The Cornea

In the simplest terms, the cornea is a fibre-reinforced fluid membrane, which its tensile strength derived from the three-dimensional organization of collagen fibers and its bulk properties derive from the inter febrile fluid pressure [1]. The cornea lacks the neurobiological [1] sophistication of the retina and the dynamic movement of the lens; yet, without its clarity, connective tissue that acts as the primary infectious and structural barrier of the eye infections [2].

The cornea has five principal layers which, from anterior to posterior, are the epithelium, Bowman’s, stromal, Descemet’s and endothelium. Bowman’s and Descemet’s layers are basement membranes for the cellular epithelium and endothelium layers, respectively. The stromal occupies 90% of the cornea’s thickness and like other soft, highly hydrated and charged collagenous tissues such as cartilage and intervertebral disc, is a polyelectrolyte gel consisting of a mixture of interacting fluid, solid and ionic phases. Water is the principal component of stromal which saturates the collagen solid phase solvates the ionic phase and accounts for about 78% of the cornea by weight [3].

In the average adult, the horizontal diameter of the cornea is 11.5 to 12.0 mm and about 1.0 mm larger than the vertical diameter. It is approximately 0.5 mm thick at the center and gradually increases in thickness toward the periphery. The shape of the cornea is prorated flatter in the periphery and steeper centrally which creates an aspheric optical system [4]. 

Figure 1: Light micrograph of normal endothelium (original magnification_100). Note the single-cell endothelial layer with a Descemet membrane of uniform thickness (epithelial surface at top of figure). Reprinted with permission of Ophthalmology.4 Copyright 2008Elsevier

Figure 2: Cross-sectional view of the corneal epithelial cell layer Reprinted with permission of Ophthalmology.4 Copyright 2008 Elsevier

Corneal shape and curvature are governed by the intrinsic biomechanical structure and extrinsic environment. Anterior corneal stromal rigidity appears to be particularly important in maintaining the corneal curvature. Organizational differences in the collagen bundles of the anterior stromal may contribute to a tighter cohesive strength in this area and may also explain why the anterior curvature resists change to stromal hydration much more than the posterior stromal, which tends to more easily develop folds . Stromal hydration also appears to affect the cornea’s response to strain and shear forces.

The human cornea consists of 5 recognized layers, 3 cellular (epithelium, stromal, endothelium) and 2 inter- face (Bowman membrane, Descemet membrane [5] (Fig - 1).

Epithelium

The epithelial surface of the cornea creates the first barrier to the outside environment and is an integral part of the tear film–cornea interface that is critical to the refractive power of the eye. Embryo logically, the corneal epithelium is derived from surface ectoderm between 5 and 6 weeks of gestation. It is composed of no keratinized, stratified squamous epithelium that is 4 to 6 cell layers thick (40 mm to 50 mm) [6].

The mucinous layer of the tear film, which is in direct contact with the corneal epithelium, is produced by the conjunctiva goblet cells and interacts closely with the corneal epithelial cell glycocalyx to allow hydrophilic spreading of the tear film with each eyelid blink.

Anatomy of the Cornea

The life span of corneal epithelium is ten days, which regularly undergoes apoptosis (programmed cell death) [7].

The tear film prohibit from interring the intercellular spaces by tight junction between neighbors surface cells.

Basal layer is the deep  cellular layer which composed of single cell layer which the only cell layer cab able of mitosis(stem cells).They are source of wing cells [8].

0.05Mm is the epithelium basement membrane thickness, once it is injured needs 4-6 weeks to heals, during this period the epithelial attachment is tends to be weak.

Bowman Layer

Its 15Mm thickness, help to maintain corneal shape once its injured can't regenerates and replaced by scar tissue.

Stroma

It provides 80-85% of the corneal thickness, it differs from other collagenous structures by its transparency due to highly arrange collagens fibers and extracellular matrix [9-10].

