TFOS DEWS II for DED diagnosis [ 19 ]: (14) ocular surface disease index [(OSDI), values from 0 to 100] [ 20 ]; (15) tear break-up time [(TBUT), expressed in seconds (s)]; (16) tear film thickness [(TFT), expressed in microns ( µ m)]; (17) tear meniscus height [(TMH), expressed in millimeters (mm)]; (18) Schirmer I test [(ST), expressed in millimeters (mm)] [ 19 ]; (19) corneal fluorescein staining [(CFS), assessed with Oxford grading score] [ 21 ]; (20) patient satisfaction (defined as “no satisfaction”, “low satisfaction”, “moderate satisfaction” and “high satisfaction”); and finally, (23) the authors’ judgment as expressed by commenting in favor or against the use of trehalose eye-drops treatments. Baseline and last-visit values for all these variables were collected in the treatment (T) and control (C) groups. Intra-group clinical outcomes were defined as “Last visit (LV)—Baseline (B) differences”. Inter-group clinical outcomes were defined as “T group (LV–B) —C group (LV–B) differences”. Mean ± SD, [range] for each variable were calculated to report intra-group and inter-group clinical outcomes. The literature that remained after full-text screening was examined to assess the quality of the studies. To avoid the risk of bias, two dependable authors created a synopsis based on the Cochrane risk-of-bias tool [ 22 ] which included the following items: (1) random sequence generation, (2) allocation concealment, (3) blinding of participants and personnel, (4) blinding of outcome assessment, (5) incomplete outcome data, (6) selective reporting and (7) other sources of bias. A third nonblinded assessor decided the quality of the studies when disagreements occurred between the two assessors. This assessment did not determine the exclusion of any study. 3. Results 3.1. Study Characteristics The study-selection process of this systematic review is presented with a flowchart diagram in Figure 1. The design of the included studies comprised prospective randomized controlled trials published between 2015 and 2021. This systematic review included 1252 patients with a mean age of 56.9 ± 10.8 years. The sex distribution was 787 females (66.1%) and 405 males (33.9%). One study did not report age and sex distribution [ 23 ]. Patient follow-up, expressed in months, ranged from 1 month [ 23 , 24 ] to 8 months [ 15 ], with a mean follow-up of 2.7 ± 2.3 months. Regarding study group intervention, six studies used Thealoz duo ® (Laboratories Théa, Ferrand, France) [ 12 , 13 , 15 , 24 – 26 ], three studies used Thealoz duo gel ® (Laboratories Théa, Ferrand, France) [ 11 , 23 , 27 ] and one study used Trehalube ® (Micro Labs, Bangalore, India) [ 28 ]. Different interventions were used in the control group, such as Hydrabak ® (Laboratories Théa, Ferrand, France) [ 12 , 13 , 23 ], Hyabak ® (Laboratories Théa, Ferrand, France) [ 12 , 15 ], Hylo gel ® (Brill Pharma, Barcelona, Spain) [ 11 ], Hylotears ® (Ursapharm, Sarrebruck, Germany) [ 28 ], Systane gel drops ® (Alcon, Barcelona, Spain) [ 11 ], Vismed ® (Brudy Lab, Barcelona, Spain) [ 25 , 26 ], Vismed gel ® (Brudy Lab, Barcelona, Spain) [ 27 ], and no intervention [ 15 , 24 ]. Three studies had conflicts of interest (supported by Laboratories Théa, Ferrand, France) [ 11 , 25 , 26 ]. More detailed study characteristics and tear-substitute compositions are presented in Table 1. J. Clin. Med. 2023,12, 7301 4 of 13 Table 1. Summary of included RCTs. Author, Date Design F/U Patients (TG/CG) Age c (TG/CG) Sex (F/M) Eyes Inclusion Criteria Intervention Control Adverse Events CoI Schmidl et al. [12] 2015 MN SM 4a60 (20/40) 42.7 ±11.6 (43.6 ±13.3/42.4 ±11) 43/17 60 DED Thealoz Duo (TH 3% and HA 0.15%) Hyabak (HA 0.15%) or Hydrabak (NaCL 0.9%) No No Wozniak et al. [11] 2016 MN SM 6a60 (20/40) 45.6 ±13.5 (45 ±13.1/45.9 ±13.8) 37/23 60 DED Thealoz Duo gel (TH 3%, HA 0.15% and CB 0.25%) Hylo gel (HA 0.2%) orSystane gel (PG 0.4%) No Yes Chiambaretta et al. [25] 2016 MT SM 3b105 (52/53) 59.2 ±12.8 (60 ±12.2/58.5 ±13.4) 86/19 105 DED Thealoz Duo (TH 3% and HA 0.15%) Vismed (HA 0.18%) No Yes Doan et al. [26] 2018 MT SM 3b105 (52/53) 59.2 ±12.8 (60 ±12.2/58.5 ±13.4) 86/19 105 DED Thealoz Duo (TH 3% and HA 0.15%) Vismed (HA 0.18%) NR Yes Caretti et al. [23] 2019 MN DM 1b60 (30/30) NR NR 60 DED Cataract Thealoz Duo gel (TH 3%, HA 0.15% and CB 0.25%) Hydrabak (NaCL 0.9%) NR No Karaca et al. [27] 2020 MN SM 4a122 (56/66) 52.9 ±12.3 (52.5 ±13.2/53.4 ±11.4) 104/18 122 DED Thealoz Duo gel (TH 3%, HA 0.15% and CB 0.25%) Vismed gel (HA 0.3%) NR No Morya et al. [28] 2020 MN SM 2b384 (192/192) 37.6 ±14.4 (36.7 ±14.8/36.1 ±13.6) 224/160 384 DED Trehalube (TH 3%, HA 0.1%) Hylotears (HA 0.1%) No No Cagini et al. [13] 2021a MN UM 1b135 (66/69) 73.7 ±4.2 (73.2 ±4.5/74.3 ±3.8) 82/53 135 DED Cataract Thealoz Duo (TH 3% and HA 0.15%) Hydrabak (NaCL 0.9%) No No Cagini et al. [15] 2021b MN UM 8b98 (33/65) 67.7 ±6.9 (65.4 ±7.2/64.8 ±5.8) 48/50 98 DED Cataract Thealoz Duo (TH 3% and HA 0.15%) No tear substitutes or Hyabak (HA 0.15%) No No Mencucci et al. [24] 2021 MN UM 1b123 (83/40) 73.7 ±9 (73.8 ±9.2/73.6 ±8.8) 77/46 123 DED Cataract Thealoz Duo (TH 3% and HA 0.15%) No tear substitutes NR No Abbreviations: CB = Carbomer; CG = Control group; CoI = Conflict of interest; DM = Double-masked; DED = Dry eye disease; F = Female; F/U = Follow-up; HA = Acid hyaluronate; M = Male; MN = Monocentric; MT = Multicenter; NaCl = Sodium chloride; NR = Not reported; PG = Polyethylene glycol; RCTs = Randomized controlled trials; SM = Single-masked; TH = Trehalose; TG = Treatment group; UM = Unmasked. aExpressed as hours. bExpressed as months. cExpressed as mean ±SD or median [IQR], years. J. Clin. Med. 2023,12, 7301 5 of 13 1 Figure 1. Flowchart study selection process according to the PRISMA statement. 