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

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.


| INTRODUC TI ON
Infertility affects 10% of couples, and nearly half of cases are due to sperm defects in the male partner. 1 There are currently no available drug therapies to improve sperm function. Reactive oxygen species (ROS) are unstable products of metabolism causing cellular damage 2 produced in the semen by leukocytes and the oxidative metabolism of spermatozoa. 3 ROS generation is a recently identified mechanism for sperm damage and measurement of ROS is a potential tool of added value in the investigation of male infertility. Previous studies have suggested that elevated semen ROS levels are associated with reduced sperm function in men with idiopathic infertility 4 and recurrent miscarriage. 5 Furthermore, many exogenous factors such as genito-urinary infections, varicocele and adverse lifestyle choices have been observed to increase semen ROS. 6 However, no current clinical guidelines support its use in routine practice. There is extensive research in the area with antioxidant therapy observed to improve semen parameters and live-birth rates in men with idiopathic infertility and varicocele-associated infertility. 7,8 Accordingly, antioxidant therapy is a novel, potential therapy to improve sperm function in men with infertility.
Nutritional supplements containing the antioxidant, L-carnitine and acetyl-L-carnitine, are readily available over the counter for men to use as empirical therapies for improving sperm function. However, published data suggest that the effects of carnitines on sperm function are unclear. Some studies suggest that the orally administered amino acid-derived antioxidant l-carnitine and acetyl-L-carnitine (LAL) increase sperm concentration and motility parameters in men with infertility 9,10 ; however, other studies suggest that carnitines do not alter sperm function. 11,12 Furthermore, some clinicians harbour concerns that infertile men without elevated semen ROS might suffer paradoxical impairment of sperm function following antioxidant therapy due to reductive stress. 13 In summary, it is common practice that men with infertility often take antioxidant therapy, but the balance of potential benefit and/ or harm from such therapy is not known.
In the absence of any pharmacological therapy for male infertility, it is important to identify which (if any) men with infertility could clinically benefit from self-administering the antioxidant carnitines to improve sperm function. We therefore conducted a single-centre prospective cohort study of routine clinical practice in men with infertility associated with reduced sperm function. Specifically, we compared changes in sperm function following carnitines therapy between participants with normal and elevated baseline semen ROS levels.

| Participants
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.  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.

| Sample analyses and ROS measurement
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)/×10 6 /mL and results were assessed against reference values. 16

| Materials
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-

| 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.

| Baseline characteristics
Forty-four infertile men with reduced sperm function were included in the study. The mean age of participants was 40 ± 1 years.
Baseline semen parameters are summarized in Table 1

NORMAL ROS GROUP (n = 29)
44 patients had abnormally elevated semen ROS levels (>10 RLU/ SEC/10 6 sperm). No significant differences in baseline characteristics were observed between men with elevated ROS levels ('High ROS' group) and normal ROS levels ('Normal ROS' group).

| Effects of antioxidant treatment on sperm function in men with infertility
We investigated whether sperm function, measured by the WHO 2010 14 criteria, changed following a 3-month course of daily LAL administration in infertile men with at least one abnormal semen analysis parameter with either normal or elevated baseline semen ROS levels.  10.6 ± 2.5, post-treatment, P = .0001 vs baseline) ( Figure 3F).

| DISCUSS ION
Semen ROS is a novel potential marker of sperm function with increasing evidence of its aetiological role in male infertility. 17 Oxidative stress may negatively affect fertility by adversely affecting sperm membrane lipid peroxidation, sperm motility, the acrosome reaction, chromatin maturation and subsequent sperm DNA fragmentation, 18   daily treatment with L-carnitine (1000 mg) and L-acetyl-carnitine (500 mg) of men with idiopathic asthenospermia. 12 Conversely, we observed that 3 months of LAL supplementation increased sperm concentration significantly by over 50%, and total and progressive motility both increased by approximately 30% when compared with baseline in the elevated ROS group. Furthermore, supplementation with carnitines significantly reduced semen ROS levels by fivefold in the elevated ROS group. However, LAL had no significant effect on sperm function or semen ROS levels in infertile men with normal semen ROS levels ≤ 10 RLU/SEC/10 6 . Results of our study may partially explain the inconsistency in the previously observed effects of carnitine therapy on male reproductive function when not stratified by baseline semen ROS levels. We also provide important mechanistic data supporting the hypothesis that LAL improves male reproductive function by reducing oxidative stress in semen of men with infertility. Our data suggest that semen ROS measurement has potential clinical utility for predicting and monitoring improvements in sperm function following LAL therapy in men with infertility.
ROS have a critical physiological role in oxidative metabolism and mitochondrial function. It is therefore plausible that excessive suppression of ROS could impair cellular function. Accordingly, it has recently been proposed that indiscriminate antioxidant therapy may be detrimental to sperm function in men with infertility by inducing a state of reductive stress. 33 Our published study is the first to investigate whether L-carnitine can worsen sperm function without elevated ROS at baseline. We did not observe any significant impairment in sperm volume, count, total or progressive motility men with infertility and normal baseline semen ROS levels. Our data therefore provide important preliminary safety data by suggesting that men without elevated semen ROS could take carnitines therapy without impairment of sperm function.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.