US 20150024385A1 as) United States a2) Patent Application Publication 10) Pub. No.: US 2015/0024385 Al Parrish (43) Pub. Date: Jan. 22, 2015 (54) PREDICTION OF FERTILITY IN MALES (52) U.S. CL CPC o.... GOIN 33/5091 (2013.01); GOIN 15/1475 (71) Applicant: WISCONSIN ALUMNI RESEARCH (2013.01); GOIN 2800/367 (2013.01) FOUNDATION, Madison, WI (US) USPC vececcsessecseesesssenscsssenseseecsesssanscnseesesaees 435/6.1 (72) Inventor: John Parrish, Mount Horeb, WI (US) (57) ABSTRACT (21) Appl. No.: 14/337,940 A method for evaluating sperm fertility. The method includes the steps of obtaining a sample of sperm from an animal of a (22) Filed: Jul. 22. 2014 species; staining the sample with a fluorescent DNA-binding , ° dye; collecting at least one image of the stained sample; Related U.S. Application Data determining an edge of a nucleusof at least one sperm within the stained sample in the at least one image; measuring an (60) Provisional application No. 61/856,828, filed on Jul. intensity of the DNA-binding dye within an area defined by 22, 2013. the edge of the nucleus ofthe at least one sperm; determining an average intensity per unit area of the area defined by the Publication Classification edge of the nucleus of the at least one sperm; comparing the average intensity per unit area to an average intensity per unit (51) Int. Cl. area for high-fertility sperm and low-fertility sperm of the GOIN 33/50 (2006.01) same species to determine if the sample has high or low GOIN 15/14 (2006.01) fertility.
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US 20150024385A1
as) United States
a2) Patent Application Publication 10) Pub. No.: US 2015/0024385 Al
Parrish (43) Pub. Date: Jan. 22, 2015
(54) PREDICTION OF FERTILITY IN MALES (52) U.S. CLCPC o.... GOIN33/5091 (2013.01); GOIN15/1475
(71) Applicant: WISCONSIN ALUMNI RESEARCH (2013.01); GOIN 2800/367 (2013.01)
FOUNDATION,Madison, WI (US) USPC vececcsessecseesesssenscsssenseseecsesssanscnseesesaees 435/6.1
(72) Inventor: John Parrish, Mount Horeb, WI (US) (57) ABSTRACT
(21) Appl. No.: 14/337,940 A method for evaluating sperm fertility. The methodincludes
the steps of obtaining a sample of sperm from an animal of a
(22) Filed: Jul. 22. 2014 species; staining the sample with a fluorescent DNA-binding, ° dye; collecting at least one image of the stained sample;
Related U.S. Application Data determining an edge of a nucleusofat least one sperm withinthe stained sample in the at least one image; measuring an
(60) Provisional application No. 61/856,828,filed on Jul. intensity of the DNA-binding dye within an area defined by
22, 2013. the edge ofthe nucleusofthe at least one sperm; determiningan average intensity per unit area ofthe area defined by the
Publication Classification edge of the nucleusofthe at least one sperm; comparing the
average intensity per unit area to an average intensity per unit
(51) Int. Cl. area for high-fertility sperm and low-fertility sperm of theGOIN 33/50 (2006.01) same species to determine if the sample has high or low
GOIN 15/14 (2006.01) fertility.
Patent Application Publication Jan. 22,2015 Sheet 1 of 7 US 2015/0024385 Al
FIG. 1
Patent Application Publication Jan. 22,2015 Sheet 2 of 7 US 2015/0024385 Al
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Patent Application Publication Jan. 22,2015 Sheet 5 of 7 US 2015/0024385 Al
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FIG. 6
US 2015/0024385 Al
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US 2015/0024385 Al
PREDICTION OF FERTILITY IN MALES
CROSS-REFERENCE TO RELATEDAPPLICATIONS
[0001] This application claimspriority to provisional appli-cation No. 61/856,828,filed Jul. 22, 2013, which is incorpo-
rated herein by referencein its entirety.
