Impact of oral contraceptives and breastfeeding on disease activity in a sample of Egyptian rheumatoid arthritis patients

Women are more affected by rheumatoid arthritis (RA) than men and the incidence of RA in women increases around the age of menopause indicating that hormonal factors may have a role in disease pathogenesis and progression. Despite several studies on the role of sex hormones and oral contraceptives (OCs) in RA patients, the effect of combined oral contraceptives on RA disease activity is still controversial, and since few studies have been performed in Egypt on this issue so we aimed to study the effect of OCs, breastfeeding, and prolactin on disease activity and their relation to the titer of autoantibodies in female Egyptian RA patients. One hundred twenty married female RA patients were classified into three groups based on the OCs use (n = 40 each); current, past and non-users and according to lactation into three groups; lactating using OCs (n = 22), lactating not using OCs (n = 30), and non-lactating (n = 68). Our patients were using combined estrogen-progestin oral contraceptives. Clinical manifestations, disease activity, and laboratory findings were determined. The clinical manifestations including arthritis, and morning stiffness were significantly reduced in current users in comparison to past and non-users. Mean DAS28 was reduced significantly (p < 0.05) in current and past users of OCs. Also, using OCs during lactation reduced the clinical manifestations significantly. Lactating women who were not using OCs had significantly high DAS28 and ESR (p < 0.05) in comparison to lactating using OCs and non-lactating. On the other hand, using OCs reduced serum prolactin significantly. Lactating women not using OCs had significantly high serum prolactin (p < 0.05) and (p < 0.001) in comparison to lactating using OCs and non-lactating respectively which was correlated with DAS28 score. Oral contraceptives may be considered in RA female patients not only as a suitable birth control method but also it has a controlling effect on disease activity even during lactation. Oral contraceptives ameliorate disease activity in RA patients

mortality [6][7][8]. Initial studies focused on comparing the prevalence of RA in both genders, while recent studies give more attention to sex hormones and reproductive factors but still their role in RA is unclear [9].
The female hormonal milieu is altered by multiple factors like breastfeeding, pregnancy, postpartum, menopause, and the use of oral contraceptives (OCs) [10,11]. It is reported that the incidence of RA in women increases around the age of menopause indicating that hormonal factors may have a role in disease pathogenesis and progression [12]. Hormonal theory may be reinforced by the fact that the incidence of RA decreases during pregnancy and RA women who get pregnant usually have disease remission [13]. However, the disease exacerbates after delivery due to the rapid decline in both estrogens and cortisol, and the significant increase in prolactin [14]. The different distribution and character of the autoimmune diseases between genders may be related to sex hormones. Prolactin is known to have an immune-stimulatory role and has a role in the pathogenesis of many autoimmune diseases. It is also related to disease relapse which occurs during pregnancy in SLE and lactation in RA patients [15].
Breastfeeding is considered as a risk factor for more aggressive inflammatory polyarthritis in women who have genetic susceptibility due to the association between HLA-DRB1 alleles and the gene of prolactin [16]. Prolactin is a hormone which has a cytokine-like action and is considered as an immune-stimulatory agent that is involved in synovial hyperplasia and inflammation [17,18], it is secreted by the pituitary gland in addition to macrophages and lymphocytes located in the synovial tissue of inflammatory arthritis patients [19]. Prolactin has a role in the severity of the disease and enhances RA joint damage [20]. Clinical trials revealed significantly high serum prolactin in RA patients suggesting that prolactin may have a significant role in disease activity and also in the disease development process [21].
Using OCs is associated with a milder form of the disease and reduced disability [22]. Chen et al. reported that the use of OCs does not decrease the incidence of RA but may reduce disease severity and progression [23]. Despite several studies on the role of sex hormones and OCs in RA patients, their effect is still controversial especially it may be affected by race and environmental circumstances, and since as far as we know, no similar studies have been performed in Egypt yet so we aimed to study the effect of both OCs and breastfeeding on disease activity and their relation to the titer of autoantibodies in RA Egyptian female patients.

