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INTRODUCTION
One of the fundamental reasons for psychological stress is socioeconomic factors1. It has been known that chronic stress is related to numerous diseases 2. Abnormalities that cause infertility, such as ovulation, implantation disorders, and tube damage, have also been connected with psychological stress 3. Many types of stress, including psychological stress, harm fertility and reproductive functions 4. There is information in the literature that stress triggers depression and anxiety 5.
Furthermore, it is stated that depression and anxiety lead to infertility 6. It has been observed that oxidative stress and pro- inflammatory cytokine production increase in depression and anxiety 7. Degeneration, infiltration, and histopathological damage , such as atretic follicles, are seen in response to stress in rat ovaries 8. Kadioglu et al. revealed that stress, which leads to infertility, increases total oxidant levels and decreases antioxidant levels in rat ovarian tissue 9. This literature recommends that drugs with antioxidant, antidepressant, anti-inflammatory, and anxiolytic effects may be beneficial in treating stress-related ovarian damage and reproductive dysfunction.
Sertraline, is an antidepressant drug with selective serotonin re-uptake inhibitor antioxidant properties 10. It has been experimentally revealed that sertraline exerts an anti-inflammatory effect by reducing tumor necrosis factor-alpha (TNF-α) levels 11. It has been suggested that the antidepressant and anxiolytic effect of sertraline is based on the suppression of excessive production of TNF-α, interleukin-6 (IL-6), interleukin-1beta (IL- 1β), and other pro-inflammatory cytokines 12,13. Cerebrolysin is another drug we would like to see for its effects on stress-related ovarian damage, infertility, and preventing delay in maternity. Cerebrolysin is a porcine brain-derived drug with neuroprotective and neurotrophic effects and contains low molecular weight peptides and amino acids 9. It has been defended that the antidepressant effect of cerebrolysin is based on its reducing oxidative stress and cytokine-related inflammation 14. In addition, it has been argued that using an antidepressant drug with a drug with neuroprotective properties increases the effectiveness of antidepressant treatment 15. All this information shows that sertraline, cerebrolysin, and a combination of both may be beneficial against increased stress, ovarian damage, infertility, and delay in achieving pregnancy in animals. This study aimed to investigate the effects of sertraline, cerebrolysin, and the combination of both on stress- induced ovarian injury, infertility, and pregnancy delay in rats.
MATERIALS AND METHODS
Animals
Seventy albino Wistar female rats provided by the Medical Experimental Application and Research Center of Ataturk University weighing between 272-288 grams at 6 months of age were utilized in our study. Rats were placed in a laboratory with a 12- hour light/12-hour dark cycle at appropriate humidity (45%) and temperature (22°C) and fed ad libitum before experimentation. Experimental applications were carried out considering the ARRIVE guidelines. The implementation of the experiment was started after the procedures were approved by the Atatürk University Animal Experiments Local Ethics Committee (date: 27.12.2018, meeting no: 13/253).
Chemicals
Sertraline was procured from PFIZER (Turkey), cerebrolysin from EVER Pharma (Austria), and sodium thiopental from IE Ulagay (Turkey).
Animal groups
The rats were randomly separated into healthy controls (HG), stress-applied control (StC), stress+sertraline (SS), stress + cerebrolysin (SC), and stress+ sertraline + cerebrolysin (SSC) groups, with 14 animals in each group.
Experiment procedure
Stress was induced in rats by the forced immobilization method (FIM). For the implementation of this experiment, all animals except the HG group were placed in the supine position; their hindlimb and forelimbs were tied and kept in this position for one hour. After one hour, sertraline (20 mg/kg) was given to the SS rat group by oral gavage. SC group was injected with cerebrolysin (2.5 mL/kg) subcutaneously. Sertraline+cerebrolysin was administered to the SSC group at the indicated doses under the same method. The HG and StC groups were given the same volume of distilled water. FIM and drug applications were continued once a day for 30 days. Six rats from each group were euthanized (ip, 50 mg/kg thiopental sodium), and the right and left ovarian tissue were removed for biochemical and histopathological examination. The rest of the animals were kept in the same environment with mature male rats for two months for pregnancy.
