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Title: Semen preparation techniques for ART


1
Semen preparation techniques for ART
  • Gülnaz Sahin, MD
  • Ege University Family Planning and Infertility
    Research and Treatment Center

2
  • The onset of clinical assisted reproduction, a
    quarter of a century ago, required the isolation
    of motile spermatozoa
  • Since the birth of Louise Brown on 25 July 1978
    and the subsequent onset of assisted reproduction
    in the human, scientists and clinicians were more
    and more urged to improve sperm separation
    techniques as the percentage of andrological
    cases increased rapidly

3
  • The first sperm separation methods were one
    or two washing procedures with subsequent
    resuspension of the male germ cells (1-3)

1. Edwards RG, Bavister BD, Steptoe PC Early
stages of fertilization in vitro of human oocytes
matured in vitro. Nature 1969 2. Edwards RG,
Steptoe PC, Purdy JM Establishing full term
human pregnancies using cleaving embryos grown in
vitro. Br J Obstet Gynaecol 1980 3. Lopata A,
Brown JB, Leeton JF, Talbot JM, Wood C In vitro
fertilization of preovulatory oocytes and embryo
transfer in infertile patients treated with
clomiphene and human chorionic gonadotropin.
Fertil Steril 1978
4
  • Researchers then described a single wash
  • followed by a swim-up procedure from the cell
    pellet.
  • Following these first reports on human
    sperm separation, more sophisticated methods were
    developed to obtain sufficient amounts of motile,
    functionally competent spermatozoa for IVF.

Mahadevan M, Baker G Assessment and preparation
of semen for in vitro fertilization. In Clinical
In Vitro Fertilization Edited byWood C, Trounson
A. Springer-Verlag, Berlin 1984
5
  • On principle, these techniques can be
    differentiated in migration, density gradient
    centrifugation and filtration
  • Swim-up (Mahadevan and Baker, 1984)
  • Percoll density gradient centrifugation (Gorus
    Pipeleers, 1981)
  • Glass-wool (Paulson Polakoski, 1977)
  • Sephadex bead filtration (Lopez et al.,1993)

6
The ideal sperm separation technique
  • should
  • be quick,
  • easy and cost-effective,
  • isolate as much motile spermatozoa as possible,
  • not cause sperm damage or nonphysiological
    alterations of the separated sperm cells,
  • eliminate dead spermatozoa and other cells,
    including leukocytes and bacterias,
  • eliminate toxic or bioactive substances like
    decapacitation factors or reactive oxygen species
    (ROS) and,
  • allow processing of larger volumes of ejaculates.

7
  • The quality of sperm samples is one of the
    factors determining successful assisted
    reproduction
  • (Ombelet et al. 2003)

8
Ejeculated semen
  • Viscous liquid composed of mixture of
    testicular, epididymal secretions containing
    spermatozoa and prostatic secretions produced at
    the time of ejeculation.
  • This seminal plasma contains substances which
    inhibit capasitation and prevent fertilization
  • Capacitation of eutherian spermatozoa is
    essential for fertilization not only in vivo but
    also in vitro, and underlies the manipulation of
    spermatozoa for clinical invitro fertilization
    (IVF).

9
The purpose
  • Concentrate the motile spermatozoa in a fraction
    which is free of seminal plasma and debris
  • Maximalize the changes of fertilization to
    provide as many normally fertilized ooctes as
    possible
  • Elemination of seminal PG, lymphokines, cytokines
    and infectious agents
  • Reduce the number of free oxygen radicals

10
Collection of sperm
  • The male partner should collect semen into a
    sterile, clearly labelled disposible plastic jar
    with in a room adjecent to the IVF laboratory
  • The time of sample collection should be recorded
    on the label
  • Semen should be prepared soon after liquefaction
  • If liquefaction delayed or specimen viscous
    mixing the specimen 11 with medium may help or
    enzymatically liquefaction can be done (ie
    a-amylase, hyolurinidase, tripsin based
    dissolving sol.)
  • 10 µl of the sample is taken to check sperm
    consentration and motility

11
  • Initial assesment of density and motility allow
    to choose the most appopriate method of sperm
    preparation
  • The choice of sperm preparation method depens
    on,
  • the motile count, ratio between
    motile/immotile count, volume, presence of
    antibodies, agglutination

12
ROS (reactive oxygen species) induced damage
  • Morphologically abnormal spermatozoa with
    retained spermatid cytoplasm and leukocytes
    present within the ejaculate generate free
    radicals in vitro
  • Centrifugation can be harmful to human
    spermatozoa (forses in excess of 800 g applied)
  • Centrifugal pelleting of unselected human sperm
    populations often resulted in the generation of
    free radical or reactive oxygen species (ROS)
    within the sperm pellet that could irreversible
    damage to the spermatozoa that can impaireven
    totally destroytheir fertilizing cability.


