Adverse effect of rheumatoid arthritis on male Wistar rat’s fertility: protective role of Costus extract

Rheumatoid arthritis (RA) is a systemic autoimmune complaint. Advanced treatments resort to the traditional herbal therapy. The aim of this study is to assess the protective effect of Costus extract on the fertility of male rats with Freund’s adjuvant-induced rheumatoid arthritis. Thirty male adult Wistar rats (190–200 g) were divided into six groups. They were subdivided into three groups; group I was the control group that received distilled water, and groups II and III received two various doses of Costus extract (200 and 400 mg/kg, respectively) for 60 days. Another three groups were subjected to RA induction via Freund’s adjuvant. Rats were injected a dose of 0.1 ml of Freund’s complete adjuvant (FCA) in the planter area of the left hind paw and then subdivided into 3 groups. Group I of RA-induced rats were given distilled water. The other two groups were given orally (200 and 400 mg/kg dosage of extract, respectively) from the 2nd day of RA induction for 60 days. Sex organ relative weight, sperm concentration assay, testicular histopathology and immunohistochemistry of androgen receptors, TNF α, and BAX protein were determined. The results showed that RA caused a significant decrease in the relative weight of sex organs and sperm count, which were relatively improved by doses of Costus (200, 400 mg/kg). RA induction caused testicular degeneration which markedly enhanced with Costus treatment as shown in histopathological sections. RA caused a reduction in %IHC of androgen receptors and increased expression level of both TNF α and BAX protein. Using IHC, it was revealed that RA caused a reduction in the expression level of androgen receptors and an increase in the expression of both TNF α and BAX protein. We can conclude that Costus speciosus had a potentially valuable role in improving fertility disorders caused by RA.


