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THE NOVEL METHOD FOR MATURE SPERM SELECTION IN ICSI (PICSI –DISH). Prof. G. Serdar GÜNALP, MD Hacettepe University , School of Medicine Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Infertility and Andrology, Ankara, TURKEY. INTRODUCTION.

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the novel method for mature sperm selection in icsi picsi dish

THE NOVEL METHOD FOR MATURE SPERM SELECTION IN ICSI (PICSI –DISH)

Prof. G. Serdar GÜNALP, MD

Hacettepe University, School of Medicine

Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, Infertility and Andrology, Ankara, TURKEY

introduction
INTRODUCTION
  • There are many biochemical parameters that might predict sperm fertility and maturity, independently from the classical semen parameters.
  • In assessments of B-type creatinekinase (CK)significantly higher sperm CK content was found in subfertile men.
    • High sperm CK activity was adirect consequence of increased cytoplasmic retantion.
    • This suggested a sperm developmental defect in terminal spermiogenesis.

Huszar and Vigue, 1993

slide3
The rate of CK activity in immature sperm is cytoplasmic remnant which is the consequence of incomplete removal of cytoplasm during spermatogenesis.
  • It is the first used biochemical marker of sperm maturation.
spermatogenetic maturation and sperm fertilizing function
SPERMATOGENETIC MATURATION ANDSPERM FERTILIZING FUNCTION
  • From the perspective of male gamete maturation, it is importantthat synthesis of the HSP70 family of proteins (HspA2 in men)is regulated developmentally and they contribute to the
    • maintenance of the synaptonemal complex and
    • meiotic function during spermatocyte development
  • The targeted disruption of the hsp70-2gene in mice caused arrested spermatogenesis and azoospermia.

Allen ,1996; Eddy, 1999; Son 1999; Dix, 1996

spermatogenetic maturation and sperm fertilizing function6
SPERMATOGENETIC MATURATION ANDSPERM FERTILIZING FUNCTION
  • HspA2expression occurs in two steps
    • First in spermatocytes as ameiotic division within the synaptonemal complex,
    • Second in elongating spermatids during terminal spermiogenesis
  • In summary, the expression levels of HspA2 are closely related to spern cellular maturity, and fertilizing potential.

Huszar, 2000; Huszar, 1998

slide7

The zona bindingcapacity is decreased in immaturespermswithlow HspA2 concentrationandcytoplasmicretention.

  • Theformation of HA bindingreceptorsduringtheremodelingprocess in normal spermiogenesisand zona bindingmay be used in the sperm selectionfor ICSI.

Huszar , 1997

spermatogenetic maturation and sperm fertilizing function8
SPERMATOGENETIC MATURATION ANDSPERM FERTILIZING FUNCTION
  • In IVF cycles; all pregnancies were foundin the "high likelihood (sperm HspA2 (>10%))" group.
  • The receiver operating characteristic (ROC) analysis showed a 100 % predictive value for failure to achieve pregnancy in therange of < 10.4% HspA2 threshold.

Huszar et al, 1992

Ergur et al, 2002

sperm maturation nuclear and cytoplasmic biochemical markers
SPERM MATURATION: NUCLEAR ANDCYTOPLASMIC BIOCHEMICAL MARKERS
  • Indevelopingspermatidifthe HspA2 expresion is lowtheaneploidy rate is high : Because
    • Synaptonemalkomplexdefect.
    • Decreased HspA2 chaperoneactivityresultinsufficient DNA repairenzymesand DNA fragmentation.
  • Net Result is:
    • Abnormal sperm morphology.
    • İncreased ROS activity.
    • İncreased DNA fragmentation.
    • Spermatogenicdefect, insufficient HBA bindingsites, anddefective ZP binding.
maturity arrested spermatozoa
MATURITY ARRESTED SPERMATOZOA

Inability to bind to the zonapellucida or fertilizationdefect via natural or IVF conception

The probable reason why such spermatozoa donot die prior to ejaculation is the presence of antiapoptoticprotein Bclx2 in the surviving germ cells

relationship between diminished arrested sperm maturity and chromosomal aneuploidies
RELATIONSHIP BETWEEN DIMINISHED/ ARRESTED SPERM MATURITY AND CHROMOSOMAL ANEUPLOIDIES
  • HspA2 defect causedsynaptonemal complex defect and immature spermatozoa.
    • Synaptic anomalies may occur in association with arrested maturity.
    • The proportions of immature spermatozoa exhibiting cytoplasmic retention were45.4 ± 3.4 versus 26.6 ± 2.2% in the semen versus Percoll group.
  • There was a close correlation between the incidences of immaturespermatozoa with cytoplasmic retention and with spermatozoaof disomic nuclei but not with diploidies.