3.2. Outcomes Regarding outcomes measures, six studies reported dry-eyesymptom outcomes [ 13 , 15 , 23 – 26 ]. Also, nine studies reported dry-eye sign outcomes [ 11 – 13 , 15 , 23 – 25 , 27 , 28 ], of which seven studies evaluated TBUT [ 12 , 13 , 15 , 23 – 25 , 28 ], six studies assessed ST [ 12 , 13 , 15 , 24 , 25 , 28 ], four studies evaluated CFS [ 13 , 15 , 23 , 25 ], three studies measured VA [ 12 , 15 , 23 ] and two studies assessed TFT [ 11 , 12 ] and TMH [ 27 , 28 ]. Patient satisfaction was reported by four studies [11,23,25,27]. Intra-group clinical outcomes are presented in Tables 2and 3. Regarding treatment groups, most of the outcomes achieved an improvement, with a mean OSDI questionnaire score of − 22.4 ± 12.2, [ − 36.7 to − 4.6] points, mean TBUT of 2.8 ± 1.2, [0.9 to 4.2] s, mean TFT 0.5 ± 0.2, [0.3 to 0.7] µ m, mean ST of 2.8 ± 1.2, [1.6 to 5.3] mm, mean CFS of − 1.1 ± 0.2, [ − 1.2 to − 0.8] points and mean VA of 10.6 ± 8.9, [ − 2.1 to 17.6] letters. TMH remained almost unchanged, with a mean value of 0.08 ± 0.1, [0.06 to 0.1] mm. Regarding the control group, most of the outcomes also achieved an improvement, with a mean OSDI questionnaire score of − 14.3 ± 10.1, [ − 28.2 to 1.2] points, mean TBUT of 0.9 ± 1, [ − 0.3 to 2.6] s, mean TFT of 0.25 ± 0.2, [0.1 to 0.4] µ m, mean ST of 1.9 ± 1.6, [ − 0.5 to 3.7] mm, mean CFS of − 0.4 ± 0.2, [ − 0.8 to − 0.2] points and mean VA 10.2 ± 7, [0.4 to 16.3] letters. TMH also remained almost unchanged, with a mean value of 0.05 ±0.1, [−0.02 to 0.1] mm. Inter-group clinical outcomes are presented in Table 4. All outcomes were in favor of the treatment group, with a mean OSDI questionnaire score of − 8.5 ± 7, [ − 20 to − 1.7] points, mean TBUT of 1.9 ± 1, [0.1 to 4.5] s, mean TFT 0.25 ± 0.1, [0.2 to 0.3] µ m, mean ST of 0.8 ± 1.4, [ − 1.1 to 3.3] mm, mean CFS of − 0.7 ± 0.1, [ − 0.8 to − 0.6] points, mean VA of 0.3 ± 2.1, [ − 2.5 to 2.3] letters and mean TMH of 0.02 ± 0.02, [0.00 to 0.02] mm. Regarding adverse events, six studies reported no adverse events after trehalose tear-substitute treatments [ 11 – 13 , 15 , 28 ]. In addition, four studies reported high patient satisfaction after trehalose tear-substitute treatments. J. Clin. Med. 2023,12, 7301 6 of 13 Table 2. Baseline, Last visit and Differences (Last visit—Baseline) outcomes in the treatment group. Author, Date Assessment OSDI (0–100) TBUT, s TFT, µmTMH, mm ST, mm CFS (0–5) VAc (0–100) Satisfaction Schmidl et al. [12] 2015 Baseline 26.1 ±5.1 5.4 ±2.1 2.4 ±0.4 NR 7.5 ±7.1 NR 83.9 ±8.2 NR Last visit NR 6.3 ±2.3 3.1 ±0.9 NR 9.1 ±9.7 NR 81.8 ± 18.3 Difference LV-B - 0.9 0.7 a- 1.6 - −2.1 Wozniak et al. [11] 2016 Baseline 50.5 ±20 3.45 ±2.1 3.5 ±0.7 NR 9.1 ±5.5 NR 86.6 ±4.7 High Last visit NR NR 3.8 ±0.6 NR NR NR NR Difference LV-B - - 0.3 - - - - Chiambaretta et al. [25] 2016 Baseline 45.4 ±17.2 5.6 ±1.9 NR NR 8 ±6.3 1.9 ±1.5 NR High Last visit 15.2 ±18.7 8.3 ±1.9 NR NR 11 ±6.2 0.5 ±1.9 NR Difference LV-B −30.2 a2.7 - - 3 −1.4 a- Doan et al. [26] 2018 Baseline 45.4 ±17.2 NR