Introduction
Infertility and gonadal dysfunction with the chronic rheumatic disorder are multifactorial (Østensen 2004;Silva and Brunner 2007). The unhealthy conditions, like malnutrition, drug abuse, and obesity in addition to female and male genital illness (Balen and Rutherford 2007), may decrease fertility (Østensen 2004 andBrunner 2007). In addition, hypothalamic-pituitary-adrenal axis dysfunction (Suehiro et al. 2008;Medeiros et al. 2009), autoimmune disorders (Silva and Brunner 2007), anti-sperm antibodies (Soares et al. 2007;Suehiro et al. 2008), and high activity of disease or chronic renal disorder and immunosuppressive medications (Latta et al. 2001;Østensen et al. 2006) can initiate reduction of fertility in patients with rheumatic sicknesses. In healthy people, reproductive ability also decreases with age in both sexes (Balen and Rutherford 2007).
Rheumatoid arthritis (RA) is a systemic disorder characterized by joint swelling, pain, function loss, joint damage, and permanent deformity if left without treatment (Tripathy et al. 2009). The spread of RA is reliable globally, influencing about 0.5-1.0% of the population (Meera et al. 2008). Although the exact cause is unknown, various hypotheses suggested that it is caused by genetic tendency and exposure Responsible editor: Mohamed M. Abdel-Daim to environmental factors like viruses (Babushetty and Sultanpur 2012). The RA has several adverse effects on the body systems, including the heart and blood vessels, lungs, kidneys, liver, and skin.
Tumor necrosis factor α (TNF α) is a crucial cytokine in acute inflammation and plays a vital role in the reproductive physiology of men (Sharkey 1998;Harada 2001). TNF α showed a wide diversity of biological actions that may impair the reproductive functions, like stimulating the immune cascade and chemotaxis of neutrophils, prostaglandin and collagenase synthesis, and possible efficacy on sperm movement and function (Farrugia and Baron 2016). The important role of TNF-α in regulating spermatogenesis and spermatozoa functions was indicated by binding to type 1 TNF-α receptor (TNFR1); TNF-α activates several transduction pathways leading to regulation of the testicular expression of several genes involved in spermatogenesis (Grataroli et al. 2000). Decreased fertility is not common among patients with the rheumatic disorder Clowse et al. 2012). Drug treatments may be considered the major cause for gonadal impairment (Freire et al. 2006). The reproductive possibility of male patients is declined by the disease directly in the testis or by immunosuppressive drugs. This reflected on decrease of both sperm count and expression of androgen receptors, accompanied by increase of %IHC of TNF α and BAX protein. (Tiseo et al. 2016).
Apoptosis is considered one of the vital cell death mechanisms. Apoptosis is controlled by different genes and molecules such as BAX proteins. The induction of BAX proteins stimulates the secretion of cytochrome c and other apoptogenic factors leading to apoptosome formation, triggering caspase-9 with caspase-3 and 7 (Aitken et al. 2011). Sperm DNA damage activated by apoptosis has been demonstrated in different mammals (Dogan et al. 2012). The balance between germ cells and Sertoli cells in the testis during spermatogenesis is achieved by apoptosis, and an imbalance in this process was denoted to cause infertility in males (Aitken et al. 2011). BAX proteins have been detected in RA (Sioud and Mellbye 1998;Kobayashi et al. 2000). These BAX proteins can form hetero-and homodimers, which are vital in adjusting apoptosis and cell cycle in RA (Hilbers et al. 2003).
Researchers are looking for traditional medicine for finding extended acting anti-inflammatory medications with fewer side effects (Ekambaram et al. 2010;Patil et al. 2010). Plant-derived drugs are still a vital resource, especially in developing countries, for severe disease therapy. It has been recorded that 60-90% of RA patients use alternative and complementary treatment (Zhao et al. 2013). In India, there are more than 2500 plants species that are used nowadays as herbal medicine (Sudha and Mathanghi 2012).
Costus speciosus Koen (Keu, crepe ginger) is considered an Indian ornamental herb and has been utilized in traditional therapy for a long period. This herb of the Costaceae (Zingiberaceae) family is well-known as keukand (Hindi) and diversified crepe ginger (English) (Srivastava et al. 2011). It is a succulent and perennial herb, up to 2.7 m in height, arising from a horizontal rhizome, found in tropical region of India (Anonymous 2007). The rhizomes and roots are ascribed to be bitter, astringent, aphrodisiac, purgative, anthelmintic, expectorant, and tonic which improves digestion, clears toxins, and is a stimulant herb. Juice of the rhizome is applied to the head for cooling and relief from headache (Gupta 2010). An alkaloid extract from Costus speciosus rhizomes is known to display papaverine-like smooth muscle relaxant and antispasmodic activities (Srivastava et al. 2012). Rhizomes are given in pneumonia, rheumatism, dropsy, urinary diseases, and jaundice, and leaves are given in mental disorders. Bruised leaves are applied in fever; decoction of stem is used in fever and dysentery (Anonymous 2007). The plant has anti-inflammatory, laxative, anti-arthritic, and anti-fungal actions. It also utilized in bronchial asthma and gout rheumatism (Khare 2007). Costus have a useful anti-arthritic efficacy as it shows good results in monitoring inflammation in adjuvant-induced arthritic rats. The medication is a hopeful anti-arthritic drug from plant extract in inflammation medication (Srivastava et al. 2012).
The current study directed to explore the effect of Costus as an anti-arthritic treatment on male rat fertility assessed by sex organ weight, sperm count, sperm abnormalities, and viability. Also, testicular androgen receptors, TNF α, and BAX protein were evaluated in the present study by immunohistochemistry.

Preparation of plant extract
The root parts of the Costus speciosus Koen plant were purchased from the local market and then subjected to morphological identification by Plant Taxonomy Department in Faculty of Sciences, Suez Canal University. The plant was extracted according to Srivastava et al. (2012), in which the parts were dried, coarsely powdered, and used for the extraction procedure. One hundred grams (100 g) of the powder was extracted using 75 % ethanol. The extract was evaporated using a rotary evaporator at 40°C under reduced pressure near dryness (gummy residue). The yield was found to be 13.2%. The dried extract was suspended in distilled water as a vehicle and utilized as an anti-arthritis drug.

