WO2001032802A1 - Procede et appareil permettant de prevoir l'infertilite masculine - Google Patents

Procede et appareil permettant de prevoir l'infertilite masculine Download PDF

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WO2001032802A1
WO2001032802A1 PCT/US2000/029839 US0029839W WO0132802A1 WO 2001032802 A1 WO2001032802 A1 WO 2001032802A1 US 0029839 W US0029839 W US 0029839W WO 0132802 A1 WO0132802 A1 WO 0132802A1
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ros
tac
oxidative stress
score
value
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WO2001032802A9 (fr
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Ashok Agarwal
Kumar Sharma Rakesh
David Roland Nelson
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The Cleveland Clinic Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

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  • the present invention relates generally to a method and apparatus for predicting male infertility, and more particularly to a method and apparatus for quantifying oxidative stress in a bodily sample obtained from a test subject.
  • Infertility is defined as the inability of a couple to initiate pregnancy following one year of unprotected sex. Semen analysis in the male can help determine whether spermatozoa are being produced or if there are any abnormalities in the seminal ejaculate and the spermatozoa themselves. As of today no single sperm parameter or laboratory test can reliably predict the quality, fertilizing ability, or ability to achieve conception in a given semen specimen. Individual semen parameters such as sperm count, motility, or morphology show poor predictive power for achieving pregnancy. Therefore, a battery of tests are necessary to determine overall semen quality. Some of these semen tests are simple whereas others are complicated and are used by only a handful of laboratories.
  • Some of the methods currently used to predict male infertility include manual semen analysis, computer assisted semen analysis, the Endtz test for Leukocytospermia, the hypoosmotic swelling test, and the ovine cervical mucus penetration tests. More advanced tests include sperm penetration assay using hamster eggs, the acrosome reaction, assessment of sperm-zona pellucida binding assay, and measurement of creative kinase More complex tests involve, identification of mannose receptors on the spermatozoa, fluorescence in situ hybridization (FISH) for chromosomal abnormalities, flow cytometry tests for extent of DNA damage, measurement of free radical generation and measurement of antioxidants.
  • FISH fluorescence in situ hybridization
  • the present invention provides a new method and apparatus for quantifying oxidative stress in a bodily sample obtained from a test subject. Such method and its result provides useful information to a trained medical professional for their diagnosis of diseases that are associated with or caused by oxidative stress. Such method is especially useful for predicting male infertility.
  • the method comprises the steps of: determining the levels of reactive oxygen species in a bodily sample obtained from the subject; determining the antioxidant capacity of the bodily sample; generating a ROS-TAC score by obtaining a weighted sum of the ROS value and the TAC value.
  • the present invention also provides a method of diagnosing infertility in a male subject.
  • the method comprises the steps of: determining the levels of reactive oxygen species in a seminal ejaculate sample obtaind from the subject, determining the levels of total antioxidant species in the sample, and calculating the ROS-TAC score of the sample.
  • a low ROS-TAC score of the sample as compared to a standard ROS-TAC score, which is determined from samples obtained from a population of healthy control subjects indicates the test subject is likely to be infertile.
  • Standard ROS-TAC values are, by way of example, mean levels, median levels, or preferably "cut-off levels, which are established by determining ROS-TAC values in comparable samples obtained from a population of normal healthy subjects.
  • the diagnostic method further comprises detecting the presence of granulocytes and, preferably, determining the concentration of sperm.
  • the present invention also relates to an apparatus for quantifying oxidative stress in a bodily sample.