BACKGROUND
[0002] The present invention relates to sperm fertility and
in particular to prediction offertility from DNAstaining
[0003] Semen quality examinationsare central role ofthe
semen-processing laboratory. Many semen quality exams
exist to evaluate semen. Howeverthesetests are often flawedbecause they are designed to find higher-rather than lower-
fertility males, or the approaches reward extreme valuesrather than those that pass a minimum threshold (Parrish et
al., 1998; 2006). In addition, fertility of bulls used in com-mercial artificial insemination of dairy cattle is likely most
dependent on non-compensable sementraits, 1.e. traits that
cannot be overcome by increasing the number of sperminseminated. Many semen quality exams, however, target the
evaluation of compensable sementraits such as the percent-age of motile, live or acrosome-intact sperm.
[0004] Research has been directed to potential non-com-
pensable defects in sperm of lower-fertility bulls that alterevents during thefirst cell cycle of the zygote and result in
changesto the timing of cell divisions and success ofembryodevelopment(Eidet al., 1994; Parrish and Eid, 1994; Parrish
et al., 2006). It has been determinedthat defects or damage insperm DNAareresponsible for these effects. Since a signifi-
cant portion ofthe sperm nucleus consists ofDNA,it has been
hypothesized that subtle changes in sperm DNA might bereflected in physical properties such as sperm nuclear shape.
As a result of research in this area, it has been demonstratedthat careful measurements of sperm head morphology can be
used to predict fertility (Parrish et al., 1998, 2006, 2012).Nevertheless, there is an ongoing need for additional methods
to assess fertility.
SUMMARY
[0005] Accordingly, disclosed herein are methodsfor pre-dicting fertility of sperm samples based on the intensity of
DNAstaining of the samples, based on the surprising obser-vation that brighter DNA staining of sperm headshasa posi-
tive correlation with decreased fertility rates.
[0006] In one embodiment, the invention provides a
method for evaluating sperm fertility. The method includes
the steps of obtaining a sample of sperm from an animal of aspecies; staining the sample with a fluorescent DNA-binding
dye; collecting at least one image of the stained sample;determining an edge of a nucleusofat least one sperm within
the stained sample in the at least one image; measuring anintensity of the DNA-binding dye within an area defined by
the edge ofthe nucleusofthe at least one sperm; determining
an average intensity per unit area of the area defined by theedge of the nucleusofthe at least one sperm; comparing the
average intensity per unit area to an average intensity per unitarea for high-fertility sperm and low-fertility sperm of the
same species to determine if the sample has high or low
fertility.
[0007] In another embodiment, the invention provides a
method for evaluating sperm fertility. The method includes
Jan. 22, 2015
the steps of obtaining a sample of sperm from an animal of aspecies, staining the sample with a fluorescent DNA-binding
dye, obtaining fluorescent intensity measurements from aplurality of sperm in the stained sample, determining an aver-
age intensity of the fluorescent intensity measurementsobtained from the plurality of sperm in the stained sample,
and comparing the average intensity to average intensities for
high-fertility sperm and low-fertility sperm of the same spe-cies to determineif the sample has high or low fertility.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 showsa fluorescent image of bovine sperm
stained with Hoechst 33342 with a line surrounding most ofthe sperm heads showingthe results of the automated edge
detection procedure that was used to obtain the outline ofthesperm nucleus. Sperm nuclei that touch another sperm, the
edge ofthe imageorhave a significant distortion were deletedfrom the analysis and do not have an outline around the sperm
nucleus.
[0009] FIG. 2 showsa phase contrast image of the bovinesperm shown in FIG. 1, where the outline of the sperm
nucleus was obtained from the Hoechst 33342 fluorescent
imageandtransferred to this image.
[0010] FIG. 3 shows a phase contrast imageofbull sperm.
[0011] FIG. 4 shows a fluorescent microscope image of
Hoechst 33342 staining for the bull sperm of FIG.3.
[0012] FIG. 5 shows the sperm heads identified from theimages of FIGS.3 and 4.
[0013] FIG. 6 shows the fluorescently-stained sperm
sample of FIG. 4 after deconvolution of the image.
with mixing until paraformaldehyde dissolves, add 0.238 gm
Hepes, adjust pH to 7.4, adjust to 100 ml with water); Parrishcitrate fixative (10 ml Paraformaldehydestock,fill to 100 ml
with 2.9% sodium citrate buffer); Hoechst 33342 stain solu-tion (5 mg/ml in water made fresh daily).
