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US20090053722A1 - Sdad assay and uses thereof - Google Patents

Sdad assay and uses thereof Download PDF

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US20090053722A1
US20090053722A1 US12/193,505 US19350508A US2009053722A1 US 20090053722 A1 US20090053722 A1 US 20090053722A1 US 19350508 A US19350508 A US 19350508A US 2009053722 A1 US2009053722 A1 US 2009053722A1
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sperm
test
decondensation
sdd
dna
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David Brown
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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D19/00—Instruments or methods for reproduction or fertilisation
    • A61D19/02—Instruments or methods for reproduction or fertilisation for artificial insemination
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction

Definitions

  • the process of fertilization involves a series of events including: a) sperm binding to, and then penetration of the oocyte zona pellucida via the acrosome reaction, b) sperm fusion with the oocyte plasma membrane, c) oocyte activation (release of the cortical granules to prevent multiple sperm from fertilizing the egg), d) sperm activation and pronucleous formation (Abou-Haila A. et al. 2000: Furlong, L. et al. 2005).
  • Sperm activation initiates post-fertilization upon entry into the ooplasm where the compacted sperm DNA undergoes many alterations as it develops into a male pronucleus.
  • the chromatin condensation acquired during spermiogenesis is reversed. Protamines are exchanged with histones reformatting the sperm chromatin allowing for pronucleus formation (Ballhom et al., Wasserman et al.).
  • the DNA synthesis phase occurs, that is followed by recondensation of the sperm chromatin in preparation for the cell division that will result in the 2-cell stage embryo (Longo et. al., (1991)).
  • SDAD sperm DNA accelerated decondensation
  • SEDS sperm earlyDNA synthesis
  • ART assisted reproductive technology
  • protocol(s) that provide a more rapid processing and assessment of sperm analyzed in sperm activation tests that include sperm DNA decondensation.
  • a rapid sperm screening assay that is predictive for human sperm reproductive and/or fertilizing capacity is provided in the present disclosure.
  • the assay provides for a highly predictive method for identifying males who produce sperm with low fertilizing capacity, or in the cases where sperm results in a pregnancy, a high probability that the pregnancy will not progress beyond the first trimester, i.e. a low probability of a take home baby.
  • the present invention identifies such males when their sperm is analyzed in a 5 minute to 10 minute in vitro assay that demonstrates a quantifiable difference between a patient's sperm, and the sperm from a known fertile male.
  • this in vitro assay is the sperm DNA accelerated decondensation (SDAD) Test.
  • the in vitro assay is the sperm early DNA synthesis (SEDS) Test.
  • the in vitro assays include the SDAD Test, SDD Test, and SEDS Test.
  • a sperm screening method is provided that is more highly predictive of a take-home baby likelihood when using an assisted reproductive technique (ART).
  • assisted reproductive technique is the intracytoplasmic sperm injection (ICSI).
  • ICSI intracytoplasmic sperm injection
  • the presently disclosed method provides a predictive value that is highly statistically significant (p ⁇ 0.01).
  • a screening method is provided that is useful in identifying male sperm donors who will have a low probability of successful ART attempts at pregnancy (success being defined as live birth), such as when using ICSI.
  • Other sperm screening assays such as the sperm DNA decondensation (SDD) Test, the sperm penetration assay (SPA), and the sperm chromatin structure assay (SCSA) have all been found to not be predictive of ICSI live birth outcome.
  • SDD sperm DNA decondensation
  • SPA sperm penetration assay
  • SCSA sperm chromatin structure assay
  • this screening method comprises obtaining a semen sample from a potential sperm donor that has been determined to provide a normal sperm assay result in a SPA and SDD Test, and assessing the sample for sperm DNA accelerated decondensation (SDAD) in a frog egg extract as described herein.
  • the sperm DNA accelerated decondensation (SDAD) is a measure of chromatin DNA decondensation evidenced after about a 5 (five) minute incubation interval in a frog egg extract.
  • a sperm sample with essentially complete sperm chromatin DNA decondesation at the five (5) minute time interval identifies an unsatisfactory sperm donor for an ICSI pregnancy attempt.
  • a screening method for identifying a male sperm donor having a low probability of an successful ART attempts at pregnancy (e.g., IUI and IVF).
  • the screening method has relatively low predictive capacity for determining a couple's success, success being defined as live birth, when sperm from the male partner is used in an ICSI attempt at pregnancy.
  • the screening method comprises obtaining a semen sample from a potential sperm donor that has been determined to have an abnormal SDD Test score a (delay in DNA decondensation when incubated in frog egg extract).
  • the delay in sperm DNA decondensation is a measure of chromatin DNA decondensation evidenced after about a 15 (fifteen) minute incubation interval in a frog egg extract.
  • a sperm sample with an abnormal response in the SDD Test identifies an unsatisfactory sperm donor for an IUI and/or IVF pregnancy attempt. However, because the SDD Test has no predictive capacity for determining if a patient will succeed in an ICSI attempt at pregnancy, such patients can go directly to ICSI attempts at pregnancy.
  • the particular sperm screening assay is the SDAD or SDD Tests, or both.
  • the automated assay is a high throughput sperm screening method that employs a 96-microtiter well plate, each plate comprising a volume of a frog egg extract.
  • each microtiter well plate may include frog egg extract containing a DNA labeling agent, such as tritiated thymidine triphosphate (when autoradiography is used to analyze DNA synthesis) or 5-Bromo-2′-deoxyuridine (BUdR), when sperm will be stained with a fluorescent tagged anti-BUdR antibody, and the DNA synthesis analyzed using an automated system/image analysis system described herein.