Gas chromatography-mass spectrometry (GC-MS)
GC-MS exploration of Costus ethanolic extract was attained through Thermo Polaris Q Ion Trap Gas Chromatography/ Mass Spectrometer (Hertfordshire, UK) as formerly illustrated by Tayade et al. (2013). The gas chromatograph was interfaced to a mass spectrometer instrument retaining the following conditions: for GC-MS detection, operating in electron impact with ionization energy of 70eV. The carrier gas used was helium (99.99%) with a constant flow of 1.21 ml/ min. Injector temperature was set at 200°C, while the mass transfer line was established at 240°C. The oven temperature was calibrated from 70 to 220°at 10°C/min, held isothermal for 2 min, and finally elevated to 300°C at 10 min. Two microliters of particular diluted samples was injected manually in the splitless mode, with a split ratio of 1:40 and a mass scan of 50-650 atomic mass unit (amu). The total running time of GC-MS is 50 min. The analysis of data was carried out by Finnigan Xcalibur data acquisition and processing software version 2.0 (ThermoQuest, LC, and LC/MS Division, USA). The relative percentage of constituents present in TC extract was indicated as the percentage of peak area normalization. The components were identified according to the spectrometer database (Wiley and NIST Libraries) and confirmed by the determination of their Kovats indices, which is used to convert retention times into system-independent constants; NIST compiles a comprehensive Kovats index database (Goel et al., 2015).

Animals and housing
This study's whole experimental procedures and protocols were reviewed by the Ethical Research Committee of Faculty of Veterinary Medicine, Suez Canal University, Egypt (approval no. 2019030). Thirty adult male albino Wistar rats were used in the current study, and their weight was between 190 and 200 g. Animals were kept in a perfect environment of 12/12 h light/dark cycle and a room temperature between 22 and 25°C in lab animal house in Faculty of Sciences, Suez Canal University. Rats received food and water ad libitum. Animals were kept in stainless steel cages of 20 cm high and 860 cm 2 foot. They were maintained for 2 weeks for acclimation to animal house circumstances.
Rats were divided into six groups, five animals for each. The first three groups subdivided into three subgroups: group I was the control group and was given the vehicle (distilled water), and groups II and III received two various dosages of Costus extract (200 and 400 mg/kg, respectively) according to Prabhu et al. (2014) orally for 60 days. The other three groups were subjected to RA induction using Freund's adjuvant. Rats were injected a dose of 0.1 ml of Freund's complete adjuvant (FCA) purchased from Sigma-Aldrich, USA, in the planter area of the left hind paw. After 2 days, FCA exhibited soft tissue puffiness around the ankle joints as a marker for arthritis and then divided into group IV of RA-induced rats which were given vehicle (distilled water). Groups V and VI were given two various dosages of extract (200 and 400 mg/kg, respectively) orally via gavage for 60 days.

Relative weights of sex organs
By the end of the study, rats were euthanized by tetrahydrofuran via inhalation and dissected organs. Testis, seminal gland, the tail of the epididymis, and prostate gland were dissected and weighed. The relative weights of an organ (organ weight/body weight × 100) were determined for each rat in both control and treated groups, according to Elgawish and Abdelrazek (2014).

Sperm count, viability, and abnormalities
The epididymis constituents were collected by cutting the cuda epididymis using surgical blades and then squeezed on a clean, sterile glass slide. This constituent was diluted five times by 2.9% sodium citrate dihydrate solution and completely mixed to assess the total sperm count. Sperm count was calculated according to the formula: the number of sperm counted × dilution factor/volume × 1000 (Sikka and Hellstrom 2016). One drop of the mixture was smeared on a glass slide and stained by Eosin Nigrosin stain to measure the viability and sperm abnormalities according to the method described by Okamura et al. (2005).

