Baseline Levels of Seminal Reactive Oxygen Species Predict Improvements in Sperm Function Following Antioxidant Therapy in Men With Infertility

Wayne Vessey; Shaghayegh Saifi; Aditi Sharma; Cassandra McDonald; Paula Almeida; Monica Figueiredo; Suks Minhas; Ashraf Virmani; Waljit S. Dhillo; Jonathan W. Ramsay; Channa N. Jayasena


Clin Endocrinol. 2021;94(1):102-110. 

In This Article



Male participants under investigation for infertility were recruited from Reproductive Clinic at Hammersmith Hospital, London, UK. Participants were included in the study if they had male factor infertility, that is failure to conceive with a female partner after at least 12 months of regular unprotected sex, with at least one abnormal semen analysis parameter using WHO criteria.[14] These included men with either oligozoospermia (sperm count < 15 million/mL), severe oligospermia (sperm count < 5 million/mL), asthenozoospermia (<40% total motility or <32% progressive motility), and/or teratozoospermia (<4% normal morphology).

In total, forty-six men were assessed, and forty-four completed the study. Patients were excluded from the study if they received previous antioxidant therapy, or had history of genital or urinary tract infection, varicocele or other intrascrotal pathology.

Ethics and Protocol

An NHS Service Evaluation of clinical practice was conducted in accordance with the National Health Service (NHS) Health Research Authority/Medical Research Council Decision Tool. Institutional ethics committee approval was not required for this NHS service evaluation.

All participants underwent assessment of semen analysis and semen ROS testing at study commencement. Then, LAL was orally self-administered by all participants on a daily basis for 3 months, immediately after which semen analysis and semen ROS testing was repeated. All semen samples were produced on site to minimize variability in analysis times. The protocol is summarized in Figure 1.

Figure 1.

Protocol diagram. Men presenting with infertility and abnormal WHO semen analysis were classified into normal reactive oxygen species (ROS) or high ROS groups. The normal ROS group had semen ROS ≤ 10 RLU/SEC/106 sperm at baseline. The high ROS group had semen ROS > 10 RLU/SEC/106 sperm at baseline. All patients self-administered 3 months of L-carnitine and acetyl-L-carnitine (LAL) therapy [Colour figure can be viewed at]

Sample Analyses and ROS Measurement

All semen analyses were performed according to WHO 2010 methodology[14] in the UK Accreditation Service (UKAS) certified laboratory at Hammersmith Hospital, London, UK following 2–7 days of sexual abstinence. ROS testing was performed on site within 20 minutes of semen production using an established in-house luminol-based colorimetric assay[5,15] as detailed: A 100mmol/L luminol (5-amino-2,3-dihydro-1,4-phthalazinedione; Sigma-Aldrich) stock solution was prepared in advance in dimethylsulphoxide (DMSO) and stored at room temperature in the dark in a foil-covered polystyrene Falcon tube. A luminol working solution (5mmol/L luminol prepared in DMSO) was freshly prepared from the stock solution each day, covered in foil, and stored in the dark, and discarded at the end of each day. Negative control samples contained 400μL phosphate-buffered saline solution (PBS) with 10 μL 5 mmol/L luminol working solution. Positive control samples contained 395μL PBS, 5μL 30% hydrogen peroxide, and 10 μL 5 mmol/L luminol working solution. The solutions were mixed gently with care to avoid any bubbles and placed in the luminometer. For measuring chemiluminescence in the semen samples, liquefied whole semen was gently mixed with the use of a plastic pipette, and 400 μL of semen was aliquoted into a 1.5 mL microfuge tube and 10 μL 5 mmol/L luminol working solution was added and mixed gently avoiding any bubbles before reading in the luminometer. Furthermore, because the reagent is light sensitive, the assay was performed with electrical lights switched off to reduce light exposure. The assay was carried out at temperatures of 20–25 degrees Celsius. Measurements were generated at 1-minute intervals over the course of ten minutes for each control and test sample. ROS levels generated were reported in Relative Light Units (RLU)/×106/mL and results were assessed against reference values.[16] Semen analysis and ROS levels were measured immediately after 90 days carnitines (Proxeed Plus) treatment. Upon completion, participants had a final consultation at the Urology Out Patient Department to review if the supplement had an impact on their spermatogenesis.


All samples were processed in an ISO 15 189 accredited laboratory. The diagnostic assessment was performed as a manual bench method and the ROS analysis was carried out using a Turner Biosystems single cuvette Modulus as outlined in previous literature.[15] The LAL-containing supplement provided was marketed under the name of Proxeed Plus supplied by Sigma-Tau HealthScience, Utrecht, The Netherlands. The supplement formulation consisted of 1000 mg of L-carnitine, 725 mg of fumarate, 500 mg of acetyl-L-carnitine, 1000 mg of fructose, 50 mg of citric acid, 50 μg of selenium, 20 mg of coenzyme Q10, 90 mg of vitamin C, 10 mg of zinc, 200 μg of folic acid and 1.5 μg of vitamin B12.

Statistical Methods

All analysis was performed with GraphPad Prism v.8. Quantitative data were assessed for normality with the D'Agostino & Pearson test, followed by paired t-testing or Wilcoxon rank-sum test. All hypothesis testing was two-tailed; P < .05 was considered statistically significant. All data are presented as standard error of mean (SEM) unless stated otherwise. Proportions were compared using Fisher's exact test.