Figure 3: Transmission electron microscopy of the human corneal stromal. A Keratocyte localized between stromal lamellae B: Higher magnification view. Showing a keratocyte in relation to collagen fibers coursing in various directions Reprinted with permission of Cornea.6 Copyright 2005 Elsevier

Figure 4: Micrograph illustrating Descemet membrane (DM) located between the posterior aspect of the corneal stromal (S) and the underlyin endothelium (EN). The anterior “banded” region (A) is secreted by the endothelial cells during fetal development and is more highly organized than the posterior “amorphous region” region” (P), which is secreted after birth. Reprinted with permission of Principles and Practice of Ophthalmology. [13] Copyright 2008 Elsevier

An additional feature of the stromal is that the ultrastructure within the organization of the lamella appears to vary based on the depth within the stromal.

Deeper layers are more strictly organized than superficial layers, and this difference accounts for the greater ease of surgical dissection in a particular planes one approaches the posterior depths of the corneal stromal. This variation in stromal organization also accounts for the differences in response to corneal edema, as mentioned previously. 

Descemet folds are the result of asymmetric swelling of the posterior stromal imposed by the structurally more rigid anterior cornea and structural restriction imposed by the limbs.

Stromal swelling is therefore directed posteriorly and results in relative flattening of the posterior surface, which can push Descemet membrane into multiple folds that become visible as striate [11-12].

Keratocytes are the major cell type of the stromal and are involved in maintaining the ECM environment. They are able to synthesize collagen molecules and glycosaminoglycans while also creating matrix metalloproteases (MMPs) dall crucial in maintaining stromal homeostasis.

Descement Membrane

At 8 weeks of gestation the endothelial cells start to secret it continuously The anterior 3 mm secreted prior to birth has a distinctive banded appearance when viewed by electron microscopy, but Descemet membrane produced after birth is unbranded and has an amorphous ultra-structural texture. Descemet can accumulate up to 10 mm in thickness with age (Figure 4).

Endothelium

The endothelium act on maintaining corneal clarity by keeping it relatively deturgesced state. It`s monolayer with honey comb mosaic from posterior view [14].

It`s 15Mm in thickness which span the posterior portion of the entire cornea and fused with trabecular [15]. The lateral membranes contain a high density of NaC, KC-ATPase pump sites [16].

The basal surface of the endothelium contains numerous hemi desmosomes that promote adhesion to Descemet membrane. Endothelial cell density and topography continue to change throughout life. From the second to eighth decades of life, the cell density declines from 3000to 4000 cells/mm2 to around 2600 cells/mm2, and the percentage of hexagonal cells declines from approximately 75% to approximately 60% [17].

Blood Supply of the Cornea

The cornea is avascular tissue 'where it met it`s need from blood components, through a tiny blood vessels at the limbs, tear film and aqueous humor.

Nerve Supply of the Cornea

The most sensitive and heavily innervated tissue in the body is the cornea, which derived from nasocillary branch, branch of ophthalmic nerve.

Overview of Gonadotropins

The gonadotropins, a family of closely related glycoprotein hormones includes the followings: follicular stimulatory hormone (FSH), luteinizing hormone (LH) which is secreted by same pituitary cells; gonadotropins and chorionic gonad tin (CG) which placental origin.

Gonadotropin-releasing hormone (GnRH) is adecapeptide secreted from the pre-optic and arcuate nuclei of the hypothalamus into the hypophyseal-portal blood vessels which transports it to the anterior pituitary. At anterior pituitary GnRH stimulates the secretion of LH and FSH, both of which play a central role in ovarian function [19].

The key hormone regulating follicular growth is FSH, while pulsatile LH appears to be involved in regulating normal follicular turnover. Our knowledge of the chemistry of the gonadotropic hormones has greatly increased our understanding of the mechanisms of mammalian reproduction. LH and FSH are synthesized in the pituitary gland, released into the systemic blood circulation, and carried to the target end organs the gonads where the production of (20) androgens and estradiol's.

