Meibomian gland secretion quality association with ocular parameters in university students during COVID- 19 restrictions

Purpose To determine if the Meibomian Gland (MG) secretion quality is associated with symptoms of ocular discomfort, hours of Video Display Terminals (VDT) use, eyelid margin abnormalities, conjunctival hyperemia, and Meibomian Gland Loss Area (MGLA) in a sample of university students. Methods An online survey that included an Ocular Surface Disease Index (OSDI) questionnaire and an extra question about hours of VDT use recruited an initial sample of 183 participants. Only 120 participants that fulfilled the inclusion criteria were scheduled for a battery of ocular surface and MG specific exam. The tests include: 1) meibometry, 2) slit lamp exploration of eyelid margin abnormalities (irregularity, hyperemia and MG orifices plugging), MG secretion quality and conjunctival hyperemia, and 3) Meibography. Results Significant positive correlations between the MG secretion quality and eyelid margin hyperemia, MG orifices plugging, MGLA, nasal conjunctival hyperemia, and temporal conjunctival hyperemia (Spearman Rho; all r>0.186, p<0.042) were found. Multivariate regression found association between OSDI with hours of VDT use (B=0.316, p=0.007), and eyelid hyperemia (B=0.434, p≤ 0.001). A statistical association between MG secretion quality and eyelid margin hyperemia, MG orifices plugging, MGLA and conjunctival hyperemia (Fisher’s exact; all p<0.039) were found. Multivariate regression found association between MG secretion quality with MG orifices plugging (B=0.295, p=0.004) and meibometry (B=-0.001, p=0.029). Conclusion Participants with higher values in MG secretion quality have higher values in eyelid margin hyperemia, MG plugging, MGLA, and conjunctival hyperemia. No direct relationship between MG secretion quality and hours of VDT use or OSDI were found.


Introduction
The coronavirus disease 2019 (COVID-19) pandemic has caused many lifestyle changes in the worldwide population [1,2]. In particular, university students were affected in the way they received their lessons, which were telematic during the lockdowns [3]. The number of hours using Video Display Terminals (VDT) by university students has risen from 4 h to more than 8 h per day due to limitations on face-toface lessons [2]. Ocular discomfort is highly correlated with the hours of VDT use because of the reduction of complete blinks, which are important to maintain the ocular surface homeostasis [4][5][6][7]. When a complete blink occurs, the apposition of the lower and the upper eyelid borders spread the tear film lipid layer all over the ocular surface [8]. The Meibomian Glands (MG) are located in the superior and inferior tarsal plate along the upper and lower eyelids with their orifices along the free eyelid margins [8]. These sebaceous glands produce the meibum, the main component of the tear film lipid layer, which plays an important role in avoiding tear film evaporation and maintaining a smooth optical surface [8,9]. Incomplete blinking reduces the meibum spreading over the ocular surface, contributes to an accumulation of fatty acids over the eyelids free border, and provokes obstruction of the MG orifices with the subsequent reduction of the MG secretion quality [10,11]. Therefore, the aim of the present study was to determine if the quality of the MG secretion is associated with symptoms of ocular discomfort, hours of VDT use, eyelid margin abnormalities, conjunctival hyperemia, and Meibomian Gland Loss Area (MGLA) in a sample of university students.

Study design
All participants were between 19 and 32 years old and were recruited from students and attendants to the Optometry Faculty to be involved in a cross-sectional study. The present study was adhered to the tents of the Declaration of Helsinki and was approved by the Bioethics Committee of the institution. Informed consent was signed by every participant to be included in the study. All participants who wanted to participate have completed an online form which could be accessed by scanning a QR code. The self-administered survey form was formed by a full Ocular Surface Disease Index (OSDI) questionnaire and an extra question about hours of VDT use ("How many hours do you use video electronic displays during a day?") [12].
A total of 183 volunteers completed the online self-administered survey. Volunteers were scheduled for an eye examination but first underwent an oral history taking to check if they could be included in the study. To be included in the study, participants had to have no history of a conjunctival, scleral, or corneal disease, active ocular disease or ocular allergy, prior eye surgery (including refractive surgery or eyelid tattooing), glaucoma, diabetes mellitus, thyroid disorders, were pregnant or breast-feeding, wore contact lenses, had a systemic inflammatory/autoimmune disease or were following any pharmacological treatment/systemic drug that can disrupt the normal function of the ocular surface. Also, participants had to be between the ages 18 and 35 years old and enrolled in the university before march of 2020. The study design was planned to create 3 groups of 40 participants each (depending on their responses about the VDT extra question): group 1-< 6 h/day; group 2-6 to < 8 h/day; and group 3 -> 8 h/day. The study design and protocol session are summarized in Fig. 1. 120 out of the 183 volunteers (mean ± Standard Deviation [SD] age 22.5 ± 2.32 years) who met the inclusion criteria were scheduled for the study session. All tests were performed in a single appointment. These tests included: meibometry, eyelid margin abnormalities, quality of the MG secretion, conjunctival hyperemia, and meibography were measured. To avoid any possible alteration, less invasive tests were performed first ( Fig. 1) [13]. All measurements were performed in the morning from 10.00 a.m. to 1.00 p.m. by the same observer. Data were masked with an alphanumeric code for the analysis by a second experienced observer.
Ocular discomfort symptomatology and hours of video displays terminals use Symptomatology of ocular surface discomfort was quantified by an online self-administered OSDI questionnaire [14,15]. Participants were categorized into 4 severity grades depending on their OSDI values [15]. For the hours of VDT use quantification, participants have completed an extra question in the online self-administered survey and were categorized into three grades. The categorization of OSDI and hours of VDT use are summarized in Table 1.

