Sperm Function Tests
Shahin Ghazali
PhD student
Yazd Reproductive Sciences Institute
Introduction:
• During recent years, apart from the light
microscopical determination of sperm count and
morphological malformation, evaluation of
function of sperm parameters has become a
powerful tool in andrological laboratories.
These assays determine:
• Biochemical parameters:
a-glucosidase
Polymorphonuclear granulocyte(PMN)-elastase
• Biological functions:
Motolity
Membrane integrity
Morphology
Zona binding
Acrosome reaction
Acrosin activity
Oolemma binding
Chromatin condensation
DNA integrity
• Sperm function test were developed to detect
abnormalities:
- In sperm survival
- Transport in the female genital tract
- Different steps of fertilization
Types:
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Vitality tests
Sperm-mucus Interaction Tests
Capacitation
Acrosome Reaction
Zona Binding assays
Hamster Ovum Penetration test
Vitality tests:
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Are used when:
Low percentage motility
Lost their flagellation
because of metabolic dysfunction or axonemal defects
Necrozoospermia (dead sperm)
Vitality tests:
• Hypo-osmotic swelling test (HOS)
• Eosin test
• Eosin-nigrosin
Hypo-osmotic swelling test (HOS)
• Simple test for integrity of plasma membrane
• Using hypo-osmotic solution
• Influx of water
• Introduced by Jeyendran et al. (1984)
Result:
Intact sperms: swelling of the tail into various sizes and shape
Dead cells: normal tail shape because of leaky membrane
Eosin Test:
• Based on the fact that eosin is excluded by live cells.
• Can be used in ICSI
• Result:
• Damaged cells: are stained specifically
Sperm-mucus Interaction Tests
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In
In
In
In
In
vivo postcoital test
vitro sperm-mucus interaction test
vitro sperm cervical mucus contact test
vitro slide test
vitro capillary test (Kremer test)
Cervical mucus:
• First barrier for sperm migration
• Highly viscous entire menstrual cycle
• Penetrable only for a few preovulatory days
• Mucus quality is very important for sperm
assessment.
In vivo postcoital test
• 9-24 h after coitus
• Examining both penetration and survival of sperm
• Using high magnification microscope
• Result:
• Presence of motile sperm= +
In vitro sperm-mucus interaction test
• Abstinence for 3 days
• Aspiration from endocervical canal
• Administrating ethinyl estradiol for insufficient volume
• Evaluation by WHO scoring system:
Volume
Viscosity
Ferning
Spinnbarkeit (elasticity)
Cellularity ( No. of leukocytes or other cells)
pH
In vitro sperm cervical mucus
contact test:
• Detecting antibodies on sperm or in mucus
• Performing immediately after semen liquifaction
• Mixing one drop of semen and mucus on a slide
• Result:
• Shaking sperm >25% = +
In vitro slide test
• A mucus drop in the center of slide
• Semen seep from the edge of cover slip
• 30 min incubation
• Result:
• Normal semen: fingerlike penetration> 90 %
In vitro capillary test
(Kremer test)
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Mucus loaded into a flat capillary tube (Sealed one end)
The open end put vertically into reservoir of semen
Incubation for 1 h at 37c
Scoring vanguard sperm at 1, 4, and 7 cm
• Result:
• Excellent to poor penetration or negative penetration
Penetrak Test
• Commercial
• Using bovine cervical mucus as a surrogate in
the penetration test.
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Androlog Mail
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The bovine mucus penetration test does measure important characteristics of
sperm function. It measures
1. the strength and effectiveness of sperm motility and
2. the surface characteristics of sperm that allow them to
interact appropriately with the fluids overlying the mucosal surfaces of the
female tract.
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However, clinically speaking, the BMPT does not add that much
information. If mucus penetration is a problem for a patient's sperm, then
chances are good that other semen characteristics will also be deficient,
and IUI, which bypasses the cervical mucus, or another ART will be
prescribed. So clinically, it may not add much to our standard Andrology
tests.
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I am still using the BMPT if physicians order it specifically, which has
become rather rare.
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I order the Penetrak Kits from Bio-Chem Immunosystems, 100 Cascade Dr.,
Allenton, PA 18001, 1-800-345-3127 (product #4913000). I last ordered them
in September, 1998.
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Erma Z. Drobnis, Ph.D.
