To observe the antioxidant effect and clinical efficacy of the method of strengthening the spleen and benefiting the kidney (sequestra seed formula) on patients with oligozoospermia. METHODS: 120 patients were divided into 2 groups according to the random number table method, 60 cases in the treatment group were treated with sequestra seed formula and 60 cases in the control group were treated with levocanidine oral solution, and the duration of treatment was 3 months in both groups. Results: The total clinical efficiency of the treatment group was 85.9%, with 6 cases of female pregnancy; the total clinical efficiency of the control group was 71.9%, with 4 cases of female pregnancy, and the clinical efficacy of the treatment group was significantly better than that of the control group (P<0.05).
There was no statistically significant difference between the two groups in the measurement of seminal plasma MDA content and SOD activity after treatment; sperm concentration, a+b level sperm and sperm viability in the treatment group improved better than those in the control group after treatment (P<0.05). Conclusion: Jian spleen and kidney method (sequestra seed formula) can enhance the antioxidant effect of spermatozoa and improve the clinical efficacy.
Oligozoospermia; Spleen strengthening and kidney benefit method; Sequoia seed formula; Chinese medicine therapy
Reactive oxygen species (ROS) are associated with physiological functions such as hyperactive sperm motility, energy acquisition and acrosome reaction, as well as pathological effects such as reduced sperm motility and oxidative DNA damage [1]. We have achieved good clinical efficacy in the treatment of oligozoospermia using the method of strengthening the spleen and benefiting the kidney (sequestra seed formula), and now we further observe its antioxidant effect in order to reveal the possible mechanism of action of sequestra seed formula in the treatment of oligozoospermia.
1 .General information
1.1 Source of cases
The cases were obtained from the outpatients of the Department of Male Medicine of the First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine from March 2011 to February 2012. Age 22-50 years old, average 31.1 years old, disease duration 1-5 years, average 1.7 years, the age and disease duration of the two groups of patients before treatment were statistically treated, the difference was not significant (P>0.05), comparable.
1.2 Western medical diagnostic criteria
According to the WHO Standard Examination and Diagnostic Manual for Infertile Couples [2], infertility without contraception for more than 12 months after marriage and normal sexual life. Semen analysis: according to WHO criteria [2]: sperm density ≤20×106/ml, a-grade motile sperm <25% or a+b-grade motile sperm <50%.
1.3 Chinese medical criteria for the identification of spleen and kidney deficiency.
The main symptoms were: dizziness and tinnitus, forgetfulness and hair loss; soreness and weakness of the waist and knees, sallow eyes, poor appetite and loose stools, fatigue, frequent nocturnal urination, poor sexual function; pale and fatty tongue or teeth marks, and sunken and weak pulse.
1.4 Exclusion criteria
(i) azoospermia and testicular dysplasia; (ii) varicocele II° or more; (iii) simultaneously taking antiepileptic, antitumor and other drugs that hinder spermatogenic function and affect sperm vitality.
1.5 Treatment methods.
The treatment group was given sequestra seed formula (empirical formula, composition: Cuscuta sinensis 20g, Fructus Lycii 10g, Fructus Ligustrum 10g, Boneset 15g, Eucommia 15g, sequestra 20g, Radix Codonopsis 15g, Rhizoma Atractylodis Macrocephalae 10g, Rhizoma Yam 15g, Ziziphi Jujube 15g, Poria 15g, Dioscorea Z 15g, Niubizi 15g), using single herbal formula granules (no decoction herbal medicine, produced by Jiangsu Jiangyin Tianjiang Pharmaceutical Ltd.); the control group was given levocanidine oral liquid orally (produced by Northeast Pharmaceutical General Factory, State Drug Administration H19990372) usage: 1g/time, 3 times/day, for 3 months. Both groups were treated for 3 months as a course of treatment, and the treatment was continued for 1 course.
1.6 Specimen collection and testing method
All subjects were required to have no history of ejaculation 2-7 days before the examination. All ejaculated semen was collected by self-stimulation in a clean and dry semen collection cup and placed in a 37℃ constant temperature water bath to observe liquefaction for examination. Detection method: Computer-assisted semen analysis (CASA) technology (Beijing Weili sperm quality testing system software) was used for a full set of relevant analyses. The remaining semen was centrifuged at 3 000 r/min for 15 min to separate seminal plasma. The seminal plasma SOD assay was performed by xanthine oxidation method and MDA assay was performed by thiobarbiturate method, according to the steps of the kit instructions.
1.7 Observation index
The sperm quality computer analysis was checked before and three months after treatment to observe the improvement of clinical symptoms and the changes of sperm parameters, and the measurement of seminal plasma MDA content and SOD activity.
1.8 Statistical methods
SPSS13.0 statistical software was used for statistical analysis, and t-test and ANOVA were commonly used for data analysis.
2, Efficacy determination criteria and results
The efficacy criteria were revised with reference to the WHO Standard Examination and Diagnosis Manual for Infertile Couples and the Clinical Research Guidelines for New Chinese Medicines [3], combined with the actual clinical situation. Clinical cure: the female partner was pregnant or the sperm density and motility were restored to normal; effective: although the sperm test was abnormal, the sperm viability and a or a + b level of viable sperm were improved by ≥ 50%, and the sperm density was improved by ≥ 20×106/ml after treatment of oligospermia; ineffective: those who did not meet the above criteria.
3 .Results
Three cases in the treatment group were shed due to going out or receiving assisted reproduction, and three patients whose spouses became pregnant during the treatment period were considered as completed cases; three patients in the control group were shed due to going out or receiving assisted reproduction. The actual evaluable cases were 114, 57 cases in the control group and 57 cases in the treatment group.
