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Predictive value of seminal oxidation-reduction potential analysis for reproductive outcomes of ICSI

Open AccessPublished:May 24, 2022DOI:https://doi.org/10.1016/j.rbmo.2022.05.010

      Abstract

      Research question

      Is seminal oxidation-reduction potential (ORP) clinically relevant to reproductive outcome?

      Design

      Prospective observational study including a total of 144 couples who had an intracytoplasmic sperm injection (ICSI) cycle between June 2018 and December 2020. The study included patients undergoing fresh ICSI cycles with autologous gametes. Cycles that had day 3 embryo transfers and cryopreservation cycles were excluded. There was no restriction on patients with severe male infertility; couples with unexplained infertility and unexplained male infertility were included, those with azoospermia were excluded. Semen analysis, seminal ORP as determined by means of the MiOXSYS system, sperm DNA fragmentation (SDF) and reproductive outcomes (fertilization, blastocyst development, clinical pregnancy and live birth) were determined.

      Results

      Seminal ORP was significantly negatively correlated with fertilization rate (r = –0.267; P = 0.0012), blastocyst development rate (r = –0.432; P < 0.0001), implantation/clinical pregnancy (r = –0.305; P = 0.0003) and live birth (r = –0.366; P < 0.0001). Receiver operating characteristic curve analysis showed significant predictive power for ORP for fertilization (≥80%; area under the curve [AUC] 0.652; P = 0.0012), blastocyst development rate (≥60%; AUC 0.794; P < 0.0001), implantation/clinical pregnancy (AUC 0.680; P = 0.0002) and live birth (AUC 0.728; P < 0.0001). Comparable results were obtained for SDF (fertilization: AUC 0.678; blastocyst development: AUC 0.777; implantation/clinical pregnancy: AUC 0.665; live birth: AUC 0.723). Normal sperm morphology showed the lowest predictive power for all reproductive outcome parameters. With male age as confounding factor, ORP (cut-off value of 0.51 mV/106 sperm/ml) has significant (P < 0.04667) effects on odds ratios for all reproductive outcome parameters. Multivariate logistic regression to investigate potential seminal and female confounding factors revealed that seminal ORP significantly (P < 0.0039; P < 0.0130) affects reproductive outcome.

      Conclusion

      Seminal ORP is relevant for good fertilization, blastocyst development, implantation, clinical pregnancy and live birth.

      Keywords

      Introduction

      Globally, nearly 70 million people are affected by infertility. According to the World Health Organization, an estimated 9% of couples worldwide are unable to conceive and up to 50% of infertility cases are attributable to male factors (
      • Boivin J.
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      International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care.
      ). Despite advances in understanding the causes of male infertility, idiopathic infertility still accounts for about 30–50% of male infertility issues (
      • Gelbaya T.A.
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      • Jeve Y.B.
      • Nardo L.G.
      Definition and epidemiology of unexplained infertility.
      ). Many causes of male infertility have been identified and can be classified into pre-testicular, testicular or post-testicular with congenital, acquired or idiopathic factors (
      • Agarwal A.
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      A schematic overview of the current status of male infertility practice.
      ,
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      • Shah R.
      Male infertility.
      ). In many of the underlying factors such as lifestyle choices, infections, varicocele, radiation or environmental pollution, oxidative stress is involved, eventually causing sperm DNA fragmentation (SDF) (
      • Agarwal A.
      • Majzoub A.
      • Parekh N.
      • Henkel R.
      A schematic overview of the current status of male infertility practice.
      ).
      Oxidative stress has been reported to play an important role in various aetiologies of male infertility including male genital tract infection/inflammation, varicocele or adverse lifestyle conditions and related diseases such as obesity or diabetes mellitus (
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      ;
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      ). Oxidative stress is induced by the imbalance between reactive oxygen species (ROS) and antioxidants. Under normal conditions, ROS and antioxidants are in balance; antioxidants neutralize excessive amounts of these highly reactive free radicals and maintain homeostasis (
      • Kothari S.
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      Free radicals: their beneficial and detrimental effects on sperm function.
      ). Under pathological conditions, when there are higher concentrations of ROS than antioxidants, ROS-mediated oxidative stress ensues. Because sperm plasma membranes have an extraordinarily high content of polyunsaturated fatty acids (
      • Henkel R.
      Leukocytes and oxidative stress: dilemma for sperm function and male fertility.
      ;
      • Parks J.E.
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      Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes.
      ), male germ cells are highly susceptible to lipid peroxidation (
      • Gharagozloo P.
      • Aitken R.J.
      The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy.
      ). Oxidative stress is also regarded as a major cause of SDF (
      • Aitken R.J.
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      Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.
      ;
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      Investigation on the origin of sperm DNA fragmentation: role of apoptosis, immaturity and oxidative stress.
      ). There is a growing body of evidence indicating important roles for seminal oxidative stress and SDF for male infertility (
      • Esteves S.C.
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      Sperm DNA fragmentation testing: summary evidence and clinical practice recommendations.
      ) and the reproductive outcomes of assisted reproduction (
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      • Arafa M.
      • Alves M.G.
      • Oliveira P.F.
      • Alvarez J.G.
      • Shah R.
      Utility of antioxidants in the treatment of male infertility: clinical guidelines based on a systematic review and analysis of evidence.
      ;
      • Esteves S.C.
      • Sanchez-Martin F.
      • Sanchez-Martin P.
      • Schneider D.T.
      • Gosalvez J.
      Comparison of reproductive outcome in oligozoospermic men with high sperm DNA fragmentation undergoing intracytoplasmic sperm injection with ejaculated and testicular sperm.
      ;
      • Sakkas D.
      • Alvarez J.G.
      Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis.
      ). In a systematic review and meta-analysis including 23 studies with a total of 6771 cycles of treatment with assisted reproductive technology (ART),
      • Deng C.
      • Li T.
      • Xie Y.
      • Guo Y.
      • Yang Q.Y.
      • Liang X.
      • Deng C.H.
      • Liu G.H.
      Sperm DNA fragmentation index influences assisted reproductive technology outcome: a systematic review and meta-analysis combined with a retrospective cohort study.
      reported that high SDF was associated with a significantly increased risk of poorer cycle outcome. In another systematic review and meta-analysis of 27 studies with 1941 patients with unexplained recurrent spontaneous abortion,
      • Yifu P.
      • Lei Y.
      • Shaoming L.
      • Yujin G.
      • Xingwang Z.
      Sperm DNA fragmentation index with unexplained recurrent spontaneous abortion: a systematic review and meta-analysis.
      reported a significant association between SDF and recurrent pregnancy loss.
      In order to predict male fertility and sperm fertilizing capacity, especially for assisted reproduction procedures, various semen parameters and sperm functions including normal sperm morphology (
      • Kruger T.F.
      • Acosta A.A.
      • Simmons K.F.
      • Swanson R.J.
      • Matta J.F.
      • Oehninger S.
      Predictive value of abnormal sperm morphology in in vitro fertilization.
      ;
      • Obara H.
      • Shibahara H.
      • Tsunoda H.
      • Taneichi A.
      • Fujiwara H.
      • Takamizawa S.
      • Idei S.
      • Sato I.
      Prediction of unexpectedly poor fertilization and pregnancy outcome using the strict criteria for sperm morphology before and after sperm separation in IVF-ET.
      ) or SDF (
      • Simon L.
      • Zini A.
      • Dyachenko A.
      • Ciampi A.
      • Carrell D.T.
      A systematic review and meta-analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome.
      ) are used and have proved to be valuable tools in the clinical work-up of male infertility. For seminal oxidative stress and ROS assessment in particular, luminometric techniques with luminol or lucigenin as chemiluminescent probes are usually used (
      • Aitken R.J.
      • Buckingham D.W.
      • West K.M.
      Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence.
      ;
      • Zorn B.
      • Vidmar G.
      • Meden-Vrtovec H.
      Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection.
      ). However, despite the simplicity of the luminometric measurement, the direct determination of ROS in semen is technically challenging, and assays are therefore not validated. For the determination of SDF, several methods with different protocols have been described. The most frequently used techniques to determine SDF and thereby predict the probability of a successful outcome of treatment with ART are the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay (
      • Henkel R.
      • Hajimohammad M.
      • Stalf T.
      • Hoogendijk C.
      • Mehnert C.
      • Menkveld R
      • Gips H.
      • Schill W.-B.
      • Kruger T.F.
      Influence of deoxyribonucleic acid damage on fertilization and pregnancy.
      ;
      • Sharma R.K.
      • Sabanegh E.
      • Mahfouz R.
      • Gupta S.
      • Thiyagarajan A.
      • Agarwal A.
      TUNEL as a test for sperm DNA damage in the evaluation of male infertility.
      ), the sperm chromatin structure assay (SCSA;
      • Jerre E.
      • Bungum M.
      • Evenson D.
      • Giwercman A.
      Sperm chromatin structure assay high DNA stainability sperm as a marker of early miscarriage after intracytoplasmic sperm injection.
      ), the comet assay (
      • Simon L.
      • Lutton D.
      • McManus J.
      • Lewis S.E.
      Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success.
      ) and the sperm chromatin dispersion (SCD) assay (
      • Muriel L.
      • Garrido N.
      • Fernandez J.L.
      • Remohi J.
      • Pellicer A.
      • de los Santos M.J.
      • Meseguer M.
      Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection.
      ). On the other hand, the different methods described for SDF actually measure different aspects of sperm DNA damage (
      • Henkel R.
      • Hoogendijk C.F.
      • Bouic P.J.
      • Kruger T.F.
      TUNEL assay and SCSA determine different aspects of sperm DNA damage.
      ;
      • Zini A.
      • Sigman M.
      Are tests of sperm DNA damage clinically useful? Pros and cons.
      ), resulting in only moderate correlation between the different methods (
      • Rex A.S.
      • Aagaard J.
      • Fedder J.
      DNA fragmentation in spermatozoa: a historical review.
      ). In addition, extensive preparation (TUNEL assay), labour-intensive procedure (comet assay), small number of evaluated cells (SCD, comet assay) and poor predictive accuracy for IVF and intracytoplasmic sperm insemination (ICSI) outcome (SCSA) result in a lack of strong evidence (
      • Cissen M.
      • Wely M.V.
      • Scholten I.
      • Mansell S.
      • Bruin J.P.
      • Mol B.W.
      • Braat D.
      • Repping S.
      • Hamer G.
      Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: a systematic review and meta-analysis.
      ;
      • Rex A.S.
      • Aagaard J.
      • Fedder J.
      DNA fragmentation in spermatozoa: a historical review.
      ;
      • Simon L.
      • Emery B.
      • Carrell D.T.
      Sperm DNA fragmentation: consequences for reproduction.
      ) and therefore lack of agreement over using these tests.
      A newly introduced method of detecting seminal oxidative stress is to measure the oxidation-reduction potential (ORP), which provides the overall balance between oxidants and antioxidants (
      • Agarwal A.
      • Roychoudhury S.
      • Bjugstad K.B.
      • Cho C.L.
      Oxidation-reduction potential of semen: what is its role in the treatment of male infertility?.
      ). This method has been shown to be a quick, easy, reliable and cost-effective tool to identify infertile men (
      • Agarwal A.
      • Henkel R.
      • Sharma R.
      • Tadros N.N.
      • Sabanegh E.
      Determination of seminal oxidation-reduction potential (ORP) as an easy and cost-effective clinical marker of male infertility.
      ). In addition,
      • Arafa M.
      • Henkel R.
      • Agarwal A.
      • Majzoub A.
      • Elbardisi H.
      Correlation of oxidation-reduction potential with hormones, semen parameters and testicular volume.
      and
      • Elbardisi H.
      • Finelli R.
      • Agarwal A.
      • Majzoub A.
      • Henkel R.
      • Arafa M.
      Predictive value of oxidative stress testing in semen for sperm DNA fragmentation assessed by sperm chromatin dispersion test.
      have shown a mediocre but highly significant positive association between SDF and seminal ORP. Because of the reported issues including labour-intensive and costly procedures with the different techniques to determine SDF, there is still a need for diagnostic tools to improve the prediction of male fertility potential. In contrast to methods for SDF determination, measurement of ORP is easy and quick, but has not yet been evaluated to predict male fertility potential in an assisted reproduction set-up. Therefore, the aim of this prospective study was to investigate the predictive capabilities of seminal ORP for reproductive outcomes (fertilization, blastocyst development, clinical pregnancy and live birth) in patients undergoing ICSI cycles and compare these with the predictive potential of a standard fluorescent TUNEL assay for SDF.

