Effects of Nigella sativa L. seed oil on abnormal semen quality in infertile men: a randomized, double-blind, placebo-controlled clinical trial.
In recent years, wide utilization of herbal drugs has encouraged scientists to determine their impressive effects on health. Since Nigella sativa L. seed (N. sativa) has many uses including infertility in traditional medicine, the effects of Nigella sativa L. seed oil on abnormal semen quality in infertile men with abnormal semen quality are of interest. This study was conducted on Iranian infertile men with inclusion criteria of abnormal sperm morphology less than 30% or sperm counts below 20 x [10.sup.6]/ml or type A and B motility less than 25% and 50% respectively. The patients in N. sativa oil group (n = 34) received 2.5 ml N. sativa oil and placebo group (n = 34) received 2.5 ml liquid paraffin two times a day orally for 2 months. At baseline and after 2 months, the sperm count, motility and morphology and semen volume, pH and round cells as primary outcomes were determined in both groups. Results showed that sperm count, motility and morphology and semen volume, pH and round cells were improved significantly in N. sativa oil treated group compared with placebo group after 2 months. It is concluded that daily intake of 5 ml N. sativa oil for two months improves abnormal semen quality in infertile men without any adverse effects.
Nigella sativa seed oil
Abnormal semen quality
Infertility resulting in involuntary childlessness is a social and clinical problem affecting couples worldwide. In a nation-wide study in France infertility was reported in 11% and 24% of couples after 12 and 24 months of unprotected intercourse (Slama et al. 2012). Among infertile couples, male factors, contribute to more than half of all cases of childlessness (Araoye 2003). Infertility does not affect the couples' life only, but it also affects the healthcare services and social environment (Zafar and Mohsin 2001). Several factors such as congenital diseases, hormonal imbalance, genetic and nutritional defects are among disorders in the normal function of testicle which causes men's infertility (Whitman-Elia and Baxley 2001). A number of antioxidants, nutritional therapies, and botanical medicines have proven beneficial in treating male infertility (Sinclair 2000; Agarwal et al. 2008; Chen et al. 1999). N. sativa is a medicinal plant which belongs to the botanical family Ranunculaceae and commonly is known as black seed (Malhotra 2004). N. sativa seed is often used as a spice, food preservative and medicine by people in Asia, Middle East and Africa (Goreja 2003). N. sativa is one of the components of Iranian traditional remedies for treatment of infertility (Salehi Surmaghi 2008). The efficacy of N. sativa oil has been reported on some reproductive traits of male chickens (Samir Bashandy 2007) and male rat's spermatogenesis and fertility (Al-Sa'aidi et al. 2009; Mukhallad et al. 2009; Abdulkarim and Al-Sardary 2009), but no clinical study has been done to investigate the effects of N. sativa oil on infertile men so far. The N. sativa oil has a wide margin of safety (Zaoui et al. 2002) and has been administered to abnormal metabolic syndrome as well as diabetic patients at the dosage of 5 ml daily for two months without any side effects (Najmi et al. 2008; Mohtashami et al. 2011). Thus in the light of the above findings, the present study was conducted to determine the effects of consumption of 5 ml daily N. sativa oil on infertile men having abnormal semen quality.
Materials and methods
N. sativa oil and mineral oil were purchased from local market in the Tehran city. N. sativa oil was the product of Barij Essence Company, Kashan, Iran. N. sativa oil had been prepared by cold press procedure as indicated in its brochure. 0.1 ml of a mixture consisting of chlorophyll and red chili pepper extract in equal proportions dissolved in oil was added to 100 ml of mineral oil and N. sativa oil in order to give color and taste to placebo. The N. sativa and mineral oils were filled in 150 ml colored bottles and marked as A and B.
Determination of total fatty acid content of N. sativa oil
The concentration of fatty acids in N. sativa oil was measured by the gas-liquid chromatography method (Duchateau et al. 1996).
Determination of chemical composition of the volatile oil of N. sativa oil
25 ml of the N. sativa oil were hydro-distilled for 4h. The distillate or volatile oil was diluted using n-hegxane and analyzed by the GC-MS using Agilent 6890 system (Adams 2007; McLafferty and Stauffer 1989).
Healthy men 20-45 years of age with infertility lasting more than one year; men not receiving any drugs for infertility during the last 3 months; men without any history of renal, hepatic or other chronic illnesses; men having semen samples abnormality (sperm abnormal morphology less than 30% or sperm count blow 20 x [10.sup.6] ml or type (A) motility less than 25% and type (B) motility less than 50%) according to WHO standard guidelines (WHO 1999).