In the ovary, the cellular contribution to steroid genesis is very different from that in the testis, and both granulose cells and theca cells contribute to steroid genesis. In the test is supporting cell lineage gives rise to Sterol cells which are nurse cells for spermatogenesis. For ovarian histogenesis, the supporting cell lineage gives rise to granulose cells. Theca cells develop from stromal steroid genic precursor cells outside the follicles and are ovarian counterparts of lending cells. The theca cells synthesize androgen in response to human chorionic gonadotropin, hCG and pituitary LH [21].

Polycystic Ovarian Syndrome

Definition and Diagnostic Criteria

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women and major cause of an-ovulatory infertility.

The diagnosis based on thee ESRHE/ASRM Rotterdam consensus meeting in 2003, which broaden the previous NIH criteria classifications of1991 [22].

At least two out of these criteria 2003 Rotterdam criteria:

• Oligo-or anovulation

• Clinical and or biochemical signs of hyperandrogenism

• Echogenic evidence of polycystic ovaries and exclusion of other causes of hyperandrogenism and anovulation [23]

Etiology

The Etiology of Pcos Is Unknown

• It is theorized that early exposure to androgen excess in utero or during neonatal, period is associated with development of the PCOS phenotype later in life [24-27]

• Genetic basis is associated with several candidate genes for insulin resistance and androgen production (eg,cytochrome p450c17,cytochrome p450c11a,and insulin receptor substrate)

• Central obesity play direct role in pathophysiology of PCOS by contributing to insulin resistance and increase androgen level

A high percentage (55-75 %) of women with PCOS have an elevated LH/FSH ratio presumably due to high levels of LH rather than reduced production of FSH. GnRH stimulation causes, indeed, excessive LH production [28] in those women. This condition may be determined by a higher frequency or amplitude of GnRH [29] it is not yet clear whether alteration of the hypothalamo-pituitary axis in PCOS is primary or secondary to alterations in steroid hormones secretion. The role of FSH is to recruit ovarian follicles and stimulate their growth: 2-5 mm follicle are sensitive to FSH whereas larger ones (6-8 mm) acquire aromatase activity and may increase estradiol (E2) and inhibit B reducing levels of FSH in late follicular stage. On the other hand, PCOS patients (having LH and FSH concentrations higher and lower than normal, respectively).

Clinical Features

The three distinguishing features include:

• Hyperandrogenism: clinical sign include hirsutism acne and hair loss [30]

• Chronic anovulation: fewer than eight episodes per a year [31]

• Ultrasonographic evidence of polycystic ovaries: The polycystic ovary was defined as having≥10 follicles (2–8 mm), anovarian volume≥12 cm3, and a bright echogenic stromal [32]

Luteiizing Hormone/Follicle Stimulating Hormone (Lh/Fsh) Ratio

A ratio>2.0 is suggestive of PCOS but it`s not highly sensitive.

Gonadotropin levels are affected by oral contraceptives [33].

Hirsutism

Define as excessive hair loss of suggests under lying hyperandrogensim it`s frequently seen on the upper lip, chest, back, chin and lower abdomen [34].

Other criteria are hyperinsulinemia, elevated insulin like factor (IGF_1), obesity.

Patients and Methods 

A case control study for 111 woman done in the ophthalmology department of AL SADDER MEDICAL CITY for the period from December 2015 to September 2016. Women included in this study were any women in her reproductive age attending the outpatient department in our hospital in Dermatology Department which has been already diagnosed as PCOS and sent for laser therapy for hirsuitism (IPL). Questionnaire for each patient include age, marital state, no. of children, address, any medical and surgical history, drug history and hormonal therapy and any eye surgery.

Their investigation was reviewed for hormonal levels and U/S criterion for PCOS.

The control group are relatives of the patients and any female that have negative history for polycystic ovary, normal hormonal assay and nomal U/S.

Exclusion Criteria

Patients with any corneal and ocular surface pathology, his- tory of eye surgery, ocular trauma, any topical medications patients with systemic diseases: DM, hypertension systemic drugs (amiodaron), metabolic disorder like cystinosis.

Patients with other endocrinopathies, pituitary adenoma, Hyperprolactinemia, pregnant ladies were also excluded.