Aitken RJ, Clarkson JS. J Androl.1988
13
ROS induced damage
  • ROS are generated both by leukocytes present in
    semen and spermatozoa
  • ROS affect not only the sperm plasma membrane by
    causing phospholipid peroxidation, but also the
    sperm DNA by causing strand breaks that can be
    revealed by various tests of sperm DNA integrity
  • spermatozoa prepared by simple washing will
    definitely be at a much greater risk of
    contributing a defective genome to the embryo and
    could underlie the increased developmental
    failure of ICSI-derived embryos after the 8-cell
    stage when the embryonic genome is activated

Saccas et al, 1997, Evenson et
al.,1999 Shoukir et al, 1998)
14
Swim-up
  • Is still used largely in IVF laboratories around
    the world.
  • Although its use among the male factor
    infertility group is very limited, the swim-up is
    still the standard technique for patients with
    normozoospermia and female infertility.
  • This procedure has several variations

15
Standart swim-up
  • The methodology of conventional swim-up is
    based
  • on the active movement of spermatozoa from
    the prewashed cell pellet into an overlaying
    medium. Typically, the incubation time is 60
    minutes.

16
Direct swim-up from liquefied semen
  • A maximum recovery is obtained by using multiple
    tubes with small volumes of semen per tube, thus
    maximizing the combined total interface area
    between semen and culture medium.
  • Mortimer suggested the use of 250 µl semen and
    500 to 600 µl culture medium per tube

17
Pellet and swim-up
  • Alternatively, semen sample may be diluted and
    centrifuged and pellet overlaid with medium
  • Useful for viscous samples
  • Not recommended when motility is very poor or
    large degree of cellular contamination and debris
  • Has disadvantage of peroxidative damage during
    centrifugation with defective sperm and white
    cells

Aitken RJ, 1990
18
The sperm select system
  • employs a high-purity preparation of 3000 kd
    sodium hyaluronate at a 1-mg/mL final
    concentration in culture medium.
  • It has been shown a significantly higher
    percentage of motile spermatozoa and, the
    achievement of a higher pregnancy rate compare
    with traditional swim-up in a clinical IVF
    program
  • However, it has been shown to increase the
    calcium influx into spermatozoa and induce
    acrosome reaction

Wikland M et al. A selfmigration method for
preparation of sperm for in-vitro
fertilization.Hum Reprod. 19872191195.
19
Migration-sedimantation
  • swim-up technique combined with a sedimentation
    step
  • spermatozoa swim up directly from liquefied semen
    into the supernatant medium and subsequently
    sediment in that inner cone within an hour
  • Zavos et al.proposed the use of a multi-chamber
    tube to retrieve functional spermatozoa for
    assisted reproductive techniques by means of a
    swim-up and sedimentation method.

Zavos PM et al Use of the multi-ZSC one-step
standardized swimup method recovery of
high-quality spermatozoa for intrauterine
insemination or other forms of assisted
reproductive technologies. Fertil Steril 2000,
74834-835.
20
Density gradients
  • Various gradient procedures
  • It is rapid, relatively simple to perform
  • Abnormal sperm, immotile sperm and debris can
    largely eliminate
  • Generally the recovery of motile sperm is greater
    with gradients but the percentages of sperm with
    progressive motility is usually lower than with
    swim-up

21
Density gradients
  • Colloidal silica particles (coated with
    PVPPercoll)
  • Silane coated silica particles (Isolate,
    PureSperm, SilSelect)
  • Nycodenz (iodinated hydrocarbon), Ficoll, Highly
    purified arabinogalactan

22
  • The ejaculate is placed on top of the
    density media with higher density and is then
    centrifuged for 1530 minutes
  • Highly motile spermatozoa move actively in
    the direction of the sedimentation gradient and
    can therefore penetrate the boundary quicker than
    poorly motile or immotile cells, thus, highly
    motile sperm cells are enriched in the soft
    pellet at the bottom

23
Density gradients
  • Percoll was withdrawn from clinical use by its
    manufacturer in 1996
  • PVP-coated silica that can have deleterious
    effects on sperm membranes, and may affect
    subsequent fertilization events (De vos et al,
    1997) because of high endotoxin levels
  • Products contain silane-coated silica particles,
    adjusted for the osmolarity with polysucrose ,
    have very low toxicity. All these replacement
    products are non-irritating and are approved for
    human in vivo use.