[0049] Slide Preparation
[0050] Straws containing sperm samples (0.25 ml or 0.5ml) were thawed at 37° C. for 60 sec and the contents wereexpelled into 1.5 ml microcentrifuge tubes. The sperm werediluted 1:1 in 2.9% Sodium Citrate dihydrate solution to afinal volume of500 or 1000 ul respectively. Ifthe volume was1000 ul, then 500 ul of the diluted sperm sample was placedin a new tubefor staining of sperm. The sperm cells werestained by adding 2.5 wl ofHoechst-33342 stain solution andincubated at 37° C. for 30 min. After incubation, 250 ul of2.9% Sodium Citrate solution was addedto each tube, each ofwhich wasthen centrifuged at 6,000xg for 15 sec. The super-natant containing excess stain and extender was removed byaspiration. The sperm pellets were resuspendedwith 650 ul ofParrish citrate fixative and incubated for 3-5 min at roomtemperature. The fixed samples were briefly vortexed andcentrifuged as above, the supernatant removed, and the spermpellet resuspended with 750 ul ofwater. The sperm pellet waswashed a second time with 750 wl water and centrifuged asabove, and then finally resuspended with 500 ul water andvortexed. Then, a 10 ul of sample was placed onto a micro-scopeslide and gently spread out to make homogenous spermdistribution, and allowed to air dry completely on a slidewarmer at 37-39° C. Next, a 3.5 ul drop of the DABCOmounting solution was placed on top ofthe sample to preventfluorescent fading. An 18x18 mm coverslip was added on topand the edges were sealed with clear fingernail polish.
[0051] The procedures for using fresh bovine sperm are thesame as for frozen-thawed semen as described above exceptfor sperm dilution to start. Sperm are diluted to either aninsemination dose or 40x10° sperm/mlin citrate buffer withBSA(FractionV BSA at 3 mg/mlin standardcitrate buffer).Semen can also be diluted with egg yolk- or milk-basedextenders, instead ofBSA andthenprocessed as described forfrozen-thawed semencitrate buffer.
[0052] Image Collection
[0053] Sperm cells were imaged on a Nikon Microphotwith phase contrast and epifluorescent microscopy (excita-tion 365+20 nm,dichromatic mirror 400 nm, emission >400nm); images were collected using a 40x objective, 1.25xmagnifier. A QIClick monochrome camera operating in 8 bitmode and using an exposuresetting of 62.5 msec was used tocollect a tiff format image that was then saved for furtherimageanalysis.
[0054] Image Analysis Procedures for Bovine Sperm
[0055] Images were analyzed with NIH ImageJ version
1.47m using custom macros to implement the proceduresdescribed below. The following describes how an image pair
is analyzed, where the imagepair includes a phase (p) imageand a Hoechst 33342 (h) intensity image.
[0056] 1. Thresholding is applied to the Hoechst image and
sperm nuclear object identified.
[0057] a. Duplicate the image and renameit (e.g. ‘h-1’).
[0058] b. Apply ‘Unsharp Mask’ with radius=20 and
mask=0.60 to duplicate image.
Jan. 22, 2015
[0059] c. Apply ‘Autothreshold’ to the duplicate imageselecting the ‘IsoData’ method with the ‘Dark back-
ground’ box checked.[0060] d. Apply ‘Convert to Mask’, followed by ‘Dilate,’
followed by ‘Erode.’[0061] e. Use ‘Analyze Particles’ with the following set-
tings: size=700-4500; circularity=0.5-1.0;
show=Masks;and with the ‘Exclude... ,’ ‘Clear... ,’and ‘Add...’ boxes checked.
[0062] f. Renameresulting image(e.g. ‘hmask’) and runthe ‘Fill Holes’ routine on the resulting image.
[0063] g. Delete any sperm nuclei that do not appear to
be thresholded correctly. Indications that a given spermnucleusis not thresholded correctly can includeholes in
the edge of the sperm head, additional area in a part ofthe sperm head due to overlapping of another sperm
headortail in the image, presence offluorescent debris,or an object that has a shape not consistent with the
sperm head of the species underinvestigation.