  • a DNA labeling agent such as tritiated thymidine triphosphate (when autoradiography is used to analyze DNA synthesis) or 5-Bromo-2′-deoxyuridine (BUdR)
  • a DNA labeling agent such as tritiated thymidine triphosphate (when autoradiography is used to analyze DNA synthesis) or 5-Bromo-2′-deoxyuridine (BUdR)
  • a DNA labeling agent such as tritiated thymidine triphosphate (when autoradiography is used to analyze DNA synthesis) or 5-Bromo-2′-de
  • a method for using the SDD Test to identify an unsatisfactory sperm donor whose abnormal score is determined to be related to the patient also having a varicocele(s). When identified, such individuals have been found to benefit from a varicocelectomy.
  • hypodecondensed sperm is defined as sperm with a 2-fold increase in size over that observed in the fully decondensed sperm.
  • a “successful” pregnancy is defined as a live birth resulting from a pregnancy achieved using an assisted reproductive technique (ART).
  • the term, “reproductively challenged couple” is defined as a human male and a human female that have been involuntarily infertile for 1 or more years. Approximately forty percent (40%) of these couples are infertile due to male factor(s), forty percent (40%) are infertile due to female factor(s), and 20% are infertile due to combined male and female factors.
  • take-home baby is defined as a live human birth.
  • SEDS sperm early DNA synthesis
  • HSAA human sperm activation assay
  • SDAD sperm DNA accelerated decondensation assay
  • ROS reactive oxygen species
  • PBS phosphate buffered saline
  • ICSI intracytoplasmic sperm injection
  • NBT nitroblue tetrazolium
  • ROS reactive oxygen species
  • SDFA sperm DNA fragmentation assay
  • H 3 -TTP tritiated thymidine triphosphate
  • FIG. 1 Lipid peroxidation (LPO) in H 2 O 2 treated sperm.
  • LPO was determined using a colorimetric assay for malondialdehyde (MDA) and 4-hydroxyalkenals (Oxford Biochemical Research). Each bar is the mean ⁇ SD. Means are significantly different (p ⁇ 0.001 using ANOVA: 1) when comparing the control untreated sperm with sperm treated with 10, 50 and 100 ⁇ M H 2 O 2 (black asterisks), and 2) when comparing the lipid peroxidation in each dose at two exposure times (15 min and 1 hr). Black crosses indicate significant differences.
  • FIGS. 2A-E and 2 a - e Pictorial representation of the effects of H 2 O 2 on human sperm activation. All sperm were photographed using phase contrast microscopy, a 60 ⁇ objective, and were printed at the same magnification.
  • 2 A-E HSAA normal responses in untreated control sperm incubated in egg extract.
  • 2 a - e Sperm exposed to 100 ⁇ M H 2 O 2 for 1 hr and then incubated in egg extract. The arrows point to examples of abnormal sperm activation responses. Bar represents 10 ⁇ m.
  • 2 B, b Sperm decondensation after a 5 min incubation in egg extract with a partially decondensed sperm ( 2 B) and partially and fully decondensed sperm ( 2 b ).
  • 2 C, c Sperm decondensation after a 15 min incubation in egg extract with typical fully decondensed sperm ( 2 C), and hyperdecondensed sperm (c; see white arrows).
  • 2 D, d Examples of recondensed sperm after a 2 hr incubation in the egg extract.
  • FIG. 3A-3B Effects of hydrogen peroxide dose-time exposure on human sperm activation. Bar is the mean ⁇ SD of sperm decondensation after a 5 and 15 min incubation in egg extract. Asterisks indicate significant difference when comparing H 2 O 2 treated sperm after a 15 min ( 3 a and 3 c ) and 1 hr ( 3 b and 3 d ) exposure with untreated control sperm using ANOVA. p values ⁇ 0.05 were considered as significant.
  • FIG. 4 Effect of ROS dose-time exposure on human sperm activation. Bar is the mean ⁇ SD.
  • Sperm recondensation was scored after a 2 and 3 hr incubation in the egg extract. Asterisks indicate significant differences when comparing H 2 O 2 treated sperm after 15 min and 1 hr exposures with control untreated sperm using ANOVA, p values ⁇ 0.05 were considered significant. Since there were no differences in sperm scored after a 2 and 3 hr incubation in the egg extract, these results were combined. Recondensation and hyperdecondensation are significantly different in sperm exposed to 50 and 100 ⁇ M H 2 O 2 for 15 min ( 4 a ) and 1 hr ( 4 b ).
  • 3 H-TTP incorporated into sperm DNA were determined in sperm exposed to 0, 10, 50 and 100 ⁇ M H 2 O 2 for 15 min and 1 hr.
  • 3 H-TTP incorporation was scored after a 5 and 15 min incubation in egg extract.
  • Thymidine incorporation into sperm was scored as follows: unlabeled (less than 5 black granules), medium label (from 5 to 25 black granules), and heavy label (more than 25 black granules). Each value is the mean ⁇ SD. Means are significantly different when the p values were ⁇ 0.05 when comparing with control untreated sperm using ANOVA.
  • FIG. 6 a - 6 d DNA synthesis in H 2 O 2 treated sperm incubated in egg extract for 2 and 3 hr.
  • 3 H-TTP incorporation was scored in sperm exposed to 0, 10, 50 and 100 ⁇ M H 2 O 2 for 15 min and 1 hr.
  • 3 H-TTP incorporation was determine after a 2 and 3 hr incubation in the egg extract. After a 2 and 3 hr incubation in the egg extract sperm with increased medium and heavy label was observed in sperm exposed to 50 and 100 ⁇ M H 2 O 2 for 15 min ( 6 a and 6 c ) and 1 hr ( 6 b and 6 d ).