Men with azoospermia, gonad abnormalities, varicocelle and urinary retention and infections; those who have taken hormone treatment; men with genital accessory gland infection; men with recent medical or surgical illnesses; those who have time limitation (e.g. those whose wives are above 35) and cigarette smokers.
Eighty Iranian infertile men 20-45 years old, referred to the Infertility Center of Mahdieh Hospital in Tehran, Iran were selected. All patients were initially visited by urologist, interviewed and questioned about their sexual behavior, history of prior surgical interventions or childhood diseases such as cryptorchidism that affect fertility and family history of infertility. A written informed consent was obtained from each participant. The ethics committee of the Infertility Center of Mahdieh Hospital approved the protocol. The study was double-blind and block randomization was used for allocation of the patients to the N. sativa oil and placebo groups. Forty patients participated in each group of the study. However, 34 patients in each group finished the study (Fig. 1). The sample size calculated to detect 21 x [10.sup.6] sperm/ml difference of sperm count between N. sativa oil and placebo groups, considering type 1 error = 0.05 and 80% power was 34 patients per group. The patients in N. saliva oil and placebo groups received N. sativa oil and mineral oil respectively each at the dose of 2.5 ml, two times a day orally, for 2 months. All participants were requested to report any adverse effects.
At baseline and after 2 months, the semen samples were obtained from the patients in both groups, 48-72 h after the patients last sexual contact for determination of semen volume, round cells and pH and sperm count, motility and morphology according to WHO guidelines (WHO 1999).
Semen quality assessment
All semen parameters were primary outcome.
The patients' semen samples were collected (after 72 h abstinence from ejaculation) in the laboratory.
Semen smear was prepared from mixture of 10 [micro]l semen and 10 [micro]l of Giemsa stain on slide, dried in air and washed under running tap water and then dried in air. Sperm count (sperm density) was determined by routine laboratory procedure and expressed as million/ml of semen.
Quantitative motility (expressed as percentage) was determined by counting both motile and immotile spermatozoa per unit area. Sperm motility was expressed as class A (fast moving forward), class B (slow moving forward), class C (moving at same place), Immotile (with no moving) and class A + B (moving fast forward and moving slow forward) motilities.
The semen smear was prepared from mixture of 10 [micro]l semen and 10 [micro]l of Giemsa stain on slide, dried in air and washed under running tap water and then dried in air. Sperm morphology (normal/abnormal) expressed as percentage was assessed under oil (Immersion oil) at 100x magnification of microscope using ocular micrometer (ocular micrometer should be calibrated with stage micrometer to measure the exact size).
Other semen parameters
Semen volume was measured by Falcon tube and expressed as ml. Semen pH was determined by using pH meter. Semen round cells was determined by counting round cells such as white blood cells or germinal cells and expressed as percentage.
The trial was registered in Iranian Registry of Clinical Trial with the number IRCT201011205204N1.
Chi squared and t-tests were used for data analyses and p < 0.05 was considered as significant.
The values for fatty acid contents of the N. sativa oil are presented in Table 1.
The N. sativa oil contained 1.1% volatile constituents. The chemical compositions of the volatile oil component of N. sativa oil are presented in Table 2.
No adverse effects were reported. The groups were matched in regard to demographic data including age, duration of infertility and body mass index (Table 3). The baseline blood levels of all parameters were not significantly different between the two groups (Table 4).
Results indicate that 12 patients in N. sativa and 15 patients in placebo group had sperm counts below 20 x [10.sup.6] ml and 15 patients in N. sativa and 15 patients in placebo group had abnormal sperm morphology less than 30%. In N. sativa group 17 patients and placebo group 20 patients had type A motility less than 25% and in each groups 21 patients had B motility less than 50%.
In N. sativa oil group sperm count, morphology and motility improved significantly (p = 0.001, p = 0.011 and p = 0.001 respectively) compared with placebo group at the end of study (Table 4). Furthermore, in N. sativa oil group semen round cells, volume and pH also improved significantly (p = 0.025, p = 0.001 and p = 0.001 respectively) compared with placebo group at the end of study (Table 4). Also, different classes of sperm motility also improved significantly in N. sativa oil group compared with placebo group at the end of study (Table 5).