NR NR NR NR NR NR Last visit 15.5 ±13.9 NR NR NR NR NR NR Difference LV-B −29.9 a------ Caretti et al. [23] 2019 Baseline 31.2 ±12.3 3.4 ±1.1 NR NR NR 0.9 ±0.6 66.4 ±4.5 High Last visit 5.1 ±7.9 6.6 ±2.1 NR NR NR 0.00 84 ±6.3 Difference LV-B −26.1 a3.2 a- - - −0.9 a17.6 a Karaca et al. [27] 2020 Baseline NR NR NR 2.3 ±0.85 NR NR NR High Last visit NR NR NR 2.4 ±0.76 NR NR NR Difference LV-B - - - 0.1 - - - Morya et al. [28] 2020 Baseline NR 6.1 ±3.4 NR 0.43 ±0.2 6.1 ±2.7 NR NR NR Last visit NR 9.5 ±3.3 NR 0.49 ±0.3 11.4 ±3.2 NR NR Difference LV-B - 3.4 a- 0.06 5.3 a- - Cagini et al. [13] 2021a Baseline 8.2 ±7.9 8 ±2.5 NR NR 11.8 ±3 2.3 ±0.5 NR NR Last visit 1.2 ±1.9 12.2 ±3.2 NR NR 14.6 ±3.4 1.5 ±0.5 NR Difference LV-B −7a4.2 a- - 2.8 a−0.8 a- Cagini et al. [15] 2021b Baseline 44.5 ±27.4 4.9 ±2.2 NR NR 7.1 ±3.2 1.3 ±0.8 71.2 ±8.3 NR Last visit 7.8 ±4.5 9.1 ±1.3 NR NR 9.5 ±1.3 0.1 ±0.4 87.5 ±6.6 Difference LV-B −36.7 a4.2 a- - 2.4 a−1.2 a16.3 Mencucci et al. [24] 2021 Baseline 20.4 ±7.2 6.03 ±1.7 NR NR 10.5 ±5.6 NR NR NR Last visit 15.8 ±7.3 7.3 ±1.3 NR NR 12.2 ±8.7 NR NR Difference LV-B −4.6 a1.3 a- - 1.7 - - Mean ±SD b−22.4 ±12.2 2.8 ±1.2 0.5 ±0.2 0.08 ±0.1 2.8 ±1.2 − 1.1 ± 0.2 10.6 ±8.9 B = Baseline; CFS = Corneal fluorescein staining; LV = Last visit; NR = Not reported; OSDI = Ocular surface disease; SD = Standard deviation; ST = Schirmer test; TBUT = Tear break-up time; TFT = Tear film thickness; TMH = Tear meniscus height; VA = Visual acuity. a Statistical significance level p< 0.05. b Mean ± SD values of the difference LV-B for each variable. cEarly treatment diabetic retinopathy study (ETDRS) letter score. Table 3. Baseline, Last visit and Differences (Last visit—Baseline) outcomes in the control group. Author, Date Assessment OSDI (0–100) TBUT, s TFT, µmTMH, mm ST, mm CFS (0–5) VA c (0–100) Satisfaction Schmidl et al. [12] 2015 Baseline 27.1 ±4.8 4.9 ±2.1 2.5 ±0.4 NR 12.1 ±9.5 NR 84.7 ±3.2 NR Last visit NR 5.4 ±2.8 2.9 ±0.5 NR 14.8 ±10 NR 85.1 ± 18.3 Difference LV-B - 0.5 0.4 a- 2.7 - 0.4 Wozniak et al. [11] 2016 Baseline 50.4 ±17.9 3.6 ±1.7 3.5 ±0.7 NR 9.9 ±8 NR 87.1 ±3.6 High Last visit NR NR 3.6 ±0.05 NR NR NR NR Difference LV-B - - 0.1 - - - - Chiambaretta et al. [25] 2016 Baseline 46 ±18.7 4.9 ±1.5 NR NR 6.8 ±4.7 2.1 ±1.6 NR High Last visit 20.4 ±16.4 7.5 ±1.2 NR NR 10.5 ±6.1 1.3 ±1.5 NR Difference LV-B −25.6 a2.6 - - 3.7 −0.8 a- Doan et al. [26] 2018 Baseline 46 ±18.7 NR NR NR NR NR NR NR Last visit 17.8 ±13.9 NR NR NR NR NR NR Difference LV-B −28.2 a------ J. Clin. Med. 2023,12, 7301 7 of 13 Table 3. Cont. Author, Date Assessment OSDI (0–100) TBUT, s TFT, µmTMH, mm ST, mm CFS (0–5) VA c (0–100) Satisfaction Caretti et al. [23] 2019 Baseline 21 ±12.7 4.1 ±1.5 NR NR NR 0.65 ±0.5 68.3 ±3.8 High Last visit 11.2 ±11.4 5.1 ±1.4 NR NR NR 0.28 ±0.4 84.6 ±6.5 Difference LV-B −9.8 a1 - - - −0.3 16.3 a Karaca et al. [27] 2020 Baseline NR NR NR 2.4 ±0.7 NR NR NR High Last visit NR NR NR 