24
Density gradients
  • Density gradients protect the sperm from trauma
    of centrifugation
  • High proportion of functional sperm can be
    recovere
  • Two or three step gradients are simple and highly
    effective in preparing motile sperm from
    suboptimal samples
  • In general longer centrifugation time increases
    the recovery of both motile and immotile sperm
  • Debris, round cells, abnormal forms never reach
    the bottom of the tube because of their low
    density

25
Density gradients
  • Gradients with larger volumes result in improved
    filtration, but decreased yield
  • Three layers of mini-gradient improve filtration,
    recovery of sperm from severely oligospermic
    samples
  • Samples wiht large amont of debris should be
    distributed in smaller volumes over several
    gradients
  • Severely asthenoozospermic samples,with normal
    density can also be distrubuted over a series of
    mini-gradients

26
Density gradients
27
Mini-gradient(95/70/50)
  • Make layers with 0.3 ml f each sol95,70,50
  • Dilute semen 11 with culture medium, centrifuge
    at 200 g for 10 minutes
  • Resuspend the pellet in 0.3 ml culture medium and
    layer over mini-gradient
  • Centrifuge at 600 g, for 20-30 minutes
  • Recover the pellet, resuspend in 0.5 ml culture
    medium and assess count and motility. Proceed
    exactly as for two step gradient prep either
    centrifuge at 200 g for 5 min and resuspended
    pellet, or layer over the pellet for a swim up

28
Sedimantation method or layering under paraffin
oil
  • Useful for samples with very low counts and poor
    motility
  • Very effective in removing debris, requires
    several hours of preparation
  • Wash the sample by dilution and cent.twice
  • Resuspend the pellet in a reduce volume of medium
  • Layer this suspansion under paraffin oil in a
    small petri dish, place in a desiccator and gas
    with 5 CO2
  • Leave at room temp for 3-24 hours.
  • Carefully aspirate motile sperm by using a fine
    drawn pipette

29
Glass wool filtration
  • Motile spermatozoa are separated from immotile
    sperm cells by means of densely packed glass wool
    fibres
  • Potential risks of the technique damages of the
    spermatozoa or the occurrence of glass wool
    fragments in the filtrate essentially depend on
    the kind of glass wool used and on the intensity
    of the washing prior to the filtration.
  • uses the whole volume of the ejaculate and
    therefore yields a significantly higher total
    number of motile spermatozoa and do not required
    prewash step
  • it can also be used for patients with oligo-
    and/or asthenozoospermia

Henkel R et al., J Assist Reprod Genet
1994 Berger T et al., Fertil Steril 1985
30
Glass wool filtration
  • glass wool filtration has been shown to
    eliminate leukocytes to an extent of up to 90,
    this effect significantly contributes to a
    reduction of free radicals in the ejaculate
  • Glass wool filtration like the density
    gradient centrifugation with PureSperm or the
    migration sedimentation technique significantly
    selects normally chromatin-condensed spermatozoa

31
Semen preparation techniques for intrauterine
inseminationBoomsma CM, Heineman MJ, Cohlen BJ,
Farquhar C
  • Summary
  • The effectiveness of specific sperm preparation
    techniques for increasing pregnancy rates in
    subfertile couples undergoing intrauterine
    insemination (IUI) is unknown
  • Semen preparation techniques are used in assisted
    reproduction to separate sperm, which have a
    normal appearance and move spontaneously, from
    the fluid portion of the semen in which the sperm
    are suspended. It is known that white blood
    cells, bacteria and dead sperm in semen can
    produce oxygen radicals that can impair
    fertilization of the egg. This review found that
    there is insufficient evidence to recommend any
    specific sperm preparation technique for
    subfertile couples undergoing intrauterine
    insemination (a procedure which places sperm
    directly into the uterus) as there were no
    differences in pregnancy rates. More research is
    needed.

