Introduction
Pupil size is a valuable parameter with important clinical implications. Its measurement can help detect abnormalities, and the knowledge of its normal ranges is important to the optical industry (1).
Pupil size is also a significant element in the optical quality of the eye. Increased pupil size increases the high-order monochromatic aberrations resulting in the reduction of image quality (2).
Smaller pupil size is affected by diffraction; however, the depth of focus increases with a

decrease in pupil size (3).
Moreover, pupil size plays an important role in optical designs, particularly bifocal or multifocal contact lenses to achieve optimal visual performance at all viewing distances under various illuminance conditions (4).
Pupil size is also considered a key factor for optimum optical quality in refractive surgeries (5).
Moreover, several studies assessed the relationship between the ablation zone and pupil size with a focus on night vision problems

following refractive surgeries (6-9).
This study aimed to determine the influence of age and gender on pupil size under different illuminance conditions in emmetropic and myopic healthy eyes using Keratograph 4 software.

Methods
The study protocol was approved by the Research Ethics Committee of Mashhad University of Medical sciences, Iran. Moreover, it was conducted according to the tenets of the Declaration of Helsinki. informed consent was obtained from all subjects before data collection.
Among all subjects who admitted to the optometry clinic for a routine examination, 221 healthy eyes of 221 subjects were recruited after a comprehensive ophthalmologic examination, including best-corrected visual acuity using the Snellen acuity test, slit-lamp examination to rule out any ocular abnormalities, and refraction measurement using auto-refractometer (AR-610, Nidek Co, Ltd, Tokyo, Japan) which was refined with subjective refraction.
Patients with any history of previous ocular or refractive surgery, ocular or systemic disease, or any history of ocular or systemic drugs, which might affect the pupil size, were excluded from the study. Emmetropia was defined as a mean spherical equivalent equal to and between +0.50 and -0.50 D. Moreover, myopia was defined as a spherical equivalent of -0.75 D or worse.
Pupil size was evaluated using Keratograph 4 software (OCULUS, Wetzlar, Germany).
The pupillometry component of the Keratograph 4 software provides continuous pupil imaging and evaluates pupil diameter under different light conditions through an infrared-sensitive camera.
Continuous video signals were sent to the computer and pupillary edges were recognized; accordingly, the pupil size measurement was calculated using this software. All participants were examined by the Pupillogram program, which included a sequence of 9.8 seconds of the scotopic stimulus (0.1 lux) and 0.2 seconds of the photopic stimulus (150 lux) repeated 5 times automatically.
At the end of the procedure, the software provided maximum and minimum values (scotopic and photopic) of pupil diameter and presented the graphical diagram of pupil behavior under different illuminous conditions (10).
Pupillometry was performed for all participants following dark adaptation periods of at least 1 min under scotopic conditions. Ambient lighting condition was the same through all measurements (<0.1 lux) measured with the digital photometer.
Patients were asked to blink completely just before capturing to obtain a uniform smooth tear film over the cornea.
An experienced examiner performed all pupillometry measurements through the automatic mode.
The data were analyzed in SPSS software (version 11.0, IBM, Beijing, China) through descriptive statistics (mean±SD).
Furthermore, the independent student t-test was employed to detect differences between gender and refractive groups in terms of pupil size.
Additionally, the correlation between pupil size and age was performed using Pearson’s analysis. A p-value less than 0.05 was considered statistically significant.

Results
Out of 221 participants who completed the study, 122 (85 female) and 99 (66 female) cases were myopes and emmetropes, respectively. The mean ages of the subjects in the myopic and emmetropic groups were 26.03±6.98 and 28.05±10.17 years, respectively (P=0.08).
Moreover, the mean squared error values were -2.18±1.69 D (range: -6.88 to -0.63D) and -0.11±0.27 D (range: -0.50 to +0.50 D) in the myopic and emmetropic groups, respectively.
Analysis of pupil size based on refractive groups revealed larger pupil diameter in myopes, compared to emmetropes in both illuminance conditions.
The mean pupil sizes of the myopic and emmetropic refractive groups were 3.48±0.99 and 3.04±0.77 mm in photopic condition, as well as 6.28±0.83 and 5.82±0.79 mm in scotopic condition, respectively (P<0.001).
No statistically significant difference was observed between males and females in terms of pupil size at any illumination settings. The mean pupil sizes were 3.27±0.90 and 3.32±0.97 mm in photopic females and males (P=0.71), respectively. Moreover, the corresponding values were 6.10±0.80 and 6.02±0.93 mm in scotopic females and males, respectively (P=0.55). 

Discussion
Pupil size is an important factor in designing bifocal or multifocal contact lenses as well as refractive surgeries to achieve optimal visual performance. The present investigation can be a fair representation of pupil diameter as a function of gender and age under different lighting conditions in emmetropic and myopic healthy eyes using Keratograph 4 software.
According to our findings, myopes had larger pupil diameter, compared to emmetropes in both illuminance conditions. However, no significant difference was observed between males and females in terms of pupil size at any illumination settings. On the other hand, a significant inverse association was found between age and pupil size under each illumination level.
Considering the refractive error, our results are in line with the findings of previous studies, which demonstrated a larger pupil size in myopes, compared to the hyperopes and emmetropes in different illumination conditions (11,12).
Cakmak et al. evaluated pupil sizes using ocular Wavefront analyzer in the mesopic condition and reported larger pupil sizes in myopes, compared to those in hyperopes (11).
Maqsood et al. also stated larger pupil sizes in myopes than emmetropes; however, it was not significant (13).
This could be explained by more accommodative
demand in emmetropes or hyperopes than myopes, as well as consecutive synkinesis between

pupils that lead to relatively greater pupillary constriction.
Other studies found that pupil size was not significantly influenced by refractive errors (14-16). Controversies in results could be attributed to the measurement device, illuminance condition, or accommodation status.
Regarding gender, our results confirmed previous studies that found no significant differences between males and females in terms of pupil size (1,11,17).
The assessment of pupil size using an ocular wavefront analyzer in mesopic conditions revealed that pupil size was independent of gender (11).
Moreover, the investigation of pupil size through a pupilometer under dark-adapted condition showed that gender had no effects on the pupil size to a statistically significant degree (17).
Considering age, according to the results of previously conducted studies, our finding showed that pupil size would be smaller with increasing age (14,17-24).
This could be due to the age-related decrease in the amplitude of accommodation, which causes further accommodative effort to see (19).
Contrary to the aforementioned findings, one study noted an increase in the pupil diameter with age in individuals aged one month to 19 years (1).Another study also exposed no statistically significant differences between the pupil diameters and age in children aged 3-4 years (25).Compared to the studies involving young children, the difference in the correlation between age and pupil diameter could be attributed to the population age.

Conclusion
The results of the present study suggest larger pupil size in myopes, compared to emmetropes in each illumination level. Additionally, advanced age significantly affects pupil diameter in any illumination conditions. Moreover, gender had no effects on the pupil size to a significant degree. Since pupil diameter is an imperative factor in visual performance, contact lenses are effective in this regard, especially in ocular surgeries (refractive or cataract).
Therefore, the possible benefits of this approach are the improvement of the patients’ management and better interpretation of the results for making clinical decisions.

Acknowledgment
This work was supported by the Deputy of Research of Mashhad University of Medical Sciences, Iran (grant code: 930431).

Conflict of Interest
The authors declare no conflict of interest.