Histopathology
Samples from testis were obtained from all control and experimental groups. They were immediately put in 10% formalin saline to be fixed and then organized utilizing standard hematoxylin and eosin stain measures as designated by Bancroft et al. (1996).

Immunohistochemistry
It was managed according to Abdelrazek et al. (2016). The formalin-fixed and paraffin-fixed testes were cut into 5μm sections and mounted on positively charged slides to estimate androgen receptors, TNF α, and BAX protein levels. Sections were dewaxed, rehydrated, and autoclaved at 120°C for 10 min in 10 Mm citrate buffer (pH 6). After washing with phosphate buffer saline (PBS), endogenous peroxidase was blocked using 0.3% H 2 O 2 in methanol for 15 min. Slides were washed in PBS again, and blocking was performed by adding blocking buffer and incubated for 30 min at room temperature. Primary monoclonal rat antibodies for androgen receptors (Cat. No. MA1-150, Thermo Fisher Scientific Co., USA), TNF α (Cat. No. PRC3014, Thermo Fisher Scientific Co., USA), and polyclonal rat antibodies for BAX (Cat. No. MABC 1597, Sigma-Aldrich, USA) were added after dilution by PBS (2μg/ml for androgen receptors, 1:50 for both TNF α and BAX protein). The slides were washed three times for 3 min each with PBS. Biotinylated polyvalent secondary antibody (Cat. No. 32230, Thermo Scientific Co., UK) was applied to tissue sections and co-incubated for 30 min. The slides were washed three times for 3 min. The reaction was visualized by adding Metal Enhanced DAB Substrate Working Solution to the tissue and incubated for 10 min. The slides were washed two times for 3 min each with wash buffer. Counterstaining was performed by adding a suitable amount of hematoxylin stain to the slide to cover the entire tissue surface (Bancroft and Cook 1994). For quantitative analysis, the intensity of immunoreactive parts was used as a criterion of cellular activity. Measurement was done using an image analyzer (ImageJ program). The total field and immunohistochemical (IHC) stained areas were calculated, and the percentage of IHC stained area was calculated as follows: %IHC stained area = IHC stained area/total area × 100.

Statistical analysis
One-way and two-way ANOVA fulfilled statistical analyses via utilizing statistical software package, SPSS, version 20. The 2-factor ANOVA with the type III sum-of-squares method by means of multivariant general liner models (GLM) was used to investigate the effect of RA model-and dose-dependent of Costus outcomes and their interaction on biochemical and immunological parameters. Results were shown as mean ± SE, and the P<0.05 were expressed as statistically significant.

Gas chromatography-mass spectrometry
The phytochemical study was carried out by GC-MS analysis of Costus extract, and the chromatogram identified thirty-two phytochemicals as constituents (Figure 3.1) of these compounds; dehydrocostuslactone acid was the major compound (62.10 %) followed by costunolid (22.43%), dihydrodehydrocostus lactone (8.15), 1,8,11,14-heptadecatetraene, (Z,Z,Z) (2.83 %), and valerenol (1.80). Remaining constituent chemical compounds were less than 1%. The components, their retention times, their molecular formula, and molecular weight are summarized in Table 1. Table 2 showed that the relative weight of testis-induced RA showed a significant (P<0.05) decrease in relative testicular weight compared to control. Treatment of RA via 200 and 400 mg/kg of Costus extract significantly (P<0.05) increased relative testicular weight when compared to RA groups, and there was no significant change between dose 200 and 400 mg/kg. However, values did not return to normal values of the control group. Same results regarding the relative weight of the tail of epididymis were observed; treatment of RA with Costus (200, 400 mg/kg) significantly (P<0.05) increased the relative weight of tail of epididymis that returned to normal levels. Concerning the relative weight of the seminal vesicle, no significant change was observed among all groups, whereas in the prostate gland, there was a significant (P<0.05) decline in relative prostate gland weight in the induced RA group compared to the control one. No change was observed in the relative weight of the prostate gland after Costus extract treatment of RA-induced rats (200, 400 mg/kg).

