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J Assist Reprod Genet. 2015 May;32(5):757-64. doi: 10.1007/s10815-015-0454-x. Epub 2015 Mar 7.

Reactive Oxygen Species (ROS) in human semen: determination of a reference range.

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Andrology Department, The Doctors Laboratory, London, W1T 4EU, UK,



High levels of reactive oxygen species (ROS) are a leading cause of male factor infertility. Measurement of ROS has been hampered by a lack of standardisation and confounding variables including choice of controls and sample selection. This study aimed to determine a reference range for ROS in human semen.


Semen samples were obtained from men attending for routine semen analysis who gave informed consent for the study. Samples were assigned groups: Group 1 (N = 94) normal semen parameters, no leucocytospermia; Group 2 (N = 100) abnormal semen parameters, no leucocytospermia; Group 3 (N = 41) any semen parameters with leucocytospermia. ROS levels were assayed in fresh neat semen using a chemiluminescence assay measured in a single tube luminometer. Data are reported in relative light units (RLU)/sec/10(6) sperm


ROS levels were significantly different between Groups 1, 2 and 3 (19.75 ± 8.12, 95.03 ± 33.63, 890.17 ± 310.23 RLU/sec/10(6) sperm respectively; p < 0.001). Group 3 gave the highest value confirming this group as the optimum choice for positive controls. The reference range < 24.1 RLU/sec/10(6) sperm was determined by ROC analysis that differentiates a reference population (Group 1) from a positive control group (Group 3), optimising the sensitivity and specificity (80.5 and 87.2% respectively) of the test.


We have determined a reference range for ROS in human semen and identified a patient population that falls outside the normal range. This simple, cost effective assay can be incorporated into routine diagnostic testing to aid in the diagnosis of male infertility, especially with regard to unexplained infertility.

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