[0064] h. Use ‘Create Selection’ command to make theperimeter of all sperm nuclei in the image from step f
(hmask') an object. Lay this object on the p image tocheckifobjects are the same approximate size and shape
ofthe sperm head in the p image.Ifany ofthe objects are
not similar between the p and hmask image, then showthe ‘hmask’file and delete any non-correct sperm nuclei.
Renamethe ‘hmask’ imageto‘all’.
[0065] i. Save the ‘p’, ‘h’, and‘all’ imagesto a subfolder
(e.g. called ‘mfiles’) as separate images (e.g. 2001 tiff,2002.tiff, 2003.tiff, respectively) and extending the
naming convention with more imagesets evaluated.
[0066] 2. Obtaining intensity values and obtaining meanvalues:
[0067] a. Access the appropriate ‘mfiles’ folder for a
particular sample.
[0068] b. Set ‘Measurements’ for mean. Additional mea-
sures of the intensity of the object (nucleus) and itsdispersion can also be selected as shown in Table 2 and
include median, standard deviation, skewness, and kur-tosis. It is also possible to select other measures that
describe the object such as perimeter and ellipse. The
perimeteris simply the outside boundary ofthe selectedsperm head. The ellipse option fits an ellipse to the
sperm head and the majoraxisofthe ellipseis the lengthand minor axis the width. The perimeter, length and
width may be expressed in tum orother suitable units.
[0069] c. Select the first ‘all’ image (e.g. 2003 tiff), use‘Analyze Particles’ with: size=O-Infinity, circularity—0.
00-1.00, show=Nothing, and with the ‘Display ... ’,‘Exclude ...’, ‘Include... ’, and ‘Add...’ boxes
checked.
[0070] d. Data per sperm is saved in a results table.
[0071] e. The next‘all’ image (e.g. 2006.tiff) is opened
and the abovesteps are repeated. This continues until nomore ‘all’ images exist. The results are saved and evalu-
ated within Statistical Analysis System (SASInc.).
[0072] f. Within SAS, 100 randomly-selected spermfrom those evaluated are selected and means from those
selected sperm are obtained for the various measure-ments.
[0073] Bull Sperm Measurement Results
[0074] This Example involves evaluating sperm from twopopulations ofbulls that represent extremesoffertility, with a
difference of8.8% fertility between the two populations. This
US 2015/0024385 Al
is the largest difference that can be obtained on a populationof bulls used for commercial insemination. There were 53
bulls in the high fertility group and 54 in the low fertilitygroup. The meantsem number of breedings used to deter-
mine fertility for the high and low fertility groups was23684324 and 1124+137, respectively.
[0075] Samples were collected and analyzed as described
above. Results comparing mean intensity of Hoechst 33342staining in the sperm head and other parameters measured on
the ‘h’ image are shownin Table 2. There were differences inmean intensity, standard deviation of intensity, skewness of
intensity, kurtosis ofintensity, median ofintensity, area ofthe
sperm head, perimeter, and width between sperm from thetwofertility groups (p<0.05). Surprisingly, the low fertility
bulls have an increasedintensity and variation between spermheads. The low fertility bulls also have sperm with a smaller
area, perimeter, and width.
TABLE 2
The mean + SEM forbulls in the twofertility groups for mean intensity
(INT,in arbitrary fluorescence units) and other measures of sperm head
characteristics determined directly from ImageJ.
Fert1 (high) Fert2(low) p value
Criteria N=53 N=54 (ANOVA)c
Fertlity Group 4.1+0.1 -4.7+0.3 —
Mean INT 99 #3 109 #3 0.0078Std of Mean 19.0 + 0.6 22.2 + 0.6 0.0002INTSkewness of 0.0088 + 0.0316 0.1741 + 0.0278 0.0001INTKurtosis of -0.2681 + 0.0483 -0.0879 + 0.0484 0.0097INTMedian of INT 100 #3 110 #3 0.0138Area (microns) 31.3 £.2 30.4 40.2 0.0017
Perimeter 23.08 + 0.07 22.79 + 0.09 0.0151
(microns)
Length 8.96 + 0.03 8.85 + 0.05 0.0611(microns)
Width 4.45 £0.02 4.37 £0.02 0.0041(microns)
Example 2
Sperm Nuclear Structure of Boars is Impacted by the
Summer Environment
[0076] The experiments in this Example involved the
evaluation of boar semen collected over the summer of2012.During the summer, it is known that boar semen declines in
fertility in response to increases in summer temperatures(Flowers, 1997). The summer of 2012 was extreme in Wis-
consin with daily high temperatures during the period of our
study exceeding 90° F. on 38 days comparedto an average of12 days in a normalyear. In Table 3, it can be observedthat
boar sperm nuclei change in their ability to have their DNAstain with Hoechst 33342 that includes an increase in mean
fluorescent intensity as well as increases in the length andwidth of the sperm heads.