  • the apparatus is a microprocessor for calculating the ROS-TAC score.
  • the apparatus also comprises a means for measuring the reactive oxygen species in a bodily sample. More preferably, the apparatus further comprises a means for measuring the total antioxidant content of the sample.
  • Figure 2 illustrates an example of a receiver operating characteristic (ROS) curve.
  • ROS receiver operating characteristic
  • FIGS 3 and 4 illustrate examples of receiver operating characteristic (ROS) curves correlated with fertility as a predictor.
  • ROS receiver operating characteristic
  • the present invention provides a method of determining whether cells that are in contact with a bodily fluid in a test subject are experiencing oxidative stress and are, therefore, likely to be malfunctioning.
  • the method comprises the steps of determining the levels of reactive oxygen species (ROS) in a body sample from the test subject; determining the antioxidant capacity, preferably, the total antioxidant capacity (TAC), of the body sample; and then correlating these values to obtain an
  • ROS-TAC score A low ROS-TAC score in a sample obtained from an individual test subject as compared to the ROS-TAC score of comparable samples obtained from a population of healthy control subjects indicates that the cells and components in the bodily sample are experiencing oxidative stress.
  • the body sample is a fluid such as seminal ejaculate, blood, cerebrospinal fluid, follicular fluid, or endometrial fluid.
  • the ROS-TAC score of a seminal ejaculate obtained from a test subject is a good predictor of male infertility.
  • the ROS-TAC score of a follicular fluid or peritoneal fluid sample obtained from a test subject may also be a good predictor of female infertility.
  • the ROS-TAC score of a cerebrospinal fluid sample obtained from a test subject may be a good diagnostic marker for a neurological disease in the subject.
  • the ROS levels be measured in the wash spermatazoa and the total nonenzymatic antioxidant capacity (TAC) in the seminal plasma be determined.
  • TAC total nonenzymatic antioxidant capacity
  • the number of granulocytes in the sample also be determined. This value is useful for distinguishing between oxidative stress that is due to an infection, which is treatable with antibiotics, and oxidative stress that is due to abnormal or defective spermatazoa, which may be treatable by administering antioxidants to the test subject.
  • levels of reactive oxygen species in a body sample are well known in the art. Examples of such methods are the tetrazolium nitroblue method which uses ferricytochrome C to directly measure the levels of ROS and an indirect method which measures lipid peroxidation levels in the sample.
  • levels of ROS are measured by a chemiluminescence assay using luminol(5-amino-2, 3 dihydro-1, 4 pthalazinedione) as the probe.
  • Luminol reacts with the free radicals in the sample thereby converting the chemical signal into an electrical signal which is recorded as counted photons per minute (cpm).
  • Chemiluminescent assay can be used to measure ROS in cellular tissues or body fluids containing cellular components that are capable of generating reactive oxygen species.
  • the levels of total nonenzymatic antioxidant species are determined using a chemiluminescence assay, more preferably an enhanced chemiluminscence assay.
  • Enhanced chemiluminescence assay for TAC can be used to screen tissue homogenates and in a variety of biological fluids including cerebrospinal fluid, urine, saliva, synovial fluid, peritoneal fluid, follicular fluid and serum.
  • Semen samples are obtained from normal healthy volunteers or patients. Semen specimens are allowed to liquefy, preferably by incubating the sample for 30 minutes at 37°C.
  • the sample is assayed to determine whether granulocytes are present in the semen.
  • an assay that is used to measure the number of white blood cells in a sample is the Endtz or myeloperoxidase test, which is well known in the art.
  • ROS Reactive Oxygen Species
  • TAC Total Antioxidant Capacity
  • ROS-TAC Score The development of a scale to measure oxidative stress is based on utilizing two measures of oxidative stress, reactive oxygen species (ROS) and total antioxidant capacity (TAC). These two measures are negatively correlated.