The rats that were found to be pregnant were taken into separate cages and fed. Rats that did not become pregnant and give birth during this period were considered infertile. In addition, the time from the day the female rats were placed in the same cage with the male rats to the day they gave birth was determined. The delay in maternity was determined by subtracting the standard gestational period (21 days) from this period.
Biochemical analyses Preparation of samples
Ovarian tissues were weighed. Tissues were made up to 2 mL with 1.15% potassium chloride solution for MDA determination and phosphate buffer with pH=7.5 for GSH determination and homogenized in an icecold medium, it was centrifuged for 15 minutes (10000 rpm, +4 °C). The supernatant portion was utilized as an analysis sample.
Malondialdehyde (MDA) and Total Glutathione (tGSH) Analysis
Malondialdehyde was measured to establish the oxidation level, tGSH was measured to determine the antioxidant level. MDA measurements were made according to the method defined by Ohkawa et al.16. tGSH measurement was performed in line with the method defined by Sedlak and Lindsay 17.
Tumor Necrosis Factor-ɑ (TNF-α), Interleukin 1-β (IL-1β), and Interleukin-6 (IL-6) Analysis
Tumor Necrosis Factor-ɑ, IL -1β, and IL -6 were measured to assess the pro-inflammatory status. The samples were weighed, and then all tissue was cut. These samples were then snap-frozen with liquid nitrogen and homogenized using a mortar and pestle. After the samples were melted, they were kept at 2-8 °C. PBS (pH 7.4), 1/10 (w/v) was added; after this, vortexed for 10 seconds, centrifuged at 10000 xg for 20 minutes, and supernatants were aliquoted. TNF-α, IL -1β, and IL -6 levels were measured using a commercial kit (Eastbiopharm Co Ltd ELISA kit, China).
Histopathological Examination
After the tissue samples were defined in 10% formaldehyde solution, they were washed in the cassettes under tap water for 24 hours. They were then treated with conventional-grade alcohol to remove water from the tissues, passed through xylol, and embedded in paraffin. Sections of 4-5 microns were taken from these paraffin blocks and subjected to hematoxylin-eosin staining. Tissues were examined under a light microscope, and then photographs were taken (Olympus® Inc. Tokyo, Japan, DP2-SAL firmware program). Ovarian tissues were evaluated regarding congestion, hemorrhage, degeneration in follicle cells, water accumulation in follicle cells, cystic changes in follicles, and edema. The severity of histopathological findings was graded from 0-3 (0-normal, 1-mild injury, 2-moderate injury, and 3-severe injury). Histopathological evaluation was performed by a pathologist who was blinded to treatment and group allocations.
Statistical Analysis
This study used the “IBM SPSS 22® (Armonk, NY: IBM Corp.)” program for statistical analysis. Since the biochemical data were numeric, the analysis was done with one-way ANOVA, Tukey was used as a post hoc test, and the results were presented as Mean ± Standard error (X±SEM). Since the histopathological data were sequential, Kruskal- Wallis was preferred for analysis, and then the Dunn’s test was used. Data were presented as Median (Minimum-Maximum), p<0.05 was accepted as statistical significance.
RESULTS
Biochemical Results MDA and tGSH Analysis
The levels of MDA in the StC group (5.87±0.02) were significantly higher than in the HG (1.34±0.06), SS (2.85±0.02), SC (3.45±0.08) and SSC (1.52±0.06) groups as can be seen in Fig. 1A (p˂0.001). The increase in MDA levels in the SSC group was observed to be significantly lower than in the SS and SC groups (p˂0.001). For MDA, the HG and SSC groups were similar (p=0.123). The amount of tGSH measured in the ovarian tissue of the StC (1.30±0.05) group was observed to be significantly lower than the values measured in the HG (6.00±0.19), SS (3.64±0.08), SC (2.37±0.05) and SSC (5.42±0.18) groups (p˂0.001). In the treatment combination group, inhibition in the decrease of tGSH was more significant than in the sertraline and cerebrolysin groups (Fig. 1B).