Methods
In this study, the data for 120 married female RA patients were collected from the department of rheumatology, from September 2019 to March 2020. Our patients were diagnosed according to the 2010 ACR revised criteria for RA [24]. Patients with concomitant systemic diseases and other autoimmune diseases including SLE, scleroderma, mixed connective tissue disease, Sjogrenʼs syndrome, polymyositis, pregnancy, patients receiving hormonal therapy, and patients with a history of thromboembolic events were excluded. Written informed consent was taken from all patients, and ethical approval was obtained from the ethical committee of faculty of medicine.
Patients were classified into three groups based on OCs use; patients currently using OCs (current users), patients who stopped using OCs for at least 1 year (past users), patients who never used OCs (non-users) with 40 patients in each group. Our selected patients were using combined estrogen-progestin oral contraceptives for a minimum duration of 6 months. Lactating patients started using OCs 6 weeks postpartum. According to lactation, our patients were classified into three groups; the first group included twenty-two lactating patients using OCs (Lact. using OCs), the second group, included 30 lactating patients not using OCs (Lact. not using OCs), the third group, included 68 non-lactating patients (non-lactating).
Our patients were evaluated for demographic data (age > 18 years, disease duration 2-8 years), current medications and clinical manifestations including morning stiffness, arthritis, and extra-articular manifestations including; rheumatoid nodules, Raynaud's phenomena, and carpal tunnel syndrome, in addition to the laboratory findings including, erythrocyte sedimentation rate, CBC, RF, and ACPA were determined. RF was determined using RF-latex slide agglutination kit (SPINREAC T, Spain) according to the manufacturer's protocol. ACPA was quantified using ELISA technique for human IgG autoantibodies to cyclic citrullinated peptides in the serum according to the manufacturer company protocol (Demeditec Diagnostics GmbH, Germany). Serum prolactin was determined using the Quantikine® ELISA kit according to the enclosed protocol provided by R&D Systems, Inc.
Disease activity was assessed using DAS28. Visual analog scale (VAS) was used by patients through scoring their pain ranging from 0 (no pain) to 100 (the worst pain imaginable). Tender and swollen joints were counted and ESR was recorded. Regarding the DAS28 score, disease activity was classified as remission (< 2.6), low disease activity (2.6-3.2), moderate disease activity (3.2-5.1), and high disease activity (> 5.1).

Statistical analysis
SPSS, version 20 (IBM Corp., Armonk, USA) was used for the data analysis. We used the Kolmogorov-Smirnov test to check the normal distribution of data. Data and results were expressed in mean with standard deviation or SEM in normally distributed data. Mann-Whitney U test was used to compare two groups for not normally distributed data and independent t test for normally distributed data.

