Effect of Typha capensis (Rohrb.)N.E.Br. rhizome extract F1 fraction on cell viability, apoptosis induction and testosterone production in TM3-Leydig cells.

Typha capensis (Rohrb.)N.E.Br. (bulrush) is used by traditional healers in Southern Africa to treat male reproductive problems. This study aimed at investigating the effects of T. capensis on TM3-Leydig cells. T. capensis rhizome crude extract obtained from autumn, winter, spring and summer harvest was fractionated using HPLC into four fractions, and TM3-Leydig cells were incubated with different concentrations of the F1 fraction (0.01, 0.02, 0.1, 1, 10 and 100 µg/ml) for 24, 48 and 96 hr respectively. The following parameters were evaluated: cell morphology, viability (MTT assay), testosterone production (testosterone ELISA test), apoptosis (Annexin V-Cy3 binding) and DNA fragmentation (TUNEL assay). Results revealed that the summer harvest obtained the highest amount of extract. The F1 fraction of all harvests was the most effective. This fraction significantly enhanced testosterone production in TM3 cells in a dose-dependent manner with maximum effect at 0.1 µg/ml. At higher concentrations, lower testosterone production was observed. Cell viability including apoptosis was not affected at concentrations used by the traditional healers to treat patients. This study shows that T. capensis enhanced testosterone production and might be useful to treat male infertility and ageing male problems.

Reactive oxygen species impact on sperm DNA and its role in male infertility.

Reactive oxygen species (ROS) have been extensively studied as a cause of male infertility. Excessive levels of ROS coupled with a deficiency in antioxidants can lead to oxidative stress (OS), which in turn can lead to nuclear and mitochondrial DNA damage, telomere shortening, epigenetic alterations and Y chromosomal microdeletions. In this review, we discuss how OS induces DNA damage and the types of DNA damage that can occur. We also briefly touch on the clinical consequences of OS-induced DNA damage.

Determination of seminal oxidation-reduction potential (ORP) as an easy and cost-effective clinical marker of male infertility.

Oxidative stress (OS) is an important contributing factor to male infertility. While previous methods to measure seminal OS are time-consuming and limited to the use of freshly produced semen, oxidation reduction potential (ORP) is easier and quicker to perform and can also be used in frozen semen. Therefore, this study evaluated the clinical utility of ORP as a potential marker of male infertility. ORP was measured in semen samples from 293 patients and 15 fertile controls and categorised according to WHO criteria as normozoospermic, oligozoospermic, asthenozoospermic, teratozoospermic and oligoasthenoteratozoospermic. Receiver operating characteristic (ROC) curves were generated to differentiate these categories. Semen parameters were significantly different when subjects were grouped as control and patients or between the patient and normozoospermic group for concentration and morphology. ORP levels were significantly different between the control and normozoospermic group. When subjects were grouped based on concentration, motility, morphology or a combination of these, the area under the ROC curve, sensitivity, specificity, positive predictive value and cut-off values were significantly different. These differences were significant when combined with ORP and grouped with any two sperm abnormalities. In conclusion, ORP is a quick, easy, cost-effective and reliable marker of semen quality as well as oxidative stress for use in a clinical setting.

Evaluation of reference values of standard semen parameters in fertile Egyptian men.

The reference values of human semen, published in the WHO's latest edition in 2010, were lower than those previously reported. The objective of this study was to evaluate reference values of standard semen parameters in fertile Egyptian men. This cross-sectional study included 240 fertile men. Men were considered fertile when their wives had recent spontaneous pregnancies with time to pregnancy (TTP) ≤12 months. The mean age of fertile men was 33.8 ± 0.5 years (range 20-55 years). The 5th percentiles (95% confidence interval) of macroscopic semen parameters were 1.5 ml for volume and 7.2 for pH. The 5th percentiles of microscopic parameters were 15 million/ml for sperm concentration, 30 million per ejaculate for total sperm count, 50% for total motility, 40% for progressive motility, 62% for vitality, 4% for normal sperm forms and 0.1 million/ml for seminal leucocyte counts. In conclusion, fertile Egyptian men had higher reference values of sperm total motility, progressive motility and vitality, and lower reference values for total sperm counts as compared to those determined by the latest edition of the WHO laboratory manual in 2010. Other semen parameters were identical to those defined by the WHO 2010 manual.

Human sperm handling in intracytoplasmic sperm injection processes: In vitro studies on mouse oocyte activation, embryo development competence and sperm oxidation-reduction potential.

Polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) are routinely used in handling spermatozoa for intracytoplasmic sperm injection (ICSI). As there are still concerns about possible adverse effects on the embryo, this study investigated sperm handling in a mouse ICSI model to (i) evaluate oocyte activation after injection of spermatozoa selected for rotational or linear motion in PVP; (ii) assess the effect of sperm selection in PVP, HA and medium on oocyte activation; (iii) examine the effects of PVP and HA on parthenogenetic oocyte activation and embryo development; and (iv) assess the oxidation-reduction potential (ORP) of spermatozoa exposed to PVP, HA or medium. Oocyte activation was higher when spermatozoa exhibited rotational motion rather than linear motion (79% vs. 52%; p = .05). There was no difference in oocyte activation and embryo development after parthenogenetic oocyte activation after sperm injection using PVP, HA or medium-incubated spermatozoa. PVP-selected spermatozoa exhibited lower (p < .0001) ORP levels than using HA. Thus, results indicate that the sperm handling method and the type of medium used impact ICSI outcomes. Overall, sperm incubation in PVP, HA and medium yields similar outcomes with regard to oocyte activation and embryo development. However, PVP provides more antioxidative protection than HA and should therefore be preferred for sperm manipulation.

Interpretation of semen analysis using WHO 1999 and WHO 2010 reference values: Abnormal becoming normal.

Reference values of WHO 1999 manual were used for the interpretation of semen analysis until 2010 when new reference values were introduced which have lower cut-off compared to WHO 1999. Therefore, several men who previously were diagnosed abnormal based on their semen analysis have now become normal using new reference values. This study was conducted on semen analyses of 661 men from Middle East region and Pakistan. All semen analyses were reviewed using WHO 1999 and WHO 2010 criteria. Results showed that based on new criteria, 19% of the population changed classification from abnormal to normal when all normal semen parameters were considered. When at least one or more abnormal semen parameters were considered, of the total 661, 44% (288) of the population changed its classification from abnormal to normal with shift from WHO 1999 to 2010 criteria. These findings show that using new cut-off values, many more men are considered normal, but using old criteria (WHO 1999), the same men would be classified as abnormal. This warrants further discussion over the investigations and management plans for patients whose semen analyses fall below WHO 1999 but above WHO 2010 cut-offs.

Association between promoter methylation of MLH1 and MSH2 and reactive oxygen species in oligozoospermic men-A pilot study.

MLH1 and MSH2 are important genes for DNA mismatch repair and crossing over during meiosis and are implicated in male infertility. Therefore, the methylation patterns of the DNA mismatch repair genes MLH1 and MSH2 in oligozoospermic males were investigated. Ten oligozoospermic patients and 29 normozoospermic donors were analysed. Methylation profiles of the MLH1 and MSH2 promotors were analysed. In addition, sperm motility and seminal reactive oxygen species (ROS) were recorded. Receiver operating characteristic (ROC) analysis was conducted to determine the accuracy of the DNA methylation status of MLH1 and MSH2 to distinguish between oligozoospermic and normozoospermic men. In oligozoospermic men, MLH1 was significantly (p = .0013) more methylated compared to normozoospermic men. Additionally, there was a significant positive association (r = .384; p = .0159) between seminal ROS levels and MLH1 methylation. Contrary, no association between MSH2 methylation and oligozoospermia was found. ROC curve analysis for methylation status of MLH1 was significant (p = .0275) with an area under the curve of 61.1%, a sensitivity of 22.2% and a specificity of 100.0%. This pilot study indicates oligozoospermic patients have more methylation of MLH1 than normozoospermic patients. Whether hypermethylation of the MLH1 promoter plays a role in repairing relevant mismatches of sperm DNA strands in idiopathic oligozoospermia warrants further investigation.

Cumene hydroperoxide induced changes in oxidation-reduction potential in fresh and frozen seminal ejaculates.

Oxidation-reduction potential (ORP) is a newer integrated measure of the balance between total oxidants (reactive oxygen species-ROS) and reductants (antioxidants) that reflects oxidative stress in a biological system. This study measures ORP and evaluates the effect of exogenous induction of oxidative stress by cumene hydroperoxide (CH) on ORP in fresh and frozen semen using the MiOXSYS Analyzer. Semen samples from healthy donors (n = 20) were collected and evaluated for sperm parameters. All samples were then flash-frozen at -80°C. Oxidative stress was induced by CH (5 and 50 µmoles/ml). Static ORP (sORP-(mV/106 sperm/ml) and capacity ORP (cORP-µC/106 sperm/ml) were measured in all samples before and after freezing. All values are reported as mean ± SEM. Both 5 and 50 µmoles/ml of CH resulted in a significant decline in per cent motility compared to control in pre-freeze semen samples. The increase in both pre-freeze and post-thaw semen samples for sORP was higher in the controls than with 50 µmoles/ml of CH. The change from pre-freeze to post-thaw cORP was comparable. The system is a simple, sensitive and portable tool to measure the seminal ORP and its dynamic impact on sperm parameters in both fresh and frozen semen specimens.