  • Sperm exposed to 50 and 100 ⁇ M H 2 O 2 for 1 hr had an abnormal increase in the number of sperm with medium incorporation of 3 H-TTP and decrease in the number of sperm with heavy incorporation ( 6 d ).
  • FIG. 7 Data fields of images captured and analyzed using an image analysis system
  • FIG. 8 Comparison of SDD Test results obtained when scoring in real time manually using phase contrast microscopy, with results obtained when scoring using an image analysis system (fluorescence microscopy).
  • FIG. 9 Pictorial representation of autoradiographes of control untreated sperm ( 9 A- 9 E) and sperm exposed to 100 ⁇ M H 2 O 2 for 1 hr ( 9 a - e ) assayed in the HSAA in the presence of 3 H-TTP. All the pictures were photographed using bright microscopy 60 ⁇ objective. The black bar represents 10 ⁇ M, and black arrows point to abnormal sperm activation. All pictures were printed at the same magnification.
  • the invention relates to methods for assessing sperm fertility through the use of a sperm DNA accelerated decondensation (SDAD) Test in combination with a delayed sperm DNA decondensation (SDD) Test, as well as a method for identifying the most appropriate assisted reproductive technology (ART), thus individualizing the treatment for reproductively challenged couples.
  • SDAD sperm DNA accelerated decondensation
  • SDD delayed sperm DNA decondensation
  • ART assisted reproductive technology
  • the invention also provides a high-through put method for clinical sperm assessments, wherein the SDAD and SDD Tests are performed as part of an automated system employing fluorescence microscopy.
  • the SDD Test has no predictive value for ICSI presumably due to the way sperm is now prepared for ART that includes a density gradient separation.
  • the SDAD Test does identify a group of patients that will have little chance for success in ICSI ART attempts at pregnancy, and is the only test available that can identify such patients. However, this group of patients are all normal in the SDD Test, so the SDD Test (an abnormal score) can now be used to determine patients who should be directed immediately to ICSI, as this is their most probable and reasonable chance for a successful pregnancy.
  • the invention also provides a method to identify males who can benefit from a varicocelectomy. Such individuals, upon finding their SDD Test results are abnormal, and having a Urologist find a varicocele(s) will benefit from a varicocelectomy. After the varicocelectomy is performed, the patient will be given 3-6 months to heal. When an improvement using the SDD Test score is found, such individuals now have an improved chance for fathering children by natural conception or ART.
  • Lipid peroxidation of the sperm cellular membrane was determined using the Oxford Biomedical Research Kit Fr 12.
  • Lipid peroxidation (LPO) measurements and HSAA responses were analyzed in each treatment to determine the effect(s) H 2 O 2 concentration- and time exposure on human sperm activation responses.
  • Semen ejaculate was collected in a sterile jar by masturbation after 3-5 days of sexual abstinence from fertile males who had previously been shown to produce sperm that responded normally in the HSAA.
  • Sperm were isolated from the seminal plasma as previously described in Brown et al., 1995.
  • PBS phosphate buffered saline
  • This technique is based on the reaction of a chromogenic reagent, N-methyl-2 phenylindole, with MDA and 4-hydroxyalkenals at 45° C.
  • MDA chromogenic reagent
  • 4-hydroxyalkenals One molecule of either MDA or 4-hydroxyalkenals reacts with 2 molecules of N-methyl-2 phenylindole to produce a stable chromophore that can be quantified at 586 nm.
  • the HSAA was performed as described by Brown et al. 1995. Briefly, sperm are washed and permeabilized using lysolecithine.
  • the reaction mix was prepared; 8 ⁇ l of sperm suspension (200,000 sperm) was added to 100 ⁇ Lof frog egg extract that contained 8 ⁇ l of 3 H-TTP (8 ⁇ Ci). At 5 and 15 min, and 2 and 3 hr incubation times in the egg extract, 5 ⁇ l aliquots were taken, wet mounts prepared, and phase contrast microscopy used to determine the percentage of sperm that were fully decondensed and recondensed, respectively, scoring 50 nuclei per slide. At the 5 and 15 min, and 2 and 3 hr time points, 25 ⁇ l aliquots of the reaction mix was removed and mixed with 75 ⁇ l PBS.
  • cytoprep slides Two 50 ⁇ l cytopreps were made for each aliquote as described by Brown et al., 1992. The dried cytopreps were fixed in one part acetic acid, and three parts cold methanol, and Giemsa stained for 1 hr followed by a 10 min wash in Giemsa buffer. Slides were examined using light microscopy and analyzed using Metamorph software. Autoradiography was performed using cytoprep slides as described by Brown et al. 1995.
  • the means and standard deviation from (untreated) control sperm was compared with sperm treated with 10-100 ⁇ M H 2 O 2 at two exposure times (15 min and 1 hr) for: lipid peroxidation values, sperm activation, decondensation at 5 and 15 min, DNA synthesis at 5 and 15 min and 2 and 3 hr, and recondensation at 3 hr.
  • ANOVA Sigma Stat analysis of the data was performed, and p values ⁇ 0.05 were considered as significant.
  • the present example is provided to demonstrate the utility of the HSAA and the 5-minute time point SDAD Test, an addition to the HSAA, in the identification of damage resulting from exposure to ROS.
  • Lipid peroxidation (LPO) measurements and HSAA responses were analyzed in each treatment to determine the effect(s) of H 2 O 2 concentration and time exposure on human sperm activation responses.
  • Semen ejaculate was collected in a sterile jar by masturbation after 3-5 days of sexual abstinence from fertile males who had previously been shown to produce sperm that responded normally in the HSAA.
  • Sperm was isolated from the seminal plasma as previously described in Brown et al. 1995.
