The Effect of Amniotic Membrane Transplantation or Conjunctival Autografts on the Tear Mucins MUC5A and MUC2 After Pterygium Resection: A Six-Month Follow-Up

Abstract

Purpose: Pterygium is an ocular surface disease characterized by the invasion of fibrovascular tissue from the bulbar conjunctiva to the cornea and is associated with abnormal tear function caused by changes in tear composition and osmolarity. In this study, the effect of two different surgical techniques to remove primary pterygium: conjunctival autograft surgery (CAG) and amniotic membrane transplantation (AMT), on changes in MUC2 and MUC5AC tear mucins concentration were evaluated.

Methods: Forty-four patients (>18 years old) with primary unilateral pterygium (> 1.0 mm long, measured from the limbus to the apex on the cornea) were randomly enrolled, and assigned to the AMT or CAG group by using the permuted block technique. Patients with systemic inflammatory diseases or other eye comorbidities were excluded from the study. Tear break-up time (TBUT) and best-corrected visual acuity (BCVA) assessments were performed before surgery and at 1, 3, and 6 months after surgery. Tears were collected concurrently with the clinical evaluations, and MUC2 and MUC5AC concentrations were subsequently measured by means of ELISA.

Results: At 6 months after CAG or AMT, TBUT and BCVA were significantly lower (P < 0.05) in comparison with the baseline values in the study subjects. The tear mucin concentrations of both MUC2 and MUC5AC were significantly higher (P < 0.0001) in patients with pterygium before any surgical procedure than in healthy individuals. The concentration of MUC2 increased at 1 and 3 months after CAG surgery and decreased at 6 months; however, the MUC2 concentration decreased on the AMT group in all time point measurements. Interestingly, the MUC5AC concentration significantly increased at 1 month after AMT or CAG and then decreased at 3 and 6 months after surgery. Finally, an inverse correlation was found between both MUC2 and MUC5AC tear mucins concentration and the TBUT.

Conclusions: These results suggest that pterygium excision via both CAG or AMT changes the concentrations of the tear mucins MUC2 and MUC5AC during the evaluated times, and these changes could affect tear film stability and clinical recovery after pterygium treatment.

Translational Relevance: The tear film stability during pterygium excision was evaluated to determine adequate treatments.

Introduction

Pterygium is a common disease of the ocular surface that is characterized by the invasion of fibrovascular tissue from the bulbar conjunctiva into the cornea.¹ This eye disorder is more prevalent in some special populations, such as farmers, fishermen, and people living in dry and sunny climates. Although the etiology of pterygium is not fully understood, prolonged exposure to ultraviolet B radiation is thought to promote its development, and hereditary features, chronic inflammation, microtrauma, and heat are other possible contributing factors.² The preocular tear film is a three-layered structure consisting of an outermost layer of Meibomian lipids, a middle aqueous layer produced by lacrimal glands, and an inner, hydrated mucus layer composed of a fluid-filled meshwork of glycoproteins produced by goblet cells that lubricate and protect the ocular surface. Cumulative evidence indicates that abnormal tear function is a risk factor for pterygium development.^3–5 The main treatment for pterygium is surgical excision; unfortunately, the most daunting challenge of pterygium surgery is its recurrence. Surgical techniques for which recurrence rates are lower and minimal complications occur postoperatively include the covering of scleral defects with conjunctival autografts (CAGs) or amniotic membrane transplantation (AMT).^6,7 Previous reports on bilateral or unilateral pterygium have shown that inadequate tear film is related to this disease. According to the results of Roka and colleagues, the mean Schirmer’s test I, mean basal secretion, and mean tear break-up time (TBUT) were lower in patients with pterygium than in control subjects. These findings suggest that inadequate tear film stability is associated with pterygium and could be considered an important parameter for post excision prognosis.⁸ Similarly, it has been reported that pterygium excision can improve tear osmolality and tear film function, thus decreasing the incidence of dry eye syndrome (DES) after surgery.^9,10 

However, the relationship between pterygium and an unstable tear film is difficult to define; for example, Li and colleagues reported that altered tear film and abnormal crystallization, showed by reduced TBUT and tear-ferning test, can be related to a decrease in goblet cells in patients with pterygium.^11,12 In this sense, it has been reported that a low goblet cell density is related to disturbances in tear compounds, such as MUC2 and MUC5AC mucins, and these mucin alterations can cause tear dysfunction and contribute to the development of DES.^13,14 

The present study was conducted to determine whether surgical removal of primary pterygia via CAG or AMT can cause any changes in the ocular tear film concentrations of MUC2 and MUC5AC during a 6-month follow-up period. 

