Methods for diagnosing human male infertility

• US 5,919,621 A
• Filed: 04/29/1996
• Issued: 07/06/1999
• Est. Priority Date: 10/24/1991
• Status: Expired due to Fees

• Current Assignee: David B. Brown
• Original Assignee: David B. Brown
• Inventors: Brown, David B.
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US08/640,251
• Time in Patent Office: 1,163 Days
• Field of Search: 435/6, 435/91, 435/806, 435/810, 424/561, 935/77
• US Class Current: 435/6.19
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: G01N 2800/367 Infertility, e.g. sperm disorder, ovulatory dysfunction
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/689 related to pregnancy or the gonads
• CPC Class Codes: Y10S 435/806 Fertility tests
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A method for determining the capacity of a human sperm to fertilize a human egg is described by assessing sperm activation events in an in vitro assay using a non-mammalian egg extract, particularly a Xenopus laevis frog egg extract. Fertilizing capacity is assessed as a comparison of sperm decondensation, DNA synthesis and/or sperm recondensation as between a test sperm sample sperm and fertile sperm, such as a sperm sample from a proven fertile human male. The method employs results from the in vitro assay to also determine relative sufficiency or insufficiency of a sperm sample for fertilizing a human egg in human couples with a history of a diagnosed unexplained infertility from standard infertility diagnostic tests. The method may also be used to screen human sperm donors in human artificial insemination programs. A fixed-slide cytoprep sperm analysis of decondensed sperm chromatin, as between a sperm test sample and a sperm sample from a proven fertile human male, may also be used to confirm in vitro decondensation results of the infertility or fertility of a particular human male. A kit is also provided for testing male sperm samples for human egg fertilizing capacity.

Claims

1. An in vitro method for screening for abnormal human sperm as part of a regimen for assessing human sperm fertilizing capacity comprising:

obtaining a human sperm sample and isolating a number of sperm therefrom to provide a test sample;

incubating the test sample in a frog egg extract capable of supporting human sperm chromatin decondensation, DNA synthesis and chromatin recondensation;

monitoring the test sample for sperm chromatin decondensation and detecting an amount of test sperm decondensation; and

assessing the amount of human test sperm decondensation of the test sample and the amount of sperm chromatin decondensation of a human sperm control sample of proven fertile human sperm in the frog egg extract,

wherein a test sample demonstrating about 80% or less decondensed sperm chromatin relative to the control sample amount of sperm chromatin decondensation provides a screen for abnormal human sperm.

3. The method of claim 1 or 2 wherein the steps of incubating the sperm test sample in the frog egg extract comprise:

permeabilizing the sperm test sample in a lysolecithin containing medium to provide permeabilized test sperm;

chemically reducing the permeabilized test sperm in an isolation medium containing dithiothreitol to provide reduced sperm nuclei;

incubating the reduced sperm nuclei in a Xenopus laevis frog egg extract which includes a detectable labeling compound for about 15 minutes to allow decondensation of the reduced sperm nuclei; and

comparing the sperm decondensation of the test reduced sperm nuclei to decondensation of control reduced sperm nuclei of a human male of proven fertility.

4. The method of claim 1 or 2 wherein the non-mammalian egg extract is Xenopus laevis frog egg extract.

5. The method of claim 4 wherein the test sample is incubated in the presence of ³ H-TTP for about 2 hours at about 19°

C.

6. The method of claim 1 or 2 wherein the sperm test sample is permeabilized in a medium containing about 0.05% lysolecithin and about 1 μ

g/ml soybean trypsin inhibitor for about 5 minutes at about 22°

C.

7. The method of claim 3 wherein the detectable labeling compound is ³ H-TTP included at a concentration of about 80 μ

Ci/ml in the Xenopus laevis frog egg extract.

8. The method of claim 3 wherein the permeabilized test sperm is reduced in a an isolation medium containing about 50 mM dithiothreitol for about 45 minutes at a temperature of between about 0°

C. to 4°

C.

9. The method of claim 3 wherein the sperm decondensation is monitored for about 10 minutes.

10. The method of claim 3 wherein the test sample is washed in a nuclear isolation medium consisting of about 200 mM sucrose, about 2.4 MM MgCl₂, about 10 mM Tris HCl, and about 5 mM maleic acid at a pH of about 7.4 before permeabilizing.