  • the ROS-TAC score is derived from a weighted sum of ROS and TAC.
  • ROS-TAC score In Predicting Fertility Based on the ROS-TAC score in normal healthy fertile males who have initiated pregnancy in the last one year, the probabilities of initiating a natural (unassisted) pregnancy in a given population of infertile population can be predicted based on the assumption that there is no female-factor involved.
  • the ROS-TAC score is independent of the semen quality, sperm concentration, percent motility or any other sperm motion characteristics
  • ROS-TAC score has a good ability to predict pregnancy in couples who have no female factor and are trying natural intercourse only (no assisted reproductive method such as in vitro fertilization) to achieve pregnancy. This score is independent of all other diagnostic tests of semen. It is simple, accurate and cost-effective. The score seems to be a more reliable predictor of fertility across the board in patients with various clinical diagnoses of male infertility. .
  • ROS-TAC score can be used as a screening tool for infertility risk in patients prior to their physical examination. Those infertility patients with abnormal oxidative stress may be treated with antioxidant supplementation. Application Of ROS-TAC To Other Diseases
  • ROS farnesoid lipids
  • DNA proteins, lipids, and DNA are all substrates for ROS attack
  • ROS levels can cause a variety of diseases and even death.
  • ROS has been implicated in astherosclerosis, cardiovascular disease, cardiac ischemia-reperfusion injury, aging, cancer, inflammation/sepsis and a variety of neurodegenerative disorders such as Alzheimer's disease.
  • Leukocytospermia is defined as the presence of at least 1 X 10 6 WBC/mL of ejaculate.
  • Luminol 5-amino-2, 3 dihydro-1, 4 pthalazinedione
  • test control has 40 X 10 6 sperm/mL, and patient has 20 X 10 6 /mL sperm concentration. Wash and adjust sperm concentration to 20 X 10 6 /mL. Take 7 tubes, one blank, 2 assay control and 4 test, two for test 1 (Donor) and 2 for test (patient). Add 400L buffer to the first 3 tubes and 400L of sperm suspension to tube 4 through 7. Prepare 5 mM luminolin dimethyl sulfoxide and add 10L to all tubes except tube 1. With the luminometer in the integrated mode, measure luminescence for 15 minutes. A table of typical values is shown in Table 1. TABLE 1
  • the ROS for Test-control is 20.0 X 10 4
  • the ROS for Patient is 330.5 X 10 4 cpm.
  • Signal reagent consists of assay buffer, and tablet A and B containing luminol, enhancer and oxidant.
  • a constant source of ROS is produced by horseradish peroxidase-linked immunoglobulin Ig. (HRP). 4. To prepare working HRP solution, add 20 ⁇ L of HRP to 4.9
  • Standard Trolox concentrations 150, 100 and 50M
  • seminal plasma after diluting 10 times are run for measuring chemiluminescence in the kinetic mode.
  • Data from the luminometer files is entered into an Excel spreadsheet and the average recovery time and the delay times are calculated to give average recovery time for each concentration.
  • a slope is generated and the equation for straight line is obtained. This information is then plotted for the actual test readings to calculate the average recovery time for test samples.
  • Average recovery time for 3 standard concentrations of 150, 100 and 50M are
  • log (ROS+1) is denoted as 'X' and TAC will be denoted as 'Y.' 2.
  • the ROS-TAC score was developed and scaled using a sample of normal, healthy males. First, both TAC and log (ROS +1) were standardized to z-scores so that both would have the same variability. A 'z-score' takes the original value and subtracts the mean of the sample, it is then divided by the standard deviation. This created a distribution of a mean of 0 and standard deviation of 1 for both variables.
  • the generalized equations are: for log (ROS + 1):
  • ROS-TAC score 50 +('Principal component' x 'Z')
  • ROS-TAC score 50 +(Principal component x 10.629).
  • ROS-TAC score with ROS and TAC are shown in FIG. 1.
  • the following is an example of the calculation based on a hypothetical specimen:
  • ROS-TAC score 50 +[(-0.707 X -0.46)+(0.707 X 1.59)] X 10.629
  • ROS-TAC score 50 + [(0.11) + (1.12)]
  • X 10.629 50 + 15.43 65.44