  • PBS phosphate buffered saline
  • the remaining portion was centrifuged at 1500 g for 15 min at room temperature.
  • the pellet was analyzed in the HSAA.
  • This technique is based on the reaction of a chromogenic reagent, N-methyl-2 phenylindole, with MDA and 4-hydroxyalkenals at 45° C.
  • MDA chromogenic reagent
  • 4-hydroxyalkenals One molecule of either MDA or 4-hydroxyalkenal reacts with 2 molecules of N-methyl-2 phenylindole to produce a stable chromophore that can be quantified at 586 nm.
  • H 2 O 2 concentration 50 and 100 ⁇ M
  • time exposure are the main factors that induced abnormal decondensation responses including: 1) the unique observation of early recondensation, and 2) the novel phenomenon of hyperdecondensation.
  • Hyper-decondensed sperm occurred in 30 ⁇ 2% and 45 ⁇ 3% of the sperm treated with 50 and 100 ⁇ M H 2 O 2 , respectively (1 hr exposure, FIG. 4 b ). Recondensed sperm were observed in 98 ⁇ 2% of both the control, and 10 ⁇ M H 2 O 2 -treated sperm (15 min and 1 hr exposure). Recondensation occurred in 66 ⁇ 2.3% and 57 ⁇ 2% of the sperm exposed to 50 and 100 ⁇ M H 2 O 2 respectively (15 min exposure, FIG. 4 a ). Recondensation occurred in 70 ⁇ 3% and 54 ⁇ 2% of the sperm exposed to 50 and 100 ⁇ M H 2 O 2 for 1 hr, respectively ( FIG. 4 b ).
  • the DNA synthesis was determined by autoradiography as previously described in Materials and Methods.
  • FIGS. 5 a, b and 9 b 3 H-TTP incorporation into sperm DNA after a 5 min incubation in egg extract.
  • FIGS. 5 a, b and 9 B No sperm were positive for 3 H-TTP incorporation in the control and 10 ⁇ M H 2 O 2 -treated sperm after 15 min and 1 hr exposures.
  • FIGS. 5 a, b and 9 B Sperm classified as having medium 3 H-TTP labeling were observed in 18 ⁇ 3.5% and 36 ⁇ 3% of the 50 ⁇ M H 2 O 2 -treated sperm after a 15 min and 1 hr exposure, respectively.
  • FIGS. 3 a 15 min H 2 O 2 exposure; and 3 b : 1 hr H 2 O 2 exposure).
  • the hyperdecondensation effect can be explained by the massive influx of the activation factors contained in the extract, through the leaky membrane. Damage to the plasma membrane, when lipid peroxidation of polyunsaturated fatty acids occurred, results in an increase in the permeability of the membranes. Also, the ‘oxidative chromatin pre-relaxation’ may accelerate sperm nuclear decondensation (Ollero M et al. 2000; Kemal Duru N et al. 2000; Saleh R A et al. 2002). These findings are in agreement with the results of Brown, et al., 1987 who showed that nuclear factors contained in the egg extract regulate sperm decondensation.
  • sperm was incubated in an active fraction of proteins isolated from the frog egg extract that contained a 70-fold enrichment of partially purified decondensation activation factors. Full decondensation of the sperm nuclei occurred within 5 minutes, and no recondensation was observed, indicating that the recondensation proteins were removed or inactivated during the purification of the decondensation proteins (Brown D B et al. 1987; Brown D B et al. 1991).
  • Some abnormal sperm decondensation responses are believed to be a result of exposure to reproductive toxicants that directly affect the chromatin such that there is a delay or an enhancement of in vitro decondensation, depending upon the type of exposure.
  • exposure to alkylating agents causes a delayed decondensation (Perreault S et al. 1987 et al. Sawyer D et al. 1995; Sawyer et al. 1998).
  • Exposure to ROS induced an increase in the kinetics of decondensation (accelerated decondensation).
  • a spectrophotometric assay was used to measure the amount of MDA and hydroxyalkenals in sperm treated with 0, 10, 50 and 100 ⁇ M hydrogen peroxide (H 2 O 2 ) for 15 min and 1 hr.
  • the means were significantly different in 50 and 100 ⁇ M H 2 O 2 -treated sperm (15 min), and 10, 50 and 100 ⁇ M H 2 O 2 -treated sperm (1 hr), when compared to the untreated control sperm ( FIG. 1 ).
  • FIGS. 6 a and b 3 H-TTP incorporation into sperm DNA after a 2 hr incubation in egg extract.
  • FIGS. 6 c and 6 d 3 H-TTP incorporation into sperm DNA after a 3 hr incubation in egg extract.
  • FIGS. 6 c and 6 d Sperm with medium labeling were observed in approximately 16.5 ⁇ 2% and 14.5 ⁇ 3% of sperm exposed to 50 and 100 ⁇ M H 2 O 2 for 15 minutes ( FIG. 6 c ), respectively.
  • Approximately 25 ⁇ 3% and 40 ⁇ 4% of the sperm exposed for 1 hr to 50 and 100 ⁇ M H 2 O 2 had medium labeling respectively ( FIG. 6 d ).
  • Heavily labeled sperm were found in 98 ⁇ 2% of the control and 10 ⁇ M H 2 O 2 -treated sperm after 15 min and 1 hr exposures ( FIGS. 6 c and 6 d ).
  • FIG. 8 demonstrates the observed correlation between Phase Contrast (Scoring done manually) and Fluorescence (Scoring done by an image analysis system with auto focus) analysis of 8 patient semen samples in th SDD Test.
  • X Fluorescent Score
  • Y Phase Contrast score
  • the hyperdecondensation effect can be explained by the massive influx of the activation factors contained in the extract, through the leaky membrane.