Clinical Assistant Professor
Obstetrics & Gynecology and Surgery/Urology
Director, Fertility Laboratories
University of Missouri-Columbia
3211 S. Providence Dr.
Columbia, MO 65201 U.S.A.
Tel: (573) 882-7176
Fax: (573) 884=4492
drobnise@health.missouri.edu
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A simplified approach to sperm-cervical mucus interaction testing using a
hyaluronate migration test
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D. Mortimer1, S.T. Mortimer, M.A. Shu and R. Swart Endocrine/Infertility Clinic, Department of Ob/Gyn, University of Calgary, Health
Sciences Centre 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada
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Correspondence: 1To whom correspondence should be addressed
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Fiffy-one comparisons were made of human sperm migration into capillary tubes
containing either human cervical mucus (‘Kremer test’) or a synthetic mucus
substitute consisting of a 5 mg/ml solution of sodium hyaluronate (average mol. wt
2 x 106) in a phosphate-buffered medium.
The results of these two tests were highly significantly correlated and dependent
upon the same sperm characteristics reflecting sperm progressive ability
(including the specific movement characteristic of lateral head displacement
amplitude), morphological normality and cellular vitality as well as the concentration
of these more functional cells in the semen.
The result of the hyaluronate migration test, in conjunction with the mucus quality
measures of Insler score and pH, allowed a 92.2% correct prediction of the Kremer
test outcome (90.9% of normal tests and 93.1% of abnormal tests). In this data set,
these values also corresponded to the sensitivity and specificity of the analysis,
respectively. From these studies, we propose the hyaluronate migration test as a
useful adjunct to routine semen analysis, sperm movement analysis and the
more traditional in-vitro tests of sperm-cervical mucus interaction in the
diagnostic investigation of infertile couples. It effectively assesses the mucuspenetrating potential of a semen sample without the need for relatively large
quantities of midcycle cervical mucus; it will therefore augment (as an internal
control), although not necessarily replace, the homologous Kremer test and reduce
the quantity of both patient and donor mucus needed for comprehensive
crossedhostility format testing of sperm — mucus interaction
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Capacitation
• Cell surface changes
• Hyperactivated motility
• Vigorous nonprogresive motion with forceful
amplitude bending
1
• Sperm wash
• Incubation in albumin-containing culture
• Very simple
• No need for oocyte or mucus
2
• Using CASA
3
• Chlortetracycline-staining
• Detection by fluorescence microscopy
• Acrosome-reacted sperm show a staining pattern different from that
of capacitated sperm with acrosomes still intact
2
• Using CASA:
• Distinguish hyperactivated from nonhyperactivated
sperm, mainly by:
the high curvilinear velocity
low linearity
large value of the amplitude of lateral head displacement of
the former
CASA:
• Several comercial manufactures provide CASA system:
Hamilton Thorne
Hosbon sperm Tracker
• Parameter settings:
The user responsible for cheking and set up
Use video tapes as quality control
Preparation of samples:
• Maintain at 37c
• Standard concentration is 25-50* 10 6
• Using Dulbecco’s PBS-glucose-BSA medium
Pic page 106
CASA terminology:
• VCL = curvilinear velocity( micron/s) Time-average velocity of a
sperm head along its actual curvilinear path, as perceived in two
dimensions in the microscope .
• VSL = straight line velocity( micron/s) Time-average velocity of a
sperm head along the straight line between its first detected position
and its last .
• VAP = average path velocity( micron/s) Time-average velocity of a
sperm head along its average path. This path is computed by
smoothing the actual path .
• BCF = beat cross frequency( beats/s) The average rate at which the
sperm's curvilinear path crosses its average path .
• LIN = linearity The linearity of a curvilinear path .
• STR straightness The linearity of the average path .
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medeaLAB CASA
A new system for objective semen analysis which offers:
Focus on routine requirements
Real-time processing for simultaneous assessment of up to 200 moving objects
Sperm tracking for the duration of several seconds
Attractive price as well as reliable support and after-sales service .
Key features :
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Automated analysis of sperm motility and morphology. Calculation of a complete
spermiogram in under 30 seconds, multiple recordings for each sample are possible
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Storage of patient data and sample recorded on template in MS Access™ and SQL-Server
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Classification of sperm motility according to WHO categories
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Print of spermiogram by means of user-defined HTML templates
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Automatic export of data into MS Excel™ for further statistical analysis
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Storage of images from morphological analysis
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Full RecDate database integration (Germany only)
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Use of standard hardware reduces investment need
MAIN SOFTWARE FEATURES:
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Acquisition of images and clips in AVI format from an imaging device.