[0077] The data from boarsis similar to the bull data, except
that the bull sperm data was correlated with known femalefertility information. For boars, on the other hand, fertility is
inferred from a known seasonal decline in fertility. As shown
above,low fertility bulls had high mean fluorescentintensityoftheir sperm nuclei which is what is demonstrated below to
occurto boar sperm nucleias the period ofsummerinfertility
Jan. 22, 2015
occurred. In contrast to bulls, in which higherfertility spermhave larger heads that those of low fertility groups, boar
sperm nuclei increased in length and width during a period inwhichit is expected that fertility decreases. Low fertility bull
sperm decreased in length and width of the heads. This maybe due to the differences in geometry ofthe boar sperm as they
are more tubularthan bull sperm. The reason for the changes
in sperm nuclear parameters of bulls and boars is unclear atthe present time. This may be due to nuclear condensation
during spermatogenesis or changes to condensation ofnucleithat occurs during passage of sperm throughthe epididymis.
The differences in sperm nuclearintensity, length, and widthprovide the means howeverto identify bulls of differentfer-
tility. For boar ejaculates, the differences in nuclear intensity,
length, and width provide the meansto identify a male suf-fering from summerheatstress and, by correlation with the
knowndeclinein fertility over the course of the summer, themeansto predict lowerfertility.
[0078] Materials and Methods
[0079] Semen was collected at a commercial boar stud inSouthern Wisconsin from Jun. 18, 2012 to Nov. 2, 2012. The
numberofboars collected each week and the Wednesday datefor a particular week are listed in Table 3. The number of
boars from which samples were collected/week ranged from
45-60 over the course ofthe summerof2012. Boars were onlythose that were usedfor single sire insemination.It is known
thatfertility of boars declines over the course of the summerwith peak declines occurring from July-August. Thus the
samples represent the period of time whenfertility of thesespecific boars are expected to decline.
[0080] Media[0081] The following mediaare required in the preparationof samples: 2.9% Sodium Citrate Buffer (2.9 gm sodium
citrate dihydrate, 90 ml distilled water, adjust pH to 7.4, adjust
final volume to 100 ml); Hepes buffered saline (0.238 gmHepesfree acid, 0.9 gm NaCl, 90 mldistilled water, adjust pH
to 7.4, adjust final volume to 100 ml with distilled water);DABCO mounting media (25 mg 1,4-Diazabicyclo[2.2.2.]
octane Triethylenediamine (DABCO), 100 ul Hepes bufferedsaline, mix until dissolved, combine with 900 ul glycerol);
Kaya A. Quantitative sperm shape analysis: What can this
tell us about male fertility. 2012. 24” Meeting of theNational Association ofAnimal Breeders, Columbia Mo.
(in Press).
US 2015/0024385 Al
[0120] Zwald N R, Weigel K A, Chang Y M, Welper R D,Clay JS. Genetic selection for health traits using producer-
recorded data. IJ. Genetic correlations, disease probabili-ties, and relationships with existing traits. Journal of dairy
science 2004a, 87:4295-4302.
[0121] Zwald N R, Weigel K A, Chang Y M, Welper R D,
Clay JS. Genetic selection for health traits using producer-
recordeddata. I. Incidencerates, heritability estimates, andsire breeding values. Journal of dairy science 2004b,
87:4287-4294.[0122] Peddinti D, Nanduri B, Kaya A, Feugang J M, Bur-
gess SC, Memili E. Comprehensive proteomic analysis ofbovine spermatozoa of varying fertility rates and identifi-
cation of biomarkers associated with fertility. BMC Syst
Biol. 2008, February 22;2:19. doi: 10.1186/1752-0509-2-19.m
[0123] Flowers W L. Management of boars for efficientsemen production. J. Reprod. Feral. Suppl. 1997, 52:67-
2.