  • the ‘oxidative chromatin pre-relaxation may accelerate sperm nuclear decondensation (Ollero M et al. (2000)); Kemal Duru N et al. (2000); Saleh R A et al. (2002).
  • Some abnormal sperm decondensation responses are believed to be a result of exposure to reproductive toxicants that directly affect the chromatin such that there is a delay or an enhancement of in vitro decondensation, depending upon the type of exposure.
  • exposure to alkylating agents causes a delayed decondensation (Perreault S et al. (1987), Sawyer D et al. (1995); Sawyer et al. (1998)), while exposure to ROS induced an increase in the kinetics of decondensation.
  • the 3 H-TTP incorporation results of sperm exposed to 50 and 100 ⁇ M H 2 O 2 for 15 min and 1 hr show that sperm had abnormal 3 H-TTP incorporation including: a) early DNA synthesis after a 5 and 15 min incubation in the egg extract, and b) increase in the number of sperm positive for medium label after 2 and 3 hr incubations in the egg extract.
  • the abnormal 3 H-TTP incorporation was H 2 O 2 concentration and exposure time dependent.
  • DNA synthesis is engineered totally by factors present in the egg extract; one of these factors is the enzyme DNA polymerase ⁇ .
  • DNA repair activity Since sperm does not have DNA repair capacity (Ramos L et al. (2001), the DNA repair activity must be provided by egg cytoplasm. DNA repair activity has been demonstrated in several mouse models for male infertility, and in biopsies taken from infertile males in which DNA integrity is compromised suggests that the repair of DNA-oxidative damage occurs between sperm decondensation and pronucleus formation, and that the DNA repair is dependent on oocyte cytoplasmic factors (Baarends et al., (2001), Ochsendorft F R (1999)). The autoradiography results in this present study are in agreement with the above observations.
  • the base excision DNA repair pathway is used to repair DNA oxidative damage.
  • DNA repair mechanisms have been involved not only in the repair of DNA damage in developing germ line cells, but also to enhance specialized gene expression during mammalian gametogenesis (Baarends W et al. (2001)).
  • SDD Sperm DNA Decondensation
  • SPA Sperm Penetration Assay
  • IVF In Vitro Fertilization
  • ICSI Intracytoplasmic Sperm Injection
  • the SDD Test measures the fraction of cells fully decondensed after a 15 minute incubation in frog egg extract, measuring delayed decondensation, while the SPA measures the sperm capacitation index (SCI), the average number of sperm penetrations per zona-free hamster oocyte.
  • SCI sperm capacitation index
  • the objective of this study was to review the pregnancy outcome in ICSI cycles in relation to the SPA and the SDD Test results using the best sperm looking at SCI improvement over SCI from semen sperm, and semen sperm looking at delayed DNA decondensation in comparison to that observed for sperm from a fertile male, and determine if either of the 2 parameters being evaluated have predictive capacity for determining success in ART.
  • ODR delivery rate
  • Ejaculates were divided into 3 portions, one used for the SPA, the second for the SDD Test and the third frozen for use in a future ICSI cycle.
  • the data consisted of 53 ICSI cycles. Cycles were grouped according to abnormal v normal score in the SPA ( ⁇ 14v ⁇ 14 SCI) and the SDD ( ⁇ 80% v ⁇ 80%).
  • a gradient preparation was used for sperm preparation for the SPA as well as ICSI. Standard methods were used for ovarian stimulation and embryo culture. Odds ratios (OR) and positive predictive value (PPV) for failure were calculated, and the statistical significance was assessed by the Chi 2 distribution.
  • the SDD is not predictive of ODR in IVF with ICSI cycles.
  • the SPA is not predictive of ODR in IVF with ICSI cycles.
  • the males were from couples where the male partners are admitted for andrology evaluation including the SPA in preparation for IVF. Measurement will be attempted on the very same ejaculates provided for ICSI attempts at pregnancy in IVF.
  • microorganisms viral, bacterial or fungal such as Mycoplasma/Ureaplasma, Chlamydia trachomatis , Bacterial Vaginosis
  • viral, bacterial or fungal such as Mycoplasma/Ureaplasma, Chlamydia trachomatis , Bacterial Vaginosis
  • female infertility Past or Current
  • Autoimmune disease (lupus, RA, MS, Diabetes, Hashimoto, etc.) determined while interviewing the infertile couple.
  • LAC Lupus anti coagulant
  • ACA Anti cardiolipin Antibodies
  • APA Anti phospholipid antibodies
  • NK Natural Killer Cells
  • RIP Reproductive immunophenotyping
  • AMA Anti Microsomal Antibodies
  • Factor V coagulation
  • Plasma Blood for workup for the female will be sent up to 6 weeks prior to beginning the cycle.
  • One plasma is to be spun and separated for Factor V (sent at ambient temperature) and LAC (sent at ambient temperature), one heparinized whole blood for cellular immunology —NK and RIP (sent ambient temp) and one test-tube with serum spun and separated for ACA, APA and AMA (sent at ambient temperature).
  • Factor V sent at ambient temperature
  • LAC sent at ambient temperature
  • NK and RIP sent ambient temp
  • test-tube with serum spun and separated for ACA, APA and AMA sent at ambient temperature.
  • Age Patients/donors younger than 25 yr or older than 45 yr.
  • the prospective study will enroll 60 patients who fulfill the selection criteria.
  • Standard andrology work-up including concentration of sperm, morphology of sperm, volume of ejaculate, and motility of the sperm in all specimens used for the study.
  • Sperm DNA decondensation in the SDD Test (15 minutes).
  • the study will determine the relationship of SDD and/or SPA results and pregnancy results with ART cycles with ICSI.
