In vitro effects of aqueous extract of unfermented rooibos on human spermatozoa.

The inability to conceive is a baleful experience for thousands of couples worldwide. Among different well-known reproductive techniques, medicinal plants have been utilized to treat male infertility. Medicinal plants, provide a therapeutic alternative, which is available and affordable for infertile couples. We investigated the direct effect of unfermented rooibos aqueous extract on human spermatozoa. Semen samples (n = 50) collected from donors and patients consulting for fertility were reassigned as normal (n = 22) and abnormal (n = 28) samples based on the outcome of the baseline semen analysis, using the World Health Organization (WHO) cut off value. Semen samples were allowed to liquefy and subsequently washed with human tubular fluid in bovine serum albumin medium. The samples were then treated with aqueous extracts of unfermented rooibos (0, 0.15, 1.5, 15, 150 µg/ml) at 37°C for 1 h and assessed thereafter. Sperm motility, vitality, DNA fragmentation, intracellular reactive oxygen species and mitochondrial membrane potential in both groups remained unchanged (p > 0.05). However, aqueous extract of unfermented rooibos (only at 1.5 µg/ml) significantly increased capacitation and acrosome reaction in the abnormal sample group (p > 0.05). Unfermented rooibos aqueous extract had no deleterious impact on human spermatozoa's function and might be attributed to its antioxidant properties.

Protocol for developing a core outcome set for male infertility research: an international consensus development study.

STUDY QUESTION: We aim to develop, disseminate and implement a minimum data set, known as a core outcome set, for future male infertility research. WHAT IS KNOWN ALREADY: Research into male infertility can be challenging to design, conduct and report. Evidence from randomized trials can be difficult to interpret and of limited ability to inform clinical practice for numerous reasons. These may include complex issues, such as variation in outcome measures and outcome reporting bias, as well as failure to consider the perspectives of men and their partners with lived experience of fertility problems. Previously, the Core Outcome Measure for Infertility Trials (COMMIT) initiative, an international consortium of researchers, healthcare professionals and people with fertility problems, has developed a core outcome set for general infertility research. Now, a bespoke core outcome set for male infertility is required to address the unique challenges pertinent to male infertility research. STUDY DESIGN SIZE DURATION: Stakeholders, including healthcare professionals, allied healthcare professionals, scientists, researchers and people with fertility problems, will be invited to participate. Formal consensus science methods will be used, including the modified Delphi method, modified Nominal Group Technique and the National Institutes of Health's consensus development conference. PARTICIPANTS/MATERIALS SETTING METHODS: An international steering group, including the relevant stakeholders outlined above, has been established to guide the development of this core outcome set. Possible core outcomes will be identified by undertaking a systematic review of randomized controlled trials evaluating potential treatments for male factor infertility. These outcomes will be entered into a modified Delphi method. Repeated reflection and re-scoring should promote convergence towards consensus outcomes, which will be prioritized during a consensus development meeting to identify a final core outcome set. We will establish standardized definitions and recommend high-quality measurement instruments for individual core outcomes. STUDY FUNDING/COMPETING INTERESTS: This work has been supported by the Urology Foundation small project award, 2021. C.L.R.B. is the recipient of a BMGF grant and received consultancy fees from Exscentia and Exceed sperm testing, paid to the University of Dundee and speaking fees or honoraria paid personally by Ferring, Copper Surgical and RBMO. S.B. received royalties from Cambridge University Press, Speaker honoraria for Obstetrical and Gynaecological Society of Singapore, Merk SMART Masterclass and Merk FERRING Forum, paid to the University of Aberdeen. Payment for leadership roles within NHS Grampian, previously paid to self, now paid to University of Aberdeen. An Honorarium is received as Editor in Chief of Human Reproduction Open. M.L.E. is an advisor to the companies Hannah and Ro. B.W.M. received an investigator grant from the NHMRC, No: GNT1176437 is a paid consultant for ObsEva and has received research funding from Ferring and Merck. R.R.H. received royalties from Elsevier for a book, consultancy fees from Glyciome, and presentation fees from GryNumber Health and Aytu Bioscience. Aytu Bioscience also funded MiOXYS systems and sensors. Attendance at Fertility 2020 and Roadshow South Africa by Ralf Henkel was funded by LogixX Pharma Ltd. R.R.H. is also Editor in Chief of Andrologia and has been an employee of LogixX Pharma Ltd. since 2020. M.S.K. is an associate editor with Human Reproduction Open. K.Mc.E. received an honoraria for lectures from Bayer and Pharmasure in 2019 and payment for an ESHRE grant review in 2019. His attendance at ESHRE 2019 and AUA 2019 was sponsored by Pharmasure and Bayer, respectively. The remaining authors declare no competing interests. TRIAL REGISTRATION NUMBER: Core Outcome Measures in Effectiveness Trials (COMET) initiative registration No: 1586. Available at www.comet-initiative.org/Studies/Details/1586. TRIAL REGISTRATION DATE: N/A. DATE OF FIRST PATIENT'S ENROLMENT: N/A.