Storage of textual data, images and clips in the built-in MultiMedia Catalog
database. You can take the advantage of all the powerful features of
MultiMedia Catalog in application to semen analysis. The database is a
perfect tool for archiving of patient data, analysis results, video clips and
images, fast and flexible search and spectacular reporting with images,
graphs and other features adjusted to your particular needs. Herewith our
database can serve the needs of virtually any laboratory or research
institution.
Automated analysis of sperm concentration and motility on native
samples according to WHO recommendations.
Automated morphology analysis on stained samples according to strict
Krueger’s criteria.
Manual assessment of concentration of white blood cells, immature
germ cells, round cells.
Manual measurements for special research or other individual tasks.
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Computer assisted semen analyzers in andrology research and
veterinary practice.
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Verstegen J ,Iguer-Ouada M ,Onclin K.
University of Liège, Department of Animal Clinical Sciences, Small Animal Reproduction Bd Colonster 20, B44, B 4000 Liège Belgium.
Abstract
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The evaluation of sperm cell motility and morphology is an essential parameter in the
examination of sperm quality and in the establishment of correlations between sperm
quality and fertility. Computer-assisted sperm analysis (CASA) allows an objective
assessment of different cell characteristics: motion, velocity, and morphology.
The development and problems related to this technology are raised in this review,
paying particular attention to the biases and standardization requirements absolutely
needed to obtain useful results. Although some interesting results, mainly in humans,
have already been obtained, many questions remain, which have to be answered to
allow for further development of this technology in veterinary medicine, clinical
fertility settings, physiological, and toxicology research activities. The main problem
is related to the standardization and optimization of the equipment and
procedures. The different CASA instruments have all demonstrated high levels of
precision and reliability using different sperm classification methodology. Their
availability gives us a great tool to objectively compare sperm motility and
morphology and to improve our knowledge and ability to manipulate spermatozoa.
Acrosome Reaction
Tests for acrosome reaction:
• Triple stain
• Trypan blue
• Fluoroscent lectins and antibodies
Triple stain:
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The sperm suspensions from the swim-up were diluted with an equal volume of the
defined medium containing 0.2% trypan blue, incubated at 37‫؛‬C for 15 minutes,
smeared on prewarmed glass slides, and air dried. The slides were rinsed in water
and blotted. The smears on slides were fixed in 3% glutaraldehyde solution in 0.2M
phosphate buffer at room temperature for 45 minutes, rinsed in water, and air dried.
The smears were stained in 0.5% Bismark Brown solution in 30% ethanol at
40‫؛‬C for 10 minutes, rinsed briefly in water, and air dried. Finally, the smears were
stained with Rose Bengal to evaluate acrosomal status, following distinction of
live cells from dead ones using trypan blue. After staining, the slides were
examined at 1,000x under phase-contrast microscopy and spermatozoa were
classified into the following four categories:17
.1live spermatozoa with acrosome reaction - light rose postacrosomal regions and
white "acrosomal regions;"
.2dead spermatozoa with normal acrosomes - blue postacrosomal regions;
.3dead spermatozoa with abnormal acrosomes (i.e.; degenerative acrosome
reactions) - blue postacrosomal regions with white "acrosomal regions;"
.4live spermatozoa with normal acrosomes - light rose postacrosomal regions and
pink acrosomes.
• .1a live spermatozoon acrosome reacted;
.2a dead spermatozoon with an intact
acrosome ;
.3a dead spermatozoon with degenerative
acrosome reaction ;
.4live spermatozoa with intact acrosomes ;
5and 6. live spermatozoa with acrosome
partially intact .
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Reproductive Biology
A triple-stain technique for evaluating normal acrosome
reactions of human sperm
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Prudence Talbot, Richard S. ChaconDepartment of Biology, University of California, Riverside, CA
92521
• Abstract
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A triple-stain technique has been developed to score normal acrosomereacted human sperm in fixed smears. Live and dead sperm are first
differentiated using the vital stain trypan blue. Sperm are then fixed in
glutar-aldehyde, dried onto slides, and the postacrosomal region and
acrosome are differentiated using Bismark brown and Rose
Bengal. Slides are examined at 1,000 × with a bright-field microscope and
assessed for (1) the percentage of sperm that were alive at the time of
fixation and (2) the percentage of sperm that had undergone normal
acrosome reactions.