Effect of Cissampelos capensis rhizome extract on human spermatozoa in vitro.

Cissampelos capensis is commonly known by the Afrikaans name 'dawidjies' or 'dawidjieswortel'. C. capensis is the most important and best-known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western regions of South Africa. Among numerous other ailments, it is traditionally taken to treat male fertility problems. Yet, no studies have investigated the effects of this plant or its extracts on human spermatozoa. The aim of study was to investigate the effects of C. capensis extracts on sperm function. A total of 77 semen samples were collected. Spermatozoa were washed with HTF-BSA medium and incubated with different concentrations of C. capensis (0, 0.05, 0.5, 5, 50, 200 µg ml(-1) ) for 1 h at 37 °C. Sperm motility, vitality, acrosome reaction, reactive oxygen species (ROS), capacitation, Annexin V binding, DNA fragmentation and mitochondrial membrane potential (Δψm ) were determined. While viability, Annexin V positivity and Δψm were not affected, the percentages of ROS-positive, TUNEL-positive, capacitated and hyperactivated spermatozoa increased significantly and dose-dependently. It is concluded that the alkaloids present in the extract of C. capansis rhizomes triggered sperm intrinsic superoxide production leading to sperm capacitation and DNA fragmentation.

Phytoandrogenic properties of Eurycoma longifolia as natural alternative to testosterone replacement therapy.

The testosterone deficiency syndrome (TDS) is characterised by numerous symptoms, including low libido, increased fat mass, fatigue, erectile dysfunction or osteoporosis, and up to 80% of men will experience some kind of ageing males' symptoms. This is caused by the age-depending decline in serum testosterone levels with concentrations being about 40-50% lower in men older than 60 years compared with young men. This significant decline in testosterone levels is further closely linked with medical conditions such as obesity, metabolic syndrome, diabetes or hypertension. The conventional way of treating TDS is the testosterone replacement therapy (TRT), for which preparations are on the market. Apart from the beneficial effects of TRT, significant adverse side effects have been described, and prostate cancer (PCa) as absolute contraindication is debated. Eurycoma longifolia (Tongkat Ali; TA) is natural alternative to TRT and has been shown to restore serum testosterone levels, thus significantly improving sexual health. This includes significant positive effects on bone health and physical condition of patients. In addition, a significant antihyperglycaemic effect and cytotoxicity against PCas cells has been shown. Thus far, at therapeutic concentrations, no significant side effects of the treatment were obvious. Therefore, TA might be a safe alternative to TRT.

In vivo effects of Eurycoma longifolia Jack (Tongkat Ali) extract on reproductive functions in the rat.

An aqueous extract of Eurycoma longifolia (Tongkat Ali; TA) roots is traditionally used to enhance male sexuality. Because previous studies are limited to only few sperm parameters or testosterone concentration, this study investigated the in vivo effects of TA on body and organ weight as well as functional sperm parameters in terms of safety and efficacy in the management of male infertility. Forty-two male rats were divided into a control, low-dose (200 mg kg(-1) BW) and high-dose (800 mg kg(-1) BW) group (n = 14). Rats were force-fed for 14 days and then sacrificed. Total body and organ weights of the prostate, testes, epididymides, gastrocnemius muscle and the omentum were recorded. Moreover, testosterone concentration, sperm concentration, motility, velocity, vitality, acrosome reaction and mitochondrial membrane potential (MMP) were assessed. Whilst TA decreased BW by 5.7% (P = 0.0276) and omentum fat by 31.9% (P = 0.0496), no changes in organ weights were found for the prostate, testes and epididymides. Testosterone concentration increased by 30.2% (P = 0.0544). Muscle weight also increased, yet not significantly. Whilst sperm concentration, total and progressive motility and vitality increased significantly, MMP improved markedly (P = 0.0765) by 25.1%. Because no detrimental effect could be observed, TA appears safe for possible treatment of male infertility and ageing male problems.

