Archives of Immunology Research and Therapy

Abstract

Rheumatoid arthritis (RA) is a common autoimmune disease that causes stiffness, pain, and swelling in the joints in the morning. If the disease gets worse, the joints can become deformed. This can make it harder for joints to work normally, which can have a big effect on the patient's daily life and quality of life. The main drug used to treat rheumatoid arthritis (RA) is methotrexate (MTX). As an immunosuppressant, it stops B cells from activating incorrectly in the body, stops the production of cytokines that aren't supposed to be there, and eases the symptoms that come with that. However, some cases have shown that using MTX by itself is not the best way to treat pain; it usually needs to be combined with other medicines. Iguratimod (IGU) is a new disease-modifying antirheumatic drug (DMARD) that can reduce inflammation and change the immune system. This is a small molecule drug made from 7-methanesulfonylamino-6-phenoxychromones. It has two amide groups. The purpose of this study was to find out how well MTX and IGU work together as a treatment choice for women with mild to severe RA. It also looked into how this medicine changes the way ovaries work, which gives doctors a medical basis for making decisions about clinical care.

Materials and Methods:

We selected 80 female patients diagnosed with moderate to severely active rheumatoid arthritis (RA) as volunteers for our research. The patients were recruited in our department from January 2020 to January 2022. To be eligible for the research, participants must meet the rheumatoid arthritis categorization criteria as out by the 2009 American College of Rheumatology (ACR) and the European Alliance of Associations for Rheumatology (EULAR). Age: Individuals aged 20 to 50 who have experienced regular menstruation for approximately 3 months if under 40 years old (with a menstrual cycle of 21-35 days and a duration of 2-7 days), and exhibit normal sex hormone levels in the follicular phase, as assessed on the 3rd day of menstruation; Have not had any pharmacological therapy in the preceding six months; The DAS28-ESR score exceeds 3.2. The subsequent conditions will exclude volunteers from the study: (1) A verified history of ovarian cysts, polycystic ovary syndrome, endometriosis, ovarian surgery or radiation therapy, and sex hormone therapy for purposes unrelated to the study; (2) A history of contraceptive use within the preceding six months; (3) Currently pregnant or breastfeeding; (4) A diagnosis of other underlying conditions such as diabetes and hypertension; (5) A history of malignant neoplasms; (6) A history of existing gynecological infectious diseases, such as pelvic inflammatory disease. Intolerance to methotrexate, iguratimod, and meloxicam. The clinical trial obtained permission from the Hospital Ethics Committee, and all participants were adequately informed and consented.

Allocation

Among the 80 patients with moderate to severe active rheumatoid arthritis (RA), they were divided into two cohorts: one cohort got methotrexate (MTX) therapy, while the other cohort received a combination of MTX and iguratimod (IGU). Each group comprised 40 participants

Research Methods

Treatement

The MTX group was administered a daily dosage of 15mg of both MTX and meloxicam. The MTX + IGU cohort was administered a regimen of MTX + IGU at a dose of 25 mg bi-day, in conjunction with meloxicam at a dosage of 15 mg daily. Both groups were administered an initial dosage of 7.5 mgqw of MTX, which was then increased after one week to a maximum of 15 mgqw. Hydroxychloroquine was delivered as a therapeutic intervention after three months.

Outcome measures

The count of inflamed joints (swollen joint count or SJC), count of uncomfortable joints (tender joint count or TJC), duration of morning stiffness, and length of the menstrual cycle were recorded at week 0 and week 24, respectively. The patients

underwent assessments to evaluate their erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), high-sensitivity C-reactive protein (hsCRP), hepatic and renal function, and cytokine concentrations (including IL-6 and TNF-α). Furthermore, the DSA28-ESR score was calculated. Indicators of ovarian reserve, including serum anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2), were assessed on the third day of the menstrual cycle. The specialists in B ultrasonography performed assessments of antral follicle count (AFC) and ovarian volume (OV).

Statistical method

We used SPSS 22 to examine the data. For measurement data that did not follow a normal distribution, it was given as x ± s. To compare the pre- and post-intervention data, a paired t-test was employed. When comparing groups, the Mann-Whitney U test is utilized, and the median P50 (P25, P75) is employed to indicate individuals that do not follow a normal distribution. Statistical significance was defined as a p-value below 0.05.