Kovanci, 2001; Egozcue, 2002

slide13
SPERM HEAD SHAPE AND SPERM MATURITY: SHAPE OR DIMENSIONAL PROPERTIES DO NOT FACILITATE THE ICSISELECTION OF HAPLOID SPERMATOZOA
  • In a recent study, 1286 individual spermatozoa from 15 men were evaluated;

Celik-Özenci, 2004

testing of sperm maturity by hyaluronic acid binding test
TESTING OF SPERM MATURITY BYHYALURONIC ACID BINDING TEST
  • Hyaluronan , themainstructure of cumulusoophorussurroundingoocyteplaythemain role in theselection of functional sperm.
  • Hyaluronanbinded sperm has lowcytoplasmicinclusions, residualhistonproteinsandlow rate of chromosomalaneuploidy.
  • HBA is an importantbiomarkerformatureandfunctional sperm selection.
  • Nowadays, HBA is available as a commercial kit.
slide16

TESTING OF SPERM MATURITY BYHYALURONIC ACID BINDING

  • The formation of the HA receptor,similar to the sperm zona-binding site(s)is regulated by theremodelling of the plasma membrane during spermiogenesis.
  • Mature spermatozoa may selectively bind to solid state HA.
  • Spermatozoa bind head-first to HA with the acrosomal region.
  • Spermatozoa with advanced levels of capacitaion or acrosomereaction do not exhibit HA-binding.

Huszar, 2003; Çelik-Özenci, 2004

testing of sperm maturity by hyaluronic acid binding
TESTING OF SPERM MATURITY BYHYALURONIC ACID BINDING
  • HA containingmediumincreasedsperm velocity, retention of long-term motility and viability offreshly ejaculated, as well as cryopreserved/thawed humanspermatozoa
  • Theseimprovements occurs as a receptormediatedresponse to HA:
    • there was an immediate increase in spermtail cross-beat frequency and sperm velocity upon HAexposure;
    • spermatozoawitharrestedmaturitywere not stimulated by HA.

Huszar, 1990; Sbracia, 1997

slide18

TESTING OF SPERM MATURITY BYHYALURONIC ACIDBINDING

  • HA-bound mature spermatozoa are viable (non-viable spermatozoa do not bind).
  • HA-bound spermatozoa are devoid of
    • cytoplasmic retention,
    • persistent histones
    • DNA fragmentation, and
    • the apoptotic marker caspase 3

Huszar, 2003;Çelik-Özenci, 2004, Aioki, 2006; Borini, 2006

sperm ha binding test
SPERM HA BINDING TEST
  • Based on the fact that mature spermatozoa selectively bind tosolid state HA.
  • Sperm binding occurs within 8-10 min.
  • The assessment of HA binding is based on theproportions of bound spermatozoa with increased tail crossbeatfrequency versus the unbound swimming spermatozoa that do not 'perceive" the HA.
  • There was a close correlation negatively between sperm CKactivity and HA binding

(r = -0,78. p< 0.001).

Huszar, 2003

assesment of hba
ASSESMENT OF HBA
  • For HBA assesment, commerciallyavailable kit areused.
  • Add 10 μl semen sampletothecentre of HBA circle.
  • Put lamel above semen samplewithoutbubbleformation.
  • Wait 15 min.
  • A total of 100 motile sperm arecounted.
  • HBA bindingratio = Thenumber of motile sperm bound / Total progressive sperm count.
sperm ha binding test21
SPERM HA BINDING TEST

Because the clinical utility of sperm CK activity and HspA2concentrations were already established in intrauterineinsemination (IUI) and IVF studies,

forfurtherconclusionnewstudiesareneeded.