Experiments are included that show that trypan blue is a reliable stain for
dead sperm and that Rose Bengal stains only sperm having intact
acrosomes. This technique may have applications in experimental and
clinical studies on sperm capacitation, acrosome reactions, and fertilization
in laboratory and domestic animals as well as in man.
Fluoroscent lectins and antibodies
• Most common probes:
• Peanut lectin, which lables the outer acrosomal
membrane.
• Pea lectin, which lables the acrosomal content
• Antibody:
• CD46, which targets the inner acrosomal membrane
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Upper panels )Laser scanning confocal fluorescence images of sperm. Triple
labeling of sperm with FITC-Pisum sativum lectin for acrosome (green), TOTO-3
iodide for DNA (blue), and Nile red for membrane lipid (red) incubated in the
absence( A )and presence of WHI-05( B ,)WHI-07( C ,)and N-9( D )for 3 h. An
intense acrosomal staining with FITC-lectin (green), nuclear staining with TOTO-3
(blue), and plasma membrane staining (red) of the sperm tail region with Nile red are
apparent. In acrosome-intact sperm, the acrosomal region of the sperm heads
exhibited a uniform, bright green fluorescence. In acrosome-reacted sperm, green
fluorescence was either absent or restricted to the equatorial segment of the sperm
heads. Sperm exposed to 1% DMSO alone( A ,)WHI-05( B ,)and WHI-07( C )did not
reveal increased acrosome reaction at 3 h of incubation. Sperm exposed to 100 μM
of N-9 under identical conditions revealed only acrosome-reacted sperm( D( )original
magnification ×1000 .)Middle panels )High-resolution low-voltage scanning
electron micrographs of sperm incubated in the absence( A )′and presence of 100
μM each of WHI-05( B ,)′WHI-07( C ,)′and 10 μM CaI( D )′for 3 h (×18 000
magnification). Postfixation with OsO 4and ruthenium red yielded very smooth
plasma membrane over the acrosome-intact sperm head. The smooth acrosomal
surface is delimited from the postacrosomal region by a equatorial band( A .)′Sperm
exposed for 3 h to WHI-05 and WHI-07, respectively, reveal intact acrosome with
various degrees of ruffling of the plasma membrane. CaI-treated sperm reveal
vesiculation, blebbing, and loss of the plasma and acrosomal membranes and wellpreserved postacrosomal membrane .Bottom panels )Transmission electron
micrographs of human sperm incubated in the absence( A )′′and presence of 100 μM
each of WHI-05( B ,)′′WHI-07( C ,)′′or 10 μM CaI( D )′′for 3 h (×18 000 magnification).
The plasma membrane is present over the sperm head. Both acrosomal and
postacrosomal membranes are clearly visible after 3 h of incubation with WHI-05 and
WHI-07. Note the complete loss of acrosome in CaI-treated sperm( D .)′′Figure
reproduced at 84% of original .
• Sperm-zona binding in human is species pecific
• Human zonae obtained from surplus oocytes in ivf
• Oocyte used fresh or after storage in high salt concentration
• It is the best single predictor for IVF success
• Recombinant ZP3 and ZP2 (responsible for primary and secondary
sperm binding)
• zona binding ability was a useful addition to routine
laboratory assessments used to estimate sperm fertility.
• Hemizona test
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ZP is cut into exactly two halves using micromanipulator
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One half is incubated with the patient’s capacitated sperm
The other half with the fertile donor’s sperm as control
Result:
Binding ability (hemizona index) = number of bound
sperm/bound donor sperm * 100
Competetive binding assay
• Both patient and donor sperm for control are labled with
different flurochrome:
Green with fluorescein isothiocyanate
Red with tetramethyl rhodamine isothiocyanate
Result:
Binding rate( ratio of patiant/donor sperm) reflects
the binding capacity
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Summary
Systems Biology in Reproductive Medicine
,1997Vol. 38, No. 2, Pages 127-131
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The Hemizona Assay: A Simplified Technique
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M. Janssen ,1,2W. Ombelet ,†1,2A. Cox ,1,2H. Pollet ,1,2D. R. Franken 1,2and E. Bosmans 1,2
1The Genk Institute for Fertility Technology, ZOL Campus St. Jan, Genk, Belgium
2Tygerberg Hospital, Reproductive Biology Unit, Department of Obstetrics and Gynecology, Tygerberg, South Africa
†Correspondence :W. Ombelet, Genk Institute for Fertility Technology, ZOL-Campus St. Jan, Schiepse Bos 6, B-3600, Genk, Belgium
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The hemizona assay is an important diagnostic tool in assessing human
sperm fertilizing potential. Previous hemizona assay results have proven
that this functional test is a good predictor of fertilization in vitro and can be
used in clinical practice to supply additional information in male factor
subfertility cases. The objective of this study was to compare two methods
for cutting human zona pellucida into equal halves (manual handcutting
versus micromanipulation) in order to examine the necessity of an
expensive micromanipulator in performing this assay. Comparable results
for recovery rate, diameter size of the hemizonae, and sperm binding were
achieved with both methods. According to these results, the use of an
expensive micromanipulator is not essential in performing the
hemizona assay.