  • Specimens will be allowed to liquefy by incubating up to 1 hr at room temperature (not less than 22° C.). Specimens will be evaluated on-site in the andrology lab for basic parameters, and then will be prepared as determined from the initial evaluation of the correlation of the sperm preparation in the preliminary study. This will be used in the SDD Test, the SPA, and ICSI attempts at pregnancy.
  • the sperm from the 60 patients will be split into 3 aliquots:
  • Specimen aliquots will be kept in the refrigerator at 2-8° C., until shipped cold by packing with a coolpak. Specimens must be sent no later than 9 days after sample collection. The samples will be analyzed in the SDD Test within 14 days of sample collection. Any sample arriving such that it cannot be analyzed within the 14 day QC window will be rejected, and another sample requested.
  • the SPA test will be performed as a modified version to Johnson et al. (1990) as described below.
  • Semen will be washed and concentrated by gradient method. After gradient preparation the sperm will be resuspended in sperm wash medium and diluted 1:1 with Test Yolk Buffer (TYB). The sperm/TYB mixture will then be slowly cooled to 2-8° C. and stored at this temperature for 2-3 days. After this time, sperm wash medium at 37° C. will be added to the cold sperm/TYB mixture, providing a thermal shock. After the sperm/TYB has incubated for 30 minutes at 37° C., the mixture will be centrifuged for 10 min at 600 g. The supernatant will then be removed, up to 1.0 mL sperm wash medium added, and the sperm allowed to incubate for 60 minutes at 37° C. The sample will then be adjusted to a concentration of 5 million total motile sperm/mL.
  • TYB Test Yolk Buffer
  • Frozen hamster ova will be utilized for the SPA. Straws containing ova will be thawed at RT for 2 min in a horizontal position. The straws are then shaken at the crimped end to vigorously mix the sucrose column with ova. The straws will then be incubated, cotton end down, in a 37° C. water bath for 3 min followed by 3 min at RT, cotton end up. Using a pushrod, the ova are then dispensed into a 35 mm culture dish and washed twice in sperm wash medium. The ova will be transferred to fresh sperm wash medium to rest for 10 min at RT.
  • the ova will be washed three times in trypsin (1%) and incubated at RT in the 3rd trypsin drop until the zona is almost depleted ( ⁇ 5 min). The ova are then washed twice in sperm wash medium. The ova will be transferred to fresh sperm wash medium to rest for 5 min at RT.
  • ova Six-seven ova will be placed into each of two 100 ⁇ L drops of sperm wash medium covered with oil containing 250,000 total motile sperm and allowed to incubate for 3.5 hr at 37° C. After incubation, the ova will be washed in sperm wash medium to dislodge any loosely bound sperm. The ova will then be placed on a microscope slide with cover slip applied so to flatten the ova for penetration assessment. The number of penetrations (clear zone with discernible tail) will be counted and the Sperm Capacitation Index (SCI) calculated by dividing the total number of penetrations by the total number of ova scored.
  • SCI Sperm Capacitation Index
  • the test About two million sperm per sample will be washed and permeablized. After four extensive washes with special buffers, the sperm will be treated with dithiotreitol (DTT)-containing buffer. The treated sperm will then be incubated with frog egg extract to induce sperm DNA decondensation. At 15 min 50-100 sperm (the number of sperm that can be score in 5 min) from a fertile male will be scored, and then this process will be repeated for each patient using phase-contract microscopy. The percentage of sperm undergoing full decondensation is recorded. The raw data will be normalized with negative control decondensation value yielding the reportable value as % of the control.
  • DTT dithiotreitol
  • control specimen is a normal, SDD pre-tested specimen from a sperm bank, or a specimen obtained from an individual who has produced 4 or more ejaculates containing sperm that respond normally at the 15 min time point.
  • This control serves as negative control when used in the complete SDD Test protocol, Patient positive controls that have been identified as being abnormal in routine testing of patient samples sent to Repromedix for analysis in the SDD Test are frozen and used as the positive control.
  • Determination of the best cutoff value predicting pregnancy failure or specific pregnancy outcomes will be done by ROC analysis. Specificity, Sensitivity, PPV and NPV as well as Accuracy will be evaluated using the selected cutoff values.
  • ODR delivery rate
  • the males were from couples where the male partners are admitted for andrology evaluation including the SPA in preparation for IVF. Measurements were performed using sperm from the same ejaculate used for each couple's ICSI attempt at pregnancy.
  • microorganisms viral, bacterial or fungal such as Mycoplasma/Ureaplasma, Chlamydia trachomatis , Bacterial Vaginosis
  • viral, bacterial or fungal such as Mycoplasma/Ureaplasma, Chlamydia trachomatis , Bacterial Vaginosis
  • female infertility Past or Current
  • Autoimmune disease (lupus, RA, MS, Diabetes, Hashimoto, etc.) determined while interviewing the infertile couple.
  • LAC Lupus anti coagulant
  • ACA Anti cardiolipin Antibodies
  • APA Anti phospholipid antibodies
  • NK Natural Killer Cells
  • RIP Reproductive immunophenotyping
  • AMA Anti Microsomal Antibodies
  • Factor V coagulation
  • Plasma Blood for workup for the female will be sent up to 6 weeks prior to beginning the cycle.
  • One plasma is to be spun and separated for Factor V (sent at ambient temperature) and LAC (sent at ambient temperature), one heparinized whole blood for cellular immunology —NK and RIP (sent ambient temp) and one test-tube with serum spun and separated for ACA, APA and AMA (sent at ambient temperature).