Meibometry
Meibometry was performed with the Meibometer MB 560 (Courage-Khazaka electronic GmbH, Cologne, Germany) [16][17][18][19]. This device is a photometer unit connected to a computer that shows the recorded data. To collect the meibomian secretion, the manufacturer`s protocol was followed by using a matt synthetic tap which is also provided by them. The matt synthetic tape was bent and brought in contact with the central portion of the eyelid margin for 10 s. The meibomian secretion sample collected on the matt synthetic tape was let to dry for 1 min before it was introduced into the meibometer for measurement. Average mean was calculated from three readings of the same tape on the photometry unit. Data was measured in Meibometer Units (MU) [16,18].

Ocular surface examination under slit lamp
An exhaustive ocular surface examination under a Topcon SL-D4 (TOPCON Corporation, Tokyo, Japan) slit lamp with a video camera DC-4 attached was performed on every participant. Videos of each participants' ocular surface were recorded for the posterior analysis by the second masked observer. Ocular surface exploration examined the eyelid abnormalities, quality of the MG secretion and conjunctival hyperemia. Different settings were used on the slit lamp for the thorough examination of each ocular structure.

Eyelid margin abnormalities
For the eyelid margin observation, white diffuse light and 16 × magnification were used on the slit lamp. A video of the lower eyelid was recorded, and the following parameters were analyzed: eyelid margin irregularity, hyperemia, and plugging of the MG orifices. Eyelid abnormalities were categorized using the Arita et al. [20] grading scale (Table 1).

Meibomian gland secretion quality
To stimulate the MG secretion, digital pressure was applied for 30 s along the length of the lower eyelid where the MG are located [21]. For video capturing, 16 × and 25 × magnification, and diffuse white light were set on the slit lamp. A single video was recorded to capture the examination of the quality of the MG secretion of the lower eyelid. The quality of the MG secretion was analyzed following Bron et al. [22] grading scale (Table 1 and Fig. 2).   [28,29]. Due to the non-continuous distribution of the data, correlations between values were assessed by performing the Spearman Rho Correlation test [28]. Since data were categorical variables, Fisher's exact test was performed to assess the association between them [28,29]. Differences in meibometry between quality of the MG secretion groups were studied by   a Kruskal-Wallis test, while differences by pairs were assessed using the Mann-Whitney U test [28]. Multivariate linear regression was performed on the studied parameters and expressed with the coefficients B and p value.

Results
Descriptive and demographic statistics of the sample The present study analyzed a total sample of 120 participants with a mean age (± SD) of 22

Association between the DED symptomatology and studied parameters
Multivariate regression showed that the DED symptomatology was significantly associated with higher values of hours of VDT use (coefficients B = 0.316, p = 0.007) and higher values of eyelid margin hyperemia (coefficients B = 0.434, p ≤ 0.001). No other studied parameter showed any association (Table 3). No statistical relationship was found between the quality of the MG secretion and eyelid margin irregularity (Fisher's exact test; p = 0.139). Contingency Table 5 shows significant variation in nasal and temporal conjunctival hyperemia among the different MG secretion quality grades, it occurs in both areas: nasal (Fisher's exact test; p = 0.023), and temporal (Fisher's exact test; p = 0.039). MGLA grade significantly varied as the quality of the MG secretion varied (Fisher's exact test; p = 0.013).

Association between the gender and studied parameters
Multivariate regression showed that the quality of the MG secretion was significantly associated with higher values of the MG orifice plugging (coefficients B = 0.295, p = 0.004) and lower values of meibometry (coefficients B = − 0.001, p = 0.029). No other studied parameter showed any association (Table 6).