3.1 Evaluation of total clinical efficacy
In the control group, 57 patients were cured in 16 cases, effective in 25 cases, ineffective in 16 cases, and female pregnancy in 3 cases, with a total clinical efficiency of 71.9%; in the treatment group, 57 patients were cured in 23 cases, effective in 26 cases, ineffective in 8 cases, and female pregnancy in 6 cases, with a total clinical efficiency of 85.9%. After 3 months of treatment, the total effective rate of the treatment group was significantly higher than that of the control group with statistical significance (P<0.05).
3.2 The results of seminal plasma MDA content and SOD activity measurement
The results of the measurement of seminal plasma MDA content and SOD activity before and after 3 months of treatment in the two groups are shown in Table 1.
3.3 Comparison of sperm concentration, sperm motility, viability and normal morphology rate
The sperm concentration, a+b sperm and sperm viability in the treatment group improved better than those in the control group after treatment (P<0.05), and the normal morphology rate was not statistically significant when comparing the two groups (see Table 2).
3.4 Sex hormone six changes
There was no statistically significant change in the six sex hormones, namely folliculogenic hormone (FSH), luteinizing hormone (LH), estradiol (E2), progesterone (P), testosterone (T) and pituitary lactogen (PRL), before and after treatment in both groups (P>0.05).
3.5 Follow up
Both groups were followed up for 3 months, 6 cases of female pregnancy in the treatment group; 3 cases of female pregnancy in the control group, 1 case of abortion with fetal arrest after 52 days of female pregnancy and semen analysis of malformed spermatozoa in the male.
4. Discussion
Oligospermia is a common cause of male infertility, and the European Society of Reproduction states that male infertility may be due to a variety of factors, such as endocrine disruption caused by long-term stressful environmental factors, reactive oxygen species and genetic defects [4,5].
Studies have confirmed that excessive production of reactive oxygen species (ROS) is one of the most important causes of male infertility, and that ROS cause infertility through three main pathways: inhibition of ATP production and sperm motility; production of lipid peroxides, which damage sperm cell membranes and sperm cell ultrastructure; and damage to sperm DNA [5,6]. Under normal conditions, the production and scavenging of ROS are in a balanced state due to the protective effects of antioxidants and antioxidant enzymes in the reproductive system (e.g., epididymis) and spermatozoa themselves, as well as the presence of antioxidants and antioxidant enzymes in seminal plasma after ejaculation; when ROS production exceeds the scavenging capacity of the above antioxidant system, it will attack spermatozoa and produce various toxic effects on spermatozoa and biological macromolecules. When ROS production exceeds the scavenging capacity of the antioxidant system, it will attack the spermatozoa and produce various toxic effects on spermatozoa and biological macromolecules, resulting in what is called oxidative stress in free radical biology.
Malondialdehyde (MDA), a product of sperm lipid peroxidation, is the main product of the peroxidation reaction between reactive oxygen species and cellular polyunsaturated fatty acids, and is an indicator of the severity of lipid peroxidation damage, and the concentration of reactive oxygen species can be understood by measuring malondialdehyde [6]. Clinical antioxidants such as levocanidine oral solution, vitamin E and vitamin C are mostly used to treat oligo- and hypospermia in men, and it has also been reported that traditional Chinese medicine can enhance the activity of antioxidant enzymes in the body, improve the function of epididymis, and ultimately protect sperm from oxidative damage.
According to traditional medicine, oligozoospermia belongs to the category of “low sperm”, “clear sperm” and “cold sperm” in Chinese medicine. We believe that spermatozoospermia is mainly due to kidney deficiency, and is related to the liver and spleen, as well as damp-heat, liver depression, blood stasis, etc. Treatment advocates strengthening the spleen and benefiting the kidneys as the fundamental method. The spleen is the essence of the postnatal period, which transports water, grain and essence, and is the source of biochemical energy and blood. If the spleen and stomach are healthy, the qi and blood will be enriched, the essence will have a source of production, and the seeds will be expected. If the spleen and stomach are healthy, the essence of water and grain can be collected and returned to the kidneys, and if the essence is strong, the seeds will flourish. Clinical practice is also common in patients with deficiency of both spleen and kidney and deficiency of qi and blood. Therefore, we established “two deficiencies of the spleen and kidney” as the basic pathology of oligozoospermia, and formed the herbal formula “Sequoia seed formula” to treat oligozoospermia with the method of strengthening the spleen and benefiting the kidney [7,8].
It consists of Cuscuta sinensis, Fructus Lycii, Radix et Rhizoma chinensis, Radix et Rhizoma sequestri, Rhizoma Bone, Eucommia, Radix Codonopsis, Rhizoma Atractylodis Macrocephalae, Rhizoma Polygonatum and Fructus Jujubae. Modern pharmacological research has confirmed that the flavonoids contained in Cuscuta have estrogen-like effects on the reproductive system with free radical scavenging, antioxidant effects, and improve the immune system of the body’s pharmacological effects; Lycium barbarum mainly contains Lycium barbarum polysaccharide with enhanced immune regulation; Chasteberry mainly contains chasteberry glycosides, oleanolic acid, dextro mannitol and trace elements Zn, on the body’s growth and development and immune system immunity It plays an important role in the growth and development of the body and the immune system. Sequoia and Eucommia have reduced the basal metabolism of tissue respiration, improved oxygen utilization, and reduced damage to the body by free radicals.
Our previous clinical observation showed that the treatment of male infertility with “sequestra seed formula” has better efficacy compared with Huang Jing Zangyu capsule, and the improvement of sperm vitality and viability of patients is better than that of Huang Jing Zangyu capsule group.