  • Factor V sent at ambient temperature
  • LAC sent at ambient temperature
  • NK and RIP sent ambient temp
  • test-tube with serum spun and separated for ACA, APA and AMA sent at ambient temperature.
  • Age Patients/donors younger than 25 y or older than 45 years.
  • the prospective study will enroll 60 patients who fulfill the selection criteria.
  • Standard andrology work-up including concentration of sperm, morphology of sperm, volume of ejaculate, and motility of the sperm in all specimens used for the study.
  • Specimens will be allowed to liquefy by incubating up to 1 hr at room temperature (not less than 22° C.). Specimens will be evaluated on-site in the andrology lab for basic parameters, and then will be prepared as determined from the initial evaluation of the correlation of the sperm preparation in the preliminary study. This will be used in the SDAD Test, and ICSI attempts at pregnancy.
  • the sperm from the 60 patients will be split into 3 aliquots:
  • Specimen aliquots will be kept in the refrigerator at 2-8° C., until shipped cold by packing with a coolpak. Specimens must be sent no later than 9 days after sample collection. The samples will be analyzed in the SDAD Test within 14 days of sample collection. Any sample arriving such that it cannot be analyzed within the 14 day QC window will be rejected, and another sample requested.
  • Semen will be washed and concentrated by gradient method. After gradient preparation the sperm will be resuspended in sperm wash medium and diluted 1:1 with Test Yolk Buffer (TYB). The sperm/TYB mixture will then be slowly cooled to 2-8° C. and stored at this temperature for 2-3 days. After this time, sperm wash medium at 37° C. will be added to the cold sperm/TYB mixture, providing a thermal shock. After the sperm/TYB has incubated for 30 min at 37° C., the mixture will be centrifuged for 10 min at 600 g. The supernatant will then be removed, up to 1.0 mL sperm wash medium added, and the sperm allowed to incubate for 60 min at 37° C. The sample will then be adjusted to a concentration of 5 million total motile sperm/mL.
  • TYB Test Yolk Buffer
  • the SDAD Test will be performed as described by Brown et al. (1992, 1995) with the new addition of the 5 minute time point to score for accelerated DNA decondensation.
  • sperm About two million sperm per sample will be washed and permeablized. After four extensive washes with special buffers, the sperm will be treated with dithiotreitol (DTT)-containing buffer. The treated sperm will then be incubated with frog egg extract to induce sperm DNA decondensation. At 5 min 50-100 sperm (the number of sperm that can be score in 5 min) from a fertile male will be scored, and then this process will be repeated for each patient using phase-contract microscopy. The percentage of sperm undergoing full decondensation is recorded. The raw data will be normalized with negative control decondensation value yielding the reportable value as % of the control.
  • DTT dithiotreitol
  • control specimen is a normal, SDD pre-tested specimen from a sperm bank, or a specimen obtained from an individual who has produced 4 or more ejaculates containing sperm that respond normally at the 15 min time point. This control serves as negative control when used in the complete SDD Test protocol.
  • Patient positive controls that have been identified as being abnormal in routine testing of patient samples sent to Repromedix for analysis in the SDD Test are frozen and used as the positive control.
  • Determination of the best cutoff value predicting pregnancy failure or specific pregnancy outcomes will be done by ROC analysis. Specificity, Sensitivity, PPV and NPV as well as Accuracy will be evaluated using the selected cutoff values.
  • VX varicocelectomy
  • the SDD Test may also be a useful marker of improved fertility potential of sperm after a varicocelectomy.
  • sperm DNA integrity testing has been reported to predict failure of sperm with assisted reproductive technology (ART), regardless of the method of insemination. It has been reported that sperm DNA structure tests (SCSA/SDFA) can identify reduced chances for success in some ART methods, such as intrauterine insemination (IUI). However, these tests are not deemed useful for others, such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Similarly, the sperm DNA decondensation (SDD) test, a sperm function test, has previously been shown to predict failure in subsequent ART attempts, regardless of method. The goal of this example is to demonstrate that the SDD Test has a predictive capability in current IUI and IVF insemination protocols.
  • the SDD will identify male patients with reduced chances of success with IUI and IVF who may benefit from earlier consideration of
  • a method that provides a novel 96 well plate with etched glass at the bottom of each well with a transparent array of micron-sized wells, with sperm analysis as one application is provided.
  • This plate will be used in the practice of the present example to accommodate the running of 8 assays at the same time and then transfer stained, fixed sperm into 8 wells at a time speeding up the processing of the samples.
  • the image analysis system can then be used to analyze 500 sperm per well without the need of a technician individually scoring each test in real time using phase contrast microscopy. This system is being used to provide a way to let the fixed stained sperm settle into the wells, such that both accelerated decondensation (5 min time point) and delayed decondensation (15 min time point) may be determined.
  • a 25 ul aliquot of the extract/sperm cocktail is placed in the next 8 wells of the 96 well plate, and 1 ul of Hoescht 33258 stain (1 ul of stock Hoescht 33258 stain in 1 ml of water; 1:1,000 dilution) is added to each well and fixation solution is added in5 ⁇ l aliquots per well (5 times) mixing the mixture in the well after each addition of fixative.
  • the fixation solution is made by mixing a EM Grade paraformaldehyde, 16% w/v, Distilled Water, 100% v/v mix 1:1 with Phosphate Buffered Saline (PBS).
  • PBS Phosphate Buffered Saline
  • Semen aliquots will be kept in the refrigerator at 2-8° C., until shipped cold by packing with a coolpak provided with shipping supplies. Specimens must be sent no later than 9 days after sample collection, and must be shipped on Monday, Tuesday, Wednesday or Thursday so they will not arrive on the weekend. The samples will be analyzed in the SDAD and/or the SDD Tests within 14 days of sample collection. Any sample arriving such that the sample can not be examined within the 14 day QC window will be rejected, and another sample requested.