      Materials and methods

      Setting and subjects

      The current prospective observational study was approved on 13 January 2017 by the Biomedical Ethics Committee (reference number: BM/16/5/18) of the University of the Western Cape, Bellville, South Africa, and all participating clinics. The study was conducted from June 2018 to December 2020. A total of 144 patients (one cycle each that was included in the study) undergoing ICSI treatments at Drs Aevitas Fertility Clinic, Vincent Palotti Hospital, Pinelands, South Africa; Cape Fertility Clinic, Claremont, South Africa; and Tygerberg Fertility Clinic, Institute for Reproductive Medicine, Tygerberg, South Africa, were enrolled in this study and gave informed consent. The study included consenting patients undergoing fresh ICSI cycles with autologous gametes. Cycles that had day 3 embryo transfers and cryopreservation cycles were excluded. There was no restriction on the inclusion of patients with severe male factor infertility; couples with unexplained infertility and unexplained male infertility were included, while those with azoospermia were excluded. On the female side, patients with primary and secondary infertility, unexplained infertility, polycystic ovaries, endometriosis, tubal factor, myomectomy, as well as ovarian insufficiency and advanced maternal age, were included.

      Study procedures

      Patients were instructed to abstain from sexual intercourse 2 to 3 days prior to providing the semen sample. The ejaculated semen samples were produced by masturbation, collected in a sterile specimen container and kept at 37°C until liquefaction was observed. Andrological diagnostics (i.e. manual semen analysis, testing for SDF and determination of seminal ORP) were conducted in 180 µl (50 µl for standard semen analysis; 30 µl for ORP; 100 µl for SDF) of the semen samples that were provided for the ICSI procedure within 30 min of liquefaction. The rest of the sample was either subjected to density gradient centrifugation or swim-up based on total motile count before the ICSI procedure (Figure 1).
      Figure 1
      Figure 1Study design and project methodology. ICSI = intracytoplasmic sperm injection; ORP = oxidation-reduction potential; SDF = sperm DNA fragmentation.

      ART procedure

      Stimulation protocol

      For ovarian stimulation, the flexible antagonist protocol was used, consisting of daily gonadotrophins (300 IU FSH in a step-down fashion to 150 IU) for 5 days beginning on day 3 of the menstrual cycle. Administration of gonadotrophin-releasing hormone (GnRH) antagonist (0.25 mg) was started by s.c. injection when the leading follicle measured 14 mm or more. Ovulation was induced by s.c. or i.m. administration of 10,000 IU human chorionic gonadotrophin (HCG) or 200 µg GnRH agonist when the lead follicle was ≥18 mm and at least two other follicles were ≥16 mm in size. Oocyte retrieval was performed within 34–36 h after HCG administration. Luteal phase support was achieved by vaginal progesterone preparation if HCG was used.

