Effect of Carob Supplement on Spermogram Parameters and Sexual Function of Infertile Men Referred to the Infertility Center, Hamadan, Iran, 2019: A Randomized Controlled Trial

As defined by the WHO, infertility is couples’ inability to conceive after one year of unprotected intercourse. Infertility is one of the most common problems in the world, experienced by about 15% of couples. About 25% of Iranian couple’s experience primary infertility in their marital life. It is due to a male factor and sperm problems in 50% of the cases. Infertility and its associated individual and social problems are an important issue for couples, because the cause of male infertility is pathologically detectable only in 40% of cases ( 1 ). Therefore, infertility treatment is more difficult in men than in women, especially in developing countries where treatment is associated with high cost. Some studies have indicated the harmful effects of environmental factors such as toxins, pesticides and radiations on male infertility and suggested that toxins and pesticides can reduce sperm concentration ( 2 ) .The major causes of male infertility are congenital or acquired anomaly of the genitourinary system, malignancies, urogenital infections, increased scrotal temperature (such as varicocele), endocrine disorders, genetic abnormalities, and immunological problems. However, infertility is idiopathic in 30–40% of infertile men, but it may have a variety of causes, including environmental pollution, Oxygen free radicals, and genetic and epigenetic abnormalities ( 3 ). Male infertility may have other factors, such as vas deferens obstruction, sperm problems (low count, low motility, dysmorphology), and sperm allergy. A male factor is involved in about half of all infertility cases. The presence of the male factor is often based on abnormal sperm parameters (azoospermia to oligozoospermia). Impaired sperm production and function and damaged spermatogenesis process are among the most common causes of male infertility. Trauma or anatomical defects in the genital system and the use of certain drugs to treat diseases can lead to impaired sperm production and consequently male infertility ( 4 ). The overproduction of reactive oxygen species (ROS) by leukocytes as well as abnormal sperm in semen and consequent oxidative stress have recently been identified to be among the causes of male infertility . Free radicals are made up of different metabolic pathways in each aerobic cell, but their main source in male seminal fluid is white cells and abnormal and immature sperm. Studies have shown that infertile men are more likely to have lower antioxidant capacity than fertile men. Researchers have sought to prevent reduced motility, increased mortality and DNA damage due to oxidative stress in infertile people using antioxidants ( 5 ). Carob or Ceratonia siliqua L is a 7- to 12-meter tall tree belonging to the Legominasae family. Its leaves are a combination of yellow, red or purple flowers with no petals. Its fruit is a curved pod light brown in color, 10 to 30 cm long and containing 12 to 16 hard seeds. Carob is native to the Mediterranean region and is found in southern Syria. It grows rapidly in India, many Mediterranean regions, California, and Fars, Iran ( 6 ). Studies on the Ceratonia siliqua seed chemicals have shown that it is high in fiber, polyphenol compounds, arachidonic acid, lignin, fats, proteins, carbohydrates, calcium, potassium and phosphorus. They also contain aspartic acid, glutamic acid, linolenic acid, linoleic acid, vitamin E, beta-sitosterol, silica, iron, and magnesium. These compounds can have different effects on the body of animals. Carob seeds are also known as a potential natural source of antioxidants and their antioxidant activity is associated with phenolic compounds and the presence of substances ( 7 ). Sexual desire has an undeniable effect on marital life and its stability. Any factor that disrupts a healthy marital relationship is classified as a sexual disorder ( 8 ). The WHO considers sexual health to be the integration and consistency among the mind, feeling, and the body, which leads the social and intellectual aspects of human beings towards the development of their personality and leads to the establishment of communication and love. Therefore, any disorder that leads to inconsistency and consequent dissatisfaction with sexual relationship can lead to sexual dysfunction. There has been little research on the effects of carob seed supplementation on male reproduction, so the present study has tried to explore the effect of carob seed supplementation on changes in sperm parameters, sex hormone levels, and sexual function in infertile men, the results of which may prove helpful in fertility treatment and family planning. This study was completed in accordance with SCARE 2020 guidelines ( 9 ).