  • the SDAD and SDD tests About two million sperm per sample are washed and permeablized as described in the Brown et. al. papers (1992, 1995). After four extensive washes with special buffers, sperm are treated with a buffer containing dithiotreitol (DTT). The DTT-treated sperm are then incubated with frog egg extract to induce sperm DNA decondensation.
  • DTT dithiotreitol
  • SDAD After a 5 min incubation in egg extract, an aliquot of the sperm/egg extract mixture is placed on a glass slide and a cover slip is placed on top of the mixture. Approximately 50-75 sperm are scored in real time during a 5 min period of time, and the percentage of fully decondensed sperm is determined using phase contrast microscopy. The percentage of sperm fully decondensed is recorded.
  • the raw data is normalized with a negative control (normal male as described below) decondensation value yielding the reportable value that is the percentage of the control sperm that have fully decondensed at 5 min. Any value less than 120% of the control is considered normal. Greater than 120% of the control is considered abnormal, and patients with such scores have a poor chance of having a successful ICSI attempt at pregnancy. This value is based on the results shown in FIG. 8 .
  • the negative control specimen is from a male who has produced 3 ejaculates that have normal decondensation at 15 min (typically 96 ⁇ 2 percent SD).
  • Frozen donor sperm from a sperm bank can also be used, but the preferred negative control sample is an ejaculate kept at 4° C. for up to a month and used whenever needed during its 1 month shelf life.
  • This control serves as a negative control when used in performing both the SDAD and SDD Test protocol.
  • Positive controls sperm that fails in the SDD Test
  • Aliquots of these samples will be maintained for use as our positive control. The same protocol will be followed for the SDAD Test when it is offered commercially.
  • the present example demonstrates the clinical relationship of results obtained using the Phase Contrast and the Fourescent Image Analyses methods in using the SDAD assay.
  • the present example also describes the equipment and presents clinical data in the form of captured images, and demonstrates the strong correlation, and hence predictive clinical value, between the Phase Contrast and Fourescent Image Analysis as tools in monitoring and screening a human sperm sample.
  • MetaMorph Premier Acquisition system including workstation and hardware control capabilities for motoized microscope components, stage, camera, shutters as well as additional capabilities that can be used for future hardware integration.
  • CoolSnap HQII scientific-grade digital camera and driver CoolSnap HQII scientific-grade digital camera and driver.
  • Prior NanoScanZ 200 um Piezo Stage System Includes sample holders for slides, dishes and microplates and DAQ Board For Piezo drive integration.
  • the present example describes the protocol performed by the liquid handling robotics that mimics the manual operation.
  • the present example also describes the equipment needed for this procedure, and provides a comparison between a test result run manually and one run on the automated system, on the same specimen(s).
  • the equipment is basically a liquid handling robotics that can handle the following functions:
  • Mimicking the various steps in the manual process can then be done as in the example protocol as follows. However, this procedure is one example, but an automated approach is not limited to using the same concentrations, incubation times or steps as below. Thus, some steps may be performed in a different order, or perhaps eliminated altogether dependent on the particular conditions, etc., under consideration when running a particular sample batch, or dependent upon what materials are most readily available or convenient.

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CN101550447B (zh) * 2009-04-30 2011-04-20 张丽红 一种用末端转移酶介导生物素标记扩散染色质检测精子dna碎片的方法
JP2022506625A (ja) * 2018-11-02 2022-01-17 アンドロヴィア ライフサイエンシーズ， エルエルシー 精子の受精能獲得を決定することによる男性受精能の状態の特定、及びコンパニオン回収キット
JP2022531174A (ja) * 2019-04-30 2022-07-06 アンドロヴィア ライフサイエンシーズ， エルエルシー 精索静脈瘤に罹患している男性における、生殖アプローチの特定におけるｃａｐ－ｓｃｏｒｅ（商標）の使用

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RU2657609C1 (ru) * 2017-08-08 2018-06-14 Федеральное государственное бюджетное образовательное учреждение высшего образования "Московская государственная академия ветеринарной медицины и биотехнологии - МВА имени К.И. Скрябина" (ФГБОУ ВО МГАВМиБ - МВА имени К.И. Скрябина) Способ определения индекса фрагментации днк сперматозоидов у животных-производителей

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US5770363A (en) 1991-10-24 1998-06-23 Brown; David B. Methods for diagnosing human male infertility
US5919621A (en) 1991-10-24 1999-07-06 Brown; David B. Methods for diagnosing human male infertility
US5358847A (en) 1991-10-24 1994-10-25 Brown David B Method of screening for human sperm abnormalities as part of a regimen for assessing fertilizing capacity based upon reduced rates of chromatin decondensation and DNA synthesis
US5480772A (en) * 1993-02-03 1996-01-02 Brandeis University In vitro activation of a nucleus
US20090053722A1 (en) 2007-08-21 2009-02-26 David Brown Sdad assay and uses thereof

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CN101550447B (zh) * 2009-04-30 2011-04-20 张丽红 一种用末端转移酶介导生物素标记扩散染色质检测精子dna碎片的方法
JP2022506625A (ja) * 2018-11-02 2022-01-17 アンドロヴィア ライフサイエンシーズ， エルエルシー 精子の受精能獲得を決定することによる男性受精能の状態の特定、及びコンパニオン回収キット
JP2022531174A (ja) * 2019-04-30 2022-07-06 アンドロヴィア ライフサイエンシーズ， エルエルシー 精索静脈瘤に罹患している男性における、生殖アプローチの特定におけるｃａｐ－ｓｃｏｒｅ（商標）の使用

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