      Semen processing for ICSI

      For sperm processing, standardized methods conforming to
      World Health Organization
      WHO Laboratory Manual for the Examination and Processing of Human Semen.
      were followed across the participating clinics. The preferred method for sperm processing was determined by the initial evaluation of semen parameters and quality of the semen sample produced. Two basic methods, swim-up and density gradient centrifugation (Sil-Select; FertiPro, Harrilabs, Cape Town, South Africa), were used. For the swim-up, ORIGIO Sperm Wash medium was used, while Sperm Preparation Medium (CooperSurgical, Ferring, Cape Town, South Africa) was used for wash procedures and Sil-Silect Plus™ (FertiPro; 40%/80%) for the density gradient centrifugation.

      Oocyte retrieval procedure

      Oocytes were retrieved under conscious sedation (i.v. Dormicum®, Hoffmann-La Roche Ltd, Basel, Switzerland) using transvaginal ultrasound guidance. Follicular fluid was aspirated using sonar-guided ultrasound, and examined for the presence of cumulus–oocyte complexes (COC) both macroscopically and microscopically. The oocytes within the COC were superficially graded to infer maturity (immature, germinal vesicles [GV] or metaphase I [MI] oocytes; mature, metaphase II [MII]). Post-grading, the COC were collected, washed in Quinn's AdvantageTM Medium with HEPES (SAGETM), and transferred to a FertTM (ORIGIO®) medium.

      ICSI and embryo culture

      All embryology procedures were performed by a fully trained and experienced embryologist. For this study, all included oocytes were injected by standard ICSI. For all preparatory and culture procedures, Quinn's Advantage™ (Harrilabs) sequential culture medium range was used including HEPES-buffered medium, sperm preparation medium, fertilization/cleavage and blastocyst medium, oil for tissue culture, hyaluronidase and polyvinylpyrrolidone. A standardized, routine method was used for all patients. After incubation for 16–18 h, oocytes were assessed for fertilization (visualization of two pronuclei [2PN]). Embryos were individually cultured and evaluated for embryo quality (blastomere morphology, size and percentage of fragmentation) on day 2 and day 3 post-insemination. Culture to the blastocyst stage (day 4 to day 6 post-insemination) was performed in SAGE one-step culture medium (CooperSurgical). Embryo culture was performed in Planer BT37 incubators (Planer, Sunbury-on-Thames, UK) under 5% O2 and 7.2% CO2.

      Embryo transfer and reproductive outcome parameters

      Embryo transfers were performed using Wallace® Classic Embryo Transfer Catheters (CooperSurgical) under standard ultrasound guidance (
      • Sallam H.N.
      • Sadek S.S.
      Ultrasound-guided embryo transfer: a meta-analysis of randomized controlled trials.
      ) on day 5. According to embryo transfer guidelines published by the Southern African Society for Reproductive Medicine and Gynaecological Endoscopy (SASREG), patient history and conforming to national legislation requirements, no more than three embryos were transferred by a reproductive medicine specialist and clinical embryologist. Embryos were assessed and graded on day 5 (blastocyst stage). Embryo grading was performed under high magnification according to a modified grading system (
      • Richardson A.
      • Brearley S.
      • Ahitan S.
      • Chamberlain S.
      • Davey T.
      • Zujovic L.
      • Hopkisson J.
      • Campbell B.
      • Raine-Fenning N.
      A clinically useful simplified blastocyst grading system.
      ).
      As study outcome parameters, fertilization (number of oocytes with 2PN stage/total MII oocytes) and blastocyst formation rates (developed blastocysts per fertilized oocyte), as well as the clinical parameters implantation (proportion of embryo transfers with at least one gestational sac visualized on ultrasound per total number of embryo transfers), clinical pregnancy (as determined by fetal heartbeat), miscarriage (premature loss of the fetus up to 23 weeks) and live birth (delivery of at least one live baby) rates were recorded. For the classification of good and poor fertilization and blastocyst development, the benchmark values for the key performance indicators (fertilization: ≥80%; blastocyst development: ≥60%) according to the Vienna consensus (
      ESHRE Special Interest Group of Embryology and Alpha Scientists in Reproductive Medicine
      The Vienna consensus: report of an expert meeting on the development of ART laboratory performance indicators.
      ) were used. For implantation, clinical pregnancy and live birth, classification for receiver operating characteristic (ROC) curve analyses was performed on a yes/no basis. Clinical pregnancy and live birth were analysed per embryo transfer.

      Andrology laboratory procedure

      Semen analysis

      All semen samples were assessed for sperm concentration and motility according to
      World Health Organization
      WHO Laboratory Manual for the Examination and Processing of Human Semen.
      criteria with standard quality control measures being applied. For the determination of normal sperm morphology, a smear of 10 µl semen was applied on a slide, stained according to the Papanicolaou staining protocol and evaluated according to strict criteria (
      • Menkveld R.
      • Stander F.S.H.
      • Kotze T.J.vW.
      • Kruger T.F.
      • van Zyl J.A.
      The evaluation of morphological characteristics of human spermatozoa according to stricter criteria.
      ).

      Oxidative stress

      Seminal oxidative stress was determined by means of measurement of the ORP using the MiOXSYS system (Aytu Bioscience, Englewood, CO, USA). In brief, 30 µl of liquefied semen were placed on the sample port of the ORP sensor. After about 3–4 min, the result could be read in millivolts (mV) at the display, then normalized according to the sperm concentration and expressed as mV/106 sperm/ml (
      • Agarwal A.
      • Panner Selvam M.K.
      • Arafa M.
      • Okada H.
      • Homa S.
      • Killeen A.
      • Balaban B.
      • Saleh R.
      • Armagan A.
      • Roychoudhury S.
      • Sikka S.
      Multi-center evaluation of oxidation-reduction potential by the MiOXSYS in males with abnormal semen.
      ).

      SDF

      SDF was measured using the fluorometric TUNEL assay (Promega Corporation, Madison, USA) according to the protocol described previously (
      • Henkel R.
      • Hajimohammad M.
      • Stalf T.
      • Hoogendijk C.
      • Mehnert C.
      • Menkveld R
      • Gips H.
      • Schill W.-B.
      • Kruger T.F.
      Influence of deoxyribonucleic acid damage on fertilization and pregnancy.
      ). A total of 300 spermatozoa were evaluated for TUNEL positivity under an epifluorescence microscope (Zeiss, Oberkochen, Germany) at 1000 × magnification. Then, the percentage of green fluorescing spermatozoa (TUNEL-positive) was calculated and recorded. Positive and negative controls were run in parallel with the samples.

      Statistical analysis

      MedCalc® Statistical Software version 20.009 (MedCalc Software Ltd, Ostend, Belgium) was used for the statistical analysis. After testing for normal distribution using the chi-squared test, non-parametric tests were employed for the statistical evaluation. Spearman's rank correlation coefficient was used to determine the numerical correlations for analysed parameters. To determine the predictive capabilities of ORP, SDF and normal sperm morphology for fertilization, blastocyst development rates, clinical pregnancy and live birth, ROC curve analyses were generated according to the DeLong method (
      • DeLong E.R.
      • DeLong D.M.
      • Clarke-Pearson D.L.
      Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach.
      ).
      In order to investigate whether seminal ORP was independently influencing the reproductive outcome, the female variables (age, primary and secondary infertility, unexplained infertility, polycystic ovaries, endometriosis, tubal factor, myomectomy, ovarian insufficiency and advanced maternal age) were included in a stepwise logistic regression model. In addition, a stepwise logistic regression model was used to determine the influence of semen factors (sperm concentration, motility, normal sperm morphology and seminal ORP) on fertilization, blastocyst development, implantation, clinical pregnancy and live birth. Considering that the data for cycles with implantation and clinical pregnancy are the same, these were reported together as ‘implantation/clinical pregnancy’.
      As a sub-analysis, men were categorized according to their age as <37 years or ≥37 years by using the median of the male age. The Cochran–Mantel–Haenszel test was used to calculate odds ratios for male age. A P-value of <0.05 was considered significant.