2,4 ±0.4 NR NR NR Difference LV-B - - - 0.1 - - - Morya et al. [28] 2020 Baseline NR 5.4 ±3.4 NR 0.45 ±0.2 6.1 ±2.7 NR NR NR Last visit NR 7.7 ±3.3 NR 0.47 ±0.3 9.8 ±3.2 NR NR Difference LV-B - 2.3 a- 0.02 3.7 a- - Cagini et al. [13] 2021a Baseline 9.3 ±9.4 10 ±3.8 NR NR 13.1 ±4.4 2.2 ±0.5 NR NR Last visit 4.8 ±3.4 9.7 ±3.5 NR NR 12.6 ±4 1.9 ±0.6 NR Difference LV-B −4.5 a−0.3 a- - −0.5 −0.2 a- Cagini et al. [15] 2021b Baseline 37.1 ±19.8 5.2 ±2.2 NR NR 7.2 ±3.1 1.1 ±0.7 73.2 ±8.1 NR Last visit 20.4 ±13.1 6.1 ±1.3 NR NR 8.4 ±1.5 0.7 ±0.4 87.2 ±5.8 Difference LV-B −16.7 a0.9 a- - 1.2 a−0.4 a14 Mencucci et al. [24] 2021 Baseline 21.1 ±6.7 6.7 ±1.7 NR NR 11.7 ±6.4 NR NR NR Last visit 22.3 ±6.4 6.48 ±1.4 NR NR 12.4 ±8.3 NR NR Difference LV-B 1.2 −0.22 - - 0.7 - - Mean ±SD b−14.3 ±10.1 0.9 ±1 0.25 ±0.2 0.05 ±0.1 1.9 ±1.6 − 0.4 ± 0.2 10.2 ±7 B = Baseline; CFS = Corneal fluorescein staining; LV = Last visit; NR = Not reported; OSDI = Ocular surface disease; SD = Standard deviation; ST = Schirmer test; TBUT = Tear break-up time; TFT = Tear film thickness; TMH = Tear meniscus height; VA = Visual acuity. a Statistical significance level p< 0.05. b Mean ± SD values of the difference LV-B for each variable. cEarly treatment diabetic retinopathy study (ETDRS) letter score. Table 4. Inter-group differences [(T group LV-B)—(C group LV-B)] as to outcomes. Author, Date Assessment OSDI (0–100) TBUT, s TFT, µm TMH, mm ST, mm CFS (0–5) VA b (0–100) F/A Schmidl et al. [12] 2015 T difference LV-B - 0.9 0.7 a- 1.6 - −2.1 F C difference LV-B - 0.5 0.4 a- 2.7 - 0.4 Difference T-C - 0.4 0.3 a-−1.1 - −2.5 Wozniak et al. [11] 2016 T difference LV-B - - 0.3 - - - - F C difference LV-B - - 0.1 - - - - Difference T-C - - 0.2 - - - - Chiambaretta et al. [25] 2016 T difference LV-B −30.2 a2.7 - - 3 −1.4 a- F C difference LV-B −25.6 a2.6 - - 3.7 −0.8 a- Difference T-C −4.6 0.1 - - −0.7 −0.6 - Doan et al. [26] 2018 T difference LV-B −29.9 a------ F C difference LV-B −28.2 a------ Difference T-C −1.7 - - - - - - Caretti et al. [23] 2019 T difference LV-B −26.1 a3.2 a---−0.9 a17.6 a F C difference LV-B −9.8 a1 - - - −0.3 16.3 a Difference T-C −16.3 2.2 - - - −0.6 1.3 Karaca et al. [27] 2020 T difference LV-B - - - 0.1 - - - F C difference LV-B - - - 0.1 - - - Difference T-C - - - 0.00 - - - Morya et al. [28] 2020 T difference LV-B - 3.4 a- 0.06 5.3 a- - F C difference LV-B - 2.3 a- 0.02 3.7 a- - Difference T-C - 1.1 - 0.04 1.6 - - Cagini et al. [13] 2021a T difference LV-B −7a4.2 a- - 2.8 a−0.8 a- F C difference LV-B −4.5 a−0.3 a- - −0.5 −0.2 a- Difference T-C −2.5 4.5 - - 3.3 −0.6 - J. Clin. Med. 2023,12, 7301 8 of 13 Table 4. Cont. Author, Date Assessment OSDI (0–100) TBUT, s TFT, µm TMH, mm ST, mm CFS (0–5) VA b (0–100) F/A Cagini et al. [15] 2021b T difference LV-B −36.7 a4.2 a- - 2.4 a−1.2 a16.3 F C difference LV-B −16.7 a0.9 a- - 1.2 a−0.4 a14 Difference T-C −20 3.3 - - 1.2 a−0.8 a2.3 Mencucci et al. [24] 2021 T difference