Aqueous leaf extract of Moringa oleifera reduced intracellular ROS production, DNA fragmentation and acrosome reaction in Human spermatozoa in vitro.

The effects of aqueous leaf extract of Moringa oleifera (MO) on human sperm functions and integrity was studied in vitro. Semen was obtained by masturbation after 3-5 days' abstinence from 34 healthy donors in Western Cape, South Africa. Liquefied semen was washed in human tubular fluid supplemented with 1% bovine serum albumin (HTF-BSA;1:5) with 10 min centrifugation at 300 g. Sperm suspensions were subsequently incubated with MO extract (0.625, 6.25, 62.5 and 625 µg/ml) for 1 hr, where HTF-BSA served as control. Sperm motility, vitality, DNA fragmentation, reactive oxygen species production, mitochondrial membrane potential, capacitation and acrosome reaction were assessed. Sperm motility, vitality, mitochondrial membrane potential and capacitation remained unchanged (p > .05). A dose-dependent decrease in sperm reactive oxygen species production (p < .0001), DNA fragmentation (p < .0001) and acrosome reaction (p < .001) was observed. An increase in the percentage of non-capacitated sperm (p < .01) was noted at 625 µg/ml. The antioxidant properties of MO actively maintained basic sperm functions, inhibited excess sperm free superoxide production and preserved acrosome reaction and DNA integrity. Further studies are needed to confirm the effect of aqueous MO leaf extract on fertility potential.

Metabolic syndrome is associated with increased seminal inflammatory cytokines and reproductive dysfunction in a case-controlled male cohort.

PROBLEM: The impact of metabolic syndrome (MetS) associated systemic inflammation on the male reproductive tract requires further investigation. METHOD OF STUDY: A cross-sectional case-controlled study design consisting of a control group (n=32) and a MetS (n=42) group was used. Variables include MetS diagnostic criterion, serum C-Reactive Protein (CRP), routine semen analysis, spermatozoa mitochondrial membrane potential (MMP) and DNA fragmentation (DF), as well as TNF-α, IL-1ß, IL6 and IL8 concentrations in serum and semen. RESULTS: Serum and seminal levels of TNF-α, IL-1ß, IL6 and IL8 were all significantly increased in the MetS group. Ejaculation volume, sperm concentration, total sperm count, progressive and total motility and vitality were significantly decreased and sperm with abnormal MMP and DF were increased in the MetS group. CONCLUSION: The results suggest that MetS is associated with decreased fertility parameters in males, as well as local reproductive tract inflammation, in the absence of leukocytospermia.

An Update on Oxidative Damage to Spermatozoa and Oocytes.

On the one hand, reactive oxygen species (ROS) are mandatory mediators for essential cellular functions including the function of germ cells (oocytes and spermatozoa) and thereby the fertilization process. However, the exposure of these cells to excessive levels of oxidative stress by too high levels of ROS or too low levels of antioxidative protection will render these cells dysfunctional thereby failing the fertilization process and causing couples to be infertile. Numerous causes are responsible for the delicate bodily redox system being out of balance and causing disease and infertility. Many of these causes are modifiable such as lifestyle factors like obesity, poor nutrition, heat stress, smoking, or alcohol abuse. Possible correctable measures include foremost lifestyle changes, but also supplementation with antioxidants to scavenge excessive ROS. However, this should only be done after careful examination of the patient and establishment of the individual bodily antioxidant needs. In addition, other corrective measures include sperm separation for assisted reproductive techniques. However, these techniques have to be carried out very carefully as they, if applied wrongly, bear risks of generating ROS damaging the germ cells and preventing fertilization.