Materials and Methods

Study Groups

This was a randomized, controlled, single-center trial developed at the Institute of Ophthalmology Conde de Valenciana Foundation. The study adhered to the tenets of the Declaration of Helsinki, was approved by the Institutional Ethics Committee Board (Registration Number: CEI-023-20160830), and was registered on www.clinicaltrials.org with the number NCT04385446. Patients with primary unilateral pterygium located nasally were enrolled in the study. Pterygium was defined as abnormal fibrovascular growth of the conjunctiva invading the cornea. After providing signed informed consent, the patients were randomly assigned to the AMT group or the CAG group by using the permuted block technique. 

Inclusion criteria included patients older than 18 years residing in the Metropolitan Area of Mexico City exhibiting symptoms, such as eye redness, sensation of grit or foreign object in the eye, and having a pterygium longer than 1.0 mm measured from the limbus to the apex on the cornea. 

Exclusion criteria included subjects with systemic inflammatory conditions, such as diabetes mellitus, collagenopathies, or autoimmune diseases, as well as lacrimal gland obstruction or other ocular comorbidities. 

Sample Size

A power analysis was performed by the formula for two independent means using the G*power software (University of California – Los Angeles [UCLA], Los Angeles, CA, USA). The analysis demonstrated that the data of 22 patients with pterygium in each group were required to achieve the following power: one-tailed (1 − β) = 0.80 and α = 0.05, assuming an effect size of 0.80 due to the lack of previous studies investigating the use of both mucins as an outcome in the treatment of pterygium (see CONSORT Flowchart in Fig. 1). 

Figure 1.

 

CONSORT Flowchart.

Pterygium Resection Surgeries

The same surgeon (author A.N.C.) performed all the surgeries, to avoid variability. Briefly, the selected eye was topically anesthetized by using 0.5% tetracaine hydrochloride (Ponti-Ofteno, Sophia, Jalisco, Mexico); afterward, the head and body of the pterygium, as well as Tenon's capsule, were resected. To cover the surgical bare bed, AMT or CAG was placed at the site of pterygium removal. Amniotic membrane fragments were obtained from the Institute of Ophthalmology “Conde de Valenciana” Amniotic Membrane Tissue Bank (COFEPRIS: 14-TR-09-015-0006). In both surgical procedures, tissues were secured with 10–0 nylon stitches (Ethicon; Johnson & Johnson, Mexico City, Mexico). After surgery, topical tobramycin and dexamethasone (Trazidex; Sophia, Jalisco, Mexico) were applied for 4 hours during the first week; additionally, carboxymethyl cellulose eye drops (Refresh Tears; Allergan, US) were applied every 4 hours for 30 days. The stitches were removed 10 days after surgery in both the AMT and CAG groups. 

Postsurgical Clinical Evaluation

Postoperative evaluations were performed at 1, 3, and 6 months after surgery, and a baseline measurement was performed for all the recruited subjects. The post-surgery evaluation included the measurement of best-corrected visual acuity (BCVA) using the Snellen chart and assessing tear quality by measuring TBUT with the fluorescein test. The TBUT was considered as the time required for dry spots to appear on the corneal surface after blinking in the presence of fluorescein staining, and was reported as the mean value of three measurements. The BCVA was measured on a Snellen chart; however, the values were converted to their equivalent logMAR scale to achieve statistical analysis. The ophthalmologist (author L.G.H.) who performed the clinical evaluation was blinded to any surgical treatment. 