      Results

      A total of 144 patients were recruited for the study, out of whom 143 underwent ICSI treatment and were included in the analysis; in one case no oocytes were obtained. In addition, cryopreservation cycles and day 3 embryo transfers were excluded. The patient cohort comprised 51 couples with primary infertility and 93 with secondary infertility. In detail, 45 patients were recorded with unexplained infertility, 43 women with polycystic ovary syndrome, 31 with endometriosis, 25 with tubal factor, 3 after myomectomy, 14 with ovarian insufficiency, 21 poor responders and 73 women of advanced maternal age (>35 years). Ninety-three patients presented with multiple of these conditions. The summary statistics of patient age, semen parameters (sperm concentration, total motility and normal sperm morphology), SDF and ORP along with fertilization and blastocyst development rates are presented in Table 1. Overall, 134 women out of 143 (93.7%) underwent embryo transfer with mean ± SD: 1.8 ± 0.5 embryos per transfer and implantation was observed in 54 patients (40.3%). The embryo transfers resulted in 54 (40.3%) clinical pregnancies and 39 (29.1%) live births, while 15 (11.2%) miscarriages occurred. In 5 out of 143 (3.5%) cases, no fertilization occurred and in 4 out of 138 (2.9%) cases with fertilized oocytes no blastocysts were obtained.
      Table 1Summary statistics of the main parameters analysed in this study
      ParameterNo. of cyclesRate (%)MinimumMaximumMedianMean ± SD
      Female age (years)14427.046.035.034.8 ± 3.9
      Male age (years)14428.051.037.036.5 ± 4.3
      Sperm concentration (106/ml)1442.0198.050.053.5 ± 35.9
      Motility (%)1445.088.053.051.9 ± 19.0
      Normal sperm morphology (%)1440.023.04.05.3 ± 4.1
      ORP (mV/106 sperm/ml)1440.0225.30.50.9 ± 2.2
      TUNEL-positive spermatozoa (%)1444.062.022.524.3 ± 13.4
      Fertilization rate (%; per oocyte injected)14396.50.0100.080.073.5 ± 22.2
      Blastocyst development rate (%; per fertilized oocyte)13897.10.0100.071.467.9 ± 24.1
      Implantation (rate per transfer)13440.30.0100.00.040.3 ± 49.2
      Clinical pregnancy (rate per transfer)13440.30.0100.00.040.3 ± 49.2
      Live birth (rate per transfer)13429.10.0100.00.029.1 ± 45.6
      Implantation = cycles with ≥1 gestational sac, clinical pregnancy = cycles with ≥ foetal heart beat and live birth = cycles with ≥1 live baby delivered.
      ORP = oxidation-reduction potential; TUNEL = terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling.
      Table 2 compares the mean values of seminal ORP and SDF in the cases where no fertilization, blastocyst formation, implantation/clinical pregnancy or live birth, respectively, occurred with those from patients with fertilization, blastocyst formation, implantation, clinical pregnancy and live birth. All comparisons except for ORP (fertilization: P = 0.6924 and blastocyst development: P = 0.0964) and SDF (fertilization: P = 0.0914 and blastocyst development: P = 0.2109) showed significant differences (ORP: implantation/clinical pregnancy, P = 0.0004; live birth, P < 0.0001) (SDF: implantation/clinical pregnancy, P = 0.0012; live birth, P = 0.0001). Table 3 reports the mean values of seminal ORP and SDF of cases with no fertilization and no blastocyst formation, compared with those cases where the fertilization and blastocyst formation rates were ≥80% and ≥60%, respectively. While no difference between the groups was found for ORP (fertilization rate P = 0.9593; blastocyst formation rate P = 0.0847), the differences for SDF were significant (fertilization rate P = 0.0188; blastocyst formation rate P = 0.0346).
      Table 2Comparison of ORP and SDF in cases with or without fertilization, blastocyst formation, implantation, clinical pregnancy or live birth
      ParameterORP (mV/106 sperm/ml)P-valueSDF (%)P-value
      Fertilization rate (0%) (n = 5)0.83 ± 0.820.692434.20 ± 14.380.0914
      Fertilization rate (>0%) (n = 138)0.98 ± 2.2223.91 ± 13.34
      Blastocyst formation rate (0%) (n = 4)2.66 ± 2.230.096431.25 ± 13.840.2109
      Blastocyst formation rate (>0%) (n = 134)0.93 ± 2.2123.47 ± 13.19
      Implantation / clinical pregnancy (no) (n = 80)0.87 ± 0.670.000426.56 ± 13.890.0012
      Implantation / clinical pregnancy (yes) (n = 54)1.00 ± 3.4018.88 ± 10.61
      Live birth (no) (n = 95)0.86 ± 0.65<0.000126.53 ± 13.630.0001
      Live birth (yes) (n = 39)1.09 ± 4.0016.63 ± 9.53
      Values reported as mean ± SD.
      Implantation = cycles with ≥1 gestational sac, clinical pregnancy = cycles with ≥ foetal heart beat and live birth = cycles with ≥1 live baby delivered.
      The significance was calculated using the Mann–Whitney test.
      P-values <0.05 indicate statistical significance.
      ORP = oxidation-reduction potential; SDF = sperm DNA fragmentation.
      Table 3Comparison of ORP and SDF in cases with no fertilization or blastocyst formation to cases with fertilization and blastocyst formation rates higher than or equal to benchmark rates
      ParameterORP (mV/106 sperm/ml)P-valueSDF (%)P-value
      Fertilization rate (0%) (n = 5)0.83 ± 0.820.959334.20 ± 14.380.0188
      Fertilization rate (≥80%) (n = 73)0.59 ± 0.4319.81 ± 10.80
      Blastocyst formation rate (0%) (n = 4)2.66 ± 2.230.084731.25 ± 13.840.0346
      Blastocyst formation rate (≥60%) (n = 98)0.83 ± 2.5419.87 ± 10.57
      Values reported as mean ± SD.
      Benchmark rates according to ESHRE Vienna consensus (
      ESHRE Special Interest Group of Embryology and Alpha Scientists in Reproductive Medicine
      The Vienna consensus: report of an expert meeting on the development of ART laboratory performance indicators.
      ).
      The significance was calculated using the Mann–Whitney test.
      P-values <0.05 indicate statistical significance.
      ORP = oxidation-reduction potential; SDF = sperm DNA fragmentation.
      Sperm preparation of the 144 semen samples was performed in 43 (29.9%) samples with standard swim-up and in 101 (70.1%) with density gradient centrifugation. No difference in fertilization (swim-up: n = 43; 70.9% versus density gradient centrifugation: n = 100; 74.6%; P = 0.3113), blastocyst development (swim-up: n = 42; 70.3% versus density gradient centrifugation: n = 96; 66.8%; P = 0.4506), and implantation/clinical pregnancy rates (swim-up: n = 41; 35.4% versus density gradient centrifugation: n = 93; 28.5%; P = 0.2983) as well as for live birth (swim-up: n = 41; 39.0% versus density gradient centrifugation: n = 93; 24.7%; P = 0.0945) was recorded between the two sperm separation techniques.
      The correlations between the male parameters analysed in this study are shown in Table 4. As expected, significant positive correlations were found between sperm concentration and motility (r = 0.522; P < 0.0001) and normal morphology (r = 0.466; P < 0.0001), as well as between normal morphology and motility (r = 0.496; P < 0.0001). There were also highly significant positive correlations between the percentage of TUNEL-positive spermatozoa and seminal ORP (r = 0.665; P < 0.0001) as well as male age (r = 0.328; P = 0.0001). Furthermore, seminal ORP showed a weak, but significant (r = 0.268; P = 0.0012) positive association with male age. On the other hand, significant negative correlations were found between male age and sperm concentration (r = –0.215; P = 0.0097), normal morphology (r = –0.252; P = 0.0023) and motility (r = –0.271; P = 0.0010), between sperm concentration and ORP (r = –0.678; P < 0.0001) and the percentage of TUNEL-positive spermatozoa (r = –0.467; P < 0.0001), between ORP and motility (r = –0.424; P < 0.0001), and normal morphology (r = –0.472; P < 0.0001), as well as between the percentage of TUNEL-positive spermatozoa and normal morphology (r = –0.415; P < 0.0001) and motility (r = –0.428; P < 0.0001).
      Table 4Correlation between basic semen parameters, rate of TUNEL-positive spermatozoa, seminal ORP and age
      ParameterConcentration (106/ml)Normal morphology (%)Motility (%)ORP (mV/106/sperm/ml)TUNEL-positive (%)
      Normal morphology (%)r

