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Sperm Function Tests Shahin Ghazali Ph. D student Yazd Reproductive Sciences Institute Sperm Function Tests Shahin Ghazali Ph. D student Yazd Reproductive Sciences Institute

Introduction: • During recent years, apart from the light microscopical determination of sperm count 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 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 • Sperm function test were developed to detect abnormalities: - In sperm survival - Transport in the female genital tract - Different steps of fertilization

Types: • • • Vitality tests Sperm-mucus Interaction Tests Capacitation Acrosome Reaction Zona Binding Types: • • • Vitality tests Sperm-mucus Interaction Tests Capacitation Acrosome Reaction Zona Binding assays Hamster Ovum Penetration test

Vitality tests: • Are used when: Low percentage motility Lost their flagellation because of Vitality tests: • 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 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 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. 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 • • • In In In vivo postcoital test vitro sperm-mucus Sperm-mucus Interaction Tests • • • 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 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 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 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) p. H

In vitro sperm cervical mucus contact test: • Detecting antibodies on sperm or in 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 • 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) • • Mucus loaded into a flat capillary In vitro capillary test (Kremer test) • • Mucus loaded into a flat capillary tube (Sealed one end) The open end put vertically into reservoir of semen Incubation for 1 h at 37 c 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 Penetrak Test • Commercial • Using bovine cervical mucus as a surrogate in the penetration test.

 • Androlog Mail • The bovine mucus penetration test does measure important characteristics • Androlog Mail • 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. • • • 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. • I am still using the BMPT if physicians order it specifically, which has become rather rare. • 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. • 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 [email protected] missouri. edu

 • A simplified approach to sperm-cervical mucus interaction testing using a hyaluronate migration • A simplified approach to sperm-cervical mucus interaction testing using a hyaluronate migration test • D. Mortimer 1, 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 T 2 N 4 N 1, Canada • Correspondence: 1 To whom correspondence should be addressed • 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 p. H, 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 • •

Capacitation • Cell surface changes • Hyperactivated motility • Vigorous nonprogresive motion with forceful Capacitation • Cell surface changes • Hyperactivated motility • Vigorous nonprogresive motion with forceful amplitude bending

1 • Sperm wash • Incubation in albumin-containing culture • Very simple • No 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 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 • 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 37 c • Standard concentration is 25 -50* Preparation of samples: • Maintain at 37 c • Standard concentration is 25 -50* 10 6 • Using Dulbecco’s PBS-glucose-BSA medium

Pic page 106 Pic page 106

CASA terminology: • VCL = curvilinear velocity) micron/s) Time-average velocity of a sperm head 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.

 • medea. LAB CASA A new system for objective semen analysis which offers: • medea. LAB 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 : • Automated analysis of sperm motility and morphology. Calculation of a complete spermiogram in under 30 seconds, multiple recordings for each sample are possible • Storage of patient data and sample recorded on template in MS Access™ and SQL-Server • Classification of sperm motility according to WHO categories • Print of spermiogram by means of user-defined HTML templates • Automatic export of data into MS Excel™ for further statistical analysis • Storage of images from morphological analysis • Full Rec. Date database integration (Germany only) • Use of standard hardware reduces investment need

MAIN SOFTWARE FEATURES: • • Acquisition of images and clips in AVI format from MAIN SOFTWARE FEATURES: • • Acquisition of images and clips in AVI format from an imaging device. Storage of textual data, images and clips in the built-in Multi. Media Catalog database. You can take the advantage of all the powerful features of Multi. Media 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.

 • Computer assisted semen analyzers in andrology research and veterinary practice. • • • Computer assisted semen analyzers in andrology research and veterinary practice. • • • Verstegen J , Iguer-Ouada M , Onclin K. University of Liège, Department of Animal Clinical Sciences, Small Animal Reproduction Bd Colonster 20, B 44, B 4000 Liège Belgium. Abstract • 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 Acrosome Reaction

Tests for acrosome reaction: • Triple stain • Trypan blue • Fluoroscent lectins and Tests for acrosome reaction: • Triple stain • Trypan blue • Fluoroscent lectins and antibodies

Triple stain: • • • The sperm suspensions from the swim-up were diluted with Triple stain: • • • 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. 2 M 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, 000 x under phase-contrast microscopy and spermatozoa were classified into the following four categories: 17 . 1 live spermatozoa with acrosome reaction - light rose postacrosomal regions and white "acrosomal regions; " . 2 dead spermatozoa with normal acrosomes - blue postacrosomal regions; . 3 dead spermatozoa with abnormal acrosomes (i. e. ; degenerative acrosome reactions) - blue postacrosomal regions with white "acrosomal regions; " . 4 live spermatozoa with normal acrosomes - light rose postacrosomal regions and pink acrosomes.

 • . 1 a live spermatozoon acrosome reacted; . 2 a dead spermatozoon • . 1 a live spermatozoon acrosome reacted; . 2 a dead spermatozoon with an intact acrosome ; . 3 a dead spermatozoon with degenerative acrosome reaction ; . 4 live spermatozoa with intact acrosomes ; 5 and 6. live spermatozoa with acrosome partially intact.

 • Reproductive Biology • Prudence Talbot, Richard S. Chacon. Department of Biology, University • Reproductive Biology • Prudence Talbot, Richard S. Chacon. Department of Biology, University of California, Riverside, CA 92521 A triple-stain technique for evaluating normal acrosome reactions of human sperm • Abstract • • 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 Fluoroscent lectins and antibodies • Most common probes: • Peanut lectin, which lables the outer acrosomal membrane. • Pea lectin, which lables the acrosomal content • Antibody: • CD 46, which targets the inner acrosomal membrane

 • Upper panels (Laser scanning confocal fluorescence images of sperm. Triple labeling of • 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 Ca. I) D (′for 3 h (× 18 000 magnification). Postfixation with Os. O 4 and 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. Ca. I-treated sperm reveal vesiculation, blebbing, and loss of the plasma and acrosomal membranes and well-preserved 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 Ca. I) 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 Ca. I-treated sperm) D. (′′Figure reproduced at 84% of original.