Tear Collection

Tear collection was performed 1 day before surgery, which was considered the baseline, and successive tear collection was performed at 1, 3, and 6 months after surgery. Tear samples were obtained only from the affected eye prior to postsurgical clinical evaluation. The tears were collected according to a previously described method, with slight modifications.¹⁵ Briefly, 100 µL of balanced salt solution (Alcon Laboratories, Inc., Fort Worth, TX, USA) was instilled on the eye surface, and each subject was asked to blink thrice; subsequently, the lower lid was pulled downward, and a borosilicate glass capillary was positioned in the conjunctival sac and held horizontally with the final collection of the liquid inside of the glass capillary. To transfer the maximal volume of the tear, the capillary was introduced to a microfuge tube and centrifuged at 14,000 rpm for 15 seconds at 4°C. The tear samples were stored at −80°C until further use. Age- and sex-matched healthy volunteers (n = 44) were invited to participate in the study to obtain control tear samples and to measure the normal tear mucin values. 

Total Protein Concentration

The total protein concentration of the collected tears was quantified by using a micro-Bradford protein assay (Bio-Rad, Hercules, CA, USA). Briefly, a standard curve was generated with a standard protein solution by using dilutions of 1, 2, 4, 8, and 10 µg/mL. Afterward, 10 µL of each tear sample and all of the standard dilutions were pipetted into a microplate well, after which 200 µL of diluted Coomassie Brilliant Blue G-250 dye reagent was added to each well according to the manufacturer's instructions. Finally, the samples and dye reagent were mixed and incubated at room temperature (RT; 25°C) for 5 minutes in the dark. The absorbance was immediately measured at 595 nm in a plate spectrophotometer (Multiskan Ascent; Thermo Labsystems, Asheville, NC, USA). 

The protein concentration of the tear samples was calculated via interpolation on the standard curve. First, a standard curve was generated by plotting the absorbance values (y-axis) against the concentration in µg/mL (x-axis) of standard dilutions; by using the equation of the line (y = m x + b), the protein concentration was calculated in all of the tear samples, considering y = absorbance, m = the slope of the line, b = the y-intercept, and x = the protein concentration in µg/mL. 

Mucin Determination

Mucin quantification was performed with a direct enzyme-linked immunosorbent assay (ELISA). First, 1 µg of tear proteins per well was adsorbed in a 96-well flat bottom microplate during 24 hours at 4°C (Corning, Corning, NY, USA) by using carbonate-bicarbonate buffer (pH of 9.5). Afterward, the plates were washed twice with washing buffer (0.05% Tween in phosphate-buffered saline solution [PBS] at a pH of 7.4) and then incubated with blocking buffer (1% bovine serum albumin [BSA] in PBS) for 2 hours at RT. For recognition of the mucins MUC2 and MUC5A, the plates were incubated for 2 hours at RT with the primary antibodies mouse anti-human MUC5AC (Invitrogen, Carlsbad, CA, USA) or mouse anti-human MUC2 (Abcam, Cambridge, UK). Afterward, the plates were washed twice, and the samples were subsequently incubated with the secondary goat anti-mouse peroxidase-conjugated antibody for 1 hour at RT and washed twice with washing buffer. Colorimetric detection was performed incubating the plates with a solution of 3,3′,5,5′-tetramethylbenzidine (TMB) and H₂O₂ (R&D Systems, Minneapolis, MN, USA) for 20 minutes at RT in the dark. The enzymatic reaction was stopped with 2.5 N sulfuric acid, and the plates were subsequently read at 450/540 nm in a plate spectrophotometer. Each sample was analyzed in triplicate. The results are reported as absorbance units (AU; 450 nm)/µg of total tear protein. The graphical results are reported as the mean ± standard error (SE). 

Statistical Analysis

Statistical analyses were performed by using GraphPad Prism statistical software (version 8.0.2), and P < 0.05 was considered to indicate statistical significance. The Mann–Whitney U test, χ² test, or Fisher's exact test were performed to analyze differences in demographic data and pterygium recurrence. The rest of the data were normally distributed according to the Shapiro‒Wilk and Kolmogorov‒Smirnoff tests, and Student's t-tests were performed to compare the healthy and pterygium groups. ANOVA was performed for comparisons among the mucin concentrations, the TBUT, and BCVA values among the evaluated time periods. Pearson's correlations were calculated between the baseline values of TBUT and the levels of MUC2 and MUC5A. 

