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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.

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Presentation on theme: "THE NOVEL METHOD FOR MATURE SPERM SELECTION IN ICSI (PICSI –DISH) Prof. G. Serdar GÜNALP, MD Hacettepe University, School of Medicine Department of Obstetrics."— Presentation transcript:

1 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

2 INTRODUCTION There are many biochemical parameters that might predict sperm fertility and maturity, independently from the classical semen parameters. In assessments of B-type creatine kinase (CK) significantly higher sperm CK content was found in subfertile men. High sperm CK activity was a direct consequence of increased cytop l asmic retantion. This suggested a sperm developmental defect in terminal spermiogenesis. Huszar and Vigue, 1993

3 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.


5 SPERMATOGENETIC MATURATION AND SPERM FERTILIZING FUNCTION From the perspective of male gamete maturation, it is important that 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 hsp70-2 gene in mice caused arrested spermatogenesis and azoospermia. Allen,1996; Eddy, 1999; Son 1999; Dix, 1996

6 SPERMATOGENETIC MATURATION AND SPERM FERTILIZING FUNCTION HspA2 expression occurs in two steps First in spermatocytes as a meiotic 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

7 The zona binding capacity is decreased in immature sperms with low HspA2 concentration and cytoplasmic retention. The formation of HA binding receptors during the remodeling process in normal spermiogenesis and zona binding may be used in the sperm selection for ICSI. Huszar, 1997

8 SPERMATOGENETIC MATURATION AND SPERM FERTILIZING FUNCTION In IVF cycles; all pregnancies were found in the "high likelihood (sperm HspA2 (>10%))" group. The receiver operating characteristic (ROC) analysis showed a 100 % predictive value for failure to achieve pregnancy in the range of < 10.4% HspA2 threshold. Huszar et al, 1992 Ergur et al, 2002

9 In developing spermatid if the HspA2 expresion is low the aneploidy rate is high : Because Synaptonemal komplex defect. Decreased HspA2 chaperone activity result insufficient DNA repair enzymes and DNA fragmentation. Net Result is: Abnormal sperm morphology. İ ncreased ROS activity. İ ncreased DNA fragmentation. Spermatogenic defect, insufficient HBA binding sites, and defective ZP binding. SPERM MATURATION: NUCLEAR AND CYTOPLASMIC BIOCHEMICAL MARKERS


11 MATURITY ARRESTED SPERMATOZOA Inability to bind to the zona pellucida or fertilization defect via natural or IVF conception The probable reason why such spermatozoa do not die prior to ejaculation is the presence of antiapoptotic protein Bclx2 in the surviving germ cells

12 RELATIONSHIP BETWEEN DIMINISHED/ ARRESTED SPERM MATURITY AND CHROMOSOMAL ANEUPLOIDIES HspA2 defect caused synaptonemal complex defect and i mmature spermatozoa. Synaptic anomalies may occur in association with arrested maturity. The proportions of immature spermatozoa exhibiting cytoplasmic retention were 45.4 ± 3.4 versus 26.6 ± 2.2% in the semen versus Percoll group. There was a close correlation between the incidences of immature spermatozoa with cytoplasmic retention and with spermatozoa of disomic nuclei but not with diploidies. Kovanci, 2001; Egozcue, 2002

13 SPERM HEAD SHAPE AND SPERM MATURITY: SHAPE OR DIMENSIONAL PROPERTIES DO NOT FACILITATE THE ICSI SELECTION OF HAPLOID SPERMATOZOA In a recent study, 1286 individual spermatozoa from 15 men were evaluated; Celik-Özenci, 2004 Head sizeDisomic spermatozoa Mean diploidy ratio Small27%3% Intermediate23%8% Large50%89% Sperm shapeDisomic or diploid spermatozoa Symmetrical18% Asymmetrical18% Irregulary41% Amorphous98%

14 Evaluation of1286 İndividual spermatozoa Kruger strict criteria Haploid (900) 2 4% Disomic (256)0% Diploid (130)1% Sperm shape Kruger strict criteria Symmetrical 26% Asymmetrical 3% Irregulary 1% Amorphous 0%

15 TESTING OF SPERM MATURITY BY HYALURONIC ACID BINDING TEST Hyaluron a n, the main structure of cumulus oophorus surrounding oocy t e play the main role in the selection of functiona l sperm. Hyaluron a n binded sperm has low cytoplasmic inclusions, residual histon proteins and low rate of chromosomal aneuploidy. HBA is an important biomarker for mature and f u nctional sperm selection. Nowadays, HBA is available as a commercial kit.