Effect of the metabolic syndrome on male reproductive function: a case-controlled pilot study.

The metabolic syndrome (MetS) is a constellation of various risk factors. This study aimed to investigate the effect of MetS on testosterone and progesterone, and semen parameters, in a case-controlled pilot study. Male patients (n = 54) had body mass index, waist-to-hip ratio (WHR) and blood pressure recorded. Blood was analysed for HDL cholesterol, triglycerides and glucose. Saliva was assayed for free testosterone and free progesterone. Ejaculates were analysed for volume, sperm concentration, total sperm count, motility, vitality, mitochondrial membrane potential (MMP), DNA fragmentation and leucocyte concentration. Participants were divided into the control group (n = 28) and the MetS group (n = 26). Differences were found between the groups for body mass index, WHR, blood pressure, high-density lipoprotein (HDL), triglycerides and glucose. The MetS group showed significant reductions in sperm concentration (P = 0.0026), total sperm count (P = 0.0034), total motility (P = 0.0291), sperm vitality (P = 0.002), MMP (P = 0.0039), free testosterone (P = 0.0093) and free progesterone (P = 0.0130), while values for DNA fragmentation increased (P = 0.0287). Results indicate that patients with MetS have compromised sperm parameters in the absence of leucocytospermia. A reduction in free progesterone suggests that steroidogenesis cascades may be compromised. It is hypothesised that a systemic pro-inflammatory state with oxidative stress associated with MetS may provide a novel explanation.

Sequential analysis of sperm functional aspects involved in fertilisation: a pilot study.

The development of diagnostic techniques in andrology as a second level of approach to the diagnosis of male factor infertility has enthused the focus of researchers on the development of a sequential diagnostic programme for these men. Semen samples of 78 men form couples undergoing in vitro fertilisation therapy were used in the study. The semen samples were used to test sperm functional aspects known to interfere with fertilisation. These tests included semen profile, DNA integrity, apoptosis, chromatin packaging, acridin orange staining, zona binding capacity, zona-induced acrosome reaction (AR). Results were correlated with fertilisation outcome. Statistical analyses of the recorded data were carried out using a logistic regression analysis model on all sperm functional tests. A negative and significant association with the fertilisation rates was recorded for DNA damage (r = -0.56; P ≤ 0.0005). A positive significant correlation was recorded between fertilisation rates and sperm with normal DNA (r = -0.57, P ≤ 0.0004), and zona-induced AR (r = 0.33, P ≤ 0.002). Diagnostic andrology can be regarded as a mandatory part of the male factor patient's work-up schedule to assist clinicians with the most suitable therapeutic modality to follow.

Standardised water-soluble extract of Eurycoma longifolia, Tongkat ali, as testosterone booster for managing men with late-onset hypogonadism?

In most countries, millions of people are relying on herbal medicines as remedy for numerous ailments. In South-East Asia, Eurycoma longifolia Jack, also known as 'Malaysian ginseng' or Tongkat ali, is used to combat stress and disease and to improve physical strength. Moreover, the compounds of the roots of this plant are reported to have aphrodisiac and testosterone enhancing effects in the rat. Considering that human studies are not available, 76 of 320 patients suffering from late-onset hypogonadism (LOH) were given 200 mg of a standardised water-soluble extract of Tongkat ali for 1 month. The Ageing Males' Symptoms (AMS) according to the standardised rating scale and the serum testosterone concentration were taken. Results show that treatment of LOH patients with this Tongkat ali extract significantly (P < 0.0001) improved the AMS score as well as the serum testosterone concentration. While before treatment only 10.5% of the patients did not show any complaint according to the AMS scale and 35.5% had normal testosterone levels, after the completed treatment 71.7% and 90.8% of the patients showed normal values, respectively. Thus, Tongkat ali extract appears to be useful as a supplement in overcoming the symptoms of LOH and for the management of hypogonadism.

Effect of Eurycoma longifolia Jack (Tongkat ali) extract on human spermatozoa in vitro.

