Introduction

Dry eye disease is a disease induced by multiple factors, accompanied by increased tear film permeability and ocular surface inflammation1. Patients often experience pain, irritation symptoms, and visual impairment2. From the perspective of epidemiology, research shows that the prevalence rate of dry eye disease among people aged 65–84 in the United States is as high as 14.6% whilt that in China is higher than in the United States3. The prevalence rate of dry eye disease among women over 50 years old is about twice that of men4. Some research has shown that sex hormone receptors can be found in the conjunctiva, cornea, and meibomian gland, and indicate that dry eye disease is related to sex hormone deficiency, while estrogen deficiency may lead to meibomian gland dysfunction5,6. Farida EH has reported that elderly patients, especially perimenopausal female patients, have a higher probability of developing severe dry eye disease in the cornea due to changes in estrogen secretion in the body, and patients experience more pronounced eye discomfort7,8,9.

At present, the main methods for treating dry eye disease include artificial tears, anti-inflammatory agents, secretagogues, obstruction of the lacrimal passage, autologous serum, therapeutic contact lenses, and meibomian gland massage10,11. In recent years, autologous serum has achieved certain therapeutic effects in the treatment of ocular surface diseases. The biochemical characteristics of autologous serum are similar to normal tears, and its active ingredients such as vitamin A and epithelial growth factor can promote the proliferation, differentiation, and maturation of ocular surface epithelial cells. At the same time, the blood contains multiple antibacterial factors, which can enhance the anti infection ability and bacterial inhibition effect of ocular surface to a certain extent, protecting ocular surface cells from damage. Furthermore, compared with artificial tears, it has the advantages of being natural and non allergenic, and is considered a better substitute for tears12,13. Research has shown that the use of estrogen as a substitute therapy for dry eye disease has certain effects, but its specific effects and mechanisms are still unclear14,15. Unfortunately, there is a lack of reports on the effectiveness of estrogen replacement therapy combined with autologous serum in the treatment of severe dry eye disease in perimenopausal women. Therefore, this study aims to analyze the effectiveness of estrogen replacement therapy combined with autologous serum therapy in controlling severe dry eye disease in perimenopausal women based on pathology and protein levels, providing new ideas for the clinical treatment of dry eye disease.

Methods

General information

1249 perimenopausal female patients with dry eye disease who visited Ziyang Hospital of West China Hospital of Sichuan University from January 2021 to December 2023 were selected, with an average age of (50.36 ± 3.76)years. At the initial diagnosis, symptoms of dry eye disease include (at least one positive): dryness, foreign body and/or sanding sensation, burning sensation, stinging sensation, visual fatigue, heaviness of eyelids, and redness of the eyes. All cases were excluded from other eye diseases, with or without menstrual disorders or menopausal duration less than 1 year. Serum luteinizing hormone (LH), progesterone (PG), estradiol (E2), prolactin (PT), and follicle stimulating hormone (FSH) tests were performed, which were consistent with the diagnosis of perimenopause; After 2 weeks of routine treatment, there was no significant improvement in symptoms.

Diagnostic criteria for perimenopausal women

The diagnostic criteria for perimenopausal women are strictly based on the World Health Organization’s definition of perimenopausal women, which refers to the cessation of menstruation for 12 months due to physiological ovarian atrophy. During this process, owing to the decline in ovarian function, the secretion ability of estrogen gradually decreases, and the body undergoes many physiological and psychological changes16.

Inclusion criteria: (1) Age > 40 years old; (2) Menopause for more than one year without the intention of menstruation; (3)Improved female menopausal self-assessment scale (Kuperman score) ≥ 10 points; (4) Not taking any medication for menopausal syndrome within 3 months; (5) The uterus is intact.

Exclusion criteria: (1) Individuals with contraindications to menopausal hormone therapy (MHT); (2) Patients with endometrial hyperplasia or cervical diseases requiring diagnosis and treatment; (3) Uterine fibroids cannot exclude malignant lesions; (4) Uterine or (and) ovarian resection patients; (5) Suffering from other serious systemic diseases; (6) Unable to follow up regularly.

Exit criteria: (1) Patients with poor compliance and interrupted treatment; (2) New patients with other illnesses who can not continue treatment; (3) Patients who add other drugs or therapies on their own that affect the efficacy evaluation.