Cochrane Reviews March 2004
32
Which procedure for IUI?Swim-up/Gradient?
Boomsma CM, Heineman MJ, Cohlen BJ, Farquhar C
.Cochrane Reviews March 2004
33
Which procedure for IUI?Swim-up/Gradient?
Cochrane Database of Systematic Reviews, 2004
34
Sperm processing
  • Samples with an acceptable number of motile sperm
    ( gt 20 millions / ml ) can be processed
    efficiently by sperm wash twice and swim-up.
  • Poor quality semen samples should be processed
    using density gradient centrifugation DGC.
    Morshedi M et al, 2003

35
Sperm preparation for ICSI
  • Combination of methods can be use
  • Extremely oligospermic/asthenozoospermic samples
    cannot be prepared by density centrifugation or
    swim-up

36
High-speed centrifugation and washing
  • Cryptozoospermic samples which must be prepared
    for ICSI can be centrifuged directly at 1800 g
    for 5 minutes or diluted with medium and then
    centrifuged at 1800 g for 5 min.
  • Wash the pellet with small volume of medium(0.5
    ml) and centrifuge at 200 g for 5 min.
  • Recover this pellet in a minimal volume of
    medium(20-50 µl), and overlay with mineral oil

37
Sperm preparation for ICSI Extremely
oligospermic/asthenozoospermic samples
  • 1. Centrifuge the whole sample, 1800 g, 5minutes,
    wash with medium, and resuspend the pellet in a
    small volume of medium
  • 2. Apply sample directly to the injection dish,
    without PVP,or add an aliquot suspension to a
    drop of HEPES buffered medium without PVP
  • 3. If possible, use the injection pipette to
    select a moving sperm with apparently normal
    morphology from this drop and transfer it into
    the PVP or into another drop of HEPES buffered
    medium
  • 4. If there is debris attached to the sperm,
    clean it by pipetting the sperm with the
    injection pipette
  • 5. It may sometimes be helpful to connect the
    sperm droplet to another small medium droplet by
    means of a bridge of medium, and allow motile
    sperm to swim out into the clean droplet

38
No motile sperm
  • It may possible to see slight tail movement in a
    medium drop without PVP.
  • If absolutely no motile sperm are found, immotile
    sperm may be used. The fertilization rate with
    immotile sperm is generally lower. Previous
    assesment with a vital stain may be helpful
    before deciding ICSI treatment

39
No motile sperm
  • To date, two different approaches for the
    distinction between live and dead spermatozoa
  • the initiation of motility as sign of vitality
    by means of stimulants
  • the identification of live spermatozoa according
    to their membrane integrity by means of the
    hypo-osmotic swelling test (HOS test).

40
PDE inhibitor
  • As pentoxifylline stimulates motility without
    altering the sperm membrane, it appeared as an
    ideal substance to initiate motility in immotile
    spermatozoa
  • This method was successfully used to identify
    live testicular and epididymal spermatozoa and
    live births are reported

Terriou P et al., J Assist Reprod Genet 2000,
17194-199. Nodar F et al, Fertil Steril 1999,
711149-1152.
41
HOS (hypo-osmotic swelling test)
  • Assesses the osmoregulatory ability of the sperm,
    and the functional integrity of its membranes
  • Can be used to discriminate viable sperm from
    non-viables in a sample which has zero motility
  • In hyposmotic environment sperms react by
    swelling of the tail. Dead spermatozoa whose
    plasma membranes are no longer intact do not show
    tail swelling

Jeyendrn RS, Van der Ven HH, et al. (1984)J
Reprod Fertil 70219228.
42
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43
No motile sperm
  • Shown significantly elevated fertilization and
    pregnancy rates when oocytes were injected with
    HOS-positive sperm
  • Sperm immotility is significantly positively
    correlated with sperm DNA fragmentation, the
    probability to select such DNA-damaged
    spermatozoa for ICSI is higher
  • According to present knowledge, sperm DNA
    fragmentation might cause an impaired embryonic
    development and early embryonic death
  • Therefore, a careful examination and counselling
    of the patients seems mandatory, and
    fertilization with ICSI should not be performed
    at all cost

Henkel R et al, RBM Online 2003, 7474-484.
Asch R et al, Hum Reprod 1995 Jurisicova A
et al, Mol Hum Reprod 1996 Simerly C et al.,
InGenetics of Human Male Fertility Edited by
Barratt C et al, Paris 1997258-286. Aitken RJ
et al, Biol Reprod 1998, 591037-1046.
44
Abnormal heads
  • In cases with 100 abnormal head anomalies, the
    fertililization and implantation rates lower,
    individual judgment should be applied to each
    case, with several assessment of several
    different semen samples
  • Globoozospermia,100 head anomaly, where all
    sperm lack an acrosomal cap debate continues as
    to whether it is ethically advisable to offer
    treatment to these man