  • ROS-TAC score The area under a ROC curve ranges from 50% to 100%, with 100% indicating a perfect predictor and 50% indicating random chance, or no predictive ability.
  • the ROC curves indicated that using a cutoff of '45' maximized the sum of sensitivity and specificity in the vasectomy reversal group (see Kolettis et al, 1999, attached). This cutoff provided 73% sensitivity and 82% specificity of the ROS-TAC score to predict fertility.
  • the probability of successful pregnancy was estimated at less than 10% for low values of the ROS-TAC score ( ⁇ 30), but increased with the increase in the ROS-TAC score.
  • the expected pregnancy rates for a patient with a ROS-TAC score of 30 would be 13.9%, 21.0%, and 31.6% for 12, 24, and 3 6 months respectively; whereas a score of 50 would have expected pregnancy rates of 35.1%,48.9%, and 54.3 % over the same intervals.
  • Receiver-operating characteristic (ROS) curves provided the sensitivity and specificity over the entire range of ROS-TAC scores, and '45' provided the maximum value of the sum of sensitivity + specificity.
  • An example of a Receiver-operating characteristic (ROS) curve is shown in FIG. 2.
  • Sensitivity is the ratio of "true positives” divided by the sum of (true positive + false negatives). In this case, it is, within the subset of all the fertile men, what percentage had ROS-TAC scores over '45.' Since the sensitivity was 73%, therefore, 27% of the fertile males had R O S-TAC scores below 45.
  • Specificity is the ratio of "true negatives” divided by the sum of (true negative + false positives). In this case, it is, within the subset of all the infertile men, what percentage had ROS-TAC scores below '45.' Therefore 18 % of the infertile males had ROS-TAC scores above 45, because the specificity was 82%.
  • ROS reactive oxygen species
  • TAC vasectomy
  • a male infertility clinic of a tertiary care center A male infertility clinic of a tertiary care center.
  • ROS-AC score a composite index of seminal oxidative stress, was a significant predictor of fertility.
  • a ROS-TAC score of 45 or greater had a positive predictive value of 73 % in predicting fertility.
  • Oxidative stress has been shown to be an important cause of male infertility (5-7).
  • ROS reactive oxygen species
  • Reactive oxygen species are highly unstable free oxygen radicals, including hydrogen peroxide, hydroxyl radical, and superoxide anion. These radicals are produced in small amounts by normal spermatozoa and are implicated in signal transduction. Elevated seminal ROS, however, can cause sperm dysfunction through lipid peroxidation of the polyunsaturated fatty acids in the sperm membrane (5-7).
  • Semen specimens were obtained by masturbation after a minimum of 2 days of sexual abstinence.
  • Computer-assisted semen analysis was performed on all specimens, with use of a Motion Analysis VP-50 semen analyzer (Motion Analysis Corporation, Santa Rosa, CA). All counts were verified manually.
  • Ten semen characteristics, including volume, sperm concentration, percent motility, and sperm morphology, were assessed according to the criteria defined by the World Health Organization, and by Kruger's strict criteria (16, 17).
  • the Endtz test myeloperoxidase staining was performed on all specimens to measure the concentration of granulocytes (18). Reactive Oxygen Species Measurement
  • phthalazinedione Sigma Chemical Co., St. Louis, MO
  • dimethylfulfoxide Sigma Chemical Co.
  • Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble tocopherolanalogue, was added as the standard at concentrations
  • chemiluminescence output between 2 and 3 x 10 cpm
  • desired level of chemiluminescence output between 2 and 3 x 10 cpm
  • One hundred microliters of the prepared seminal plasma was added immediately to the signal reagen and HRP, and the chemiluminescence was measured. Suppression of chemiluminescence and the time from the addition of seminal plasma to 10% recovery of the initial chemiluminescence were recorded. Antioxidant capacity was expressed as molar Trolox equivalents.
  • Fertility defined as pregnancy resulting from intercourse within 1 year after vasectomy reversal
  • infertility defined as no pregnancy from intercourse during the year after vasectomy reversal
  • CI 95 confidence intervals
  • ROS-TAC score 50 +(Principal component x 8.97)