The participants’ mean age was 34.83 ± 6.22 years in the carob group, and 33.67 ± 5.82 years in the placebo group. The majority of participants in all groups had less than a high school diploma and was mostly self-employed. A comparison of the variables presented in Table 1 did not show a statistically significant difference among the groups. Comparison of demographic and contextual characteristics in groups * Independent t test, ** Chi-square test and the rest: Fisher’s exact test A comparison of seminal fluid parameters at the pre-intervention stage showed that 96.3% of the carob group members and 78.3% of the placebo group members had normal semen volume. About 93% of the carob group subjects had normal sperm counts, while this rate was 70% in the placebo group. In terms of progressive motility, sperm shape and WBC number, the majority of subjects in the groups were in normal condition. None of these parameters showed a statistically significant difference between the two groups (P>0.05) ( Table 2 ). The results of multivariate analysis showed that by controlling the values of semen at the pre-intervention stage, the normal volume of semen in the carob group was 9% less than that in placebo, and these results were statistically significant (P0.05), the rate of normal sperm counts increased by 17%, which was also statistically significant (P<0.05) ( Table 3 ). Comparison of semen analysis results before intervention between groups * Chi-square test and the rest of Fisher’s exact test Comparison of semen analysis results after intervention between groups * Chi-square test and the rest of Fisher’s exact test Inter-group and intragroup comparisons of the results revealed that although the mean scores in the carob group after intervention were higher than that in placebo in the subscales of orgasm function, IIEF, satisfaction with sexual intercourse, sexual desire, overall satisfaction, and erectile function, no significant statistical difference was observed in these subscales (P>0.05) ( Table 4 ). A comparison of the level of hormones presented in Table 5 demonstrated that the two groups did not have a statistically significant difference at the pre-intervention stage (P>0.05) ( Table 5 ). The results of multivariate analysis in terms of hormone status showed that by controlling hormone levels at the pre-intervention stage, normal testosterone levels were 40% higher than its abnormal levels in placebo users and this difference was statistically significant (P<0.05). Comparison of hormones levels before the intervention between groups * Chi-square test and the rest of Fisher’s exact test Comparison of hormones levels after the intervention between groups * Chi-square test and the rest of Fisher’s exact test In the case of prolactin, the normal level of this hormone in the carob group was 37% higher than that in placebo, but this difference was not statistically significant (P>0.05). The changes in the rest of the hormones are negligible, as shown in Table 6 . Comparison within and between groups of the total score of sexual function domains before and after the intervention * Mann–Whitney U test, ** Wilcoxon Test

According to the results of this study, the use of carob supplement is recommended to improve some spermogram parameters such as viscosity and increase the normal levels of testosterone and prolactin hormones.

Nowadays, herbs are used to treat infertility with male or female factor in many countries of the world, especially in Asian countries. One of the herbal and local medicines widely used in world to treat infertility with male factor and impotence is carob. In our study, the results of comparison between the carob and placebo groups indicated normal viscosity, progressive motility of more than 32%, and normal shape of more than 4% in the carob group after the intervention as compared with the placebo. However, no significant difference was observed only in the progressive motility due to the limited sample size. Since antioxidants play a central role in the defense of cells against free radicals, a decrease in the antioxidant activity of the body’s physiological system is likely to be associated with a decrease in the quality of sperm cells ( 12 ). Numerous studies have shown that active oxygen species (AOS) can strongly affect various sperm parameters, including sperm morphology and motility. According to studies, sperm quality has decreased over the past decades. Various micronutrients are involved in sperm metabolism. The high content of unsaturated fatty acids in the plasma membrane of sperm is considered to be the reason for the high sensitivity of sperm to ROS ( 13 ). Carob has traditionally been used to increase the number of sperm in infertile men. Animal studies have shown carob extract to affect cAMP production and the activity of enzymes involved in steroidogenesis in rat testes. The use of carob has been shown to affect sex hormones, sperm production and spermatogenesis