[0124] Gibbs K M,Schindler J R, Parrish J J. Determiningthe effect of scrotal insulation on sperm production in the
boar. J. Anim. Sci. E-suppl. 2013, 91:591.[0125] Haralick R M, Shanmugam K, Dinstein I. Texture
parameters for image classification, IEEE Trans SMC 3.1973, 610-621.
[0126] Parrish J J. Bovine in vitro fertilization: In vitro
oocyte maturation and sperm capacitation with heparin.Theriogenology 2014, 81:67-73.
[0127] Various features and advantagesofthe invention areset forth in the following claims.
Whatis claimed is:1. A methodfor evaluating sperm fertility, comprising the
steps of:
obtaining a sample of sperm from an animalof a species;staining the sample with a fluorescent DNA-binding dye;
collecting at least one image ofthe stained sample;determining an edge of a nucleus of at least one sperm
within the stained sample in the at least one image;measuring an intensity of the DNA-binding dye within an
area defined by the edge ofthe nucleusofthe at least one
sperm;determining an average intensity per unit area of the area
defined by the edge of the nucleus ofthe at least onesperm; and
comparing the average intensity per unit area to averageintensities per unit area for high-fertility sperm and low-
fertility sperm of the same species to determine if the
sample has high or low fertility.
2. The method ofclaim 1, wherein staining the sample with
a fluorescent DNA-binding dye comprises staining thesample with Hoechst 33342.
3. The method ofclaim 1, wherein staining the sample with
a fluorescent DNA-binding dye further comprises applyingthe stained sample to a substrate.
4. The method ofclaim 3, wherein the nucleus ofthe at leastone sperm within the stained sample comprises a flattened
oval and wherein the flattened portion is adjacent to the sub-
strate.
5. The method of claim 1, wherein obtaining a sample of
sperm from an animal of a species comprises obtaining asample of sperm from a bull, a boar, a human,a horse, a ram,
or a dog.
Jan. 22, 2015
6. The method of claim 1, wherein collecting at least oneimage of the stained sample comprises deconvolving the atleast one image to removeout of focus information.
7. The method ofclaim 1, wherein the high-fertility spermhas a lowerintensity per unit area than the low-fertility sperm.
8. A method for evaluating sperm fertility, comprising the
steps of:obtaining a sample of sperm from an animalof a species;
staining the sample with a fluorescent DNA-binding dye;obtaining fluorescent intensity measurements from a plu-
rality of sperm in the stained sample;
determining an average intensity of the fluorescent inten-sity measurements obtained from the plurality of sperm
in the stained sample; andcomparing the average intensity to average intensities for
high-fertility sperm and low-fertility sperm of the samespecies to determine if the sample has high or low fer-
tility.9. The method of claim 8, wherein obtaining fluorescent
intensity measurements from a plurality of sperm in the
stained sample further comprises collecting at least oneimageofthe stained sample.
10. The method of claim 9, wherein determining an aver-age intensity of the fluorescent intensity measurements
obtained from the plurality of sperm in the stained sample
further comprisesdetermining an edge of a nucleus of at least one sperm
within the stained sample in the at least one image,measuring an intensity of the DNA-binding dye within an
area defined by the edge ofthe nucleusofthe at least one
sperm, anddetermining an average intensity per unit area of the area
defined by the edge of the nucleus of the at least onesperm.
11. The method ofclaim 10, wherein collecting at least oneimage of the stained sample comprises deconvolving the at
least one image to removeout of focus information.
12. The methodof claim 11, wherein staining the samplewith fluorescent DNA-binding dye further comprises apply-
ing the stained sample to a substrate.13. The method of claim 12, wherein the nucleusofthe at
least one sperm within the stained sample comprisesa flat-tened oval and wherein theflattened portion is adjacent to the
substrate.
14. The methodof claim 8, wherein obtaining fluorescentintensity measurements from a plurality ofsperm ina sample