Eurycoma longifolia (Tongkat ali; TA) is a Malaysian shrub used to treat various illnesses including male infertility. Considering that TA is used to improve male fertility and no report regarding its safety has been published, this study investigated the effects of TA extract on various sperm functions. Semen samples of 27 patients and 13 donors were divided into two groups, washed and swim-up spermatozoa, and incubated with different concentrations of TA (1, 10, 20, 100, 2000 µg ml(-1) ) for 1 h at 37 °C. A sample without addition of TA served as control. For washed spermatozoa, significant dose-dependent trends were found for vitality, total motility, acrosome reaction and reactive oxygen species-positive spermatozoa. However, these trends were only significant if the highest concentrations were included in the calculation. Contrary, the increase in the percentage of acrosome-reacted spermatozoa with increasing TA concentrations is very significant (P < 0.0001), and a significant difference (P = 0.0069) to the control could even be recorded at 20 µg TA per ml. For swim-up spermatozoa, no trend could be observed. Results indicate that the TA extract has no deleterious effects on sperm functions at therapeutically used concentrations (<2.5 µg ml(-1) ). However, at very high concentrations, TA may have harmful effects in vitro.

Accurate sperm morphology assessment predicts sperm function.

Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.

Typha capensis (Rohrb.)N.E.Br. (bulrush) extract scavenges free radicals, inhibits collagenase activity and affects human sperm motility and mitochondrial membrane potential in vitro: a pilot study.

The biodiversity in South Africa provides more than 30,000 higher plants, of which more than 3000 are used by traditional healers to treat diseases. Typha capensis (bulrush) is one of the medicinal plants used in South Africa to treat male fertility problems. Considering that South African traditional healers have been recognised by Law and the health benefits of T. capensis have not been scientifically investigated yet, this study aimed at investigating the in vitro effects of aqueous extracts from this plant on male reproductive functions. Both leaves and rhizomes of T. capensis were dried, infused with distilled water and freeze-dried. Motile sperm from 50 men were isolated by swim-up and incubated with 1 µg ml(-1) aqueous extract of Typha rhizome for 1 h at 37 °C. Vitality, motility, sperm production of reactive oxygen species and mitochondrial membrane potential were analysed in the test sample, a control and in the pellet from the swim-up. Results showed that the rhizome extract had significant (P < 0.0001) negative effects on all parameters. The extracts from the leaves and rhizomes revealed dose-dependent inhibitory activity for collagenase and free radical formation. No inhibitory activity for elastase was found. The inhibitory activity for collagenase might indicate possible anti-cancer effects.

Leucocytes and intrinsic ROS production may be factors compromising sperm chromatin condensation status.

Considering that the final protection of the DNA against major assaults in terms of chromatin condensation is finalized in the epididymis, it is not known how sperm production of reactive oxygen species (ROS) and inflammatory processes can contribute to protamine deficiency that is predetermined in the testes. Therefore, this study aimed at investigating relationships between poor chromatin condensation, morphology, ROS production, DNA damage and the impact of the presence of leucocytes. In 70 patients, sperm DNA status was determined using TUNEL and chromomycin A(3) (CMA(3)) assays, and ROS-production by means of dihydroethidine. Morphology was evaluated according to strict criteria. The percentage of CMA(3)-positive spermatozoa and leucocyte concentration (r = 0.178, P = 0.0377) as well as percentage of ROS-positive spermatozoa (r = 0.3010; P = 0.012) correlated significantly. Particularly, patients with leucocyte counts >0.5 x 10(6) ml(-1) exhibited higher CMA(3) positivity. No association was found between CMA(3) positivity, TUNEL positivity and sperm morphology. While P- (poor prognosis: 0-4% normal morphology) and G-pattern (good prognosis: 5-14% normal morphology) morphology did not differ regarding chromatin condensation, P-pattern patients had a significantly higher percentage of DNA fragmentation (P = 0.0323). As oxidative stress is associated with disturbed chromatin condensation, results suggest that the idea that under-protamination of sperm DNA will automatically lead to DNA fragmentation might have to be revisited.

Antiserum to a synthetic peptide recognizes the HTLV-III envelope glycoprotein.

In a study performed to determine which regions of the human T-cell lymphotrophic virus type III (HTLV-III) may represent vaccine candidates to prevent the acquired immune deficiency syndrome (AIDS), a synthetic peptide corresponding to amino acid sequence 735 to 752 of the precursor envelope glycoprotein of HTLV-III was used to immunize rabbits. The resulting rabbit antiserum to the synthetic peptide specifically recognized the precursor envelope glycoprotein (gp160) of HTLV-III. Human sera positive for antibody to HTLV-III reacted with this peptide. These findings indicate that synthetic peptides can be used to induce an immune response directed against a native envelope glycoprotein epitope of HTLV-III. The data are discussed in terms of using synthetic peptides to identify antigenic determinants involved in the induction of protective immunity and possibly as vaccine candidates against the etiologic agent of AIDS.