Severity grading of dry eye syndrome

This study used corneal fluorescence staining to grade the severity of dry eye syndrome. 2 μl of 1% sodium fluorescein was dropped into the conjunctival sac of the patient, and the range and degree of corneal staining were observed under slit lamp. The staining results were analyzed using a standardized scoring method: 0 points = no corneal staining; 1 point = corneal micro damage, limited to scattered spot staining of superficial epithelium, including staining range ≤ 15%; 2 points = mild corneal injury, localized or scattered staining in the center or aggregation, or peripheral staining, including staining within the range of 3 and 9 points or large dots, ranging from 16 to 30%, may affect deep epithelium, slow and mild stromal staining; 3 points = moderate corneal injury with a maximum diameter exceeding 2 mm, dense clustered block staining, large point fusion, range 31–45%, involving deep epithelium, rapid localized stromal staining; 4 points = Severe corneal injury, dense clustered block staining with a diameter exceeding 2 mm, patchy, with a range greater than 45%, affecting deep epithelium, and rapidly diffusing bright staining of the stroma. Patients diagnosed with severe dry eye with a score of 4 were selected as the study subjects and grouped for research17.

Experimental grouping

This study was randomly divided the research subjects into a normal group, a control group, an estrogen replacement therapy group (ERT), an autologous serum group (AS), and an estrogen replacement therapy + autologous serum group (ERT + AS), with 40 cases in each group, for a total of 200 cases. The normal group consisted of women of the same age who were not diagnosed with perimenopause, while the control group consisted of severely dry eye research subjects who were included in the trial but did not receive estrogen replacement therapy or autologous serum treatment. The patients were treated with a general ophthalmic clinical treatment plan.

Preparation method of autologous serum

The patient collected 20 ml of fasting venous blood in the morning, centrifuged at 3000 r/min for 15 min, carefully aspirated the upper layer of serum using a sterile needle, and diluted it with levofloxacin eye drops (eye drops to serum ratio of 4:1). Administration method: Directly drop the prepared serum into the eye, 1 drop/time, every 4 h. After corneal fluorescence staining is negative, stop the medication after 1 week.

Estrogen treatment plan

Take 2 mg of nilestriol orally every 15 days, and add 2 mg of progesterone daily to the second dose of nilestriol taken by perimenopausal patients every month for half a month. At the same time, use autologous serum locally in the eyes18,19. After 30 days of oral medication treatment, compare and analyze the changes in luteinizing hormone (LH), estradiol (E2), and follicle stimulating hormone (FSH) levels in each group of patients.

Eye surface data collection

(1) Using corneal fluorescence staining method, observe and record the analysis of the corneal surface condition of the study subjects at different time points, and collect images; (2) Observe and record the tear secretion of patients at different time points through Schirmer test; (3) Observe and record the changes in intraocular pressure of patients at different time points using a Goldman tonometer.

Pathological analysis

After 28 days of drug treatment, the endometrium of each group of study subjects was extracted, and the morphological changes of the endometrium were analyzed by HE staining.

Proteomic analysis

After 28 days of drug treatment, endometrial protein solutions were extracted from each group of study subjects, and ELISA detection kits were used to compare and analyze the changes in inflammatory factors TNF, IFN-α, and IL-12 in the endometrium of each group based on protein levels. Collect tear analysis and use ELISA detection kit to analyze the changes in dry eye related inflammatory factors PEG2, VEGF, IL-1β, and TFN-α in tears of each group. Determination of tear protein by enzyme-linked immunosorbent assay (ELISA): competitive method is used to detect the content of lysozyme (LZM), lactoferrin (LF), albumin (ALB), and secreted immunoglobulin A (sIgA) in tear samples.

Statistical methods

Using Graphpad 8.1 statistical software to process data, the measurement data conforms to a normal distribution, using mean ± standard deviation. Independent sample t-test is used for inter group comparison, and one-way ANOVA or paired sample t-test is used for intra group comparison. Inspection level: α = 0.05.