LV-B −4.6 a1.3 a- - 1.7 - - F C difference LV-B 1.2 −0.2 - - 0.7 - - Difference T-C −5.8 1.5 - - 1 - - B = Baseline; CFS = Corneal fluorescein staining; F/A = Favor/against; LV = Last visit; NR = Not reported; OSDI = Ocular surface disease; SD = Standard deviation; ST = Schirmer test; TBUT = Tear break-up time; TFT = Tear film thickness; TMH = Tear meniscus height; VA = Visual acuity. a Statistical significance level p< 0.05. bEarly treatment diabetic retinopathy study (ETDRS) letter score. 3.3. Risk of Bias The risk-of-bias summary of the included studies is presented in Figure 2. Risk-of- bias assessment was classified into three evidence-level groups: (1) studies with a low risk of bias (Schmidl et al. [ 12 ], Chiambaretta et al. [ 25 ], Doan et al. [ 26 ], Caretti et al. [ 23 ], Karaca et al. [ 27 ] and Morya et al. [ 28 ]), (2) studies with an unclear risk of bias (Wozniak et al. [ 11 ], Cagini et al. [ 13 , 15 ] and Mencucci et al. [ 24 ]) and (3) studies with a high risk of bias (no studies). The overall risk-of-bias summary of the domains used in each study is presented in Figure 3. The items used to assess the risk of bias showed an overall low risk of bias for over 50% of the included analyses. The Robvis tool (NIHR, Bristol, UK) was used to create risk-of-bias assessment figures [29]. J.Clin.Med.2023,12,xFORPEERREVIEW9of14    Figure2.Risk-of-biassummaryfortheincludedstudies,withtraffic-lightplot.Thetrafficlights representtheauthors’riskofbiasedjudgmentineachdomain(D)usedtoassessthequalityofthe studies[11–13,15,23–28].  Figure3.Overallrisk-of-biassummaryofthedomains,withbarplot.Barsrepresenttheauthors’ overallriskofbiasedjudgmentineachdomain,presentedaspercentages.  Figure 2. Risk-of-bias summary for the included studies, with traffic-light plot. The traffic lights represent the authors’ risk of biased judgment in each domain (D) used to assess the quality of the studies [11–13,15,23–28]. J. Clin. Med. 2023,12, 7301 9 of 13 J.Clin.Med.2023,12,xFORPEERREVIEW9of14    Figure2.Risk-of-biassummaryfortheincludedstudies,withtraffic-lightplot.Thetrafficlights representtheauthors’riskofbiasedjudgmentineachdomain(D)usedtoassessthequalityofthe studies[11–13,15,23–28].  Figure3.Overallrisk-of-biassummaryofthedomains,withbarplot.Barsrepresenttheauthors’ overallriskofbiasedjudgmentineachdomain,presentedaspercentages.  Figure 3. Overall risk-of-bias summary of the domains, with bar plot. Bars represent the authors’ overall risk of biased judgment in each domain, presented as percentages. 