  • Receiver operating characteristic curves were used to examine the diagnostic ability of the ROS-TAC score to predict fertility.
  • the ROC curves illustrate the sensitivity and specificity over the entire range of the ROS-TAC score.
  • the area under the curve can range from 50% to 100% with diagnostic tests that approach 100%, indicating a perfect predictor and 50%, indicating random chance or no predictive ability.
  • Statistical significance among the three groups was assessed with two-tailed tests at the P ⁇ 05 level.
  • Statistical test were performed using SAS version 6.12 (SAS Institute, Cary, NC) RESULTS
  • the mean ( ⁇ SD) are of the infertile vasectomy reversal patients was 42.7 ⁇ 1.5 years, of the fertile vasectomy reversal patients was 44 ⁇ 1.8 years, and of the healthy donors was 31.1+ 2.1 years.
  • the healthy donors were significantly younger than both groups of vasectomy reversal patients (P ⁇ .001).
  • VE vasoepididymostomy
  • W vasovasotomy
  • VSL Com s 13.89 + 0.82 10.45 £ 1.13 10.25 + 1.10 .009 .01 .008 .002 .90
  • TAC levels were 1,654 ⁇ 121 for the donors, 1,955 ⁇ 222 for the fertile reversal patients, and 1,527 ⁇ 170 for the infertile reversal patients (Table 4). The differences in TAC were not statistically significant among the groups. Based on the sample sizes of fertile and infertile patients, this study had 90% power to detect a difference of 925 Trolox equivalents between the two groups.
  • the percentage of sperm body by IgA was 60.8 % ⁇ 8.3 % in the
  • Elevated levels of seminal ROS have been demonstrated in 4 0%-88% of infertile men (11). - Elevated seminal ROS can damage the sperm membrane through lipid peroxidation, causing lower fertilization rates in vitro (20). Depressed seminal antioxidant capacity also has been implicated in male infertility. Both TAC and individual antioxidant levels have been shown to be lower in the semen of infertile men (11, 12).
  • ROS-TAC score combines these two measurements to compensate for this differing variability. Indeed, the ROS-TAC score did correlate with fertility in our study.
  • ROS-TAC score below 45 had a sensitivity for infertility of 73 % and a specificity of 82%. This index requires further validation in other populations of infertile men.
  • Empirical trials of antioxidant supplementation in infertile men have shown encouraging results. Improved semen characteristics and higher rates of fertilization in vitro have resulted from intramuscular glutathione and oral vitamin E therapy, respectively (13, 14). Furthermore, a trial of oral vitamin E in men with asthenospermia resulted in a higher pregnancy rate in the treatment group (15). Our findings demonstrate oxidative stress in men after vasectomy reversal and suggests a rationale for empiric antioxidant supplementation in this group of men as well.
  • the imbalance between reactive oxygen specks (ROS)production and total antioxidant capacity (TAC) in seminal fluid indicates oxidative stress and is correlated with male infertility.
  • a composite ROS-TAC score may be more strongly correlated with infertility than ROS or TAC alone.
  • ROS, TAC, and ROS-TAC scores in semen from 127 patients and 24 healthy controls. Of the patients, 56 had varicocele, eight had varicocele with prostatitis, 35 had vasectomy reversals, and 28 had idiopathic infertility.
  • ROS levels were higher among infertile men, especially those with varicocele with prostatitis (mean ⁇ SE, 3.25 ⁇ .89) and vasectomy reversals (2.65 ⁇ 1.01). All infertile groups had significantly lower ROS-TAC scores than control. ROS-TAC score identified 80% of patients and was significantly better than ROS at identifying varicocele and idiopathic infertility. The 13 patients whose partners later achieved pregnancies had a mean ROS-TAC score of 47.7 + 13.2, similar to controls but significantly higher than the 3 9 patients who remained infertile (35.8 + 15.0; P ⁇ 0.01).
  • ROS-TAC score is a novel measure of oxidative stress and is superior to ROS or TAC alone in discriminating between fertile and infertile men.
  • Infertile men with male factor or idiopathic diagnoses had significantly lower ROS-TAC scores than controls, and men with male factor diagnoses that eventually were able to initiate a successful pregnancy had significantly higher ROS-TAC scores than those who failed.