Results

Evaluation of clinical treatment effects on the eyes

After 28 days of drug treatment, the corneal fluorescence staining results of ERT group, AS group, and ERT + AS group showed varying degrees of recovery progress of corneal epithelium, with significantly reduced corneal staining area and intensity. Especially, the ERT + AS group showed the most significant decrease. This study further analyzed the changes in tear film rupture time (BUT), Schirmer, and intraocular pressure in different groups. The average BUT of the normal group was (15.16 ± 0.73)s, while the Control group (non treatment group) had the lowest (5.11 ± 0.45)s. After treatment with ERT (8.27 ± 0.59)s, AS (8.15 ± 0.72)s, and ERT + AS (13.37 ± 0.85)s, the BUT gradually increased, with the ERT + AS group showing the most significant increase, and the difference between the groups was statistically significant (P < 0.05). The study showed that the trend of Schirmer experiment results in each group was consistent with BUT, and the difference was statistically significant (P < 0.05). At the same time, the study showed that compared with the normal group, there was no significant difference in intraocular pressure after drug action in the other groups (P > 0.05) (Fig. 1).

Fig. 1
figure 1

Analysis of tear film rupture time (BUT), Schirmer, and intraocular pressure changes in each group after 28 days of drug treatment.

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Analysis of systemic hormone levels

As shown in Fig. 2, after 28 days of drug treatment, compared with the Control group, the ERT group and ERT + AS group showed a significant decrease in FSH and LH, and a significant increase in E2 while there was no statistically significant difference between the two groups. Besides, there was no statistically significant difference between the AS group and the Control group (P > 0.05).

Fig. 2
figure 2

Analysis of changes in FSH, LH, and E2 levels in each group after 28 days of drug treatment.

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Analysis of ocular surface inflammation

The Elisa experiment results showed that compared with the normal group, the expression intensity of inflammatory factors VEGF, IL-1β, PGE2, and TNF-α in the control group was significantly increased, and the difference between the two groups was statistically significant (P < 0.05). After treatment with ERT, AS, and ERT + AS, the expression of inflammatory factors VEGF, IL-1β, TNF-α, and PGE2 gradually decreased, with the ERT + AS group showing the most significant downregulation of inflammatory factor expression, and the difference was statistically significant (P < 0.05) (Fig. 3).

Fig. 3
figure 3

Analysis of changes in ocular surface inflammatory factors VEGF, IL-1β, PGE2, and TNF-α in each group after 28 days of drug treatment.

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Analysis of tear components

As the Fig. 4 showed, the expression intensity of LZM, LF, ALB and sIgA in the control group was significantly decreased, and the difference between the two groups was statistically significant (P < 0.05). After treatment with ERT, AS, and ERT + AS, the expression of inflammatory factors LZM, LF, ALB and sIgA gradually increased, with the ERT + AS group showing the most significant upregulation, and the difference was statistically significant (P < 0.05).

Fig. 4
figure 4

Analysis of changes in tear components LZM, LF, ALB and sIgA in each group after 28 days of drug treatment.

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Endometrial HE staining analysis

This study further extracted endometrium from each group for HE staining after 28 days of drug action. As shown in Table 1, compared with the normal group, the endometrium thickness of the Control group, ERT group, AS group, and ERT + AS group before treatment was significantly thinner, and there was no statistically significant difference between the groups (P < 0.05). After giving intervention measures, the endometrium thickness of the ERT group and ERT + AS group increased significantly, but there was no difference in the endometrium thickness of the AS group before and after treatment (P > 0.05). Further morphological analysis revealed that compared to the normal group, the Control group had loose endometrial connective tissue, larger gaps, cytoplasmic vacuolization, and uterine gland atrophy. After intervention with ERT and ERT + AS, the endometrial connective tissue was tightly arranged, the uterine gland structure was dense, the endometrial morphology improved, and there was a single layer of columnar epithelium covered by circular uterine glands. However, there was no significant change in the endometrial morphology before and after treatment in the AS group (Figs. 5 and 6).

Table 1 Changes in endometrial thickness in each group before and after treatment.
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Fig. 5
figure 5

HE staining of endometrial connective tissue before and after treatment.

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Fig. 6
figure 6

HE staining of endometrial glandular tissue before and after treatment.

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Changes in the modified Kuperman score during the treatment cycle

As shown in Table 2, compared with the normal group, the Kuperman scores of the Control group, ERT group, AS group, and ERT + AS group19 were significantly increased before treatment, but there was no statistically significant difference among the groups. After intervention, with the prolongation of treatment time, the Kuperman scores of the ERT group and ERT + AS group gradually decreased. At 4 weeks of treatment,the Kuperman scores in these groups reached their lowest point, but there was no statistically significant difference between the ERT group and ERT + AS group (P > 0.05), and there was no statistically significant difference in the AS group before and after treatment (P > 0.05).