Sperm count, viability, and abnormalities
In RA-induced rats, the count of sperm cells significantly (P<0.05) decreased compared to other groups. RA treatment with 200 and 400 mg/kg significantly increased sperm cell count than RA group but not returned to the control group value. Regarding the sperm abnormalities, there was a significant (P<0.05) rise in sperm abnormalities in RA-induced rats than in control. Treatment of RA with 200 and 400 mg/kg significantly (P<0.05) decreased sperm abnormalities compared to the RA group. However, it did not return to the control values. The sperm viability % significantly (P<0.05) decreased in RA-induced group more than the control. Treatment of RA rats with 200 and 400 mg/kg significantly (P<0.05) increased sperm viability % than RA group but not returned to the control value, whereas there was a dose-dependent increase ( Table 3).

Histopathology of the testis
The testis of the control group consisted of convoluted seminiferous tubules in the stroma with Leydig cells. Each seminiferous tubule showed normal cell lines lined with a stratified epithelium of spermatogenic cells besides Sertoli cells and showed the different phases of spermatogenesis, primary and secondary spermatocytes with spermatids forming then finally spermatozoa. Between the tubules, the interstitial cells of Leydig and interstitial connective tissue were seen. Histopathological changes in seminiferous tubules of Costus (200, 400 mg/kg) showed typical tissue architecture with abundant spermatozoa formation. RA-induced group showed sloughing, disrupted cell arrangement, a moderate number of tubules were lined with moderate to few numbers of spermatogenic cells, and a large number of spermatids exhibited degeneration, necrosis and vacuolation. The interstitial space was enlarged due to edema, congestion of blood vessels, and tubular atrophy in the seminiferous tubules. Most of the a RT retention time of the compounds based on GC-MS peaks; compounds are listed in order of their elution from a TG5MS column tubules showed thickening and hyalinization of their basement membranes, hyperplasia of Sertoli cells, and lined with few spermatogenic cells characterized by degeneration. Vacuolization and necrotic changes with sloughing coagulation of spermatids at the center of tubules were also observed. Treatment with Costus (200, 400 mg/kg) showed mild to moderate degeneration of germ cells in seminiferous tubules; mature spermatozoa could be observed in the lumen of a moderate to a large number of tubules ( Figure 1).

Immunohistochemistry
Concerning the results of androgen receptors, a significant decrease (p<0.05) in the %IHC of androgen receptors in the testicular tissue of RA induced rats compared to control groups. Treatment with Costus (200, 400 mg/kg) exhibited a significant rise (P<0.05) in the %IHC of androgen receptors than RA group to a level nearly equal to that of the testis of control (Figure 2) rats. Both TNF α ( Figure 3) and BAX ( Figure 4) exhibited significant (P<0.05) elevation of %IHC in the RA-induced group compared to the control. Treatment with Costus (200, 400 mg/kg) exhibited a significant reduction (P<0.05) in the %IHC of TNF α to a level lower than RA group but not returned to the level of the control group. The % IHC of BAX in RA-induced rats treated with Costus (200, 400 mg/kg) revealed a significant reduction (P<0.05) than that of the RA non-treated group (Table 4).
Regarding the results of immunostaining for androgen receptors, control and Costus (200, 400 mg/kg) groups showed normal %IHC of androgen receptors. RA group showed scanty to minimal expression. RA Costus (200, 400 mg/kg)-treated groups showed dose-dependent expression ( Figure 2). The immunostaining for both TNFα and BAX proteins in the testis showed that control and Costus (200, 400 mg/kg) groups had minimal TNFα and BAX protein immune reactive spermatogenic cells. The RA group showed the intense positive immune reaction of both spermatids cells and macrophages in the interstitial tissue. RA Costus (200, 400 mg/kg) showed downregulation of the immune reaction ( Figure 3 and Figure 4, respectively).