• Comparison of two methods to obtain hemizonae pellucidae for
sperm function tests
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R. Sánchez1, C. Finkenzeller, W.-B. Schill and W. Miska2 Department of Dermatology and Andrology, Justus
Liebig University Gaffkystrasse 14, 35392 Giessen, Germany 1Department of Preclinical Science, La Frontera
University Temuco, Chile
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Correspondence: 2To whom correspondence should be addressed
• The development of a new generation of diagnostic techniques has
provided objective data on the physiological function of the
spermatozoon. The hemizona assay has been considered the best
predictor for in-vitro fertilization results. Its clinical application has
been limited to the availability of human oocytes and the use of a
special micromanipulator. Here, the sperm binding capacity of handsectioned hemizonae was compared with that of oocytes bisected
by a micromanipulator.
• The results obtained in parallel assays showed no statistical
differences between the two methods. Therefore, the bisection of
oocytes by hand is useful for hemizona assays even in normal
clinical laboratories.
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Neural cadherin is expressed in human gametes and participates in sperm–oocyte
interaction events
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C. I. Marín-Briggiler*, L. Lapyckyj*, M. F. González Echeverría†, V. Y. Rawe‡, C. Alvarez Sedó‡ and M. H. Vazquez-Levin* *Instituto de
Biología y Medicina Experimental, National Research Council of Argentina (CONICET), University of Buenos Aires, Buenos Aires,
Argentina , †Centro Médico Fertilab, Buenos Aires, Argentina , and ‡Centro de Estudios en Ginecología y Reproducción, Buenos Aires,
Argentina
Correspondence to Mónica H. Vazquez-Levin, Instituto de Biología y Medicina Experimental, CONICET, Vuelta de Obligado 2490, Room
B16 & B24, ZIP Code: 1428ADN, Buenos Aires, Argentina. E-mail:
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ABSTRACT
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Neural cadherin (N-cadherin) is a transmembrane glycoprotein involved in calcium-dependent
cell–cell adhesion and signalling events. The previous evidence shows N-cadherin expression in
the human gonads and gametes; however, N-cadherin subcellular localization in human
spermatozoa and oocytes, and its involvement in fertilization remain to be
characterized. In this study, expression of N-cadherin in human spermatozoa and testis was
confirmed by RT-PCR and protein forms were identified using Western immunoblotting. Ncadherin localization in testicular and ejaculated spermatozoa, in cells that had undergone
capacitation and acrosomal exocytosis, as well as in oocytes was assessed using
immunocytochemistry. Participation of the adhesion protein in fertilization was evaluated using the
HemiZona Assay (HZA) and the zona pellucida (ZP)-free hamster oocyte sperm penetration assay
(SPA). Both the N-cadherin transcript and the mature protein form (135 kDa) were found in
spermatozoa and testis. The protein was mainly immunolocalized in the acrosomal region of
testicular, non-capacitated and capacitated spermatozoa, and was found in the equatorial
segment after acrosomal exocytosis. N-cadherin was also detected in oocytes, in conjunction
with β-catenin, a member of the adhesion complex. Sperm incubation with anti N-cadherin
antibodies did not affect their ability to interact with homologous ZP in the HZA; by contrast,
presence of the antibodies in the SPA led to a significant (p < 0.01) reduction in the percentage of
penetrated oocytes. In conjunction, results indicate that N-cadherin is a sperm protein of
testicular origin localized in cellular regions involved in gamete interaction. N-cadherin
would not participate in sperm–ZP interaction, but it would have a role in sperm–
oolemma adhesion/fusion events.