Huszar, 2002; Ergur, 2002; Huszar, 1990, 1992

icsi sperm se l ection by ha binding
ICSI SPERM SELECTION BY HA BINDING
  • Theremay be differences in semen attributes between men who are fertileand those who failed to cause pregnancy.
  • Assessment of singlesperm attributes, even strict sperm morphology, is of limitedpredictivevalue.
  • Differentfertilizationrateswereassessedvia IVF or ICSI evenwithsyblingoocytes.
  • No semen parameters are identified that would predict whether IVF orICSI is more beneficial for a particular couple.

Ombelet, 2003; Westerlaken, 2006; Tournaye, 2002; Aziz, 2006

icsi sperm se l ection by ha binding23
ICSI SPERM SELECTION BY HA BINDING
  • The increased rate of chromosomal aberrations and otherpotential consequences of using immature spermatozoa forICSI are of major concern.
  • HA receptors in mature, butnot in immature spermatozoa, coupled with a respective devicewith an HA-coated surface, it is expected that the method willfacilitate the selection of single mature spermatozoa with highDNA integrity and low frequenciesof chromosomal aneuploidies for ICSI.
  • Sperm selection by HA is performed by PICSI dish.
icsi sperm se l ection by ha binding25
ICSI SPERM SELECTION BY HA BINDING
  • HA binding resulted in a substantial selection effect, as there was
    • a 4.3-fold decline in sex chromosome disomies
    • a 5.8-fold reduction in diploidies

Jakab, 2005

ha sperm selection genetic results
HA SPERM SELECTION : GENETIC RESULTS

Simpson, 2001; Bonduelle, 2002

icsi sperm se l ection by ha binding27
ICSI SPERM SELECTION BY HA BINDING
  • HA-selection of mature spermatozoa is likely to be independent from seminal sperm concentrations.
  • None of the groups thatusedthe HA sperm selection have reportedany adverse effect on fertilization or embryo development.

ESHRE 2005

icsi sperm se l ection by ha binding clinic results29
ICSI SPERM SELECTION BY HA BINDING: CLINIC RESULTS

Warrilow, 2006

ADVANTAGES PREGNANCY RESULTS

icsi sperm selection by ha binding is it safe
ICSI SPERM SELECTION BY HA BINDING : IS IT SAFE?
  • HA occurs normally in thefemale reproductive tract and in the cumulus oophorus.
  • HA is carried with the spermatozoa into oocytes even during in-vivo conception,
  • The removal of spermatozoa from HA may cause a few HA molecules to attachto spermatozoa, or cause an extremely small area of the sperm membrane from the region of the acrosomal cap (which is lostotherwise during the acrosomal reaction) to remain attached to the HA.

Huszar, 2007

potential benefits of the ha mediated sperm selection method for icsi offspring
POTENTIAL BENEFITS OF THE HAMEDIATEDSPERM SELECTION METHODFOR ICSI OFFSPRING
  • Following ICSI fertilization with visually selectedspermatozoa,there were increased rates of

de-novo numericalchromosomal aberrations, and also cytogenetically detectable structural chromosomal aberrations. HA may be beneficial in male associatedproblems.

  • To decrease the abortion rate of conventional ICSI (%18) by HBA selected sperm.
  • To decrease the birth defect ratio of ICSI (odds ratio of 2.77) by HBA selected sperm.
potential benefits of the ha mediated sperm selection method for icsi offspring33
POTENTIAL BENEFITS OF THE HAMEDIATEDSPERM SELECTION METHODFOR ICSI OFFSPRING
  • HA-mediated sperm selection for ICSI may reduce the risk forthe chromosomal aberrations for the offspring, and increase ICSI success rate.
  • To increase the rate of normal chromosomal arrangement in offspring of father with abnormal chromosomal arrangement.

Baltacı, 2006; Menezo, 2006

conclusion
CONCLUSION
  • HA-bindingattributes of maturespermatozoa provides a new approach in andrology testing and in ICSI sperm selection.
  • The sperm HA-binding test provides a 15-minute microscopicassay for the assessment of the proportion of spermatozoa thatwould bind to the zonapellucida.
  • Thefrequencies of chromosomal disomies and diploidies aredecreasedwithinthe normal range of conventional IVF.
  • Mature spermatozoa selected by virtue of HA-binding arealso viable, and devoid of persistent histones.
    • Reduceearlypregnancyloss.
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