Obesity is associated with increased seminal insulin and leptin alongside reduced fertility parameters in a controlled male cohort.

BACKGROUND: Obesity appears to be associated with male reproductive dysfunction and infertility, although this has been inconsistent and inconclusive. Insulin and leptin are known mediators and modulators of the hypothalamus-pituitary-testes axis, contributing to the regulation of male reproductive potential and overall wellbeing. These hormones are also present in semen influencing sperm functions. Although abdominal obesity is closely associated with insulin resistance (hyperinsulinaemia), hyperleptinaemia and glucose dysfunction, changes in seminal plasma concentrations of insulin, leptin and glucose in obese males has not previously been investigated. METHODS: This small case controlled study assessed serum and seminal concentrations of insulin, leptin and glucose in obese (BMI > =30; n = 23) and non-obese (BMI < 30; n = 19) males. Following a detailed medical history and examination, participants meeting the inclusion criteria were entered for data analysis. Body parameters such as BMI, waist and hip circumference and the waist hip ratio were measured. Serum and semen samples were collected and assayed for insulin, leptin and glucose. Semen samples also underwent a standard semen analysis, with sperm mitochondrial membrane potential (MMP) and DNA fragmentation (DF). RESULTS: Obesity was associated with increased serum and seminal insulin and leptin, with no significant difference in seminal glucose. Serum and seminal concentrations of insulin and leptin were positively correlated. Furthermore, obesity was associated with decreased sperm concentration, sperm vitality and increased MMP and DF, with a non-significant impact on motility and morphology. CONCLUSIONS: Hyperinsulinaemia and hyperleptinaemia are associated with increased seminal insulin and leptin concentrations, which may negatively impact male reproductive function in obesity. Insulin was also found to be highly concentrated in the seminal plasma of both groups. This data will contribute to the contradictive information available in the literature on the impact of obesity and male reproduction.

Tongkat Ali as a potential herbal supplement for physically active male and female seniors--a pilot study.

Tongkat Ali (Eurycoma longifolia; TA) is known to increase testosterone levels and alleviate aging males' symptoms. This study aimed at investigating TA as an ergogenic supplement for elderly people. Thirteen physically active male and 12 physically active female seniors (57-72 years) were supplemented with 400-mg TA extract daily for 5 weeks. Standard hematological parameters were taken. In addition, the concentrations of total and free testosterone, dihydroepiandrosterone, cortisol, insulin-like growth factor-1, and sex hormone-binding globulin were analyzed. As additional biochemical parameters, blood urea nitrogen and creatine kinase as parameters of kidney function and muscle damage, respectively, as well as the muscle strength by a simple handgrip test were determined. After treatment, hemoglobin, testosterone, and dihydroepiandrosterone concentrations, and the ratio of total testosterone/cortisol and muscle force remained significantly lower in female seniors than in male seniors. Hematocrit and erythrocyte count in male seniors increased slightly but were significantly higher than in female seniors. Treatment resulted in significant increases in total and free testosterone concentrations and muscular force in men and women. The increase in free testosterone in women is thought to be due to the significant decline in sex hormone-binding globulin concentrations. The study affirms the ergogenic benefit of TA through enhanced muscle strength.

Leukocytes and oxidative stress: dilemma for sperm function and male fertility.

Spermatozoa are constantly exposed to the interphase between oxidation through high amounts of reactive oxygen species (ROS) and leukocytes, and reduction by means of scavengers and antioxidants. Considering the very special functions as being the only cells with such high polarization and exerting their functions outside the body, even in a different individual, the female genital tract, the membranes of these cells are chemically composed of an extraordinary high amount of polyunsaturated fatty acids. This in turn, renders them very susceptible to oxidative stress, which is defined as an imbalance between oxidation and reduction towards the oxidative status. As a result, ROS deriving from both leukocytes and the male germ cells themselves cause a process called 'lipid peroxidation' and other damages to the sperm cell. On the other hand, a certain limited amount of ROS is essential in order to trigger vital physiological reactions in cells, including capacitation or the acrosome reaction in sperm. The treatment of patients with antioxidants to compensate the oxidative status caused by oxidative stress is highly debated as uncontrolled antioxidative treatment might derail the system towards the reduced status, which is also unphysiological and can even induce cancer. This paradox is called the 'antioxidant paradox'. Therefore, a proper andrological diagnostic work-up, including the evaluation of ROS levels and the antioxidant capacity of the semen, has to be carried out beforehand, aimed at keeping the fine balance between oxidation and scavenging of vital amounts of ROS.