Results
Our study included 120 married female RA patients. Their age range was 19-40 years and disease duration was 2-8 years. Our patients were classified according to OCs use and lactation as shown in the flow diagram (Fig. 1). The current lines of treatment for our patients are shown in Table 1, where 15.83% of the patients were on methotrexate (MTX), 13.33% were on MTX + hydroxychloroquine, 6.66% were on MTX + sulphasalazine + hydroxychloroquine, 10.83% were on leflunomide, 10% were on MTX + leflunomide, 29.19% were on corticosteroids + hydroxychloroquine, and 14.16% were on corticosteroids + sulphasalazine ( Table 1). None of our selected patients were using biological therapy as their count was extremely low so they are excluded at the start of the study. The lactating patients were using only two lines of treatment, corticosteroids + hydroxychloroquine and corticosteroids + sulphasalazine, which make it easy to compare, while in other groups different lines of therapy were used which make it difficult to compare.
Clinical manifestations for current, past, and non-oral contraceptives users As shown in Table 2, using oral contraceptives lowered the mean rank of morning stiffness between the studied groups; women currently using OCs had significantly lower mean rank of morning stiffness in comparison to past users and non-users (p < 0.05) and (p < 0.001) respectively. Also, the past users of OCs had significantly (p < 0.05) lower mean rank of morning stiffness in comparison to non-users. On the other hand, there was no significant difference between the current and past users of OCs concerning arthritis; however, past users had a significantly lower (p < 0.05) mean rank of arthritis in comparison to non-users, and a highly significant (p < 0.001) lower mean rank of arthritis for current users in comparison to non-users. Meanwhile, using OCs had no significant effect on the extra-articular manifestations between the studied groups.
Clinical manifestations in lactating women using and not using oral contraceptives, and non-lactating Using oral contraceptives during lactation significantly reduced (p < 0.05) the mean rank of morning stiffness in lactating using OCs in comparison to nonlactating. However, no significant difference was found between lactating using and not using OCs also, between lactating not using OCs and nonlactating. On the other hand, using OCs during lactation significantly reduced the mean rank of arthritis (p < 0.001) and (p < 0.01) in comparison to those who are lactating not using OCs and non-lactating respectively. Meanwhile, using OCs during lactation had no significant effect on the extra-articular manifestations in all studied groups as shown in Table 3. Effect of using oral contraceptives on disease activity, ESR, RF, and ACPA As shown in Fig. 2a, the DAS28 score reduced significantly (p < 0.05) in women currently using OCs in comparison to those who previously used OCs, the same reduction was found in DAS28 in women who previously used OCs in comparison to those who never used OCs. The DAS28 was more significantly lower (p < 0.001) in current users of OCs in comparison to those who never used OCs. Using OCs did not have any significant effect on the ESR as shown in Fig. 2b. Also, using oral contraceptives did not have any significant effect on ACPA and RF titers as on comparing the ACPA and RF titers between the three groups the p value was > 0.05.
Effect of using oral contraceptives during lactation on disease activity score and ESR As shown in Fig. 3a, lactating women not using OCs has a significantly high DAS28 (p < 0.05) in comparison to those women who are lactating using OCs and nonlactating. Meanwhile, there was a non-significant difference in the DAS28 score on comparing lactating women using OCs with those non-lactating. As regards to the effect of using OCs on the ESR, there was a significant difference (p < 0.05) between lactating women not using OCs in comparison to lactating using OCs and non-lactating. However, there was no significant difference in the ESR between women who are lactating using OCs and women who are non-lactating (Fig. 3b). As shown in Fig. 4, there was no significant difference between the effect of using different lines of treatment on the DAS28 score within the same group, (lact + OCs + C + H vs lact + OCs + C + S, p > 0.05, (lact + C + H vs lact + C+S, p > 0.05). However, there was a significant difference on comparing lactating women using OCs with lactating not using OCs in spite of using the same lines of treatment (lact + OCs + C + H vs lact + C + H, p < 0.05, (lact + OCs C + S vs lact + C + S, p < 0.05).
Effect of using oral contraceptives during lactation on serum prolactin, ACPA, and RF titers Using OCs during lactation reduced the prolactin level significantly (p < 0.05) in comparison to those women who are lactating not using OCs as shown in Fig. 5a, lactating women not using OCs has a significantly high serum prolactin level (p<0.05) and (p < 0.001) in comparison to those women who are lactating using OCs and non-lactating respectively. Moreover, the level of prolactin significantly (r = 0.771, p < 0.001 and r = 0.831, p < 0.001) correlated with DAS28 score in lactating using OCs, and lactating not using OCs respectively as shown in Fig. 5b, c. On the other hand, using oral contraceptives during lactation did not have any significant effect on ACPA and RF titers as on comparing the ACPA and RF titers between the three groups the p value was > 0.05.