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Fertil Steril. 2006 Jun;85(6):1697-707. Epub 2006 May 6.
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Predictive value of the hemizona assay for pregnancy outcome in patients
undergoing controlled ovarian hyperstimulation with intrauterine insemination.
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Arslan M, Morshedi M, Arslan EO, Taylor S, Kanik A, Duran HE, Oehninger S.
The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk,
Virginia, USA.
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Abstract
OBJECTIVE: The hemizona assay (HZA) is an established functional test that examines in vitro
sperm-zona pellucida binding capacity with high predictive power for fertilization outcome in IVF.
The objective of this study was to evaluate the value of the HZA as a predictor
of pregnancy in patients undergoing controlled ovarian hyperstimulation (COH)
and intrauterine insemination (IUI).
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DESIGN: Prospective clinical study. SETTING: Academic center. PATIENT(S): Eighty-two
couples with unexplained or male factor infertility that underwent 313 IUI cycles.
INTERVENTION(S): Basic semen analysis and HZA were performed within three months of
starting COH/IUI therapy.
MAIN OUTCOME MEASURE(S): Hemizona index (HZI) and clinical pregnancy.
RESULT(S): Overall, patients with an HZI of <30 had a significantly lower
pregnancy rate compared to patients with an HZI of > or =30 (11.1% vs. 40.6%,
respectively; P<.05; relative risk for failure to conceive: 1.5 (confidence interval 1.2-1.9)). In all
patients combined, and in the range of HZI 0-60, the duration of infertility (P=.000) and the HZI
(P=.004) were significant determinants of conception (receiver operating characteristics (ROC)
analysis). In couples with male infertility, the average path velocity and HZI were significant
predictors of conception (P=.001 and P=.005, respectively, ROC analysis). The negative and
positive predictive values of the HZA for pregnancy were 93% and 69%, respectively. Logistic
regression analysis provided models of HZI (P=.021) and duration of infertility (P=.037) with
highest predictability of conception in male factor and unexplained infertility groups, respectively.
CONCLUSION(S): The HZA predicted pregnancy in the IUI setting with high sensitivity and
negative predictive value in couples with male infertility. Results of this sperm function test are
useful in counseling couples before allocating them into COH/IUI therapy
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Anim Reprod Sci 2008 .Feb 1;104(1):69-82. Epub 2007 Jan 17.
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Porcine sperm zona binding ability as an indicator of fertility.
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Collins ED ,Flowers WL ,Shanks RD ,Miller DJ.
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Department of Animal Sciences, North Carolina State University, Raleigh, NC 27695, United States.
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Abstract
The escalated use of artificial insemination in swine has increased the importance of determining
fertility of a semen sample before it is used. Multiple laboratory assays have been developed to
assess fertilizing potential but they have yielded inconsistent results. This experiment sought to
determine the relationship between in vitro competitive zona binding ability and in vivo fertility
based on heterospermic inseminations and paternity testing. The zona pellucida binding ability
and fertility of sperm from 15 boars was assessed by comparing sperm from one boar with sperm
from other individual boars in a pairwise fashion using four ejaculates. The relationship of zona
binding ability to the mean number of piglets sired per litter for each boar as well as historic fertility
data (litter size and farrowing rate) was assessed. The in vitro competition assay consisted of
labeling sperm from each boar of the pair with a different fluorophore and incubating an equal
number of sperm from each boar in the same droplet with porcine oocytes. The competitive assay
was highly effective in ranking boars by zona binding ability (R2=0.94). Paternity testing using
microsatellite markers was used to determine the mean number of piglets sired per litter for each
boar during heterospermic inseminations. The pairwise heterospermic insemination assay was
effective in ranking boar fertility (R2=0.59). Using historical data from these boars, average litter
size and farrowing rate were correlated (r=0.81, p<0.001). However, zona binding ability was not
significantly correlated with historic farrowing rate data or historic average litter size. Boar sperm
zona binding ability was also not correlated significantly with the mean number of piglets sired per
litter following heterospermic insemination. But the number of piglets sired by each boar was
related to a combination of zona binding ability, sperm motility, normal morphology, acrosomal
integrity, and the presence of distal droplets (R2=0.70). These results suggest that zona
binding ability is not an accurate predictor of fertilizing ability when used alone; however,
when coupled with other sperm assessments, fertility may be predicted successfully.