A novel approach for the selection of human sperm using annexin V-binding and flow cytometry.

OBJECTIVE: To develop a method whereby sperm with phosphatidylserine externalization can be separated from those without this feature. Because annexin V binds phosphatidylserine, this study is using this feature to select functional spermatozoa. In addition, the relationship between annexin V binding in human spermatozoa and normal sperm morphology according to strict criteria was to be assessed. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynaecology of Stellenbosch University at Tygerberg Academic Hospital, Tygerberg, South Africa. PATIENT(S): Semen from 14 healthy sperm donors. Exclusion criterion was the presence of less than 20 x 10(6)/mL total motile spermatozoa in the original sample. MAIN OUTCOME MEASURE(S): Annexin V-negative sperm, annexin V-positive sperm, normal sperm morphology. INTERVENTION(S): An aliquot of a semen sample after double density gradient centrifugation was incubated with annexin V fluorescein isothiocyanate conjugate (FITC). Cell fluorescence signals were determined using a FACScalibur flow cytometer equipped with a FACSSort fluidic sorting module. The sorting procedure delivered two sperm subpopulations: annexin V-negative and annexin V-positive. Morphology slides were made and the sperm morphology was assessed according to strict criteria. RESULT(S): There was a significant enrichment of annexin V-negative sperm as well as morphologically normal sperm in the annexin V-negative subgroup after separation with flow cytometry. The percentage of morphologically normal sperm increased from 8.3% in the control to 11.9% in the annexin V-negative fraction, whereas the percentage of annexin V-positive sperm decreased to 5.7%. CONCLUSION(S): The annexin V-negative sperm subpopulation had morphologically superior quality sperm compared to annexin V-positive sperm. It is important to select morphologically normal sperm during intracytoplasmic sperm injection (ICSI) as it may contribute to increased implantation and pregnancy rates (PR).

Sperm preparation for ART.

The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI). Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep, Nycodenz, SilSelect, PureSperm or Isolate were developed in order to replace Percoll. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS) by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate their motility to increase the yield, a number of substances can be added to the ejaculate or the separation medium. Caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility. Recent approaches to stimulate spermatozoa include bicarbonate, metal chelators or platelet-activating factor (PAF). While the use of PAF already resulted in pregnancies in intrauterine insemination, the suitability of the other substances for the clinical use still needs to be tested. Finally, the isolation of functional spermatozoa from highly viscous ejaculates is a special challenge and can be performed enzymatically to liquefy the ejaculate. The older method, by which the ejaculate is forcefully aspirated through a narrow-gauge needle, should be abandoned as it can severely damage spermatozoa, thus resulting in immotile sperm.

Estimate of oxygen consumption and intracellular zinc concentration of human spermatozoa in relation to motility.

AIM: To investigate the human sperm oxygen/energy consumption and zinc content in relation to motility. METHODS: In washed spermatozoa from 67 ejaculates, the oxygen consumption was determined. Following calculation of the total oxygen consumed by the Ideal Gas Law, the energy consumption of spermatozoa was calculated. In addition, the zinc content of the sperm was determined using an atomic absorption spectrometer. The resulting data were correlated to the vitality and motility. RESULTS: The oxygen consumption averaged 0.24 micromol/10(6) sperm x 24h, 0.28 micromol/10(6) live sperm x 24h and 0.85 micromol/10(6) live motile sperm x 24h. Further calculations revealed that sperm motility was the most energy consuming process (164.31 mJ/10(6) motile spermatozoa x 24h), while the oxygen consumption of the total spermatozoa was 46.06 mJ/10(6) spermatozoa x 24h. The correlation of the oxygen/energy consumption and zinc content with motility showed significant negative correlations (r= -0.759; P<0.0001 and r=-0.441; P<0.0001, respectively). However, when correlating sperm energy consumption with the zinc content, a significant positive relation (r=0.323; P=0.01) was observed. CONCLUSION: Poorly motile sperm are actually wasting the available energy. Moreover, our data clearly support the "Geometric Clutch Model" of the axoneme function and demonstrate the importance of the outer dense fibers for the generation of sperm motility, especially progressive motility.