The results indicate that daily intake of 5 ml IV. sativa oil improves semen quality including sperm count, morphology and motility and semen volume, pH and round cells in infertile men having abnormal semen parameters. The effects of N. sativa oil on abnormal semen parameters in the present study agree with the previous study indicating the favorable effects of N. sativa oil on male fertility in normal and hyperlipidemie rats (Samir Bashandy 2007). Furthermore favorable effects of N. sativa oil also have been reported on spermatogenesis, infertility and some reproductive parameters in male rats and chickens (Al-Sa'aidi et al. 2009; Mukhallad et al. 2009; Abdulkarim and Al-Sardary 2009). The mechanisms underlying these effects are not clear, but antioxidant properties of N. sativa oil may be involved. In normal conditions, the reactive oxygen species (ROS) are neutralized by antioxidants of ejaculatory fluid (Lamirande and Gagnon 1999), but in case of reduction of the antioxidant property or increased production of ROS in semen for any reason, increased level of oxidative stress would be harmful to sperm parameters (Schulte et al. 2010; Aitken and Krausz 2001). There is also report indicating that ROS level is increased in semen of infertile patients (Zini et al. 1993). Thus, the favorable effects N. sativa oil on sperm parameters in infertile men observed in the present study may be due to excellent ability of N. sativa oil to inactivate free radicals (Burits and Bucar 2000). In support to this hypothesis it should be noted that the thymoquinone (Al-Wafai 2013; Ghosheh et al. 1999; Houghton et al. 1995) with antioxidative properties, present in of N. sativa oil may neutralize the ROS in semen and positively influence abnormal sperm parameters. In agreement with this hypothesis the positive effects of antioxidants like vitamin E, selenium and nutritional supplements on sperm motility of infertile men have been documented (Suleiman et al. 1996; Vezina et al. 1996; Sinclair 2000). Furthermore, the favorable effects of N. sativa oil on abnormal sperm parameters in the present study may be due to unsaturated fatty acids content of N. sativa oil (Nickavar et al. 2003). This hypothesis is supported by studies indicating significant positive correlation between the unsaturated fatty acid supplementation and enhanced sperm count, motility, and normal morphology in infertile men (Nissen and Kreysel 1983; Safarinejad 2011). Although in the present study we quantified the unsaturated fatty acid and volatile constituents of N. sativa oil that agree with previous studies, but due to study limitations such as lack of determination of male sex hormone levels, antioxidant activity and unsaturated fatty acid content of semen fluid we could not find out the possible mechanisms of action of N. sativa oil. Furthermore, due to short duration of study we could not find out the possible effects of N. sativa oil on reproductive ability of infertile men.
It conclusion, N. sativa oil improves semen quality in infertile men having abnormal semen parameters; further research seems necessary to elucidate possible mechanisms of action of N. sativa oil semen parameters and its effects on the ability of infertile men having abnormal semen parameters to reproduce.
Received 27 August 2013
Received in revised form 1 December 2013
Accepted 22 February 2014
Conflict of interest
All authors have no conflict of interest to disclose.
This study was supported by the ACECR (Iranian Academic Center for Education, Culture and Research), and Payame Noor University and Infertility Center of Mahdieh Hospital Tehran, Iran. Thanks are due to Dr. Kaveh Sultanzadeh urologist from Royan Research Institute and Clinical Center for Infertility and Reproductive Health in Tehran, for his valuable assistance.
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M. Kolahdooz (a), S. Nasri (a), S. Zadeh Modarres (b), S. Kianbakht (c), H. Fallah Huseini (c), *
(a) Department of Biology, Payamenoor University, Tehran, Iran
(b) Department of Obstetrics and Gynecology, Mahdieh Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran
(c) Pharmacology and Applied Medicine Department of Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Alborz, Karaj, Iran
* Corresponding author at: Institute of Medicinal Plants, ACECR, Karaj-Qazwain Freeway, Supa Boulevard, Jahade-Daneshgahi Research Society, Alborz, Karaj, Iran.
Tel.: +98 26 34764010 20: fax: +98 26 34764010 20.
E-mail address: email@example.com (H.F. Huseini).