*p=0.123 vs SSC group; **p˂0.001 vs SS and SC groups; ***p˂0.001 vs HG, SS, SC and SSC groups. Statistical analysis was done with one-way ANOVA, followed by the Tukey test. HG, healthy group; StC, stress-treated control group; SS, stress+sertraline group; SC, stress+cerebrolysin group; SSC, stress+sertraline+cerebrolysin group.
Fig. 1 MDA (A) and tGSH (B) levels in the ovarian tissue of study groups.
TNF-α, IL-1β and IL-6 Analysis
As seen in Fig. 2, TNF-α (Fig. 2A), IL -1β (Fig. 2B) and IL -6 (Fig. 2C) levels in the StC group (6.73±0.05, 8.05±0.17, 8.52±0.23, respectively) were significantly increased compared to HG (1.73±0.17, 2.25±0.04, 2.87±0.12, respectively), SS (3.27±0.04, 4.28±0.11, 4.89±0.03, respectively), SC (4.66±0.05, 5.81±0.04, 6.82±0.03, respectively) and SSC (2.04±0.10, 2.39±0.05, 3.14±0.03, respectively) groups (p˂0.001). The increase in TNF-α, IL -1β, and IL -6 levels was significantly lower in the combination group when compared with the SS and SC groups alone (p˂0.001). Cytokine levels were similar in SSC and HG groups (p>0.05).
*p>0.05 vs SSC group; **p˂0.001 vs. SS and SC groups; ***p˂0.001 vs. HG, SS, SC and SSC groups. Statistical analysis was done with one-way ANOVA, followed by the Tukey test. HG, healthy group; StC, stress-treated control group; SS, stress+sertraline group; SC, stress+cerebrolysin group; SSC, stress+sertraline+cerebrolysin group.
Fig. 2 TNF-α (A), IL -1β (B), and IL -6 (C) levels in the ovarian tissue of study groups.
Reproduction Test Results
Animals in the HG group gave birth within 23-26 days, as observed in Table 1. Six of the eight rats in the SS group gave birth within 27-36 days, while two did not give birth within two months. Three of the eight rats in the SC group gave birth within 33-38 days, but three did not give birth within two months. In the SSC group, all eight female rats gave birth on days 24-28. One of eight female rats in the StC group gave birth on day 49, but the remaining seven did not give birth during this time.
Table 1 Infertility and reproductive process in rats preproductiveprocess in rats.
| Groups |
Non-infertile rats n % |
İnfertile rats n % |
Reproductive process (RP) (day) |
Delay in maternity (RP-21 days) |
||
|---|---|---|---|---|---|---|
| HG (n=8) | 8 | 100 | - | - | 24.63 ± 0.42* | 3.63 ± 0.42* |
| SS (n=8) | 6 | 75 | 2 | 25 | 30.17 ± 1.45** | 9.17 ± 1.45** |
| SC (n=8) | 3 | 37.7 | 5 | 62 | 36.00 ± 1.53*** | 15.00 ± 1.53*** |
| SSC(n=8) | 8 | 100 | - | - | 25.75 ± 0.59 | 4.75 ± 0.59* |
| StC (n=8) | 1 | 12 | 7 | 88 | 49.00 | 28.00 |
*, p>0.05 vs SSC; **, p<0.05 vs SC and SSC, ***; p<0.05 vs SS and SSC. Statistical analysis was done with one-way ANOVA, followed by the Tukey test. Results are expressed as mean±standard error of the mean. HG, healthy group; StC, stress-treated control group; SS, stress+sertraline group; SC, stress+cerebrolysin group; SSC, stress+sertraline+cerebrolysin group; n, number of animals.
Histopathological findings
As seen in Fig. 3A and Table 2, no pathological findings were found in the ovarian tissue of the HG group; corpus luteum and follicle structure were observed within normal limits. Grade-3 degenerated secondary follicle and congestion were observed in the ovarian tissue of the StC group (Fig. 3E, Table 2). Moreover, grade-3 dilated congested vessels, hemorrhage, and edema were seen in the ovarian tissue of the StC group (Fig. 3F, Table 2). Vascular congestion (grade-1) and relatively normal follicle and corpus luteum structure (grade-0) were seen in the ovarian tissue of the SS group treated with sertraline (Fig. 3B, Table 2). In the SC group, mild fluid accumulation in the lumen, cystic changes (grade-1), minimal vascular congestion (grade-1), and corpus luteum damage (grade-1) were detected (Fig. 3C, Table 2). There were no histopathological signs other than mild vascular congestion (grade-1) and relatively normal corpus luteum (grade-0) in the SSC group (Fig. 3D, Table 2).