16 The formation of the HA receptor, similar to the sperm zona-binding site(s) is regulated by the remodelling 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 acrosome reaction do not exhibit HA-binding. Huszar, 2003; Çelik-Özenci, 2004 TESTING OF SPERM MATURITY BY HYALURONIC ACID BINDING

17 HA containing medium increased sperm velocity, retention of long-term motility and viability of freshly ejaculated, as well as cryopreserved/thawed human spermatozoa These improvements occurs as a receptor mediated response to HA : there was an immediate increase in sperm tail cross-beat frequency and sperm velocity upon HA exposure; spermatozoa with arrested maturi t y were not stimulated by HA. Huszar, 1990; Sbracia, 1997

18 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 TESTING OF SPERM MATURITY BY HYALURONIC ACID BINDING

19 SPERM HA BINDING TEST Based on the fact that mature spe rm atozoa selectively bind to solid state HA. Sperm binding occurs within 8-10 min. The assessment of HA binding is based on the proportions of bound spermatozoa with increased tail crossbeat frequency versus the unbound swimming spermatozoa that do not 'perceive" the HA. There was a close correlation negatively between sperm CK activity and HA binding (r = -0,78. p< 0.001). Huszar, 2003

20 ASSESMENT OF HBA For HBA assesment, commercially available kit are used. Add 10 μ l semen sample to the centre of HBA circle. Put lamel above semen sample without bubble formation. Wait 15 min. A total of 100 motile sperm are counted. HBA binding ratio = The number of motile sperm bound / Total progressive sperm count.

21 SPERM HA BINDING TEST Huszar, 2002; Ergur, 2002; Huszar, 1990, 1992 HA binding score CK activityManagement plan 80%0.18 ± 0.02 (Normal) There was no male factor infertility or need for intervention 60-80%0.50 ± 0.06 (High) IUI 60%2.8 ± 0.1 (Very high) Indicating a high concentration of sperm cells with cytoplasmic retention. I VF / ICSI Because the clinical utility of sperm CK activity and HspA2 concentrations were already established in intrauterine insemination (IUI) and IVF studies, for further conclusion new studies are needed.

22 ICSI SPERM SELECTION BY HA BINDING There may be differences in semen attributes between men who are fertile and those who failed to cause pregnancy. Assessment of single sperm attributes, even strict sperm morphology, is of limited predictive value. Different fertilization rates were assessed via IVF or ICSI even with sybling oocytes. No semen parameters are identified that would predict whether IVF or ICSI is more beneficial for a particular couple. Ombelet, 2003; Westerlaken, 2006; Tournaye, 2002; Aziz, 2006

23 ICSI SPERM SELECTION BY HA BINDING The increased rate of chromosomal aberrations and other potential consequences of using immature spermatozoa for ICSI are of major concern. HA receptors in mature, but not in immature spermatozoa, coupled with a respective device with an HA-coated surface, it is expected that the method will facilitate the selection of single mature spermatozoa with high DNA integrity and low frequencies of chromosomal aneuploidies for ICSI. Sperm selection by HA is performed by PICSI dish.


25 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

26 HA SPERM SELECTION : GENETIC RESULTS Unselected spermatozoa HA-bound spermatozoa Diploidy0.51%0.09% Sex chromosome diploidy 0.27%0.05% TOTAL0.52%0.16% Simpson, 2001; Bonduelle, 2002

27 ICSI SPERM SELECTION BY HA BINDING HA-selection of mature spermatozoa is likely to be independent from seminal sperm concentrations. None of the groups that used the HA sperm selection have reported any adverse effect on fertilization or embryo development. ESHRE 2005

28 ICSI SPERM SELECTION BY HA BINDING: CLINIC RESULTS Visually selected ICSI HA-selected ICSl Fertilization rates%69.7%67.0 Good grade embryos%56.6%51.7 Pregnancy rate%45.3%33.0 Abortion rate%7%0 Take home baby%46.5%39.0 Sanchez, 2005



31 ICSI SPERM SELECTION BY HA BINDING : IS IT SAFE? HA occurs normally in the female 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 attach to spermatozoa, or cause an extremely small area of the sperm membrane from the region of the acrosomal cap (which is lost otherwise during the acrosomal reaction) to remain attached to the HA. Huszar, 2007

32 POTENTIAL BENEFITS OF THE HA MEDIATED SPERM SELECTION METHOD FOR ICSI OFFSPRING Following ICSI fertilization with visually selected spermatozoa, t here were increased rates of d e- novo numerical chromosomal aberrations, and also cytogenetically detectable structural chromosomal aberrations. HA may be beneficial in male associat ed problems. 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.

33 POTENTIAL BENEFITS OF THE HA MEDIATED SPERM SELECTION METHOD FOR ICSI OFFSPRING HA-mediated sperm selection for ICSI may reduce the risk for the 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

34 CONCLUSION HA-binding attributes of mature spermatozoa provides a new approach in andrology testing and in ICSI sperm selection. The sperm HA-binding test provides a 15-minute microscopic assay for the assessment of the proportion of spermatozoa that would bind to the zona pellucida. The frequencies of chromosomal disomies and diploidies are decreased within the normal range of conventional IVF. Mature spermatozoa selected by virtue of HA-binding are also viable, and devoid of persistent histones. Reduce early pregnancy loss.

35 Good Bad Ugly

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