Baseline levels of seminal reactive oxygen species predict improvements in sperm function following antioxidant therapy in men with infertility.

BACKGROUND: Poor sperm function is a major cause of infertility. There is no drug therapy to improve sperm function. Semen oxidative stress is a recently identified pathway for sperm damage. Commercial antioxidants such as L-carnitine and acetyl-L-carnitine (LAL) are commonly self-administered by infertile men. However, concerns have been raised whether inappropriate LAL therapy causes reductive stress-mediated sperm damage. It is imperative to investigate whether: (1) LAL improves sperm function by reducing reactive oxidative species (ROS); (2) LAL has differential effects on sperm function between men with normal and elevated ROS. METHODS: A prospective cohort study of routine clinical practice was performed in infertile men with abnormal sperm quality. Changes in sperm function and semen ROS levels following three months of oral LAL therapy were compared between participants with baseline seminal normal ROS (≤10RLU/SEC/106 sperm; n = 29) and High ROS (>10 RLU/SEC/106 sperm; n = 15) levels measured using an established colorimetric-luminol method. RESULTS: In normal ROS group, sperm function did not change following LAL therapy. In high ROS group, LAL therapy reduced semen ROS fivefold, increased sperm count by 50% (mean count in mill/ml: 21.5 + 7.2, baseline; 32.6 + 9.5, post-treatment, P = .0005), and total and progressive sperm motility each by 30% (mean total sperm motility in % 29.8 + 5.0, baseline: 39.4 + 6.2, post-treatment, P = .004; mean progressive sperm motility in % 23.1 + 4.6, baseline: 30.0 + 5.5, post-treatment, P = .014 vs. baseline). CONCLUSIONS: We report for the first time that LAL only improves sperm quality in infertile men who have baseline high-ROS levels prior to treatment. These data have important potential implications for couples with male infertility and their clinicians.

Reduced Testicular Steroidogenesis and Increased Semen Oxidative Stress in Male Partners as Novel Markers of Recurrent Miscarriage.

BACKGROUND: Recurrent pregnancy loss, (RPL) affecting 1%-2% of couples, is defined as ≥3 consecutive pregnancy losses before 20-week' gestation. Women with RPL are routinely screened for etiological factors, but routine screening of male partners is not currently recommended. Recently it has been suggested that sperm quality is reduced in male partners of women with RPL, but the reasons underlying this lower quality are unclear. We hypothesized that these men may have underlying impairments of reproductive endocrine and metabolic function that cause reductions in sperm quality. METHODS: After ethical approval, reproductive parameters were compared between healthy controls and male partners of women with RPL. Semen reactive oxygen species (ROS) were measured with a validated inhouse chemiluminescent assay. DNA fragmentation was measured with the validated Halosperm method. RESULTS: Total sperm motility, progressive sperm motility, and normal morphology were all reduced in the RPL group vs controls. Mean ±SE morning serum testosterone (nmol/L) was 15% lower in RPL than in controls (controls, 19.0 ± 1.0; RPL, 16.0 ± 0.8; P < 0.05). Mean ±SE serum estradiol (pmol/L) was 16% lower in RPL than in controls (controls, 103.1 ± 5.7; RPL, 86.5 ± 3.4; P < 0.01). Serum luteinizing hormone and follicle-stimulating hormone were similar between groups. Mean ±SE ROS (RLU/sec/106 sperm) were 4-fold higher in RPL than in controls (controls, 2.0 ± 0.6; RPL, 9.1 ± 4.1; P < 0.01). Mean ±SE sperm DNA fragmentation (%) was 2-fold higher in RPL than in controls (controls, 7.3 ± 1.0; RPL, 16.4 ± 1.5; P < 0.0001). CONCLUSIONS: Our data suggest that male partners of women with RPL have impaired reproductive endocrine function, increased levels of semen ROS, and sperm DNA fragmentation. Routine reproductive assessment of the male partners may be beneficial in RPL.

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

PURPOSE: 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. METHODS: 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 RESULTS: 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. CONCLUSIONS: 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.

Reactive oxygen species in human semen: validation and qualification of a chemiluminescence assay.

OBJECTIVE: To standardize and validate an assay for reactive oxygen species (ROS) in human semen. DESIGN: ROS levels assayed in blanks, negative and positive control samples (30% H2O2), and human semen, with the use of a luminol-based chemiluminescence assay measured in a single tube luminometer. SETTING: Andrology laboratory. PATIENT(S): Semen samples from 19 men attending for routine semen analysis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): ROS levels reported in relative light units (RLU) per second, adjusted for sperm concentration. RESULT(S): The ROS assay equipment performed according to expectations, generating a chemiluminescence signal for positive control samples and semen samples that decayed rapidly and was captured within 10 minutes. Blanks and negative control samples gave negligible readings. There was no significant intra- or interassay variation. Interference from extraneous factors was negligible. The assay distinguished changes in ROS over a wide range of concentrations and provided consistent results between reagent batches. Working reagents remained stable for 3 months. Acceptable levels for negative and positive control samples were established to set criteria for the test passing or failing on any given day. The assay was sensitive to ambient temperature >25°C. ROS declined significantly with time after ejaculation. Mechanical agitation doubled ROS production in semen. CONCLUSION(S): These results validate the ROS assay and demonstrate that it is a highly reliable and accurate diagnostic test.