Results

The Clinical Evolution of the Patients was Similar Between the AMT and CAG Groups

A total of 44 patients with a pterygium located nasally were included in the present study. Half of the enrolled subjects received CAG, and the other half received AMT surgery. The study subjects were followed for a period of 6 months, with periodic revisions at 1, 3, and 6 months after surgery. There were no significant differences (P > 0.05) between the ages of the individuals in the evaluated groups. In the AMT group, the ages ranged from 32 to 81 years, with a mean age of 54 (±15.5) years; in the CAG group, the ages ranged from approximately 36 to 77 years, with a mean age of 50 (±13.5) years. The initial BCVA in both groups was similar at 0.46 (±0.5) and 0.40 (±0.5) logMAR in the AMT and CAG groups, respectively (see the Table). 

Table.

 

Demographic Data of Patients Participating in the Study

 

Similarly, we did not observe significant differences (P > 0.05) in pterygium size between the groups. The mean size of the pterygia in the AMT group was 2.54 mm ± 0.79 mm and 2.7 mm ± 1.0 mm on the CAG group (see the Table, Fig. 2A). 

Figure 2.

 

The clinical outcomes of the patients in both the CAG and AMT groups were similar after the 6-month follow-up. Clinical photographs before surgery and at 6 months after surgery for both the AMT and the CAG procedures. Slit-lamp nasal pterygium photograph showing the characteristic fibrovascular invasion from the bulbar conjunctiva onto the cornea (black arrows; a, c). Slit-lamp photographs at 6 months after the CAG and AMT procedures (black arrows; b, d) (A). TBUT measurements before surgery (baseline) and at 6 months after surgery in both the AMT and CAG groups. The AMT (black bars) and CAG (gray bars) groups presented with significantly (*P < 0.05 and **P < 0.01) lower TBUTs than did the healthy subjects. The dotted line represents the mean TBUT of healthy subjects. Preoperative (baseline) and 6 months postsurgery BCVA was measured in both the AMT and CAG groups. The AMT (black bars) and CAG (gray bars) groups presented with significantly (*P < 0.05) reduced BCVA values at 6 months postsurgery (B). The data are expressed as the mean ± SD (n = 44).

After surgery, three patients from the AMT group presented hematoma at the graft site, whereas two patients from the CAG group presented pyogenic granuloma. All these complications resolved spontaneously, and none of the subjects presented corneal Dellen or any other complications. In both groups, the grafts were adequately integrated into the surgical bed; moreover, graft necrosis was not observed in either group. Clinical evolution was similar in both groups during the time of the study; however, a more transparent graft and more cosmetically pleasing appearance were observed in the AMT group than in the CAG group (see Fig. 2A). Five cases of pterygium recurrence (22%) were observed in the AMT group, compared to three recurrences (13%) in the CAG group (P = 0.68). Recurrences were defined in accordance with previous reports.¹⁶ 

After clinical evaluation, the difference in the baseline TBUT values between the AMT (7.4 seconds ± 0.4 seconds) and CAG (6.3 seconds ± 0.4 seconds) groups was statistically significant (P < 0.05); interestingly, these values were lower (P < 0.05) than those of healthy subjects (12.4 seconds ± 6.8 seconds; Fig. 2B, left panel). 

At 6 months postsurgery, the TBUT values of the AMT group (5.8 seconds ± 0.3 seconds) were significantly lower (P < 0.01) than the TBUT baseline values. No significant differences were found in the TBUT values in the CAG group at baseline (6.3 seconds ± 0.4 seconds) or at 6 months postsurgery (6.2 seconds ± 0.4 seconds). At 6 months postsurgery, the TBUT values remained also lower in the CAG and AMT groups than in the healthy group (P < 0.05; see Fig. 2B, left panel). 