Fig. 3 (A-F). Histopathological examination of ovarian tissues in study groups. A. Ovarian tissue of the SG group; view of healthy corpus luteum (star) and follicle structure (arrow) within normal limits. B. Ovarian tissue of the StC group; Section showing degenerated secondary follicle (bilateral arrow), dilated congested blood vessel (straight arrow). C. Ovarian tissue of the StC group; section showing dilated congested blood vessel (straight arrow), hemorrhage, and edema (bilateral arrow). D. Ovarian gland of the SS group; Section showing congested blood vessels (arrows), the follicle (star), and corpus luteum (zigzag arrow). E. Ovarian tissue of the SC group; Follicle structure with fluid accumulation in the lumen and cystic change (arrow), section showing mild congestion (zigzag arrow). F. Ovarian tissue of the SSC group; Section, showing mildly congested blood vessel (zigzag arrow), corpus luteum (star). H&E x 200. HG, healthy group; SS, stress+sertraline group; SC, stress+cerebrolysin group; SSC, stress+sertraline+cerebrolysin group; StC, stress-treated control group. H&E x 200.
Table 2 Histopathological examination of ovarian tissues.
| Groups | Congestion | Hemorrhage |
Follicle cell degeneration |
Water accumulation in follicle cells |
Cystic change in follicles |
Edema |
|---|---|---|---|---|---|---|
| HG (n=6) | 0(0-0)* | 0(0-0)* | 0(0-0)* | 0(0-0)* | 0(0-0)* | 0(0-0)* |
| SS (n=6) | 1(0-2)* | 0(0-0)* | 0(0-1)* | 0(0-0)* | 0(0-0)* | 0(0-0)* |
| SC (n=6) | 1(0-2)* | 0(0-0)* | 0(0-0)* | 1(0-2)** | 1(1-1)** | 0(0-0)* |
| SSC (n=6) | 1(0-1)* | 0(0-0)* | 0(0-0)* | 0(0-0)* | 0(0-0)* | 0(0-0)* |
| StC (n=6) | 3(2-3)** | 3(3-3)** | 3(2-3)**- | 0(0-0)* | 0(0-0)* | 3(2-3)** |
Histopatological grading; 0-normal, 1- mild injury, 2-moderate injury, and 3-severe injury. *, p>0.05 vs other groups with the same sign; **, p<0.05 vs other groups. Kruskal Wallis test was used. Results are expressed as median (minimum -maximum). HG, healthy group; SS, stress+sertraline group; SC, stress+cerebrolysin group; SSC, stress+sertraline+cerebrolysin group; StC, stress-treated control group.
DISCUSSION
The effects of sertraline, cerebrolysin, and their combination against FIM-related stress-associated ovarian damage, infertility, and delay in achieving pregnancy in female rats were investigated in this study. Various stress factors lead to damage to all organs and tissues of the body, as can be understood from the literature 18. Previous studies have revealed that psychological or physiological stress is related to oxidative stress 19. Stress increased the levels of MDA, known as the toxic product of lipid peroxidation (LPO), and decreased the antioxidant tGSH levels in the ovarian tissue of animals, as can be observed in our results. These biochemical findings show that stress changes the oxidant-antioxidant balance in favor of oxidants in the ovarian tissue. In the literature, it has been shown that stress increases oxidants in ovarian tissue while decreasing antioxidants 20. As it is known, membrane lipids are oxidized by reactive oxygen species (ROS), and a toxic product, MDA, is formed. MDA resulting from LPO greatly disrupts the structure and functions of the cell membrane and leads to further destruction 21. As such, MDA level is known as a marker of oxidative stress and antioxidant status in patients 22. Our results, which align with these previous findings, showed that the amount of tGSH decreased significantly. At the same time, MDA increased in the ovarian tissues of animals exposed to stress. GSH is a tripeptide that can be found in most cells. GSH protects cells from the toxic effect of ROS by detoxifying hydrogen peroxide and organic oxides 23.