      P
      0.466

      <0.0001
      Motility (%)r

      P
      0.522

      <0.0001
      0.496

      <0.0001
      ORP (mV/106 sperm/ml)r

      P
      –0.678

      <0.0001
      –0.472

      <0.0001
      –0.424

      <0.0001
      TUNEL-positive (%)r

      P
      –0.467

      <0.0001
      –0.415

      <0.0001
      –0.428

      <0.0001
      0.665

      <0.0001
      Male age (years)r

      P
      –0.215

      0.0097
      –0.252

      0.0023
      –0.271

      0.0010
      0.268

      0.0012
      0.328

      0.0001
      Sample size: n = 144.
      P = P-value; r = Spearman rank correlation coefficient.
      P-values <0.05 indicate statistical significance.
      ORP = oxidation-reduction potential; TUNEL = terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling.
      Correlations between male age, standard semen parameters, advanced sperm function test (ORP and SDF) and reproductive outcomes (fertilization rate, blastocyst development rate, implantation/clinical pregnancy and live birth) in patients undergoing ICSI treatment are reported in Table 5. In the current study, all assisted reproduction parameters (fertilization, blastocyst development, implantation/clinical pregnancy and live birth) were significantly negatively correlated with ORP (r = –0.267, P = 0.0012; r = –0.432, P < 0.0001; r = –0.305, P = 0.0003; and r = –0.366, P < 0.0001, respectively). Similarly, the percentage of TUNEL-positive spermatozoa was significantly negatively associated with these parameters (fertilization: r = –0.341, P < 0.0001; blastocyst development: r = –0.450, P < 0.0001; implantation/clinical pregnancy: r = –0.280, P = 0.0010; live birth: r = –0.347, P < 0.0001, respectively). While male age was not correlated with the fertilization rate, weak but significant negative associations were observed for the blastocyst development rate (r = –0.198; P = 0.0215), implantation/clinical pregnancy (r = –0.239; P = 0.0055) and live birth (r = –0.186; P = 0.0312).
      Table 5Correlation between advanced sperm function tests, normal morphology and reproductive outcomes in patients undergoing ICSI treatment
      ParameterORP (mV/106 sperm/ml)TUNEL-positive (%)Normal morphology (%)Fertilization rate (%)Blastocyst development rate (%)Implantation / clinical pregnancy rate (%)Live birth rate (%)
      TUNEL-positive (%)r

      P

      n
      0.665

      <0.0001

      144
      Normal morphology (%)r

      P

      n
      –0.472

      <0.0001

      144
      –0.415

      <0.0001

      144
      Fertilization rate (%)r

      P

      n
      –0.267

      0.0012

      143
      –0.341

      <0.0001

      143
      0.156

      0.0630

      143
      Blastocyst development rate (%)r

      P

      n
      –0.432

      <0.0001

      138
      –0.450

      <0.0001

      138
      0.331

      0.0002

      138
      0.034

      0.6696

      138
      Implantation / clinical pregnancy rate (%)r

      P

      n
      –0.305

      0.0003

      134
      –0.280

      0.0010

      134
      0.194

      0.0245

      134
      0.119

      0.1712

      134
      0.187

      0.0304

      134
      Live birth rate (%)r

      P

      n
      –0.366

      <0.0001

      134
      –0.347

      <0.0001

      134
      0.196

      0.0232

      134
      0.104

      0.2312

      134
      0.215

      0.0124

      134
      0.780

      <0.0001

      134
      Male age (years)r

      P

      n
      0.268

      0.0012

      144
      0.328

      0.0001

      144
      –0.252

      0.0023

      144
      –0.086

      0.3060

      134
      –0.198

      0.0215

      134
      –0.239

      0.0055

      134
      –0.186

      0.0312

      134
      Advanced sperm function tests include ORP and SDF.
      Implantation = cycles with ≥1 gestational sac, clinical pregnancy = cycles with ≥ foetal heart beat and live birth = cycles with ≥1 live baby delivered.
      n = sample size; P = P-value; r = Spearman rank correlation coefficient.
      P-values <0.05 indicate statistical significance.
      ICSI = intracytoplasmic sperm injection; ORP = oxidation-reduction potential; SDF = sperm DNA fragmentation; TUNEL = terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling.
      ROC curve analyses for seminal ORP, the percentage of TUNEL-positive spermatozoa and normal sperm morphology were carried out to evaluate the predictive power of seminal ORP, SDF and normal morphology with regard to the benchmark cut-off values for fertilization (≥80%) (Figure 2a) and blastocyst development rate (≥60%) (Figure 2b), implantation/clinical pregnancy (Figure 2c) as well as live birth (Figure 2d) after ICSI. The calculations were significant (range of P-values: P = 0.0420 to P < 0.0001) for all parameters (ORP, percentage of TUNEL-positive spermatozoa and normal morphology) with respect to all end-points (fertilization and blastocyst development rates, implantation/clinical pregnancy and live birth) except for the prediction of fertilization with normal sperm morphology (AUC 0.572, P = 0.1320) (Table 6, Table 7, Table 8, Table 9). When comparing the predictive power of the tests by evaluating the areas under the curve (AUC), it appeared that the AUC for normal morphology was the lowest, ranging between 0.572 and 0.732, whereas the AUC for ORP was from 0.652 to 0.794 and that for the percentage of TUNEL-positive spermatozoa from 0.665 to 0.777. When the AUC for these different parameters were directly statistically compared, no differences were found for all end-points (fertilization, blastocyst development, implantation/clinical pregnancy and live birth) except for the comparison TUNEL-positive spermatozoa versus normal sperm morphology for fertilization (P = 0.0400; Table 6). Furthermore, while the predictive power for the three parameters tested were well below 70% for fertilization and implantation/clinical pregnancy, it was well above 70% for ORP and the percentage of TUNEL-positive spermatozoa for blastocyst development and live birth. For normal morphology, the predictive power was highest with about 70% for blastocyst development. The relevant calculated cut-off values for seminal ORP predicting good fertilization (>80%), blastocyst formation (>60%), implantation/clinical pregnancy and live birth were ≤0.709, ≤0.530, ≤0.465 and ≤0.393 mV/106 sperm/ml, respectively, with an average value of 0.51 mV/106 sperm/ml (Table 6, Table 7, Table 8, Table 9).
      Figure 2
      Figure 2Comparison of ROC curves of seminal ORP and the percentages of TUNEL-positive spermatozoa and sperm normal morphology for fertilization rate (≥80%) (A), blastocyst development (≥60%) (B), implantation/clinical pregnancy (C) and live birth (D). The data for the fertilization and blastocyst development rates were categorized according to benchmark classification for ICSI (
      ESHRE Special Interest Group of Embryology and Alpha Scientists in Reproductive Medicine
      The Vienna consensus: report of an expert meeting on the development of ART laboratory performance indicators.
      ). Calculations are based on the following numbers: fertilization: n = 143, blastocyst development: n = 138, implantation/clinical pregnancy: n = 134, live birth: n = 134. ICSI = intracytoplasmic sperm injection; ORP = oxidation-reduction potential; ROC = receiver operating characteristic; TUNEL = terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling.
      Table 6ROC curve analysis results for ORP, normal sperm morphology and SDF using the benchmark fertilization rate (≥80%) as variable for categorical classification
      VariableAUCCut-offSensitivity (%)Specificity (%)+PV (%)–PV (%)P-value
      ORP (mV/106 sperm/ml)0.652≤0.70975.357.164.769.00.0012
      Morphology (%)0.572>453.460.058.255.30.1320
      TUNEL (%)0.678≤1961.671.469.264.10.0001
      Comp. of AUCORP versus TUNEL:

      ORP versus morphology:

      TUNEL versus morphology:
      0.5151

      0.0950

      0.0400
      P-values <0.05 indicate statistical significance.
      AUC = area under the curve; ORP = oxidation-reduction potential; ROC = receiver operating characteristic; SDP = sperm DNA fragmentation; TUNEL = terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling.
      Table 7ROC curve analysis results for ORP, normal sperm morphology and SDF using the benchmark blastocyst development rate (≥60%) as variable for categorical classification
      VariableAUCCut-offSensitivity (%)Specificity (%)+PV (%)–PV (%)P-value
      ORP (mV/106 sperm/ml)0.794≤0.5366.382.590.350.0<0.0001
      Morphology (%)0.732>370.470.085.249.1<0.0001
      TUNEL (%)0.777<3488.855.082.966.7<0.0001
      Comp. of AUCORP versus TUNEL:

      ORP versus morphology:

      TUNEL versus morphology:
      0.6794

      0.2151

      0.3863
      P-values <0.05 indicate statistical significance.
      AUC = area under the curve; ORP = oxidation-reduction potential; ROC = receiver operating characteristic; SDP = sperm DNA fragmentation; TUNEL = terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling.
      Table 8ROC curve analysis results for ORP, normal sperm morphology and SDF using implantation/clinical pregnancy as variable for categorical classification
      VariableAUCCut-offSensitivity (%)Specificity (%)+PV (%)–PV (%)P-value
      ORP (mV/106 sperm/ml)0.680≤0.46562.771.359.674.00.0002
      Morphology (%)0.614>461.162.552.470.40.0278
      TUNEL (%)0.665≤1963.062.553.171.40.0005
      Comp. of AUCORP versus TUNEL:

      ORP versus morphology:

      TUNEL versus morphology:
      0.7332

      0.2376

      0.3856
      Implantation = cycles with ≥1 gestational sac.
      P-values <0.05 indicate statistical significance.
      AUC = area under the curve; ORP = oxidation-reduction potential; ROC = receiver operating characteristic; SDP = sperm DNA fragmentation; TUNEL = terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling.
      Table 9ROC curve analysis results for ORP, normal sperm morphology and SDF using live birth as variable for categorical classification
      VariableAUCCut-offSensitivity (%)Specificity (%)+PV (%)–PV (%)P-value
      ORP (mV/106 sperm/ml)0.728≤0.39361.576.852.283.00<0.0001
      Morphology (%)0.621>466.760.040.681.40.0420
      TUNEL (%)0.723≤1971.862.143.884.3<0.0001
      Comp. of AUCORP versus TUNEL:

      ORP versus morphology:

      TUNEL versus morphology:
      0.9100

      0.1183

      0.1504
      Live birth = cycles with ≥1 live baby delivered.
      P-values <0.05 indicate statistical significance.
      AUC = area under the curve; ORP = oxidation-reduction potential; ROC = receiver operating characteristic; SDP = sperm DNA fragmentation; TUNEL = terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling.
      Logistic regression with fertilization, blastocyst development, implantation, clinical pregnancy and live birth as dependent parameters and high/low seminal ORP (≤0.51 mV/106 sperm/ml), primary and secondary infertility, unexplained infertility, polycystic ovaries, endometriosis, tubal factor, myomectomy, ovarian insufficiency and advanced maternal age as independent variables showed overall model fits (P < 0.0002) with a significant (P < 0.0130) influence of ORP on all reproductive outcome parameters. Together with seminal ORP, female age was only included in the stepwise logistic regression model for blastocyst development, implantation/clinical pregnancy (P = 0.0002). Among the semen factors, high ORP values proved significant (P < 0.0018) in the overall model fit (P < 0.0045) for fertilization, implantation/clinical pregnancy and live birth.
      Calculation of odds ratios using the cut-off point for a normal ORP of ≤0.51 mV/106 sperm/ml obtained in this study and the various reproductive end-points (fertilization, blastocyst development, implantation/clinical pregnancy and live birth) resulted in significantly higher odds for patients achieving ≥80% fertilization (OR 0.4651; 95% CI 0.2385–0.9070; P = 0.0247), ≥60% blastocyst development (OR 0.1287; 95% CI 0.0517–0.3203; P < 0.0001), implantation/clinical pregnancy (OR 0.3163; 95% CI 0.1535–0.6517; P = 0.0018) and live birth (OR 0.2299; 95% CI 0.1005–0.5260; P = 0.0005), if the ORP was low, i.e. ≤0.51 mV/106 sperm/ml (Table 10). If the previously published cut-off value of 1.34 mV/106 sperm/ml (
      • Agarwal A.
      • Panner Selvam M.K.
      • Arafa M.
      • Okada H.
      • Homa S.
      • Killeen A.
      • Balaban B.
      • Saleh R.
      • Armagan A.
      • Roychoudhury S.
      • Sikka S.
      Multi-center evaluation of oxidation-reduction potential by the MiOXSYS in males with abnormal semen.
      ) was used, the results were only significant for fertilization and blastocyst development, but not for clinical pregnancy and live birth (data not shown).
      Table 10Odds ratios for various reproductive outcome points using a cut-off value for ORP of 0.51 mV/106 sperm/ml
      RateORPNumbers per group (%)Odds ratio(95% CI)P-value
      ≤0.51 mV/106/ml>0.51 mV/106/ml
      Fertilization
      <80%284270 (49.0%)0.4651

      (0.2385 to 0.9070)
      0.0247
      ≥80%433073 (51.0%)
      7172143
      Blastocyst development
      <60%73340 (29.0%)0.1287

      (0.0517 to 0.3203)
      <0.0001
      ≥60%613798 (71.0%)
      6870138
      Implantation / clinical pregnancy
      No314980 (59.7%)0.3163

      (0.1535 to 0.6517)
      0.0018
      Yes361854 (40.3%)
      6767134
      Live birth
      No385795 (70.9%)0.2299

      (0.1005 to 0.5260)
      0.0005
      Yes291039 (29.1%)
      6767134
      Implantation = cycles with ≥1 gestational sac, clinical pregnancy = cycles with ≥ foetal heart beat and live birth = cycles with ≥1 live baby delivered.
      P-values <0.05 indicate statistical significance.
      ORP = oxidation-reduction potential.
      A similar trend was observed in the odds ratios when the male age was taken into consideration as confounding factor (Table 11). It was also obvious that older men (≥37 years) with high ORP (>0.51 mV/106 sperm/ml) not only have a lower chance of fertilizing oocytes, but also a lower chance of good (≥60%) blastocyst development. Consequently, this will result in a lower chance of implantation/clinical pregnancy and live birth than in men with low ORP or young men. Overall, younger men have higher chances of a successful reproductive outcome.
      Table 11Odds ratios for various embryological end-points using a cut-off value for ORP of 0.51 mV/106 sperm/ml and male age as confounding factor
      Male ageReproductive outcome parameterORP cut-offOdds ratio for age groupOverall odds ratio(95% CI)P-value
      ≤0.51 mV/106/ml>0.51 mV/106/mlTotal no. of cases
      Fertilization ≥80%NoYesNoYes
      <37 years142614140.53850.5016

      (0.2545 to 0.9889)
      0.04667
      ≥37 years141728160.4706
      Total no. of cases28434230143
      Blastocyst development ≥60%NoYesNoYes
      <37 years3377210.24320.1447

      (0.0572 to 0.3657)
      0.00001
      ≥37 years42426160.1026
      Total no. of cases7613337138
      Implantation / clinical pregnancyNoYesNoYes
      <37 years19211990.42860.3272

      (0.1578 to 0.6786)
      0.0024
      ≥37 years12153090.2400
      Total no. of cases31364918134
      Live birthNoYesNoYes
      <37 years23172350.29410.2370

      (0.1028 to 0.5461)
      0.0005
      ≥37 years15123450.1838
      Total no. of cases38295710134
      Implantation = cycles with ≥1 gestational sac, clinical pregnancy = cycles with ≥ foetal heart beat and live birth = cycles with ≥1 live baby delivered.
      P-values <0.05 indicate statistical significance.
      ORP = oxidation-reduction potential.