in rats by influencing biochemical factors affecting Leydig cells and testosterone biosynthesis ( 14 ). Studies conducted by Alsalman et al . in 2013 ( 15 ) and Elsheikh MG et al . in 2015 ( 16 ) have revealed that the use of antioxidants improves sperm quality. A study was conducted by Greco E in 2005, finding that antioxidant administration can be effective in controlling damage to sperm DNA in the short term ( 17 ). Mahdiani et al . (2018) found that changes in the mean total antioxidant capacity and plasma malondialdehyde concentration was significant after the intervention in the carob group in comparison with the placebo group. Researchers have sought to prevent reduced motility, increased mortality and DNA damage due to oxidative stress in infertile people using antioxidants ( 10 ). Faramarzi et al . (2020) found that an increase in the progressive motility of sperm was observed after the administration of carob in the laboratory environment ( 18 ). Sufficient animal and human studies have not been carried out on the effects of the extract of this plant and the mechanisms involved, as well as its effective amounts. The results of the present study revealed that there was no statistically significant increase in many of the sexual function subscales in the carob group after intervention, and the normal levels of prolactin and testosterone were higher in the carob group than in the placebo. Sanagoo et al . (2021 ) found that the use of carob supplement did not affect the sexual function of infertile men. Carob is a rich source of iron, calcium, sodium, potassium, phosphorus and sulfur, as well as vitamins E, C, D, niacin, folic acid and pyridoxine. Its powder contains 11 phenolic compounds with large contents of pyrogallol, catechol and chlorogenic acid and small amounts of other phenolic compounds such as coumarin, cinnamic, ferulic and gallic acid. It also contains 17 fatty acids, mainly the four fatty acids of oleic, linoleic, palmitic and stearic acid ( 19 ). Mokhtari et al . (2012) studied the possible effects of carob seeds on levels of FSH, LH, testosterone and dihydrotestosterone, testicular tissue, and fertility improvement in male rats. They found that the use of carob seed extract caused a significant increase in testosterone and dihydrotestosterone concentrations and a decrease in the LH levels in experimental groups, but the FSH concentration did not show significant changes ( 20 ). Mahdiani found that changes in testosterone were positive after taking carob supplements, although the changes were not significant. Also, the change in other sex hormones was not significant in that study. The only study conducted on the effect of carob use on the sex hormones of rats revealed that the use of carob seed extract in male mice significantly increased the concentration of testosterone and dihydrotestosterone, but it decreased the LH levels ( 10 ) It seems that the increase in testosterone levels by carob is due to its direct effect on Leydig cells and in testosterone biosynthesis. These effects are probably due to the stimulation of PGE2 synthesis. In addition, carob seeds contain gamma linolenic acids and alpha linolenic acid, which can be converted to dihomo-gamma-linolenic acid and PGE2, which ultimately increases the production of cyclic adenosine monophosphate and stimulates testosterone production ( 16 ). One of the limitations of this study is the small number of samples that reduce the generalizability of the results to the whole community.

This study was designed as a two-group randomized, double-blind, placebo-controlled clinical trial ((IRCT20120215009014N284) performed on idiopathic infertile men. After obtaining permission from the Vice Chancellor for Research of Hamadan University of Medical Sciences and the code of ethics (IR.UMSHA.REC.1398.155), the present study was done in Fatemieh Infertility Center in Hamadan, from May 10 to September 9, 2019. The sample size was calculated in Stata 13 software with Sampsi module. The sample size was determined 30 for each group based on the data obtained from Mahdiani et al .’s study ( 10 ), considering the 25% loss, and based on the following information: M 1 = 17 ,   M 2 = 21 ,   Sd 1 = 4 ,   Sd 2 = 1.5 ,   α = 0.05 ,   power = 0.90 The inclusion criteria were being a man under 40 years of age with primary infertility; abnormality of at least one of the semen parameters (volume, concentration, sperm count, motility and morphology of sperm), lack of infertility-related disorders such as chromosomal abnormalities, testicular failure, varicocele, and cryptorchidism, lack of chronic diseases such as diabetes, kidney disease, infectious diseases, genital infections, thyroid, having a BMI less than 30, lack of the use of narcotic substances, alcohol and any drugs that disrupt spermatogenesis (such as methotrexate, nitrofurantoin, colchicine and chemotherapy), pituitary suppressants (such as testosterone injections, GnRh analogues), anti-androgens (cimetidine, spironolactone), use of drugs that cause ejaculation failure (alpha-blockers, antidepressants, phenothiazines), use of drugs that cause dysfunction (Beta-blockers, thiazide diuretics, metoclopramides), long-term use of drugs such as anabolic steroids, cannabis, heroin and cocaine, no history of testicular and vasodilator surgery, no contact with pesticides, heavy metals and solvents, and non-use of antioxidant supplements in the last three months ( 11 ). Infertile men with drug and alcohol use, the use of the creatinine more than twice, strenuous physical activity, infertility during the study, diet for weight loss, and change of location were not included in the study. A semen sample was initially taken from men who had referred for infertility treatment. Samples were collected in case of three-day avoidance of sexual intercourse. Incubation was performed for 30 to 60 minutes to convert the samples from bulk to liquid. To evaluate sperm parameters in accordance with the WHO standards, 200 microliters of fluid sample was examined. Computer semen analysis was used to assess sperm motility. Also, microscopic tests were performed to evaluate and determine parameters such as sperm concentration per milliliter of semen, viability and sperm morphology. The research goals and methods were explained to those who had the inclusion criteria and then written consent was obtained from all volunteers for the research. The data collection form about the general characteristics of the patients was completed. Furthermore, 10 cc of blood was taken from patients at the beginning of the study to measure their sex hormones (LH, FSH, prolactin, and testosterone) and TSH. The packages containing the capsules were coded by individuals other than the researchers as A and B, and the volunteers were randomly assigned to intervention and placebo groups. There were mixed cards with labels A and B based on the number of participants ( Figure 1 ). Flowchart of sampling By taking out the cards, the patients were divided into two groups of carob and placebo. The researcher and the patient were blind to the study groups. The first group received 1.5 grams of carob seed powder per day in three, 500 mg capsules. They received placebo capsules, which were similar in appearance to carob capsules and contained lactose, and both groups received the same routine infertility treatment in 12 weeks. The prescribed dose selection was based on a pilot study which had been conducted by the researchers. This pilot study had been performed on ten idiopathic infertile men as a supplement to carob on sperm parameters. Both carob and placebo drugs are prepared in the form of capsules by the Faculty of Pharmacy of Hamedan University of Medical Sciences. Follow-up of patients was performed by phone once every 15 days in order to control the use of capsules and prevent the loss of samples. Moreover, by counting the remaining capsules, patients who had not used more than 10% of their capsules were excluded at the end of the study. Patients were also advised not to change their diet. Finally, after the intervention, the semen samples were evaluated for spermogram and blood samples were examined for sex hormones. LH and FSH were measured using the ELISA method with the CSB E12654r kit made by the Japanese CUSABIO Company. Serum levels of Testosterone, prolactin and TSH were measured using the ELISA hormone measurement kits made by the German DRG Instruments GmbH Company with hormonal sensitivity of 0.083 ng / ml and the RIA (radioimmunoassay) with hormonal sensitivity of 0.09. It should be noted that sexual function was examined in the infertile men with the inclusion criteria in groups in two stages before and after the intervention using the IIEF. This index was completed by researchers via interviews. It contains 15 standard questions and is divided into 5 subscales, namely erectile function, orgasmic function, sexual desire, satisfaction with intercourse and overall satisfaction. It is scored from zero to five and the total score is obtained by adding the scores of the questions of each dimension. Higher score indicates the most optimal sexual function. The scores range from 15 to 75, with scores within the 15–25 range indicating low sexual function, scores within the 25–50 range indicating moderate sexual function, and scores higher than 50 indicating high sexual function. In previous studies, the reliability of the questionnaire was confirmed with the Cronbach’s alpha of 0.85%. In this study, all the data were shown as mean and frequency for quantitative and qualitative variables, respectively. Initially, the normality of data distribution was evaluated using the Kolmogorov-Smirnov test. The independent sample t-test was used to compare the mean changes of quantitative variables between the two groups. P-value less than 0.05 was considered statistically significant. Statistical analysis of data was performed using SPSS 16.