The evaluation of BCVA showed that both evaluated groups presented similar baseline logMAR BCVA values. In the AMT group, the mean baseline BCVA values was 0.46 ± 0.05, whereas in the CAG group, it was 0.40 ± 0.05, which corresponds to 20/50 values in the Snellen chart. 

Interestingly, compared with that at baseline, at 6 months postsurgery, the BCVA values were significantly lower in both the AMT and CAG groups, with values of 0.2 ± 0.01 (P < 0.05) and 0.17 ± 0.01 (P < 0.05), respectively (see Fig. 2B, right panel). 

CAG and AMT Modified MUC2 and MUC5AC Levels in the Tears of Patients With Pterygium

After tear analysis, the baseline mucin MUC2 concentration (0.21 ± 0.018 AU) was significantly higher in patients with pterygium (P < 0.0001) than in healthy individuals (0.065 ± 0.004 AU; Fig. 3A). Similarly, the baseline MUC5AC concentration in patients with pterygium was significantly (P < 0.0001) higher (0.61 ± 0.018 AU) compared with healthy individuals (0.347 ± 0.07 AU; Fig. 3B). 

Figure 3.

 

Changes in the tear levels of MUC2 and MUC5AC in pterygia and follow-up after pterygium resection. Bar graphs of tear sample quantification by ELISA for MUC2 (A) and MUC5AC (B) in patients with pterygium (black bars) and healthy individuals (white bars). The MUC2 and MUC5AC tear levels in patients with pterygium were significantly greater than those in healthy subjects. Bar graphs of MUC2 (C) and MUC5AC (D) tear level quantification by ELISA at 1, 3, and 6 months post-AMT (black bars) and CAG (gray bars) surgery. Changes in the concentrations of MUC2 and MUC5AC were significant after pterygium resection with AMT and CAG surgeries. The data are expressed as the mean ± SE (n = 44), *P < 0.05, **P < 0.01, *** P < 0.001, and ****P < 0.0001.

The AMT group exhibited consistent tear mucin MUC2 concentrations at baseline (0.21 ± 0.018), 1 month (0.18 AU ± 0.03), and 2 months (0.21 AU ± 0.04) postsurgery. Interestingly, a significant decrease (P < 0.0001) in MUC2 levels (0.07 AU ± 0.007) was observed at 6 months after AMT surgery to other time points evaluated (Fig. 3C, black bars). Conversely, we found that the CAG group had a significantly (P < 0.05) higher MUC2 levels at baseline and at 1 month (0.45 AU ± 0.08) and 3 months (0.53 AU ± 0.10) postsurgery compared to 6 months postsurgery (0.07 AU ± 0.006; see Fig. 3C, gray bars). These findings suggest that surgical interventions, such as AMT or CAG, can influence MUC2 mucin levels. 

At the beginning of the study, both the AMT (black bars) and CAG (gray bars) groups presented similar baseline levels of MUC5AC (0.74 AU ± 0.07 and 0.60 AU ± 0.06, respectively; Fig. 3D). Conversely, at 1 month postsurgery, the MUC5AC levels were significantly higher (P < 0.05) than at 6 months postsurgery in both groups, AMT (0.82 AU ± 0.07) and CAG (0.88 AU ± 0.11). At 3 months postsurgery, the MUC5AC levels in the AMT group were slightly lower (0.65 AU ± 0.05 and 0.65 AU ± 0.06, respectively) than those at baseline and 1 month postsurgery; however, no significant differences were found (see Fig. 3D). At 6 months, MUC5AC levels were significantly lower (0.47 AU ± 0.204, P < 0.05) with respect to baseline and 1 month postsurgery in the AMT group (Fig. 3D). 

TBUT was Inversely Correlated With MUC2 and MUC5AC Levels

Finally, to identify whether there were possible associations between the TBUT values and tear mucin levels, correlation analyses were performed. In these analyses, the baseline values of both the AMT and CAG groups and healthy controls were considered. As shown in Figure 4, MUC2 (r = −0.46) and MUC5AC (r = −0.53) levels were significantly negatively correlated with TBUT (P < 0.05). Taken together, these findings suggest that higher TBUT values are related to lower tear levels of both MUC2 and MUC5A mucins analyzed. 