      Discussion

      Although the WHO has standardized the procedures for conventional standard semen analysis (
      World Health Organization
      WHO Laboratory Manual for the Examination and Processing of Human Semen.
      ,
      World Health Organization
      WHO Laboratory Manual for the Examination and Processing of Human Semen.
      ), it cannot predict male fertility and fertilizing potential of spermatozoa (
      • Nagler H.M.
      Male factor infertility: a solitary semen analysis can never predict normal fertility.
      ;
      • Wang C.
      • Swerdloff R.S.
      Limitations of semen analysis as a test of male fertility and anticipated needs from newer tests.
      ). Because the fertilization process is multifactorial (
      • Henkel R.
      • Maaß G.
      • Bödeker R.-H.
      • Scheibelhut C.
      • Stalf T.
      • Mehnert C.
      • Schuppe H.C.
      • Jung A.
      • Schill W.-B.
      Sperm function and assisted reproduction technology.
      ), any given test can only provide a limited probability for successful fertilization and even less forecast a positive reproductive outcome. Consequently, conventional semen parameters provide only a limited prognosis for adequate sperm function and do not identify the cause of the infertility in about 30–50% of cases (
      • Chehab M.
      • Madala A.
      • Trussell J.C.
      On-label and off-label drugs used in the treatment of male infertility.
      ;
      • Jungwirth A.
      • Giwercman A.
      • Tournaye H.
      • Diemer T.
      • Kopa Z.
      • Krausz C.
      European Association of Urology Working Group on Male Infertility (2012); European Association of Urology guidelines on Male Infertility: the 2012 update.
      ). Hence, these cases remain idiopathic or unexplained and frequently do not receive the necessary treatment. Due to the problems of standard semen analysis in predicting male fertilizing potential, additional sperm function tests including tests for sperm DNA damage and oxidative stress, i.e. tests for SDF and ROS, have been developed.
      While the clinical value of SDF testing is gradually being acknowledged and recommended (
      • Tharakan T.
      • Bettocchi C.
      • Carvalho J.
      • Corona G.
      • Jones T.H.
      • Kadioglu A.
      • Martínez Salamanca J.I.
      • Serefoglu E.C.
      • Verze P.
      • Salonia A.
      • Minhas S.
      EAU Working Panel on Male Sexual Reproductive Health
      A clinical consultation guide on the indications for performing sperm DNA fragmentation testing in men with infertility and testicular sperm extraction in nonazoospermic men.
      ;
      World Health Organization
      WHO Laboratory Manual for the Examination and Processing of Human Semen.
      ), tests for oxidative stress are still regarded as research tests. Traditionally, seminal oxidative stress is measured by direct determination of the ROS using luminescent probes such as luminol or lucigenin (
      • Aitken R.J.
      • Buckingham D.W.
      • West K.M.
      Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence.
      ). Alternatively, the determination of the total antioxidant capacity (
      • Sharma R.K.
      • Pasqualotto F.F.
      • Nelson D.R.
      • Thomas A.J.Jr
      • Agarwal A.
      The reactive oxygen species – total antioxidant capacity score is a new measure of oxidative stress to predict male infertility.
      ) has been suggested as an indirect measure for oxidative stress. However, these techniques suffer from high variation in results and are not generally accepted for routine use. A novel galvanostatic technique measures the balance between oxidation and reduction, the ORP, and has been shown to distinguish between sperm donors and infertile patients (
      • Agarwal A.
      • Roychoudhury S.
      • Bjugstad K.B.
      • Cho C.L.
      Oxidation-reduction potential of semen: what is its role in the treatment of male infertility?.
      ). However, it is still considered experimental, as there is insufficient evidence to correlate seminal ORP with reproductive outcomes. To the best of our knowledge, the current study is the first presenting evidence for the predictive value of seminal ORP measurement with regard to reproductive end-points, namely fertilization, blastocyst development, implantation/clinical pregnancy and live birth after ICSI.
      As expected, seminal ORP was significantly negatively correlated with sperm concentration, motility and normal morphology (
      • Agarwal A.
      • Henkel R.
      • Sharma R.
      • Tadros N.N.
      • Sabanegh E.
      Determination of seminal oxidation-reduction potential (ORP) as an easy and cost-effective clinical marker of male infertility.
      ;
      • Cicek O.S.Y.
      • Kaya G.
      • Alyuruk B.
      • Doger E.
      • Girisen T.
      • Filiz S.
      The association of seminal oxidation reduction potential with sperm parameters in patients with unexplained and male factor infertility.
      ) and significantly positively with male age (
      • Nago M.
      • Arichi A.
      • Omura N.
      • Iwashita Y.
      • Kawamura T.
      • Yumura Y.
      Aging increases oxidative stress in semen.
      ) and SDF (
      • Arafa M.
      • Henkel R.
      • Agarwal A.
      • Majzoub A.
      • Elbardisi H.
      Correlation of oxidation-reduction potential with hormones, semen parameters and testicular volume.
      ). While the former associations indicate the negative impact of seminal oxidative stress on sperm functions, the latter associations, although only weak, are in line with the age-related increase in seminal ROS concentrations (
      • Cocuzza M.
      • Athayde K.S.
      • Agarwal A.
      • Sharma R.
      • Pagani R.
      • Lucon A.M.
      • Srougi M.
      • Hallak J.
      Age-related increase of reactive oxygen species in neat semen in healthy fertile men.
      ). Yet, logistic regression revealed that ORP is independent from female and other semen parameters. This not only explains the significant associations between male age and the reproductive outcomes, but also the observation that older men (≥37 years) with high seminal oxidative stress (>0.51 mV/106 sperm/ml) have lower chances of their spermatozoa resulting in successful reproductive outcome than men of the same age group with low seminal ORP values (≤0.51 mV/106 sperm/ml). This further implies that an antioxidant treatment or lifestyle changes leading to a decrease in oxidative stress may result in increased fertilization and improved blastocyst development rates.
      ROC curve analyses for normal sperm morphology, SDF and seminal ORP were performed in this study for fertilization, blastocyst development, implantation/clinical pregnancy and live birth. All tested sperm/semen parameters significantly predicted the reproductive outcome parameters included in this study with predictive powers between 61.4% (normal morphology for implantation/clinical pregnancy) and 79.4% (ORP for good blastocyst development). Notably, normal sperm morphology showed the worst predictive power for all classification variables. This might be because in this study all patients were treated with ICSI, where a selection of the best morphologically appearing vital spermatozoa for injection is performed. When the AUC were statistically compared, no differences between the AUC of the sperm/semen parameters, except between SDF and morphology for fertilization, were observed. In fact, the AUC of SDF and ORP were comparable with those reported for SDF in previous studies (
      • Cissen M.
      • Wely M.V.
      • Scholten I.
      • Mansell S.
      • Bruin J.P.
      • Mol B.W.
      • Braat D.
      • Repping S.
      • Hamer G.
      Measuring sperm DNA fragmentation and clinical outcomes of medically assisted reproduction: a systematic review and meta-analysis.
      ;
      • Muratori M.
      • Marchiani S.
      • Tamburrino L.
      • Cambi M.
      • Lotti F.
      • Natali I.
      • Filimberti E.
      • Noci I.
      • Forti G.
      • Maggi M.
      • Baldi E.
      DNA fragmentation in brighter sperm predicts male fertility independently from age and semen parameters.
      ;
      • Wiweko B.
      • Utami P.
      Predictive value of sperm deoxyribonucleic acid (DNA) fragmentation index in male infertility.
      ).