 • Sperm-zona binding in human is species pecific • Human zonae obtained from • 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 ZP 3 and ZP 2 (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 • ZP is cut into exactly two halves using micromanipulator • Hemizona test • ZP is cut into exactly two halves using micromanipulator • 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 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

 • • Summary Systems Biology in Reproductive Medicine , 1997 Vol. 38, No. • • Summary Systems Biology in Reproductive Medicine , 1997 Vol. 38, No. 2, Pages 127 -131 • The Hemizona Assay: A Simplified Technique • • M. Janssen , 1, 2 W. Ombelet , † 1, 2 A. Cox , 1, 2 H. Pollet , 1, 2 D. R. Franken 1, 2 and E. Bosmans 1, 2 1 The Genk Institute for Fertility Technology, ZOL Campus St. Jan, Genk, Belgium 2 Tygerberg 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 • 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 • Comparison of two methods to obtain hemizonae pellucidae for sperm function tests • R. Sánchez 1, C. Finkenzeller, W. -B. Schill and W. Miska 2 Department of Dermatology and Andrology, Justus Liebig University Gaffkystrasse 14, 35392 Giessen, Germany 1 Department of Preclinical Science, La Frontera University Temuco, Chile • Correspondence: 2 To 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.

 • Neural cadherin is expressed in human gametes and participates in sperm–oocyte interaction • Neural cadherin is expressed in human gametes and participates in sperm–oocyte interaction events • 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, 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 B 16 & B 24, ZIP Code: 1428 ADN, Buenos Aires, Argentina. E-mail: • • ABSTRACT • 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 Hemi. Zona 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 k. Da) 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.

 • Fertil Steril. 2006 Jun; 85(6): 1697 -707. Epub 2006 May 6. • • Fertil Steril. 2006 Jun; 85(6): 1697 -707. Epub 2006 May 6. • Predictive value of the hemizona assay for pregnancy outcome in patients undergoing controlled ovarian hyperstimulation with intrauterine insemination. • • 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. • • 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). • • • 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

 • Anim Reprod Sci 2008. Feb 1; 104(1): 69 -82. Epub 2007 Jan • Anim Reprod Sci 2008. Feb 1; 104(1): 69 -82. Epub 2007 Jan 17. • Porcine sperm zona binding ability as an indicator of fertility. • • Collins ED , Flowers WL , Shanks RD , Miller DJ. • • 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 (R 2=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 (R 2=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 (R 2=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. Department of Animal Sciences, North Carolina State University, Raleigh, NC 27695, United States.

Hamster Ovum Penetration Test (Sperm Penetration Assay) • Using hamster eggs induced to superovulate 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 A 23187 • 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 • 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.

 • Androl 2006. Feb; 29(1): 69 -75; discussion 105 -8. • Sperm function • Androl 2006. Feb; 29(1): 69 -75; discussion 105 -8. • Sperm function tests and fertility. • • 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. [email protected] 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 Relationship of Hamster Ovum Sperm Penetration Assay to Seminal Fluid Analyses in the Evaluation of Infertile Couples • • 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.

 • • Geburtshilfe Frauenheilkd 1986. Feb; 46(2): 110 -4. ]Simplified hamster ovum-sperm penetration • • Geburtshilfe Frauenheilkd 1986. Feb; 46(2): 110 -4. ]Simplified hamster ovum-sperm penetration test (HSPT) in routine • Metka MM , Karlic H , Söregi G , Kozak W , Huber JC. • • 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 sterility testing][Article in German[

Reactive oxygen species • Abnormal sperm and retained cytoplasm result in: 1 - Excessive Reactive oxygen species • Abnormal sperm and retained cytoplasm result in: 1 - Excessive generation of ROS 2 - Cytoplasmic enzyme such as cratin phosphokinase

ROS include: • • Superoxide anion Hydrogen peroxidase Hydroxyl radical Nitric oxide ROS include: • • Superoxide anion Hydrogen peroxidase Hydroxyl radical Nitric oxide

Oxidative damages: • Cellular lipids • Proteins • DNA Oxidative damages: • Cellular lipids • Proteins • DNA

Source of ROS in the ejaculate: • Spermatozoa • Leukocytes • The capacity of 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 Measurment of ROS • Using luminometer • Using probe such as luminol or lucigenin • Mixture of luminol and horseradish peroxidase • • • 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

 • • • Reactive Oxygen Species Generation in Human Sperm: Luminol and Lucigenin • • • Reactive Oxygen Species Generation in Human Sperm: Luminol and Lucigenin Chemiluminescence Probes W. Thompson; a S. E. M. Lewis ; a K. A. Mc. Kinney : 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 (H 2 O 2 O 2 -, 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 samples should allow the intra- and 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 12 myristate 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 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.

 • • • Comet assay TUNEL assay In situ nick translation Acridine orange • • • 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 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 d. UTP nick end labeling (TUNEL) is a TUNEL assay • Terminal deoxynucleotidyl transferase d. UTP 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 Test system to assess sperm DNA condensation • Aniline bluestain • Toluidin blue stain • Chromomycin A 3 stain

Test systems to assess chromosomal aberration and aneuploidy • Fluorescence in situ hybridization Test systems to assess chromosomal aberration and aneuploidy • Fluorescence in situ hybridization