Table 2 Changes in modified Kuperman score for each group during the treatment cycle.
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Analysis of endometrial inflammatory response

This study extracted endometrial tissue protein solution for Elisa experiment after 28 days of drug treatment intervention. The results showed that compared with the normal group, the expression intensity of inflammatory factors IFN-α, IL-12, and TNF-α in the control group was significantly increased, and the difference between the two groups was statistically significant (P < 0.05). After treatment with ERT, AS, and ERT + AS, the expression of inflammatory factors IFN-α, IL-12, and TNF-α in the ERT group and ERT + AS group gradually decreased, but the difference between the two groups was not statistically significant. There was no statistically significant difference between the AS group and the Control group (P > 0.05)(Fig. 7).

Fig. 7
figure 7

Analysis of changes in endometrial inflammatory response factors VEGF, IL-1β, PGE2, and TNF-α in each group after 28 days of drug treatment.

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Discussion

The oily substances secreted by the meibomian gland participate in the composition of the tear film play an important role in maintaining eye surface health and preventing dry eye syndrome20,21. Saama Sabeti22 and Pun YL23 have shown that there are estrogen alpha receptors (ER alpha) and beta receptors (ER beta) present in the meibomian gland tissues of humans and rats. Under physiological conditions, estrogen regulates the structure and function of target tissues or cells through ERα and ERβ mediation. The ovary is the main organ for estrogen secretion. Clinical studies have confirmed that during perimenopause, women may experience menstrual disorders, hot flashes, sweating, emotional instability, and other mental and neurological disorders due to ovarian dysfunction, dysfunction of the hypothalamic pituitary ovarian axis feedback regulation, and ultimately abnormal synthesis and secretion of various sex hormones. Some patients may also experience eye symptoms such as red eyes, dry eyes, and blurred vision24,25. Wang26 reports that the worldwide dry eye incidence rate is 5.5%-33.7%, of which women are higher than men, while the perimenopausal incidence rate is 58.42%. At present, the main treatment strategies for dry eye include local tear replacement, anti-inflammatory, secretion promoting, lacrimal spot obstruction, autologous serum, therapeutic corneal contact lenses, and eyelid closure. However, due to the special physiological changes of perimenopausal women, more and more studies have shown that a simple ophthalmic treatment plan is not enough to solve the eye symptoms of such patients, and a new treatment plan for dry eye in perimenopausal women is urgently needed27.

This study first analyzed the recovery of corneal epithelium in each group after 28 days of drug treatment using corneal fluorescence staining. The results demonstrated that the ERT group, AS group, and ERT + AS group showed varying degrees of recovery progress in corneal epithelium, with significantly reduced corneal staining area and intensity while the ERT + AS group showed the most significant decrease. Furthermore, the tear film rupture time (BUT) and Schirmer experiment results showed the same change trend of corneal fluorescence staining. Meanwhile, we further detected the expression of ocular inflammatory factors VEGF, IL-1β, PGE2, and TNF-α through Elisa experiments. The results showed that the expression of these inflammatory factors significantly increased in the control group. After treatment with ERT, AS, and ERT + AS, the expression of VEGF, IL-1β, PGE2, and TNF-α inflammatory factors gradually decreased, with the ERT + AS group showing the most significant downregulation of these inflammatory factor expression28,29. Further analysis of systemic hormone changes in the ERT and ERT + AS groups showed a significant decrease in FSH and LH levels, as well as a significant increase in E2 levels. Among them, the ERT + AS group showed the most significant changes, while there were no significant changes in hormones in the AS and Control groups. Further research has shown that with intervention measures, the Kuperman scores of the ERT group and ERT + AS group gradually decreased with the prolongation of treatment time. At 4 weeks of treatment, the Kuperman scores of the ERT group and ERT + AS group reached their lowest point. The above results indicated that estrogen replacement therapy + autologous serum treatment can effectively reduce the expression of dry eye related inflammatory factors in perimenopausal women, promoted corneal epithelial recovery, and improved patients’ eye symptoms and comfort14,17.