  • Various clinical diagnoses are unable to determine the underlying cause of sperm dysfunction and pathophysiology of infertility.
  • the distribution of Endtz positive specimen was: varicocele (3/5 6, 5.3 %), varicocele with prostatitis (4/8, 5 0%), vasectomy reversal (2/3 5, 5.7%), and idiopathic infertility (2/2 8, 7.1%).
  • Total antioxidant capacity was measured in the seminal plasma using an enhanced chemiluminescence assay (Kolettis et al. inter 1999). Aliquots of the seminal plasma were thawed at room temperature and assessed for their antioxidant capacity.
  • Millipore filter Allegiance Health Care Corporation, IL. USA.
  • Signal reagent was prepared using a chemiluminescence kit (Amersham Life Science, Buckingham, UK).
  • a Constant source of ROS was produced by horseradish peroxidase-linked immunoglobulin (HRP-linked lg: Amersham Life Science, Buckingham, UK).
  • HRP-lg horseradish peroxidase-linked immunoglobulin
  • Twenty microlitres of HRP-lg was added to 4.98 ml dH 2 0 and further diluted l .T to give a desired chemiluminescence output (3 x 10 7 c.p.m.).
  • Trolox (6-hydroxy-2.5.7.8-tetramethylchroman-2-carboxylic acid) a water-soluble tocopherolanalogue was added as the standard at concentrations
  • ROS-TAC score 50 +(Principal components x 10.629)
  • ROS reactive oxygen species
  • TAC total antioxidant capacity
  • Vasectomy reversal (infertile, n -23) 265" ⁇ 101 00004 138989- -72392 030 3322- ⁇ 1524 00002
  • Vasectomy reversal (ferule, n - 12) 176-086 080 187693 ; - 75082 062 4935- '1225 100
  • ROS TAC score and area under the receiver operator characteristic (ROC) curve showing the prcdicitvc value ot reactive oxygen species (ROS) total antioxidant capacity (TAC), and ROS-TAC score
  • Infertile varicocele (n - 55) 689 (570 - 807) 802(700-905) 808(713-903) 0002* 092 Infertile varicocele with prostatitis (n - 8) 948(868-100) 828(642-100) 932(804-100) 070 009 Infertile vasectDmy rcvereal ( ⁇ - 23) 846 (734 • 958) 670(513-829) 808(676-940) 043 004* Idiopathic infertility (n - 28) 743(604-881) 818(705-932) 845(741-949) 0005* 068
  • Receiver operator characteristics (ROC) curves were used to examine the diagnostic ability of ROS-TAC score to predict fertility.
  • ROC curves illustrate the sensitivity and specificity over the entire range of the ROS-TAC score. The area under the curve can range from 50% to 100% with diagnostic tests that approach 100% indicating a perfect predictor and 50%) indicating random chance, or no predictive ability.
  • the total antioxidant capacity was significantly higher in the control group (1650.93 - 532.22) than in any other infertile groups (P ⁇ 0.006), with the exception of the infertile vasectomy reversal group.
  • the TAC in fertile vasectomy reversal patients did not differ significantly from the controls. ROS-TAC score
  • the area under the curve for ROS was less than 80% in both the infertile varicocele group (68.9%>: 95 % Ci, 5 7.0 - 8 0.7%) and idiopathic infertility group (74.3 %: 95% CI. 60.4-88.1%).
  • FIG. 2 Shows predicted 1-year pregnancy rate (and 95 % confidence interval) over the range of the ROS-TAC score for men with either varicocele or vasectomy reversal from, logistic regression results.
  • the TAC measure was, greater than 8 0% successful in discriminating infertile varicocele, varicocele with prostatitis, and idiopathic infertility groups. Howerver, the area under the curve for infertile vasectomy reversal was 67.0%> (95 %> CI, 51.2 -
  • the level of the confidence interval is only slightly greater than random chance (50%>).
  • the ROS-TAC score was significantly better at discriminating infertility among all diagnostic groups versus controls than either ROS or TAC alone. For all the infertile diagnoses, the area underthe curve was greater than 80%>.
  • the probability of successful pregnancy was estimated at less than 10 % for values of ROS-TAC score less than 3 0, but increased as the ROS-TAC score increased.
  • the expected pregnancy rates for a patient with a ROS-TAC score of 3 0 would be 13.9, 21.0, and 31.6%o for 12, 24, and 36 months respectively; whereas a score of 50 would have expected pregnancy rates of 35.1, 48.9 and 5 4.3 % over the same intervals.
  • the estimated number of successful pregnancies among the 2 8 idiopathic infertility patients (assuming no female factor diagnoses) over, a 12-month interval is 5.6 pregnancies (9 5 % CI: 3.2 - 12.8). Discussion