Hamster Ovum Penetration Test
(Sperm Penetration Assay)
• Using hamster eggs induced to superovulate by injection
of hormones.
• They are denuded by removing the cumulus and zona
with hyaluronidase and trypsin, respectively.
• Overnight preincubation or
• Short preincubation plus induction of acrosome reaction
by ionophore A23187
• Diagnostic value is contraversial, probably owing to the
difficulty in optimizing the test protocol, which can lead to falsenegative results.
• Interpreting the Results:
• If the results of your sperm penetration tests
show abnormal sperm, IVF may be suggested to
help you conceive. However, so long as there
are no fertility problems indicated for the female,
some couples may also want to consider using
donor sperm with IUI.
• It is important to note, though, that test results
can be compromised if a semen sample has not
been collected properly.
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Androl 2006 .Feb;29(1):69-75; discussion 105-8.
• Sperm function tests and fertility.
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Aitken RJ.
ARC Centre of Excellence in Biotechnology and Development, Discipline of Biological Sciences,
and Hunter Medical Research Institute, University of Newcastle, Callaghan, NSW, Australia.
jaitken@mail.newcastle.edu.au
Abstract
Traditionally, the diagnosis of male infertility has depended upon a descriptive evaluation of
human semen with emphasis on the number of spermatozoa that are present in the ejaculate,
their motility and their morphology. The fundamental tenet underlying this approach is that male
fertility can be defined by reference to a threshold concentration of motile, morphologically
normal spermatozoa that must be exceeded in order to achieve conception. Many
independent studies have demonstrated that this fundamental concept is flawed and, in
reality, it is not so much the absolute number of spermatozoa that determines fertility, but
their functional competence. In the light of this conclusion, a range of in vitro tests have
been developed to monitor various aspects of sperm function including their potential for
movement, cervical mucus penetration, capacitation, zona recognition, the acrosome reaction and
sperm-oocyte fusion. Such functional assays have been found to predict the fertilizing capacity of
human spermatozoa in vitro and in vivo with some accuracy. Recent developments in this field
include the introduction of tests to assess the degree to which human spermatozoa have suffered
oxidative stress as well as the integrity of their nuclear and mitochondrial DNA. Such assessments
not only yield information on the fertilizing capacity of human spermatozoa but also their ability to
support normal embryonic development.
Relationship of Hamster Ovum Sperm Penetration Assay to
Seminal Fluid Analyses in the Evaluation of Infertile Couples
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Abstract
The males of 279 infertile couples were evaluated with hamster ovum sperm
penetration assay (SPA) and seminal fluid analysis. The mean SPA score for the total
population was 23.0% penetration with a range of 0-97%. Twenty five percent of the
patients demonstrated scores within the abnormal range (0-10%), and 15% were in
the “equivocal” range (11-14%). Comparing each individual with the total population
using linear regression analysis, it was noted that sperm concentration, percent
motility, and percent oval forms varied directly with the SPA, and the slopes of the
relationships are positive and statistically significant( p ,0.002 ,0.0001 < and 0.0001,
respectively). The relationship between SPA and volume is not statistically significant
(p .)0.354 > To determine whether the SPA could be utilized to establish appropriate
normal parameters for various components of SFA, these were analyzed in 169 men
who had SPAs of > 15%. Although most SFA values fell within the normal range for
this group, there were several exceptions, particularly with respect to percent motility
and the presence of leukocytes in the semen. Comparing the percentage of males
with abnormal SPA in groups of couples with or without a demonstrable
abnormality affecting fertility in the wife, no statistically significant differences
could be found. The value of the SPA and SFA in investigating males of infertile
couples is discussed.
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Geburtshilfe Frauenheilkd 1986 .Feb;46(2):110-4.
[Simplified hamster ovum-sperm penetration
test (HSPT) in routine
sterility testing[]Article in German]
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Metka MM ,Karlic H ,Söregi G ,Kozak W ,Huber JC.