Discussion
The number of hours using VDT by university students during the COVID-19 pandemic increased substantially and could influence their ocular surface health [2,3]. In fact, the number of complete blinks during the use of VDT was measured to be reduced  [4,5]; incomplete blinking reduces the meibum spreading over the ocular surface, leading to the accumulation of fatty acids over the eyelids free border, and causing MG orifices obstruction with the subsequent reduction of the MG secretion quality [8].
Although the main objective of this research was to analyze if the quality of the MG secretion was associated with other ocular surface parameters, results showed a positive correlation between VDT use and ocular symptomatology as well as multivariate regression showed an influence of the hours of VDT use in the DED symptomatology. Similar results to the present study were observed by García-Ayuso et al. [30] who studied 676 university students and observed higher OSDI values due to synchronous hybrid learning during the COVID-19 pandemic. Also, other researchers performed only surveys in healthy university students during the COVID-19 pandemic and observed an increase in ocular discomfort consistent with DED symptomatology [3,31,32]. Fenga et al. [6] also observed differences in symptomatology values in a sample of participants that use VDT with and without MG dysfunction. Wu et al. [33] studied 93 participants with dry eye symptomatology distributed into two VDT users groups, a positive correlation between hours of VDT use and OSDI was found. Ocular discomfort could also be due to incomplete blinks during the use of VDT; this hypothesis was supported by Portello et al. [7] which found higher values of dry eye symptomatology in participants with lower blink rates and high ratios of incomplete blinks during different visual tasks.
A normal blink rate and complete blinking could be factors that do not allow the obstruction of the MG orifices by the meibum accumulated over the eyelid border, with the consequent alteration of the quality of the MG secretion [4]. The present study found positive correlations between the quality of the MG secretion and eyelid margin hyperemia, plugging of  [33], who found significant differences between groups for eyelid margin abnormalities, quality of the MG secretion, and MGLA. Also, Zhou et al. [34] stated that lid margin score was a useful predictor for MG loss depending on the obstruction of the MG orifices. The relationship between MGLA, eyelid margin abnormalities, and the quality of the MG secretion must be considered [8], because incomplete blinking could accumulate lipids over the eyelid margin, which may change their chemical proprieties, and obstruct the MG orifices. A thicker MG secretion is more difficult to be spread over the ocular surface, and therefore cannot reach its anti-evaporative function which is the main purpose of the lipids secreted by the MGs. This finding could explain why the obstruction of the MGs orifices altered the quality of the MGs secretion and triggers the MGs destruction, which is in concordance with previous reports [34]. The use of VDT reduces the rate of complete blinks which may influence the quality of the MG secretion with the subsequent loss of ocular surface homeostasis [7]. Also, a significant relationship between nasal and temporal conjunctival hyperemia with quality of the MG secretion was found by both, Spearman Rho correlation and Fisher's exact test. Participants with thicker MG secretion showed higher values of conjunctival hyperemia, which could be due to an inefficient evaporative function of the thicker meibum because of its difficulty to be spread over the ocular surface. To the authors' knowledge, there are no other studies that evaluated conjunctival hyperemia and its relationship with the quality of the MG secretion and plugging of the MG orifices.
No differences were reported in meibometry values between the quality of the MG secretion grades. This could be because the meibometry technique only measures the quantity of lipids present on the matt synthetic tape and not their quality [16,18]. It also does not consider the thickness of the meibum which depends of the chemical quality of the lipids (polar or not). Additionally, the authors noticed that MGs does not yield all at the same time point of the day and the present study was carried out from 10 am to 7 pm, not always at the same day hour [35], which could be a limitation of the study. Nevertheless, multivariate regression identified the meibometry as a causative factor for the alteration of the quality of the MG secretion, which could explain that lower meibometry values could be due to a worse quality of the MG secretion and higher values of plugging of the MG orifices.

Conclusion
In conclusion, participants with higher values in the quality of the MG secretion have higher values in eyelid margin hyperemia, plugging of the MG orifices, MGLA, nasal conjunctival hyperemia, and temporal conjunctival hyperemia. No direct relationship between the quality of the MG secretion and hours of VDT use, or OSDI was found. Participants that spend long hours using VDT had higher values in OSDI, which can be explained by the impact of VDT in complete blinking, responsible for spreading the MG secretion over the ocular surface to avoid tear evaporation. To support the hypothesis proposed on how to complete blinking and VDT use affect the eyelid border and MGs, other parameters like blink rates, tear film break-up time, ocular protection index, and tear film lipid layer interference patterns should be measured in future research.
Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The authors have not disclosed any funding.

Conflict of interests
The authors declare no competing interests.
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