Two-way ANOVA
The two-way analysis of variance was conducted to determine the effects of dose of Costus and induction of RA on all tested parameters. Table 5 displayed a significant effect of concentration administrated of Costus (P< 0.05). Results also showed a main effect for RA, as well as significant Costus dose * RA interaction for all parameters except for epididymis weight in which Costus treatment revealed non-significant effect.

Discussion
Rheumatoid arthritis is an autoimmune disorder. The influence of rheumatic illness on fecundity and reproduction can be notable (Bazzani et al. 2015). Numerous studies have been done to investigate the traditional uses of Costus speciosus for the treatment of inflammation. An in vitro study targeted the effect of costunolide on the production of pro-inflammatory mediators and mechanisms stimulated with lipopolysaccharides (LPS) in a murine BV-2 cell culture. Costunolide attenuated the expression of tumor necrosis factor alpha (TNF-α), interleukin (IL), IL-6, inducible NO synthase (NOS), and cyclooxygenase In the same manner, the n-hexane-chloroform-soluble fraction of m e t h a n o l i c C o s tu s s p e c i o s u s ex tract co ntain ed 22,23-dihydrospinasterone, dehydrodihydrocostus lactone, dehydrocostus lactone, and dehydrocostuslactone acid which induced prominent decrease in the levels of IL-1 β, IL-6, TNF-α, prostaglandin E2, lipoxgenase-5, and COX-2 in isolated peripheral blood mononuclear cells (Al-Attas et al. 2015). The above-mentioned anti-inflammatory activities revealed by C. speciosus prove the usefulness of C. speciosus application against inflammatory disease. The present study revealed that RA caused a significant decrease in the relative weight of sex organs. The treatment of RA rats with Costus (200, 400 mg/kg) significantly ameliorated the relative weight of testis but did not reach the control value. Moreover, the latter Costus doses significantly enhanced the relative weight of the tail of the epididymis. No alteration was detected in the relative weight of the prostate gland and the seminal vesicle. The histopathology of testis showed that induction of RA caused testicular degeneration with marked depletion of spermatozoa in male Wistar rats, which markedly improved with Costus treatment. Rheumatoid arthritis caused scanty to minimal testicular expression of androgen receptors. Costus (200, 400 mg/kg)-treated groups showed a dose-dependent increase in androgen expression than the RA group.  Our results were in approval with that reported via Shiraishi et al. (2009), who found that RA caused testicular damage and may affect fertility in males. Also, Silva et al. (2010) showed that reduced fertility was popular in patients with rheumatic disorders.
Moreover, the current study revealed that RA showed a significant decline in sperm count, which was relatively improved by Costus treatment (200, 400 mg/kg). This result disagrees with that of Sari et al. (2016), who demonstrated that the aqueous extract of Costus speciosus rhizome reduced both quantity and quality of spermatozoa, which both influenced male fertility. Work done by Kagbo and Obinna (2017) on methanolic extract of Costus lucanuscianus stem recorded that such extract caused a decrease in sperm count and increased sperm cell defects. However, no defect was detected in the testicular and epididymal segments of rats in all the treated groups (Kagbo and Obinna 2017). A study by Kagbo and Obinna (2018) showed that methanolic leaf extract of Costus lucanuscianus had no significant influence on testosterone levels, testicular and epididymal weights, sperm cell count, and characteristics. The testicular and epididymal sections of rats in all the treated groups were not affected. The variation in the data among studies may be accredited to the distribution of the phytochemicals in the stem and leaf parts of the plant. The medicinal values of plants lie in their component phytochemicals, which produce specific physiological actions in a biological system. Generally, phytochemicals and nutrients accumulate in different plant parts in variable concentrations (Kagbo and Obinna 2018).