John Sheppard30 has reported that mitochondria are important organelles within cells, playing a crucial role in energy metabolism, the formation of oxygen free radicals, and the process of cell apoptosis. In the process of material metabolism, catabolism releases energy, synthesis metabolism requires energy, and the process of lipid synthesis requires the participation of energy. The cellular energy metabolism decreases after mitochondria are damaged, which affects the synthesis of glycogen and lipids31. The pathological changes in meibomian gland tissue caused by the decrease in estrogen levels in perimenopausal women may be related to the weakened expression level of ERβ or the imbalance of ERα/ERβ expression ratio, which weakens their protective effect on mitochondria32. In this study, the endometrium of each group was extracted for HE staining after 28 days of drug action. The results showed that after intervention measures, the endometrial thickness of the ERT group and ERT + AS group increased significantly, but there was no difference in endometrial thickness before and after treatment in the AS group. At the same time, morphological studies showed that compared with the normal group, the control group had loose endometrial connective tissue, larger gaps, cytoplasmic vacuolization, and uterine gland atrophy. After intervention with ERT and ERT + AS, the endometrial connective tissue was tightly arranged, the uterine gland structure was dense, and there was a single layer of columnar epithelium covered with circular uterine glands. The morphology of the endometrium improved. Besides, the expression of inflammatory factors IFN-α, IL-12, and TNF-α in endometrial tissue gradually decreased in the ERT group and ERT + AS group, and there was no statistically significant difference between the two groups. Singh33 reported that E2 was the most active estrogen, and as one of the main hormones produced by the ovaries, E2 played a crucial role in maintaining female secondary characteristics and reproductive tract function. The decline of ovarian function in perimenopausal women can lead to a decrease in E2 synthesis and secretion, resulting in a decrease in its level and physiological and pathological changes in the endometrium.

Priori34 discovered that treatment with 0.5 mg of 17β-estradiol and 0.1 mg of norgestrel acetate MHT, in combination with placebo, had an impact on bone turnover markers in postmenopausal women. The results showed a decrease in bone resorption and formation markers in the treatment group, suggesting that moderate estrogen supplementation has a bone protective effect. This study found that estrogen replacement therapy can effectively improve the current situation of estrogen deficiency in perimenopausal female patients, and had an improvement effect on endometrial function. The increase of estrogen in patients’ bodies can increase the number of mitochondria in cells, maintain the structure and function of mitochondria, increase the number of mitochondrial cristae, stabilize mitochondrial membrane voltage, and thus increase the number of ERβ in meibomian gland tissue, thereby improving the normal dry eye function of patients35. Therefore, appropriate supplementation of estrogen in perimenopausal women may play an important role in preventing meibomian gland acinar atrophy and protecting mitochondria, and its mechanism needs to be further studied36. Autologous serum containing vitamin A, fibronectin, and growth factors can promote the recovery of damaged tissues and structures in the eye, thereby maintaining tear film stability and significantly alleviating eye symptoms in patients. Therefore, for postmenopausal women with severe dry eye symptoms, if conventional ophthalmic treatment can not improve the symptoms, a combination therapy of estrogen replacement therapy and autologous serum can be used to improve the neurological and psychological symptoms of patients during menopause, while also played an important role in improving dry eye symptoms37,38.

Of course, there are still some shortcomings in the research of this article. For example, we only conducted research on severe dry eye patients in perimenopausal women, it is worth further investigating whether age and dry eye type have an impact on this result. Besides, the side effects of estrogen replacement therapy mainly include the following aspects: breast cancer risk, risk of endometrial cancer, thromboembolic diseases, liver disease, and other side effects include headache, nausea, vomiting, endometrial cancer, venous thrombosis. As for the side effects of autologous serum therapy for the eyes, it mainly includes: eye discomfort, infection, allergic reactions, elevated intraocular pressure, blurred vision, skin allergy or local hardening. Therefore, it is necessary to strictly follow the doctor’s advice for this treatment plan to avoid potential risks.

Conclusion

In summary, the combination of estrogen replacement therapy and autologous serum therapy can effectively improve the symptoms of perimenopausal women, enhance their quality of life, and have important reference value for the treatment of dry eye. However, the limitation of this study is that it did not explore in detail the specific mechanism of estrogen replacement therapy in improving sexual dysfunction in perimenopausal women, and further in-depth analysis is needed in the later stage.