  • Oxidative injury to spermatozoa is a major cause of spermatozo al dysfunction, and total nonenzymatic antioxidant defences in human seminal plasma are inversely related- to lipid peroxidation (Smith et al., 1996). Many studies have demonstrated the association of lipid peroxidation with mid-piece abnormality, decreased sperm count, motility, and loss of the capacity of the spermatozoon to undergo the acrosome reaction and fertilize (Sukcharoen, et al., 1996; Griveau and de Lannou,1997). The fertilizing ability of human spermatozoa is inversely related to the sperm ROS production (Sukcharoen et al., 1996).
  • Oxidative stress at the testicular level has also been implicated in the disruption of spermatogenesis observed during cryptorchidism, vitamin E depletion, and exposure to xenobiotics (Peltola et al., 1994; 1995).
  • TAC levels in all infertile patients.
  • the idiopathic infertility group had the lowest levels of TAC, followed by the varicocele with inflammation, and varicocele groups. These levels were significantly different (>2 SD) compared to controls. Whether TAC levels characterize the level of oxidative stress will depend on the source of ROS, i.e. whether it is produced by the abnormal spermatozoa and the neutrophils (extracellularly), or within the spermatozoa (intracellularly).
  • ROS-generating systems Two ROS-generating systems have been proposed, an NADPH oxidase-like system at the sperm plasma membrane level, and a sperm disphorase (an NADH-dependent oxido-reductase) located in the middle piece and integrated into the mitochondrial respiratory system of the spermatozoa (Aitken, 1997).
  • ROS emanates in part from abnormal spermatozoa, which are characterized by the retention of excess residual cytoplasm as a result of defective spermiogenesis (Aitken et al., 19 94). Only one-third of the ROS produced by spermatozoa is released extracellularly (Plante et al., 1994).
  • ROS scavengers such as catalase and superoxide dismutase
  • ROS production is intracellular.
  • asthenozoospermia in association with peroxidative damage to the spermatozoa demonstrated a potential value of oral vitamin E supplementation (Suleiman et al., 1996).
  • classical antioxidants would be very effective, and the TAC level may be indicative of the extent of oxidative stress.
  • the ROS-TAC score as proposed by us provides a measure derived both from the levels of ROS produced and the antioxidant levels present in a given set of patients. Furthermore, this score minimizes the variability present in the individual parameters of oxidative stress. The degree of oxidative stress can be effectively determined from the ROS-TAC score which provides a measure derived from both the levels of ROS produced and the antioxidant capacity in a given set of patients.
  • ROS-TAC score was calculated from control comprising healthy normal men who had very low levels of ROS, had initiated pregnancies in the past 2 years, and were therefore fertile.
  • the composite ROS-TAC score calculated for these men was representative of the fertile group, and any scores significantly below these were indicative of the patients being infertile.
  • ROS-TAC score was a superior discriminator between fertile and infertile men than either ROS or TAC alone. It is quite likely that neither variable alone adequately quantifies seminal oxidative stress and the combination of these two variables is better at quantifying the overall oxidative stress affecting spermatozoa.
  • ROS-TAC score a novel measure of oxidative stress as being superior to ROS or TAC alone in discriminating between fertile and infertile men.
  • infertile men with male factor or idiopathic diagnoses had significantly lower ROS-TAC scores than controls, and that men with male factor diagnoses that eventually were able to initiate a successful pregnancy had significantly higher ROS-TAC scores than those who failed.
  • male partners of couples who achieved pregnancy did not have significantly different ROS-TAC scores than controls. Therefore, the ROS-TAC score may serve as an important measure in identifying those patients with a clinical diagnosis of male infertility who are likely to achieve a pregnancy over a period of time.