      Interestingly, while cut-off values for SDF and normal sperm morphology were calculated for all reproductive outcome points in the range of what was previously published, between 19% and 36.5% for SDF and 4% for normal morphology, respectively, the cut-off point for ORP with an average of 0.51 mV/106 sperm/ml was markedly lower than those (1.34, 1.36 and 1.38 mV/106 sperm/ml) published previously (
      • Agarwal A.
      • Roychoudhury S.
      • Sharma R.
      • Gupta S.
      • Majzoub A.
      • Sabanegh E.
      Diagnostic application of oxidation-reduction potential assay for measurement of oxidative stress: clinical utility in male factor infertility.
      ,
      • Agarwal A.
      • Panner Selvam M.K.
      • Arafa M.
      • Okada H.
      • Homa S.
      • Killeen A.
      • Balaban B.
      • Saleh R.
      • Armagan A.
      • Roychoudhury S.
      • Sikka S.
      Multi-center evaluation of oxidation-reduction potential by the MiOXSYS in males with abnormal semen.
      ;
      • Arafa M.
      • Agarwal A.
      • Al Said S.
      • Majzoub A.
      • Sharma R.
      • Bjugstad K.B.
      • AlRumaihi K.
      • Elbardisi H.
      Semen quality and infertility status can be identified through measures of oxidation–reduction potential.
      ). A possible explanation for this discrepancy could be that these values were not calculated by using reproductive outcomes in an assisted reproduction programme, but by distinguishing between men with normal/abnormal semen parameters and donors/patients, respectively. However, when considering the clinical value of a specific test in andrological diagnostics, reproductive outcome parameters should be used. Therefore, a lower cut-off value for seminal ORP than the previously published ones seems reasonable and a cut-off of ≤0.51 mV/106 sperm/ml is proposed. However, at this stage, it must also clearly be stated that this value still needs to be validated in larger, more controlled studies. This value might also differ for IVF or for intrauterine insemination (IUI) because for IVF and IUI all sperm functions have to be in the optimum range to achieve a clinical pregnancy and live birth.
      In many studies trying to evaluate sperm functional tests, fertilization is used as the primary reproductive end-point without further exploring possible adverse effects in early embryo development, implantation/clinical pregnancy and live birth. However, considering that spermatozoa with DNA damage can still fertilize oocytes (
      • Henkel R.
      • Hajimohammad M.
      • Stalf T.
      • Hoogendijk C.
      • Mehnert C.
      • Menkveld R
      • Gips H.
      • Schill W.-B.
      • Kruger T.F.
      Influence of deoxyribonucleic acid damage on fertilization and pregnancy.
      ), this might not be the best end-point to assess sperm functional capability. In addition, one has also to consider that besides the fertilizing capacity of spermatozoa, blastulation depends on the quality of the oocyte as well as the culture conditions. Moreover, the occurrence of clinical pregnancy and live birth depend on a good embryo transfer, endometrial receptivity as well as on female variables such as pre-eclampsia, systemic diseases or preterm labour. Based on this, it is obvious that the AUC and the odds ratios diminished steadily from fertilization to live birth. This can clearly be seen in the current results. Therefore, later reproductive end-points were used in this study.
      Because no difference between the AUC for ORP and SDF was observed, the question arises as to which assay might be clinically most suitable. In this study, the association between SDF and seminal oxidative stress measured as ORP was confirmed (
      • Homa S.T.
      • Vassiliou A.M.
      • Stone J.
      • Killeen A.P.
      • Dawkins A.
      • Xie J.
      • Gould F.
      • Ramsay J.W.A.
      A comparison between two assays for measuring seminal oxidative stress and their relationship with sperm DNA fragmentation and semen parameters.
      ), but it should not be forgotten that DNA damage can also occur due to defective or failing DNA repair mechanisms (
      • Puzuka A.
      • Alksere B.
      • Gailite L.
      • Erenpreiss J.
      Idiopathic infertility as a feature of genome instability.
      ). Hence, both tests complement each other. However, when considering time, labour and cost, these factors seem to be in favour of the determination of ORP. As reported previously (
      • Rex A.S.
      • Aagaard J.
      • Fedder J.
      DNA fragmentation in spermatozoa: a historical review.
      ), the TUNEL assay is labour-intensive. In contrast, the analysis of seminal ORP requires only about 4 min following liquefaction. Hence, the result can be discussed with the patient on the same day alongside possible treatment options.
      Although this study clearly shows the impact of seminal oxidative stress on reproductive outcome in a wider range of male patients, the study has limitations. First, the use of ICSI might have contributed to the fact that the calculated cut-off value of ORP was lower than the previously published values because sperm functions such as motility or acrosome reaction did not play a role in the fertilization process. In contrast, for IUI and IVF, these sperm functions play an essential role as capacitation has to be triggered by a small amount of ROS. Therefore the cut-off value of ORP may be different for IUI or IVF. Second, the use of a fluorescence microscopic TUNEL assay limited the number of spermatozoa that were evaluated. Third, this study was conducted using a non-select cohort of patients in whom seminal ORP was determined and related to reproductive outcomes. Further prospective studies need to confirm the current findings to see whether the management of patients on the basis of the ORP results can improve clinical outcomes in infertile patients.
      In conclusion, this is the first study showing the impact of seminal ORP on fertilization, blastocyst development, implantation/clinical pregnancy and live birth as reproductive outcome parameters with an average cut-off value of 0.51 mV/106 sperm/ml in an ICSI programme. However further evaluation, not only by carefully establishing a physiological range of ORP for normal sperm functions, but also by conducting larger studies including IVF and IUI, are necessary to clearly establish the predictive capabilities of ORP in andrological diagnostics for assisted reproduction. An early and reliable detection of seminal oxidative stress can help to further personalize ART treatment in male infertility patients and guide clinicians in counselling patients. Such an approach could then lead to an increase in successful fertility treatments or the use of less invasive approaches, thereby leading to better cost-effectiveness in healthcare systems. However, at this stage, it must also be pointed out that the measurement of seminal ORP cannot be offered to patients with severe oligozoospermia or azoospermia. Based on these findings, it can be concluded that ORP is a useful parameter to be incorporated in the evaluation of male infertility. In order to uncover the impact of seminal ORP on other sperm functional parameters, which play an essential role in natural conception as well as for IVF or IUI, relevant studies are under way.

      Data availability

      The data underlying this article will be shared on reasonable request to the corresponding author.

      Acknowledgements

      The authors wish to thank Dr Rupin Shah, Division of Andrology, Department of Urology, Lilavati Hospital and Research Centre, Mumbai, India, for reviewing the manuscript. The authors are grateful to Aytu Bioscience, Englewood, CO, for providing the MiOXSYS system and the relevant sensors. AM was financially supported by a DAAD-NRF In-Country Scholarship (grant number: 118279).

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      Biography

      Ralf Henkel studied Biology/Chemistry in Marburg, Germany, and obtained his PhD in 1990. In 2004, he became Professor in the Department of Urology at the University of Jena, and at UWC, Bellville, South Africa, from 2005 until 2020. Since 2020, he has been at LogixX Pharma and Imperial College London, UK.
      Key message
      Seminal oxidation-reduction potential (ORP) is predictive of fertilization, blastocyst development, implantation, clinical pregnancy and live birth after ICSI. Because ORP measurement showed comparable predictive value to the TUNEL assay, it can be a quicker and cheaper alternative to the determination of DNA damage.