4. Discussion Tear film instability is considered the trigger for the ocular surface inflammatory mechanisms that lead to the signs and symptoms of dry eye [ 3 ]. Tear substitutes are usually the first line of treatment for patients with DED [ 6 ]. Therefore, new formulations that improve the tear film quality and restore the homeostasis of the ocular surface are the subjects of constant research [ 30 ]. This systematic review aimed to report the effects of trehalose tear-substitute treatments on the signs and symptoms of dry eye. 4.1. Trehalose Efficacy in DED Although there are different questionnaires to assess dry-eye symptoms, the OSDI questionnaire is the most widely used for DED studies, and it is validated in different languages. Chiambaretta et al. [ 25 ] and Doan et al. [ 26 ] reported significant OSDI score improvement in both groups, but the results were in favor of the trehalose group, with differences of − 4.6 and − 1.7 points, respectively. The control groups received hyaluronic acid (HA) 0.18% tear substitutes, which may explain the OSDI score improvements. HA is a linear polysaccharide which has been shown to be superior in increasing tear film viscosity and stability compared to other tear-substitute formulations, resulting in greater relief of dry-eye symptoms [ 31 , 32 ]. However, Doan et al. [ 26 ] reported that 78.8% of patients in the trehalose group achieved an OSDI score below 19 points, compared to 58.5% in the HA group, after 3-month follow-up. Tear film stability and volume, as well as damage to the ocular surface, are tests recommended by the TFOS DEWS II for DED diagnosis [ 19 ]. Regarding tear film stability, Schmidl et al. [ 12 ], Chiambaretta et al. [ 25 ] and Morya et al. [ 28 ] reported that the trehalose groups achieved TBUT improvements of 0.4 s, 0.1 s and 1.1 s compared to the control groups, respectively. Morya et al. [ 28 ] was the only study that reported significant TBUT improvements in the trehalose group, which may be explained by the large sample size. Regarding tear-film volume, Schmidl et al. [ 12 ] and Wozniak et al. [ 11 ] showed that the trehalose group achieved higher TFT measurements, of 0.3 µ m and 0.2 µ m, respectively, compared to those of the control groups. In addition, Schmidl et al. [ 12 ] and Wozniak et al. [ 11 ] also reported that HA 0.15% and propylene glycol (PG) 0.4% only increased TFT for 35 ± 5 min, while trehalose increased TFT for as long as 180 ± 60 min. This may be explained as being due to the interaction of trehalose with lipid membranes, which involves the surrounding hydration shell, where the unique disaccharide structure of the trehalose stabilizes the membrane by replacing water molecules and enhancing the membrane’s resilience to stress, thereby maintaining the membrane’s integrity and functionality [ 33 ]. Karaca et al. [ 27 ] reported a significant TMH improvements, of 0.1 mm, in the trehalose group. The control group also achieved significant TMH improvements because they received HA 0.3%, which may match the effect of trehalose due to its spreading, pseudoplasticity and muco-adhesion properties [ 34 – 36 ]. Karaca et al. [ 27 ] also reported that trehalose and HA 0.3% increased TMH for 90 ± 30 min. However, Morya et al. [ 28 ] showed that TMH remained unchanged in both groups after two-month follow-ups. This