Discussion
The etiology and pathogenesis of RA are still unknown; however, sex hormones may have a role in modulating disease onset and progression [14]. Females are more commonly affected than males. Progesterone and androgens have suppressive effects over the immune system, while prolactin has a stimulatory effect, and estrogen can have either effect [25]. The disease activity of RA patients improves during pregnancy due to some changes in the maternal immune system, occurring through multiple immunological mechanisms that stop after delivery leading to increased postpartum disease activity [26]. The increase in estrogen and progesterone levels during pregnancy was found to have a protective effect against both the development and activity of RA [27,28]. It is reported that combined oral contraceptives may have a tendency of controlling disease activity in RA patients [29]. Our findings revealed that using oral contraceptives significantly reduced the clinical manifestations. Women who are currently using and previously used OCs presented with less morning stiffness and arthritis. The effect of OCs was more pronounced on the DAS28 score; current users of OCs had significantly low DAS28 scores in comparison to past users and both of them had a lower score when compared with non-users. Our findings agree with Amini et al. who found that the activity and severity of rheumatoid arthritis can be improved by oral contraceptive pills [30]. Also, Brouwer et al. reported that oral contraceptives are considered the best option for RA patients as they positively affect the disease activity [31]. Fayed et al. found that RA patients who are using combined oral contraceptives have the lowest disease severity in comparison to the other methods of contraception [32]. However, using OCs did not have any significant effect on ESR; this agrees with Amini et al. [30], and it also had no significant effect on ACPA and RF titers in all studied groups.
The combined OCs contain two anti-inflammatory agents; estrogen and progesterone. The potential protective effect of OCs on RA may be explained by more than one mechanism; the first, estrogen in OCs can suppress the immune response and downregulate proinflammatory cytokines production; the second, estrogen may prevent the formation of osteoclasts and pannus formation, hence decreasing RA progression [33]. Moreover, progesterone prevents the production of immunoglobulins and pro-inflammatory cytokines [34].
Using OCs during lactation reduced the morning stiffness in lactating women using OCs in comparison to those non-lactating. However, its effect on arthritis was more pronounced where arthritis was significantly reduced on using OCs. Concerning disease activity, lactating women who are not using OCs had a significantly high DAS28 in comparison to those women who are lactating using OCs and non-lactating. Despite the wellknown role of therapy in controlling disease activity in RA patients, we suppose that the reduction in the disease activity in lactating women using OCs was more related to the use of OCs than the effect of therapy, this may be supported by the finding that the same two lines of treatment were used in the two groups but OCs using group had lower DAS28 in comparison to OCs nonusers. Regarding serum prolactin, lactating women not using OCs have significantly high serum prolactin in comparison to lactating using OCs and non-lactating. Moreover, the prolactin levels significantly correlated with DAS28 in all groups. This may be explained by the role of increased serum prolactin in enhancing postpartum disease flare in RA patients. Meanwhile, there was a non-significant difference in the DAS28 score on comparing lactating women using OCs with those non- Fig. 4 Effect of different lines of treatment in lactating women on DAS28 score. Where Lac + OCs + C + H, lactating women using OCs and treated by corticosteroids + hydroxychloroquine n = 16. Lac + OCs + C + S, lactating women using OCs and treated by corticosteroids + sulphasalazine n = 6. Lac + C + H, lactating women not using OCs and treated by corticosteroids + hydroxychloroquine n = 19. Lac + C + S, lactating women not using OCs and treated by corticosteroids + sulphasalazine, n = 11. Where * is significant difference at p < 0.05 and ns, non-significant difference lactating. Our results agree with Fayez et al. who reported that increased level of serum prolactin was found in patients with high disease activity when compared to moderate and low disease activity and serum prolactin level was significantly correlated with DAS28 and ESR [20], also, Fojtíková et al. reported that RA patients with active disease had high serum prolactin level which correlated significantly with disease activity and Larsen score [35]. Many previous studies have explored the effect of breastfeeding on disease activity during the postpartum period which is challenging for women suffering from rheumatologic diseases due to the increased risk of disease flare and limitations in the use of medications during breastfeeding [36]. Barrett et al. found that breastfeeding women with inflammatory polyarthritis developed a more aggressive disease 6 months later in comparison to previous breastfeeders and nonbreastfeeders [16]. The disease activity of the majority of RA patients increases during the first 3 months after delivery, and in few patients, it increases by 9 months; thus, women with RA might benefit from being nonbreastfeeders [15]. The prolactin release during breastfeeding could also play a role in the disease flare in the Fig. 5 Effect of oral contraceptives on serum prolactin and its correlation with disease activity. a Serum prolactin level. b Correlation between serum prolactin and DAS28 in lactating women using oral contraceptives. c Correlation between serum prolactin and DAS28 in lactating women not using oral contraceptives. Pearson's correlation was used for calculation of the correlation r = correlation coefficient, * significant difference at p < 0.05, ** high significant difference at p < 0.01, and *** very high significant difference at p < 0.001