Results

General data

With an average age of 41.90 ± 7.95 years, 80 female patients diagnosed with rheumatoid arthritis (RA) ranged in age from 20 to 50 years. The average length of time a person was sick was 12.15 ± 10.16 months. In contrast to the mean TJC of 5.66 and standard deviation of 3.25, the mean SJC was 2.53 and standard deviation was

0.67. On average, stiffness in the morning lasts for 50.42 ± 42.45 minutes. This study found that the median rheumatoid factor (RF) was 154 (47.68, 356) iu/ml, and the mean erythrocyte sedimentation rate (ESR) was 59.10 ± 19.59 mm/Hr. With a standard deviation of 0.38, the mean DSA28 score was 5.17. There were no statistically significant differences between the two groups of patients when it came to age, disease progression, swelling joint count (STC), tender joint count (TJC), duration of morning stiffness, erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), or illness Activity Score 28 (DSA28) scores before treatment.

Follow-up data

Four patients were given extra hydroxychloroquine after three months of therapy because the MTX group was shown to have limited effectiveness. On the other hand, the MTX + IGU group did not need any further treatments.

Comparison of efficacy between the 2 groups after treatment

(1) Clinical data comparison: After therapy, the group that received both MTX and IGU showed a more significant improvement in SJC, TJC, ESR, and DSA28-ESR score than the group that received just MTX. When comparing the two groups, we do not find any statistically significant differences in the prevalence or duration of morning stiffness (Figure 1).

Fig 1: the results of comparing the clinical data. A total of 28 erythrocyte sedimentation rate (ESR) units, rheumatoid factor (RF) units per milliliter, duration of morning stiffness (in minutes), tender joint count (TJC), and swelling joint count (SJC) were the variables assessed in the research. Both the methotrexate (MTX) and methotrexate plus iguratimod (MTX + IGU) groups had these characteristics assessed before and after treatment. A statistically significant difference (p < 0.05) was seen between the groups who received both MTX and IGU and the ones that received just MTX, according to the study.

The levels of IL-1β, IL-6, Il-17, and TNF-α were shown to be lower in both groups after therapy compared to their levels before treatment.The levels of IL-1β, IL-6, IL-17, and TNF-α were lower in the MTX + IGU group after treatment compared to the MTX group alone (Figure 2).

Fig 2: A comparison of cytokines is shown in Figure 2 Both the MTX and MTX + IGU groups had their IL-1β, IL-6, IL-17, and TNF-a levels checked both before and after treatment. Both the group treated with MTX plus IGU and the group treated with just MTX had a significant difference (p < 0.05) in the statistical analysis.

Comparison of ovarian functions between the 2 groups after treatment Comparison of ovarian function post-treatment between the two groups: There was no significant disparity in ovarian function between the two groups prior to treatment. Following the treatment, the group receiving a combination of MTX and IGU exhibited a higher level of AMH compared to their initial level. There was no significant statistical difference in the AMH level before and after therapy in the MTX group. The MTX + IGU group exhibited a significantly elevated level of anti-Müllerian hormone (AMH) compared to the MTX group. Nevertheless, there were no significant statistical disparities detected in FSH, LH, E2, AFC, and OV levels between the two groups, both prior to and during the treatment (Figure 3).

Fig 3: depicts the difference in ovarian function between the sexes. Before and after treatment, the MTX and MTX + IGU groups had their AMH, FSH, E2, LH, AFC, and OV levels assessed in ng/ml, mIU/ml, pg/ml, and cm3, respectively. A statistically significant difference (p < 0.05) was seen between the groups treated with MTX alone and those treated with both MTX and IGU.