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Abstract
In 67 patients, in most of whom the spermiogram did not, a priori, furnish definitive proof of fertility,
the hamster egg human sperm penetration test was performed as part of a screening programme
for infertility therapy conducted from February 1984 to February 1985. Evaluation of the test was
facilitated by identification of the decondensed spermatozoon head in the penetrated hamster egg
with a simple fluorescein staining instead of the usual aceto-lacmoid staining. The penetration rate
(= percentage proportion of the hamster ova liberated from the zona pellucida which are
penetrated within 3 hours following incubation with 1 million motile spermatozoa) was evaluated
statistically in comparison to the fertility criteria of the spermiogram (motility, density, pathoforms),
in order to verify the existence of possible fertility disturbances due to inability to penetrate
despite the motility of the spermatozoa. In 42 patients the diagnosis of infertility was
confirmed by a penetration rate of less than 10%. In 17 patients fertility was conformed
according to HSPT (penetration rate of over 20%), while in 7 patients fertility was in the lower
normal range. In the light of the authors' findings fertility confirmed by the HSPT is a
precondition of referral for extracorporeal insemination. In the light of the authors' findings
intrauterine insemination appears advisable for women whose fertility is in the lower
normal range according to HSPT
Reactive oxygen species
• Abnormal sperm and retained cytoplasm
result in:
1- Excessive generation of ROS
2- Cytoplasmic enzyme such as cratin phosphokinase
ROS include:
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Superoxide anion
Hydrogen peroxidase
Hydroxyl radical
Nitric oxide
Oxidative damages:
• Cellular lipids
• Proteins
• DNA
Source of ROS in the ejaculate:
• Spermatozoa
• Leukocytes
• The capacity of a leukocyte to generate ROS is at
least 100 times greater than that of spermatozoa
Seminal plasma:
• possess antioxidant and enzymes
Measurment of ROS
• Using luminometer
• Using probe such as luminol or lucigenin
• Mixture of luminol and horseradish peroxidase
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Washed sperm
Suspention in a simple krebs ringer medium lacking phenol red
Adding luminolin DSMO together horseradish peroxidase
Monitor chemiluminescent signal
FLMP provocation test for leukocytes
PMA provocation of ROS generation by leukocyte and sperm
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Reactive Oxygen Species Generation in Human Sperm: Luminol and Lucigenin
Chemiluminescence Probes
W. Thompson;a S. E. M. Lewis ;a K. A. McKinney :Authors
Abstract
The objective of this study was to compare measurements of reactive oxygen species (ROS)
generation from human spermatozoa in vitro using the luminol and lucigenin chemiluminescent
probes. Luminol reacts with a variety of reactive oxygen species (H2O2 O2-, OH) and allows
both intra- and extracellular ROS to be measured. Lucigenin, however, yields a
chemiluminescence that is more specific for superoxide anions released extracellularly.
Therefore, measurements made with both probes on the same samples should allow the intraand extracellular components of ROS generation to be identified. Sperm samples from 47 men
were divided into two equal aliquots, then processed by centrifugation and swim-up. Following
further division into aliquots and the addition of the two chemiluminescent probes, Phorbol 12myristate 13-acetate was added to trigger ROS release. Forty-three percent of the sperm
samples generated detectable levels of ROS. In the centrifuged preparations luminol
produced a significantly higher peak luminescence than lucigenin. However, the sperm
prepared by swim-up showed no significant differences in peak luminescence between luminol
and lucigenin.
The higher level of ROS generation produced by centrifugation may be due to membrane
disruption or possibly the use of unfractionated cell suspensions.
Extracellular ROS generation is more clinically important because surrounding healthy
spermatozoa may be damaged. Therefore the lucigenin probe may be a more useful
diagnostic tool than luminol for identifying sperm at risk of peroxidative
damage after swim up preparation. The patients identified in this way may benefit from the
addition of ROS scavengers to the culture medium in order to protect healthy sperm from
collateral damage
Detection of DNA damage in sperm
• Sperm DNA damage is predictive of fertilization
and pregnancy rate.
• If the genetic damage in the male germ cell is
severe, embryonic development stops.
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Comet assay
TUNEL assay
In situ nick translation
Acridine orange test
Sperm chromatin structure assay
Comet assay
• Single cell gel electrophoresis to evaluate DNA
integrity, including single and double-strand
breaks
• Depending amount of damageed DNA, create
smaller or bigger tail
TUNEL assay
• Terminal deoxynucleotidyl transferase dUTP nick
end labeling (TUNEL) is a method for detecting DNA
fragmentation by labeling the terminal end of nucleic
acids.
Test system to assess sperm DNA
condensation
• Aniline bluestain
• Toluidin blue stain
• Chromomycin A3 stain
Test systems to assess chromosomal
aberration and aneuploidy
• Fluorescence in situ hybridization