Endocrinological Assessment 

Includes hormonal level measurement of both LH and FSH hormones at day 3 and 4 from the menstrual cycle measured in IU for patients with PCOS in the lab of the infertility center.

LH/FSH ratio were calculated, resulted in normal ratio PCOS and high ratio PCOS patients.

Ophthalmological Assessment

• Patients underwent corneal examination by slit lamp bio microscopy in ophthalmological department of AL-SADDER MEDICAL CITY for both control and PCO patients group

• Measurements of CCT by pentagm (pachymetry) for Rt and Lt corneas for both control and disease groups expressed in Mm 

Statistical analysis 

Statistical analysis was done by using SPSS (statistical package for social sciences) version 20 in which we use independent sample t-test and pearson correlation coefficient for numerical data and chi square with odds ratio for categorical data. P value≤0.05 regarded significant. 

A total of 111 women had been included in this study. Twenty nine of them had PCO with normal LH: FSH 

Figure 5: Correlation between serum LH level and right corneal thickness

Note: r=-0.013   p=0.782

ratio (group 1), 40 had high LH:FSH ratio (group 2) and 42 control group (group 3).

Figure 6: Correlation between serum LH level and left corneal thickness

Note: r=-0.035   p=0.545

Table 1: Demographic characteristics of women under study

Table 2: Comparison between group 1 and 3 in corneal thickness

Note: Values are expressed in mean ± SD. P>0.05 

Table 3: Association between PCO (normal ratio) and corneal thickness(right)

There is no significant association between right corneal thickness and PCO (normal ratio)

Table 4: Association between PCO (normal ratio) and corneal thickness (left)

There is no significant association between left corneal thickness and PCO (normal ratio)

Figure 7: Correlation between serum FSH level and left corneal thickness

Note: r=-0.036    p=0.823

Figure 8: correlation between serum FSH level and right corneal thickness

Note: 0.014   p=0.785

Table 5: Comparison between group 2 and 3 in corneal thickness.

Values are expressed in mean ± SD. P>0.05

Table 6: Association between PCO (high ratio) and corneal thickness (right)

There is no significant association between high ratio group and right corneal thickness

Table 7: Association between PCO (high ratio) and corneal thickness (left)

There is no significant association between high ratio group and left corneal thickness

        Table 5 shows no significant difference in cornea between group 2 and 3.

        Figure 5 shows no significant correlation between serum LH and right corneal thickness.

        Figure 6 shows no significant correlation between serum LH and left corneal thickness.

        Figure 7 shows no significant correlation between serum FSH and left corneal thickness.

        Figure 8 shows no significant correlation between serum FSH and right corneal thickness.

Polycystic ovarian syndrome (PCOS) is the most common gynecological endocrinopathy [58].

PCOS appears to be associated with an increased risk of metabolic aberrations, including insulin resistance and hyperinsulinism, type 2 diabetes mellitus, dyslipidemia, cardiovascular disease, and endometrial carcinoma [59].

A total of 111 women had been included in this study. Twenty nine of them had PCO with normal LH:FSH ratio (group 1), 40 had high LH:FSH ratio (group 2) and 42 control group (group 3).

Demographic data of studied group 1: 

Demographic characteristics of women under study. Table 1 shows no significant difference between groups regarding age and marital status (P value=0.581,0.422 respectively).

Results in Table 2 show Comparison between group 1 (Patients with PCO and normal:

LH\FSH ratio) and group3 (control group) in CCT for the right cornea for each group according to study in Lithuanian population for CCT (546±13) [78] The CCT was (541.44±38.17), (537.61±33.50) and for left cornea, (544.3103±45.40619), (538.8095±34.33709) where no significant diffirence between these two groups where p value±(0.656,0.563) respectively. 

This result disagree to one recent report,corneal thickness in patients with PCO was found to be higher than amoung controls:this finding attributed to higher IGF-1 levels in patients with PCO [60].

It has been suggested that this association may give rise to more serious eye diseases such as glaucoma [61].