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Abstract

L'invention concerne un procédé et un appareil permettant de prévoir l'infertilité masculine, et notamment un procédé et un appareil permettant de quantifier le stress oxydant présent dans un échantillon corporel obtenu d'un sujet expérimental. Ces procédé et appareil fournissent à un professionnel expérimenté de la médecine des informations utiles pour le diagnostic de maladies associées au stress oxydant ou causées par ce dernier. Un tel procédé est spécialement utile pour prévoir l'infertilité masculine. Le procédé consiste à déterminer les quantités d'espèces oxygène réactives (ROS) dans un échantillon corporel, à déterminer la capacité antioxydante de l'échantillon corporel (TAC), à générer une valeur ROS-TAC après obtention d'une somme pondérée de la valeur ROS et de la valeur TAC. L'appareil quantifie le stress oxydant dans un échantillon corporel au moyen d'un microprocesseur destiné à calculer la valeur ROS-TAC. L'appareil comporte également un dispositif de mesure des espèces oxygène réactives et de la teneur totale d'anti-oxydants dans un échantillon corporel.
PCT/US2000/029839 1999-10-29 2000-10-30 Procede et appareil permettant de prevoir l'infertilite masculine WO2001032802A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU14430/01A AU1443001A (en) 1999-10-29 2000-10-30 Method and apparatus for predicting male infertility

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16240699P 1999-10-29 1999-10-29
US60/162,406 1999-10-29

Publications (2)

Publication Number Publication Date
WO2001032802A1 true WO2001032802A1 (fr) 2001-05-10
WO2001032802A9 WO2001032802A9 (fr) 2002-08-15

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PCT/US2000/029839 WO2001032802A1 (fr) 1999-10-29 2000-10-30 Procede et appareil permettant de prevoir l'infertilite masculine

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AU (1) AU1443001A (fr)
WO (1) WO2001032802A1 (fr)

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WO2003016527A2 (fr) * 2001-08-14 2003-02-27 Probiox Sa Procede permettant de detecter un stress oxydatif et trousse destinee a la mise en oeuvre de ce procede
US7981399B2 (en) 2006-01-09 2011-07-19 Mcgill University Method to determine state of a cell exchanging metabolites with a fluid medium by analyzing the metabolites in the fluid medium
WO2012059615A1 (fr) * 2010-11-04 2012-05-10 Universitat Autònoma De Barcelona Procédé pour déterminer la production d'espèces réactives de l'oxygène dans une population cellulaire
IT201800006166A1 (it) * 2018-06-08 2019-12-08 Un metodo per la valutazione dello stress ossidativo in campioni biologici e dispositivo per realizzare tale metodo

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003016527A2 (fr) * 2001-08-14 2003-02-27 Probiox Sa Procede permettant de detecter un stress oxydatif et trousse destinee a la mise en oeuvre de ce procede
WO2003016527A3 (fr) * 2001-08-14 2003-12-31 Probiox Sa Procede permettant de detecter un stress oxydatif et trousse destinee a la mise en oeuvre de ce procede
US7288374B2 (en) 2001-08-14 2007-10-30 Probiox Sa Process for the detection of oxidative stress and kit for its implementation
US7981399B2 (en) 2006-01-09 2011-07-19 Mcgill University Method to determine state of a cell exchanging metabolites with a fluid medium by analyzing the metabolites in the fluid medium
US8486690B2 (en) 2006-01-09 2013-07-16 Mcgill University Method to determine state of a cell exchanging metabolites with a fluid medium by analyzing the metabolites in the fluid medium
WO2012059615A1 (fr) * 2010-11-04 2012-05-10 Universitat Autònoma De Barcelona Procédé pour déterminer la production d'espèces réactives de l'oxygène dans une population cellulaire
ES2381721A1 (es) * 2010-11-04 2012-05-31 Universitat Autónoma De Barcelona Método para determinar la producción de especies reactivas de oxígeno en una población celular.
US9618503B2 (en) 2010-11-04 2017-04-11 Universidad Autonoma De Barcelona Method for determining the production of reactive oxygen species in a cellular population
IT201800006166A1 (it) * 2018-06-08 2019-12-08 Un metodo per la valutazione dello stress ossidativo in campioni biologici e dispositivo per realizzare tale metodo
EP3578973A1 (fr) * 2018-06-08 2019-12-11 Universita' Degli Studi di Firenze Procédé et dispositif d'évaluation du stress oxidatif dans des énchantillons biologiques

Also Published As

Publication number Publication date
WO2001032802A9 (fr) 2002-08-15
AU1443001A (en) 2001-05-14

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