2. A method for diagnosing infertility in a human male of unexplained infertility comprising:

obtaining a sperm test sample from the human male of unexplained infertility;

incubating the sperm test sample in a frog egg extract capable of supporting human sperm decondensation, DNA synthesis and chromatin recondensation; and

comparing the amount of chromatin decondensation of the sperm test sample to the amount of chromatin decondensation of a sperm control sample from a human male of known fertility,

wherein a sperm test sample demonstrating 80% or less decondensed sperm as compared to the sperm control sample is diagnostic of infertility in the human male of unexplained infertility.

11. A method for screening human sperm as part of a regimen for selecting prospective fertile human sperm donors for in vitro fertilization of a human egg comprising:

obtaining a sperm sample from a prospective human sperm donor to provide a test sperm sample;

incubating the test sperm sample in a frog egg extract containing a detectable labeling compound;

monitoring sperm chromatin decondensation of the test sperm sample;

comparing the test sperm sample chromatin decondensation to a control sperm chromatin decondensation from a human male of proven fertility; and

selecting a sperm test sample that demonstrates at least 80% of the control sperm chromatin decondensation as prospective donor sperm for in vitro fertilization of a human egg.

12. The method of claim 11 wherein sperm chromatin decondensation is monitored for at least about 15 minutes.

14. The method of claim 11 wherein the frog egg extract is Xenopus lavis frog egg extract, and incubating the test sperm sample in frog egg extract comprises the steps of:

permeabilizing a number of test sample sperm in a medium containing lysolecithin and a protease inhibitor to provide permeabilized human test sperm;

reducing the permeabilized test sperm in an isolation medium containing about 50 mM dithiothreitol for about 45 minutes at about 0°

C. to 4°

C.; and

incubating the reduced sperm in the frog egg extract for a period of time sufficient to allow chromatin decondensation to occur.

15. The method of claim 14 wherein the lysolecithin is contained at a concentration of about 0.05% and the protease inhibitor is soybean trypsin inhibitor at a concentration of about 1 μ

g/ml.

16. The method of claim 11 wherein the detectable labeling compound is ³ H-TTP or tritiated thymidine.

13. The method of clam 11 wherein the frog egg extract is a Xenopus laevis or Rana pipiens frog egg extract.

17. A method for diagnosing infertility in a human male partner of an unexplained infertile human couple comprising the steps of:

obtaining a sperm sample from the human male partner of the unexplained infertile human couple to provide a test sperm sample;

incubating the test sperm sample in a frog egg extract capable of supporting human sperm chromatin decondensation to provide test sperm chromatin decondensation;

determining the amount of test sperm chromatin decondensation;

comparing the amount of the test sperm chromatin decondensation to an amount of sperm chromatin decondensation of control sperm of a proven fertile human male; and

diagnosing infertility in the human male partner of the unexplained infertile human couple where the test sperm chromatin decondensation is 80% or less of the control sperm chromatin decondensation.

18. The method of claim 17 wherein the frog egg extract is a Xenopus laevis or Rana pipens egg extraxt.

19. The method of claim 17 wherein the test sperm sample is processed according to the following steps prior to incubating in the frog egg extract:

permeabilizing the test sperm sample in a nuclear isolation medium containing about 0.05% lysolecithin and about 1 μ

g/ml soybean trypsin inhibitor for about 5 minutes at about 22°

C. to provide a permeabilized test sperm sample;

washing the permeabilized test sperm sample with a nuclear isolation medium containing about 3% bovine serum albumin;

pelleting and washing a second time the permeabilized test sperm sample with the nuclear isolation medium containing about 0.4% bovine serum albumin to provide a washed test sperm sample;

reducing the washed test sperm sample in a volume of isolation medium including about 50 mM dithiothreitol for about 45 minutes at about between 0°

C. to about 4°

C. to provide a permeabilized reduced test sperm sample; and

labeling the permeabilized reduced test sperm sample with a detectable labeling compound contained within a volume of Xenopus laevis frog egg extract.