It is known that pro-inflammatory cytokine production plays a role in parallel with excessive oxidant production in the pathogenesis of ovarian damage, infertility, and delay in achieving pregnancy that develops due to stress and other factors 9,24. Our results, accordingly, showed that TNF-α, L -1β and IL -6 levels increased in the ovarian tissue of animals with infertility and delay in achieving pregnancy. In studies with patients, it has been reported that TNF-α, IL - 6, and other pro-inflammatory cytokines are among the factors that lead to infertility in ovarian pathologies 25. Oxidative stress and pro-inflammatory cytokines increase in psychological disorders such as depression and anxiety, as mentioned above 7.
It has been reported that stress is associated with depression, and depression may lead to ovarian dysfunction 26. Sertraline was more effective than cerebrolysis against stress-related ovarian damage, infertility, and pregnancy delay. The fact that sertraline is more effective than cerebrolysin may be due to its more significant inhibition of overproduction of oxidant and pro-inflammatory cytokines than cerebrolysin. Sertraline has antioxidant properties as mentioned hereinabove 10. Moreover, it is argued that the antidepressant and anxiolytic effect of sertraline is based on suppressing excessive production of TNF-α, L -1β, IL -6, and other pro-inflammatory cytokines 12,13. It has been documented that the antidepressant effect of cerebrolysin is due to the reduction of oxidative stress and cytokine-related inflammation 14. Cerebrolysin is also a neuroprotective and neurotrophic drug 27. The fact that the use of an antidepressant drug together with a drug with neuroprotective properties increases the effectiveness of the treatment has been reported in the literature 15. The administration of sertraline and cerebrolysin in combination suppressed oxidant and inflammatory markers in ovarian tissue better than sertraline and cerebrolysin administered alone. It also better prevented stress- related infertility and delay in achieving pregnancy in our study.
Severe histopathological injury was seen in the ovarian tissue of the stress group. In addition, mild cystic changes in the follicles and water accumulation in the follicle cells were observed in the stress and cerebrolysin group, while adding sertraline to the treatment prevented these changes. Combination therapy, sertraline, and cerebrolysin were the best suppressors of histopathological damage, respectively. It is known that oxidant and pro-inflammatory cytokines lead to hemorrhage, congestion, follicle degeneration, inflammatory cell infiltration, and necrosis in the ovarian tissue 24. Infertility and delay in achieving pregnancy developed in the stress-treated control group in which severe histopathological damage was detected in the ovarian tissue, as can be understood from our results. Ince et al. reported severe follicle degeneration in ovaries with high MDA and low tGSH levels 28. In another study by Ince et al. stated that severe degeneration was found in the ovarian follicles of animals in which sterility and delay in achieving pregnancy developed 29. It was revealed in the study of Kadioglu et al. that the stress induced by the forced immobilization method causes widespread congestion, hemorrhage, accumulation of fluid, and inflammatory infiltration in the subcapsular area in the ovaries. Infertility, delay in achieving pregnancy, decrease in the number of offspring, and intrauterine physical developmental retardation were found in the animal group with these histopathological signs 9. The stress induced by the FIM has led to oxidative and inflammatory damage in the ovarian tissue of animals, sterility, and delay in achieving pregnancy. The combination therapy of sertraline and cerebrolysin were the drugs that best prevented ovarian damage, infertility, and delay in achieving pregnancy, respectively. This information has revealed the fact that antidepressant drugs with antioxidant and anti-inflammatory effects might be useful in the treatment of stress-related ovarian damage, infertility and delay in achieving pregnancy. It has been revealed particularly that the combination of antidepressant (sertraline) and neuroprotective / neurotrophic (cerebrolysin) drug combination may be more beneficial. In line with this information sertraline, cerebrolysin and their combination may be preferred in treating stress- associated ovarian damage, infertility, and delay in achieving pregnancy. However, it is required to investigate antidepressant and neuroprotective/neurotrophic drug combinations from different groups in the future to confirm these findings.