Table 3 association between PCO with normal ratio and right CCT. 

Results shows no significant associations in Rt CCT between control and PCO (normal ratio) ,51.7% are ≤546 and 38.5% >546.as OR=0.569.

Table 4 Association between PCO (normal ratio) and Rt CCT.Theres no significant association between, Rt CCT and PCO were 51.7%≤546 and 48.3% > 546, OR=0.659.

Figure 1,2 correlation of serum LH level and Rt and Lt CCT (Group 1).

Shows no significant correlation between serum level and CCT as (r=0.137) (p=0.476).

The result shows no significant correlation between serum LH and Rt CCT. In group one this result is disagree with previous study result where they found patient with PCO had higher level of CCT in Comparison with control patient they suggest this might attributed to high levels IGF-1 which found to be higher in those patients as the cornea is possible target [61-62].

While many women with PCOS still have LH and FSH still within the 5-20 mlU/ml range, their LH level is often two or three times that of the FSH level. For example, it is typical for within the normal range of 5-20 mlU/ml). This situation is called an elevated LH to FSH ratio or a ratio of 3:1 so there is no real elevation in LH hormone in PCO patients rather than elevated ratio which might help in diagnosis [63].

Figure (3)(4) correlation between FSH level and Rt and Lt CCT. In this figure there is no significant correlation, (r=0.13 p=0.5) and (r=0.129 p=0.506).

As this due to the fact that the hormonal level of PCO remain within the normal levels for both LH and FSH. For most of the patients if we compare our result with study of normal pregnant females how shows increase in CCT versus normal non pregnant females.

As pregnant female show little changes in FSH level as the pregnancy proceed [64].

This changes in corneal thickness causes by corneal oedema as apart of the generalized oedema occurs in pregnants ladies especially in final trimester which is statistically significant only in the31-40 wk (p=0.01) [65-66]

Table 5 Comparison between group 2 and group 3 in CCT, which shows no significant difference. P=0.305, 0.485 respectively.

Table 6 association of CCT in group 2, i.e high ratio, for RT and left cornea.

Results in no significant association for both Rt and left cornea, percentage of CCT >546 is 40%,37% FOR Rt and Lf eye respectively.

This result is inconsistent with other studies for CCT in PCO as there is significant increase in the corneal thickness [62].

In the other hands other study also show significant increase in CCT and tear film instability in patients with PCO [61].

To compare our study with other study for evolution of corneal thickness during menstrual cycle which shows a significant increase in CCT for both eyes during preovulatry and ovulatory time which coincide with LH surge, their result shows increase CCT at days 1 and 3 was 541.40+-11.36 and 540.80±11.70 microne for Rt and Lf cornea respectively.

Corneal thickness was not stastically different in Rt and Lt eyes at any time (p<0.05) [67].

These changes could be secondary to hormonal influences; estrogen receptors can be found in human corneas, suggesting that estrogen may have a role in corneal physiology; [69]. Another study demonstrated that there is a significant change in corneal hydration during the normal menstrual cycle. Apparently, the change is associated with the effects of estrogen more than with progesterone. The authors believe one might assume ready access of these hormones via the aqueous humor or tear film because of their high lipid solubility. They suggest that another possibility would be an indirect action of hormones on the cornea via their effects on tear film osmolarity [68].

• There is no direct correlation between the CCT and LH/FSH ratio, this might be attributed to small studied group, we might need large sample size

• As the results are non-significant, it might be belongs to the fact that there is no increase in hormonal level and remain in normal range, but there is only increase in the ratio

Recommendations

• Further studies and increase sample size might be needed to assess the effects of LH/FSH on CCT

• Enquiry about the hormonal therapy for induction of ovulation may affect CCT

• Timing of menstrual cycle and the time of the measurement of CCT, should be take in account

• pregnant ladies have higher readings so should be excluded from the study

• Any topical and systemic hormonal therapy may affect the result, so should put in the mind

• Cautions should be taken in account for patients with PCO when they ask about LASIK surgery

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