Table 1 Fatty acid contents of fixed oil component of N. sativa oil. Fatty acids Percentage Myristic acid 0.2090 Palmitic acid 12.84 Palmitoleic acid 0.2812 Heptadecanoic acid 0.1047 Stearic acid 3.3884 Oleic acid 22.5836 Linoleic acid 58.2370 Arachidic acid 0.2250 Eicosenoic acid 0.3839 Behenic acid 0.0356 Lignoceric acid 0.0414 Table 2 Chemical compositions of volatile oil component of N. sativa oil. Compound Percentage [alpha]-Thujene 13.95 [alpha]-Pinene 3.74 2,4,10, Thujaden 0.08 Camphene 0.05 2-Heptenal(z) 0.18 Sabinene 1.52 [beta]-Pinene 3.27 [rho]-Cymene 51.62 Limonene 1.95 1,8-Cineole 0.07 [gamma]-Terpinene 0.17 cis-Thujon 0.06 (-)-cis-Sabinol 0.11 4-ol-Terpineol 0.27 Dodecane(n) 0.07 Thymoquinone 14.48 Isobornyl acetate 0.09 Carvacrol 0.96 [alpha]-Longipinene 0.28 [beta]-Longipinene 0.82 Table 3 The demographic and baseline data in N. sativa oil and placebo groups (mean [+ or -] SE). N. sativa seeds Placebo oil (N = 34) (N = 34) Duration of Infertility 4.3 [+ or -] 0.6 3.9 [+ or -] 0.6 (year) Age (year) 31.5 [+ or -] 1.1 32.1 [+ or -] 0.8 BMI (kg [m.sup.-2]) 26.3 [+ or -] 0.6 26.6 [+ or -] 0.8 p-Value Duration of Infertility 1.631 (year) Age (year) 0.711 BMI (kg [m.sup.-2]) 0.806 Table 4 Effects of 2 months N. Sativa and mineral oils consumption on semen quality in infertile men. The semen quality values of fertile men are given for reference. Values are expressed as mean [+ or -] SE. Semen quality Fertile men parameters Sperm count 98.23 [+ or -] 8.31 (million/ml) Sperm motility (%) 89.46 [+ or -] 3.62 Sperm morphology 73.23 [+ or -] 2.12 (normal/abnormal) Semen volume (ml) 4.31 [+ or -] 0.23 Semen pH 8.00 [+ or -] 0.3 Semen round cell 0.83 [+ or -] 0.01 (%) Semen quality Groups at baseline parameters Placebo N. sativa oil p-Value Sperm count 46.68 [+ or -] 5.11 43.53 [+ or -] 4.94 0.660 (million/ml) Sperm motility (%) 62.83 [+ or -] 1.56 63.45 [+ or -] 2.27 0.812 Sperm morphology 40.56 [+ or -] 3.27 39.53 [+ or -] 2.54 0.805 (normal/abnormal) Semen volume (ml) 3.73 [+ or -] 0.44 3.77 [+ or -] 0.33 0.947 Semen pH 8.44 [+ or -] 0.07 8.38 [+ or -] 0.07 0.261 Semen round cell 3.83 [+ or -] 0.69 3.32 [+ or -] 0.44 0.525 (%) Semen quality Groups after treatment parameters Placebo N. sativa oil p-Value Sperm count 42.88 [+ or -] 4.46 60.18 [+ or -] 5.16 0.014 (million/ml) Sperm motility (%) 61.66 [+ or -] 2.01 69.75 [+ or -] 2.25 0.015 Sperm morphology 37.21 [+ or -] 3.04 49.09 [+ or -] 2.27 0.003 (normal/abnormal) Semen volume (ml) 3.06 [+ or -] 0.25 4.21 [+ or -] 0.34 0.025 Semen pH 8.26 [+ or -] 0.02 8.01 [+ or -] 0.09 0.001 Semen round cell 4.23 [+ or -] 0.83 0.97 [+ or -] 0.25 0.001 (%) p-Values are the results of comparison with the placebo group at baseline and after treatment. p<0.05 was considered as significant. Table 5 Effects of 2 months N. Sativa and mineral oils consumption on sperm motility in infertile men. The semen quality values of fertile men are given for reference. Values are expressed as mean [+ or -] SE. Classes of sperm Fertile men motility (%) Class A 64.73 [+ or -] 4.12 Class B 51.43 [+ or -] 3.91 Class C 8.67 [+ or -] 1.28 Class A + B 81.32 [+ or -] 4.35 Immotile 12.74 [+ or -] 1.21 Classes of sperm At baseline motility (%) Placebo N. sativa oil p-Value Class A 20.18 [+ or -] 1.88 21.79 [+ or -] 2.31 0.590 Class B 43.26 [+ or -] 1.41 39.54 [+ or -] 2.27 0.311 Class C 9.93 [+ or -] 0.76 9.27 [+ or -] 0.70 0.182 Class A + B 62.23 [+ or -] 1.62 61.73 [+ or -] 2.41 0.314 Immotile 37.85 [+ or -] 1.56 42.54 [+ or -] 2.27 0.310 Classes of sperm After treatment motility (%) Placebo N. sativa oil p-Value Class A 19.29 [+ or -] 2.25 30.47 [+ or -] 3.503 0.010 Class B 38.33 [+ or -] 2.19 37.35 [+ or -] 2.38 0.600 Class C 11.26 [+ or -] 1.02 10.91 [+ or -] 1.02 0.213 Class A + B 58.71 [+ or -] 2.21 69.93 [+ or -] 2.37 0.002 Immotile 42.91 [+ or -] 0.53 36.26 [+ or -] 2.25 0.001 p-Values are the results of comparison with the placebo group at baseline and after treatment. p<0.05 was considered as significant.