45
Retrograde ejeculation
  • Bladder shoul emptied via catheter, approximately
    20 ml of culture medium then installed
  • After ejeculation, bladder is again emptied,
    entire sample centrifuged
  • Resulting pellet can then be resuspended in
    medium and processed on appopriate density
    gradients

46
Retrograde ejeculation
  • 1 gm bicarbonate PO night before and morning
  • Split the urine 10 ml. fractions and santrifugate
    at 300 g, 5 min
  • Resulting pellet can then be resuspended in
    medium and processed on appopriate density
    gradients

47
Azoospermia epididymal sperm
  • Can be obtained by open microscopic surgery or by
    percutaneus puncture using by needle to aspirate
    fluid
  • If large numbers of sperm are found, they can
    processed by density gradient or swim-up
  • Samples with fewer sperm can be washed and
    centrifuged with IVF medium, and concentrated
    sample is than added to microdroplets in the
    injection dish.

48
Azoospermia Testicular sperm
  • Testicular tissue is obtained either by open
    biopsy or percutaneus fine-needle aspiration
  • Place tissue into a small petri dish of warm
    culture media with hepes, albumin
  • Dissect and squeeze tubules using fine gauge
    needles
  • Transfer raw suspension to a test tube and vortex
    for 30 s
  • Depending on concentration, motility, amount of
    debris, either use directly after wash and
    resuspension or seperate on a density gredient
  • Leave sperm to incubate to allow sperm to gain
    motility up to 24 hours in culture media with
    albumin at 37C under 5 CO2
  • for same day use, prepare plate for ICSI and
    leave at 37C in Hepes buffered culture media
    with albumin

49
enzimatic digestion or mechanical preparation of
testicular tissue
  • The optimal method of obtaining suitable sperm
    from testicular tissue is still under debate
  • Mechanical preparation by mincing and
  • shredding
  • Enzimatic digestion by using collagenase
  • It has been shown no advantages one over the
    other preparation method

Verheyen et al. Hum Reprod, 1995
Salzbrunn et al. Hum Reprod, 1996
V.Baukloh on behalf of German Sociaty for
Human Repro. BiologyHum Reprod,2002
50
sperm morphology and genotype
  • Abnormally small and large sperm heads are often
    associated with aneuploidy and diploidy
  • It has been shown that morphologically abnormal
    spermatozoa can carry normal karyotypes (Martin
    and Rademaker, 1988) and can produce normal
    offspring
  • This subject is a major area of current research

51
Sperm DNA damage
  • DNA damage in human spermatozoa is negatively
    correlated with pregnancy rates in both natural
    and assisted conception cycles and has been
    linked with both increased rates of miscarriage
    and diseases in the offspring
  • (Aitken, 1999 Loft et al., 2003 Lewis and
    Aitken, 2005)

52
Sperm DNA aneuploidy
53
Sperm cromatin condensation
  • The degree of nuclear chromatin condensation can
    be assessed by various techniques such as aniline
    blue (Terquem Dadoune, 1983), acridine orange
    (Tejada et al., 1984) or chromomycinCMA3 (Bianchi
    et al., 1993)
  • Manicardi et al. (1995) and Sakkas et al. (1996)
    have indicated that in a normal semen sample CMA3
    Fluorescence can be taken at lt30, while levels
    above these values would be indicative of a semen
    sample containing spermatozoa with abnormal
    chromatin

54
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55
Hammadeh ME. Int JAndrol.200124360-368
56
Hammadeh ME. Int JAndrol.200124360-368
57
Hammadeh ME. Int JAndrol.200124360-368
  • The proportion of chromatin condensed spermatozoa
    increased after sperm processing with swim-up,
    PureSperm gradients centrifugation and glass-wool
    in comparison with the value observed in the
    native semen samples
  • This increase was significantly shown higher
    after semen separation with glass-wool in
    comparison with traditional swim-up
  • However, there were no significant differences in
    the fertilization, implantation and pregnancy
    rates of sperm prepared by means of swim-up,
    PureSperm or glasswool filtration
  • Their conclution was, glass-wool filtration could
    be used as the first choice for semen preparation
    in an ICSI programme as the natural selection is
    bypassed. Swim-up and Pure Sperm should be used
    for semen processing in IVF programme