Discussion

One novel drug for rheumatism is intramuscular injection (IGU), which takes time to take effect. It may alter the immune system and decrease inflammation [1]. It inhibits LPS-stimulated THP-1 IkappaBalpa breakdown but blocks nuclear factor-B (NF-B) activation by preventing NF-B from migrating from the cytoplasm to the nucleus. By blocking the PKC/Egr1/BLIMP1 axis, this inhibition reduces the synthesis of immunoglobulin and other inflammatory cytokines in B cells [2]. Xu et al. (2015) also showed that it increases Treg cell numbers, which hinders cell immunity. Moreover, it has many benefits, including preventing cartilage degradation, inhibiting bone resorption, and stimulating bone formation [3]; [4]. IGU is a chemical that originates from nimesulide. By blocking the metabolism of prostaglandin E2, a product of arachidonic acid, it reduces the symptoms experienced by people with rheumatoid arthritis[5]. Not only that, but it also decreases IL-1 and IL-6 production and bradykinin release. This new drug serves as an NSAID and also has long-term anti-rheumatic effects, making it unique among medications[6]. According to Ye et al. (2019), this medicine was legal in Japan and China for the treatment of rheumatoid arthritis (RA) in 2012. More than that, it was recommended as an effective option for severe RA therapy at the 2014 Asia-Pacific League of Associations for Rheumatology (APLAR) Conference RA recommendations [7]. Several studies have shown that when used as a treatment for RA, IGU has few side effects. A total of 253 patients were randomly assigned to either the IGU or placebo groups in a 2013 trial by Ishiguro et al. In comparison to the placebo group, which had an ACR 20 response of only 30.7% at week 24, the IGU group had 69.5%. Further, when comparing pre- and post-treatment scores, there was a statistically significant improvement in the following areas: ACR 50 and 70 responses, RF, HAQ-DI, and DAS28. When comparing IGU to salazosulfapyridine (SASP), a further review of the 2007 trial by Hara et al. indicated that the former had a better response rate when administered to older patients with rheumatoid arthritis. A more marked reduction in RF levels and a reduction in adverse responses were also seen with IGU. The retention rates of the two groups were compared at 36 months; the IGU group achieved 52.4% and the SASP group 32.1%. While 65.2% of those in the SASP group responded, 85.8% of those in the IGU group did so. In the IGU group, the cumulative rate of any adverse event was 16.7%, but in the SASP group, it was 46.7% [8]. Those RA patients whose symptoms do not improve after taking MTX might greatly increase their ACR 20 response rate by continuing treatment with IGU. A research conducted by Hara et al. in 2014 showed that this rate rises from 30.7% at week 24 to 72.1% at week 52. According to Tanaka et al. (2015), there was no evidence that combining IFU with MTX would significantly increase the frequency of adverse drug events. A retrospective analysis was carried out on 106 RA patients who were treated with or without methotrexate (MTX) in conjunction with intrathecal mesh (IGU). Both the IGU group and the MTX + IGU group were formed from the patients. Inoue et al. (2020) found that both groups' DAS28-CRP levels dropped significantly from their baseline values, suggesting that IGU therapy may be an effective substitute for patients who do not respond to MTX. Patients who did not respond adequately to tocilizumab (TCZ) were given medicine by intragastric syringes by Kosuke Ebina and colleagues. At week 24, 51.6% of patients had reached ACR 20, and 64.5% had obtained a moderate response. Ebina et al. (2019) found that patients who do not react well to TCZ may have an additional safe and helpful therapeutic option with IGU therapy. In this research, individuals with moderate to severe active rheumatoid arthritis (RA) who were female were studied to see what happened when methotrexate (MTX) and iguratimod (IGU) were taken together. In comparison to MTX alone, the findings demonstrated that the combination treatment considerably decreased the erythrocyte sedimentation rate (ESR), tender joint count (TJC), swollen joint count (SJC), and Disease Activity Score 28 based on ESR (DAS28-ESR) scores at week 24. Patients also had a more significant reduction in levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) as a result of the combined treatment. Taken together, these results point to a substantial therapeutic benefit of MTX and IGU. Female patients with rheumatoid arthritis (RA) had lower fertility and a higher risk of early ovarian failure, according to a research by Brouwer et al. (2019). In a study conducted by Clowse et al. (2012), it was shown that 578 women who suffered from rheumatoid arthritis (RA) were 1.5 times more likely to have infertility than the control group. A time to pregnancy (TTP) of more than 12 months was seen in 42% of women with rheumatoid arthritis (RA), which is substantially longer than the control group, according to a nationwide prospective cohort study in the Netherlands [9]. Follicle number and quality, or ovarian reserve function, is a measure of  a woman's fertility and a prerequisite for her body's regular reproductive processes. Publicated in 2020, the paper "Testing and interpreting measures of ovarian reserve: a committee opinion" delves into the evaluation and comprehension of markers of ovarian reserve within a certain time limit. Reduced egg production and follicle quality, known as ovarian reserve loss, reduces a woman's fertility, causes a deficiency in sex hormones, and eventually causes premature ovarian failure. An indication of the number of surviving follicles in the ovaries and a way to estimate ovarian reserve is anti-Müllerian hormone (AMH), a hormone that is generated by granulosa cells throughout reproductive years. Premenopausal women  with rheumatoid arthritis (RA) had a significant drop in anti-Müllerian hormone (AMH) levels, according to research by Brouwer et al. (2019) and Henes et al. (2015). Additionally, AMH levels were much lower in RA patients who tested positive for the cyclic citrullinated peptide (CCP) antibodies. Del Junco et al. (1989) found that menopause occurs at a younger age in RA patients, and our results are in line with that. It is thought that rheumatoid arthritis (RA), disease activity, age, and medicine all contribute to decreased fertility in female patients, although the exact mechanism is still unclear. A role is played by sex hormones in the onset and advancement of RA. According to Hughes and Choubey (2014), the beginning of RA is sped up by estrogen and delayed by progesterone. The hypothalamic-pituitary-ovarian axis is negatively impacted by the sex hormone level variations that occur with the onset and course of RA. These hormonal shifts also impact the local multifactor regulatory system, which may have an effect on ovarian reserve function in RA patients. A history of autoimmune disorders is present in about 20% of individuals diagnosed with premature ovarian failure. Particular autoantibodies, including those against the ovaries, nucleus, RF, cardiolipin, zona pellucida, and others, have been detected in a subset of individuals. Evidence from the past suggests that rheumatoid factor (RF) and cyclic citrullinated peptide (CCP) antibody positive RA women, even when not using cytotoxic medications, had an earlier menstrual cycle termination. According to Del Junco et al. (1989), immunological factors might have a role in the early loss of ovarian follicles. When looking at female patients with rheumatoid arthritis, Brouwer et al. (2015) used a Cox regression analysis to determine that characteristics such as age, disease activity, labor absenteeism, and the use of NSAIDs and prednisone before pregnancy were independent risk factors for protracted pregnancy. Both groups of patients with moderate to severe active rheumatoid arthritis (RA) showed a rise in anti-Müllerian hormone (AMH) levels after treatment compared to pre-treatment values. Reduced ovarian reserve function in RA patients is likely caused in part by disease activity.