Elsayed et al. Egyptian Rheumatology and Rehabilitation
(2020) 47:46 postpartum period [19]. Our results concerning disease activity during lactation agree with the previously mentioned studies; but interestingly, we found that lactating women using OCs had low disease activity in comparison to those lactating not using OCs, and their disease activity was near to that of non-lactating women; hence, OCs may be considered the most suitable contraceptive method for lactating RA women with the additional benefit of improving disease activity. As regards to the effect of using OCs on the ESR, lactating women using OCs had a significantly low ESR in comparison to those who are not using OCs.
Hormonal changes alongside changes in cytokine patterns and immune cells are all factors contributing to the postpartum disease flare in RA patients [37]. Another theory that may contribute to the postpartum RA disease flare is the prolactin hormone which increases during breastfeeding [15,38]. Prolactin is considered to have a dual action in the joint tissues, while it prevents the apoptosis of chondrocytes by activating antiapoptotic genes, activates the proliferation of synovial cells, and enhances the synthesis of IL-6, IL-8, and matrix metalloproteinase, so it is suggested that high prolactin either produced locally in joints by chondrocytes or obtained from the circulating blood may have a role in structural changes leading to the development of RA [39].
Prolactin level is normally high in lactating women but the effect of using OCs on the serum prolactin level is still controversial. Our results indicated that using OCs significantly reduced serum prolactin. Our finding agrees with Ryu et al. who found that the use of combined OCs decreased the serum prolactin level in lactating women [40], also Clendenen et al. found that parity and OCs use was associated with a decreased level of serum prolactin and ovarian cancer risk [41]. Testa et al. reported that using combined OCs insignificantly decreased serum prolactin [42]. On the other hand, our results are in contradicting with Alvarez-Tutor et al. who reported that serum prolactin significantly increased on using combined OCs [43]. There are controversies whether the content of estrogen in OCs affects serum prolactin or not [44]; Josimovich et al. found that high estrogen OCs increases serum prolactin within the first 6 months of its use but this effect is not manifested after 6 months, while low estrogen OCs either decrease serum prolactin or have no effect depending on basal prolactin value [45].
Prolactin has a strong immunomodulatory effect through stimulating the transcription of the gene of interferon regulatory factor IRF-1, and modulation of the expression of IL-12, and other pro-inflammatory cytokines like tumor necrosis factor TNF-α, and interferon (IFN)-α, it also increases the survival of both B cells and autoreactive T cells through its anti-apoptotic activities [35]. Prolactin stimulates the immune system mainly through the inhibition of the autoreactive B lymphocytes' negative selection [15]. Our findings reinforce the concept that the levels of estrogen, progesterone, and prolactin change after delivery affecting the cytokines and immune cells and this in turn modulates the RA disease activity. Meanwhile, using OCs during lactation may have a tendency of controlling disease activity in RA patients, as lactating women not using OCs had the highest prolactin level and DAS28 score and those using OCs had significantly reduced serum prolactin and DAS28 score.

Conclusion
Oral contraceptives may be considered in RA female patients not only as a suitable birth control method but also it has a controlling effect on disease activity even during lactation

Study limitations
Further studies with a big sample size are required comparing the effect of progesterone only and combined oral contraceptive pills on disease activity in RA patients.