The study revealed that RA causes a marked reduction in the %IHC of androgen receptors. This results in harmony with Pikwer et al. (2014), who found an adverse effect of RA on testosterone production. In addition, Cutolo (2009) observed that inflammation clearly down-regulated androgen receptor production. Our study confirmed that increased expression of testicular TNF α protein, as an inflammatory mediator, was associated with reduced androgen expression.
In this study, Costus speciosus extract showed a significant improvement in a dosage-dependent manner and increased the %IHC of testicular androgen receptors, which markedly decreased in RA-treated groups. The existence of alkaloids and flavonoids in Costus extract could ameliorate RA's oxidative and inflammatory damage on testes. The methanolic extract of Costus in 400 and 800 mg/kg doses showed dose-dependent action in male Albino rats as previously recorded by Srivastava et al. (2012). This amelioration seemed to be mediated by reducing testicular TNF α that promoted androgen receptors expression (Xiong and Hales 1993), as shown in our study.
TNF α %IHC was detected by several studies in the testis and suggested a paracrine mechanism of action in the normal testicular tissue (Lysiak 2004). The current study showed a remarkable increase in the %IHC of TNF α receptors in the testis of RA-induced rats, which explained that TNF α is one of the most vital cytokines concerned with RA's cascade of inflammatory reactions. This significant increase resulted in a marked reduction in basal t estosterone release besides cAMP-stimulated testosterone production (Xiong and Hales 1993). This is because of the decline in mRNA and protein levels of two cytochrome P450 enzymes essential in testosterone biosynthesis, cholesterol side-chain cleavage enzyme, and 17α-hydroxylase/C 17-20 lyase (Xiong and Hales 1993). Normally, the body blocks excess TNF α naturally, but in rheumatic disease, it is still active and creates more inflammation.
The current study revealed a significant increase in the % IHC of BAX protein in the testis of RA-induced rats compared to other groups. BAX plays an essential role in the mitochondrial apoptotic manner. Under normal circumstances, BAX is mainly cytosolic by continuous retrotranslocation from mitochondria to the cytosol facilitated by BCL2L1/Bcl-xL, preventing aggregation of toxic BAX secretion at the mitochondrial outer membrane. Under stress situations, a conformational alteration occurs to it that causes translocation to the mitochondrion membrane, leading to secretion of cytochrome c, then stimulates apoptosis, and increases triggering of Caspase-3 and then apoptosis (Edlich et al. 2011). Costus extract-enhanced apoptosis is mainly mitochondriamediated and associated with the transmembrane potential's failure, leading to the secretion of key apoptogenic molecules like cytochrome from the mitochondria. The antioxidant action of methanol extract of Costus was determined by its greater hydroxyl radical scavenging activity and free radical slaking capability (Vijayalakshmi and Sarada 2008). The main component separated from C. speciosus is diosgenin (Srivastava et al. 2011). The antioxidant phytochemicals comprising diosgenin may release the antiproliferative efficacy to methanol extract of Costus speciosus plant (Nair et al. 2014).
Rheumatoid arthritis has an adverse effect on fertility in male rats. This is reflected in the decrease of both sperm count and %IHC of androgen receptors; this reduction is accompanied by an increase of %IHC of TNF α and BAX protein. Bauerova and Bezek (2000) demonstrated that the antioxidant characteristics of Costus speciosus and its efficacy to stop the COX-2 pathway in the course of inflammation support the use of the plant extract in rheumatoid arthritis therapy.

Conclusion
From the current experimental results, it can be concluded that RA has an adverse effect on fertility in males, and the dosages of 200 mg/kg and 400 mg/kg extract of Costus specious have a potentially beneficial role in improving fertility since it improved weight of sex organs and sperm count. It also markedly improved testicular degeneration and exhibited a significant rise in the %IHC of androgen receptors and a significant reduction in the %IHC of TNF and BAX in the induced arthritic model in male rats. Costus is a promising anti-arthritic drug. However, additional research is needed to clarify the precise mechanism of action of plants in improving fertility.