20. The method of claim 19 wherein the detectable labeling compound is ³ H-TTP contained at a concentration of about 80 μ

Ci/ml within a Xenopus laevis frog egg extract.

21. The method of claim 17 wherein a test sperm chromatin decondensation of less than or equal to 85% compared to control sperm sample chromatin decondensation is diagnostic of human male infertility.

22. A toxicological screen for human sperm damage comprising:

obtaining a sample of human sperm exposed to a toxic agent to provide a test sperm sample;

incubating the test sperm sample in a frog egg extract capable of supporting human sperm chromatin decondensation to provide a test sperm chromatin decondensation;

measuring the test sperm chromatin decondensation; and

comparing the test sperm chromatin decondensation to a control sperm chromatin decondensation of proven fertile human male sperm,

wherein a test sperm chromatin decondensation less than about 80% of the control sperm chromatin decondensation provides a toxicological screen for human sperm damage.

23. The method of claim 22 wherein the non-mammalian egg extract is a Xenopus laevis frog egg extract.

24. The method of claim 22 wherein a percentage of test sperm decondensation less than 90% of fertile human sperm decondensation identifies sperm damage in the test sample.

25. A method for screening varicocele human male patients as part of a regimen for screening patients for varicocelectomy comprising:

monitoring chromatin decondensation in a sperm sample from the varicocele male and a proven fertile human sperm sample in a frog egg extract; and

comparing decondensed nuclei in the varicocele sperm sample and the proven fertile human sperm sample,

wherein a percentage of varicocele sperm decondensation less than 80% of the percentage of fertile sperm decondensation identifies a potential candidate for varicocelectomy.

26. The method of claim 25 wherein a percentage of test sample sperm decondensation less than 90% of the percentage of fertile sperm decondensation identifies a potential candidate for varicocelectomy.

27. The method of claim 25 wherein the non-mammalian egg extract is a Xenopus laevis frog egg extract.

28. A diagnostic kit for screening human males for infertility comprising:

a carrier means adapted to receive at least four container means therein;

a first container means containing a volume of dithiothreitol;

a second container means containing a volume of lysolecithin;

a third container means containing a non-mammalian egg extract capable of supporting fertile human sperm chromatin decondensation;

a fourth container means containing a control sperm sample having a capacity for human sperm activation from a proven fertile human male; and

at least two container means suitable for including therein a volume of up to 1 ml of a human sperm sample.

29. The kit of claim 28 further comprising a written instruction sheet defining diagnostic test steps.

30. The kit of claim 28 wherein the non-mammalian egg extract is a Xenopus laevis egg extract.

31. The kit of claim 28 wherein the Xenopus laevis frog egg extract is stable for at least 48 hours stored at about 4°

C.

32. The kit of claim 28 comprising at least 2 glass slides.

33. The kit of claim 28 comprising at least 6 glass slides and at least 6 filter cards.

34. The kit of claim 28 further comprising an autoradiography emulsion, an autoradiography developer, and an autoradiography fixer.

35. The kit of claim 28 comprising a container means including a volume of bovine serum albumin.

36. The kit of claim 28 comprising a Giemsa Blood Staining Solution.

37. An in vitro method for screening human sperm for abnormalities as part of a regimen for assessing human sperm fertilizing capacity comprising:

obtaining a human sperm sample and isolating a number of sperm therefrom to provide a test sample;

incubating the test sample in a frog egg extract capable of supporting human sperm decondensation, DNA synthesis and sperm recondensation;

monitoring the test sample for sperm chromatin decondensation, DNA synthesis and sperm chromatin recondensation and comparing sperm chromatin decondensation, DNA synthesis, and sperm chromatin recondensation of the test sample to a control sperm sample of a proven fertile human male,

wherein a test sample demonstrating 80% or less of the chromatin decondensation, DNA synthesis and chromatin recondensation of the control sample provides a method for screening human sperm for abnormalities as part of a regimen for assessing human sperm fertilizing capacity.

38. The method of claim 37 wherein the frog egg extract is a Xenopus larvis frog egg extract.

39. The method of claim 37 wherein sperm chromatin decondensation after a 15 minute incubation of the test sample in the frog egg extract is compared to sperm chromatin decondensation after a 15 minute incubation of the control sample in the frog egg extract.