Figure 4.

 

The TBUT was inversely correlated with the MUC5AC and MUC2 tear levels. Spearman's correlation graph between baseline TBUT values and mucin (MUC2 and MUC5AC) tear concentrations in patients with pterygium and healthy controls; MUC2 versus TBUT (A); MUC5AC versus TBUT (B). The data are expressed as the mean ± SE (n = 44), ****P < 0.0001.

Discussion

Over time, many surgical techniques for the management of pterygium have been described. However, none of these methods have achieved complete success in terms of safety and efficacy. The placement of a conjunctival autograft, usually taken from the upper bulbar conjunctiva, is the gold standard for surgical management of pterygium. In the last 10 years, advances in the understanding of the biology of the ocular surface have allowed us to determine that pterygium is a growth disorder of the ocular surface secondary to changes in limbic epithelial cells produced by exposure to UV-B radiation and intrinsic alterations of the tear film, particularly regarding qualitative and quantitative deficiencies of the mucinous layer. Knowledge of the alteration of epithelial limbal cells has led to the emergence of more complex surgical techniques, such as limbal cell transplantation, sclerokeratoplasty, and amniotic membrane transplantation. In this context, we decided to perform a comparative study of the 2 widely accepted techniques AMT and CAG for surgically removing pterygium, with the aim of determining whether there was any difference between the TBUT and the concentrations of MUC2 and MUC5AC mucins between these techniques when observed over a 6-month period. 

The BCVA was also evaluated to determine changes in visual acuity, and we observed that both the CAG and AMT surgeries improved BCVA at the end of the study. These findings are in accordance with previous reports demonstrating that there is no difference in visual acuity postoperatively between the two utilized techniques.¹⁷ Interestingly, AMT improved BCVA since the first month after surgery (data not shown); whether this finding is related to any change in the quality of the tear film is still a matter of further study. 

The TBUT value is a valuable clinical diagnostic test that is used to detect tear film abnormalities. A statistically significant difference was found in the TBUT values from patients with pterygium in the AMT group. In patients in the CAG group, no statistically significant difference was observed for TBUT values, at baseline and at the end of the study, in accordance to the findings of Kiliç and Gürler,¹⁸ wherein the limbal conjunctival autografting technique did not improve the results of tear function tests. However, these results differ from those reported by Li and colleagues, who reported of a statistically significant increase in the TBUT in the first month after surgery; conversely, the utilized technique was the bare-sclera technique.¹¹ Thus, the results are not entirely comparable. 

We observed reduced TBUT values in patients with pterygium compared with healthy controls. Similarly, as reported by Ishioka and colleagues,¹⁹ pathological conjunctival, corneal, or eyelid changes may lead to disrupted tear film function or insufficient tear film and may initiate the growth of pterygia, thus causing lower TBUT values in patients with pterygium.²⁰ Moreover, other authors have reported that clinical parameters, such as the TBUT and Schirmer I test values, can be modified by abnormalities and changes in the osmolarity of the tear film.^3,21 

Similarly, modifications of the mucin layer of the tear film are reportedly associated with reduced TBUT scores in patients with pterygium.²² Interestingly, alterations in the expression of mucins, such as MUC5AC and MUC16, are related to tear film instability and abnormal visual function in patients with dry eye discomfort.²³ 

Mucins are secreted on the surface of the eye by goblet cells that are within the conjunctival epithelium, and some of their specific functions include lubrication, maintenance of surface wetting, maintenance of tear film across the epithelium, and prevention of infections.^24,25 At least 4 subtypes of secreted mucins, including MUC2, MUC5AC, MUC5B, and MUC7, are expressed on the ocular surface; moreover, 2 of them (MUC2 and MUC5AC) are predominant in the tear film.¹⁷ In the present study, we found that patients with pterygium exhibited significantly greater concentrations of the tear mucins MUC5AC and MUC2 than healthy controls. According to our results, it has been reported that ocular surface complications and corneal ulcers in patients with Sjogren syndrome and atopic individuals are related to surface tear film instability caused by goblet cell deficits and modifications of mucin MUC5AC concentration,^26,27 suggesting the mucins as important components of tear film stability. 