Because of their effects on ovarian reserve function in particular, antirheumatic medications are a major contributor to the fall in female fertility. A number of studies have shown that compared to SLE patients who do not get CTX therapy, those who do had significantly lower AMH levels [10]; [11]. Moreover, the AMH value is related to both age and the length of time that CTX has been used. Ovarian function impairment is more likely to occur with higher cumulative dosages of tripterygium glycosides. Using a mouse model with biochemical anomalies, Ma et al. (2014) showed that tripterygium glycosides damage the gonads by causing oxidative stress. It was determined that tripterygium glycosides and CTX both negatively impact ovarian function. When treating rheumatoid arthritis, MTX is often the first line of defense [12]. The available data indicate that the effects of MTX treatment for RA on the reproductive system of females are modest. In a study conducted by Brouwer et al. (2013), it was shown that rheumatoid arthritis (RA) patients who got methotrexate (MTX) therapy at week 6 did not differ significantly from those who did not in terms of anti-Müllerian hormone (AMH) levels. Additionally, Brouwer et al. (2015) looked at pregnant women with rheumatoid arthritis and whether or not there was no correlation between previous MTX use and longer gestational length. Both the pre- and post-methotrexate (MTX) levels of follicle-stimulating hormone (FSH) and antral follicle count (AFC) were not significantly altered by a reproductive physician's evaluation for an ectopic pregnancy. During the second round of in vitro fertilization (IVF), the ovarian reserve and ovarian reactivity were evaluated to ascertain this. In the cycles following MTX, greater dosages of gonadotropin were administered, but neither the amount of oocytes retrieved nor the transplanting of high-quality embryos changed much. Thus, it seems that MTX has no effect on ovarian reserve, ovarian responsiveness, or the success rate of IVF in following cycles.

The effects of MTX treatment in conjunction with IGU on reproductive health in women have not been the subject of any research. This research is the first of its kind to find that ovarian function is not more negatively affected by MTX and IGU combination therapy than by MTX alone in female patients with moderate to severe active RA. Contrarily, AMH levels in treated individuals rise significantly above pre-treatment levels, suggesting better disease management. Studies have shown that female patients with moderate to severe active RA respond better to a combination

treatment that includes both MTX and IGU than to MTX alone. Additionally, this combination medication improves ovarian reserve function while decreasing its influence on female ovarian function, all thanks to its ability to effectively reduce RA activity.

Finding statement

Conflicts of interest

All authors confirm that there are no conflicts of interest in this study

Data availability statement

Data are available from the corresponding author under reasonable requests

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