Interestingly, the concentrations of MUC2 increased in the first and third months postsurgery in the CAG group and subsequently decreased at the end of the study. In contrast, the changes in MUC2 levels in the AMT group showed a decreasing trend compared with those at 6 months postsurgery. With respect to the MUC5AC levels, we found that the values increased in the first postoperative month after surgery for both CAG and AMT and decreased after 3 and 6 months postsurgery. Li and colleagues¹¹ reported that the mean goblet cell density is significantly increased at 1 month after pterygium excision using the bare-sclera technique; similarly, cytological changes, such as squamous metaplasia, have been reported in pterygium with increased goblet cell density over its surface.²⁸ Therefore, these changes may be responsible for the increased secretion of mucins (mainly MUC5AC) in the tear film, which is consistent with our findings. However, although changes in MUC2 expression have been previously reported, MUC2 is usually expressed at lower levels in conjunctival tissue than is MUC5AC,¹³ which can explain the decreased levels of this mucin in our samples. 

One of the most relevant findings of this study was that both MUC5A and MUC2 mucin levels decreased in both the CAG and AMT groups at the end of the trial. The present study used the two best-performing techniques for pterygium surgery (the CAG and the AMT). The purpose of placing either a conjunctival autograft or amniotic membrane in the bed where the pterygium has been removed is to cover the defect and to reduce the rate of recurrence. In the present study, CAG and AMT were found to be equally effective at restoring the ocular surface after pterygium resection, and the restoration of tear film stability was determined by mucin production. These results agree with those of Clearfield and colleagues²⁹ who reported that pterygium excision combined with limbal-conjunctival autograft transplantation can partially restore the tear film to a normal state and that the tear film functions are stable for 4 weeks after surgery. 

We found a negative correlation between mucin MUC5AC and MUC2 levels and the clinical value of the TBUT, in contrast with the findings of Zhan et al.,³⁰ who reported of a positive correlation between MUC5AC and TBUT in a cohort of 66 patients with dry eye. However, it has recently been described that there is an association between decreased TBUT and increased MUC5AC expression in elderly people. These results are like ours because the mean ages of both AMT and CAG groups were 54 and 50 years, respectively, thus indicating the importance of these parameters as clinical biomarkers.³¹ 

The main contribution of our findings to the scientific field is the measurement of 2 mucins MUC5AC and MUC2 that are present in tear film, and the analysis of their concentration changes before and after 2 different surgical techniques of pterygium resection. Moreover, as it has been mentioned in other reports, changes in mucins can be related to the pathogenesis of ocular surface diseases such as dry eye,^30,32 atopic keratoconjunctivitis,³³ and limbal stem cell deficiency.³⁴ 

One of the limitations of this study was the fact that MUC5AC and MUC2 concentrations were only measured quantitatively. However, for further studies, we propose the use of other more reliable techniques to assess goblet cells function in patients with pterygium, such as imprint cytology or confocal microscopy. Comparisons of these methods before and after pterygium surgery could provide valuable insights into their efficacy, warranting exploration in subsequent studies. 

Acknowledgments

The authors are thankful for the support from the Conde de Valenciana Foundation. We would like to acknowledge Mohamed Ali Pereyra Morales and Oscar Vivanco Rojas for their invaluable technical support. In addition, we want to thank Biotechnologist Enya Angélica de la Torre Galván and Jonathan Daniel Zagal Cardoso for their extensive review of the manuscript. 

Supported by PAPIIT-DGAPA-UNAM IN210224; CONACYT-Paradigmas- 319469. SECTEI/159/2023 and Conde de Valenciana Foundation. 

Disclosure: A. Nava-Castañeda, None; L. Garnica-Hayashi, None; N. Santiago-Rea, None; E. González-Mondragón, None; B. Buentello-Volante, None; F.S. Magaña-Guerrero, None; Y. Garfias, None