58
  • Density gradient centrifugation separate
    spermatozoa according to their density and
    favours the isolation of the motile and normal
    morphological spermatozoa (Mortimer et al,1999)
  • Differential gradient sperm separation method
    using Percoll is superior to the swim-up method
    for selecting sperm with normal morphology
    (Prakash et al.,1998)
  • Sakkas et al. (2000) investigated the efficiency
    of the PureSperm, Percoll and swim-up preparation
    techniques to eliminate spermatozoa with nuclear
    anomalies. They indicated that both the PureSperm
    and Percoll techniques can enrich the sperm
    population by separating those with nicked DNA
    and with poorly condensed chromatin

59
  • Henkel et al (1994), demonstrated that glass-wool
    filtration results in a significant higher
    percentage of normal chromatin condensed
    spermatozoa compared with the ejaculate
  • Soderlund Lundin (2000) was also demonstrated
    that PureSperm-treated spermatozoa resulted in
    similar fertilization and pregnancy rates as
    using spermatozoa obtained after the swim-up
    procedure.

60
(MACS) annexinV magnetic-activated cell sorting
separation
  • Successful fertilization requires a sperm plasma
    membrane with normal integrity and function
    (Flesch et al., 2000)
  • The plasma membrane is one of the key structures
    in spermatozoa of infertile men displaying
    apoptotic features(Glander and Schaller, 1999)
  • The phospholipid phosphatidylserine (PS), which
    is normally present on the inner leaflet of the
    plasma membrane, becomes externalized to the
    outer leaflet (Vermes et al., 1995), The
    externalization of PS is currently accepted as a
    membrane marker for early apoptosis (Martin et
    al., 1995)

61
MACS
  • Annexin V is characterized by high affinity for
    PS and does not have the ability to pass the
    intact sperm membrane. Therefore, annexin V
    binding to spermatozoa characterizes disturbed
    integrity of the sperm membrane(Glander and
    Schaller, 1999),
  • Colloidal super-paramagnetie microbeads (50 nm
    in diameter) conjugated with annexin V have been
    shown to separate the dead and apoptotic
    spermatozoa by magnetic activated cell sorting
    (MACS), Cells exposing PS bound to these
    microbeads (annexin positive) are enriched to
    high extent within a column containing iron balls
    when placed in a very strong magnetic field.
    Cells with intact membranes remain unlabelled
    (annexin negative), and pass freely through the
    column (Miltenyi et al., 1990 von Schonfeldt et
    al., 1999)

62
MACS
  • The combination of MACS with density gradient
    centrifugation (DGC) in a single sperm
    preparation protocol results in spermatozoa with
    superior quality (Said et al., 2005a)
  • In general, MACS is a feasible and safe method
    that may be used to provide a high-quality sperm
    fraction (Glander et al., 2002 Grunewald et al.,
    2001 Paasch et al., 2003b, 2005)
  • The high sperm recovery rate following advocates
    the use of this protocol as sperm preparation
    technique prior to assisted reproduction.
    Separating a distinctive population of
    nonapoptotic spermatozoa with intact membranes
    and subjecting it to IVF or ICSI is a step
    further in optimizing the outcome of assisted
    reproduction
  • Nevertheless, future experiments using animal
    models that evaluate embryo viability and genetic
    integrity would still be needed before the
    technique could be applied to human cases.

63
PICSI
  • Hyaluronan (H) is a major constituent of the
    cumulus oophorous matrix and may play a critical
    role in the selection of functionally competent
    sperm during in vivo fertilization.
  • The in vitro selection of sperm for ICSI is
    critical and may influence the developmental
    potential of the resulting embryo.
  • Research has demonstrated that specific motile
    sperm attach to H and that such H-bound (HB)
    sperm carry enhanced levels of developmental
    maturity, sperm chromatin integrity and normal
    morphology. HB sperm may therefore contain
    increased levels of functional competence.
  • The PICSI plate provides microdrops of H for
    sperm selection

Gabor Huzsar,MD Yale University K. C.Worrilow,
H. T. Huynh, et al. ASRM 2007 October OP
64
PICSI
  • Authors suggests that the use of HB-sperm in ICSI
    may allow the isolation of sperm with potentially
    enhanced levels of functional competence, thereby
    exerting a positive paternal influence on
    preimplantation embryogenesis. The statistically
    significant reduced levels of fragmentation,
    increased cpr and decreased mr associated with
    the use of PICSI-derived embryos is promising.

K. C.Worrilow, H. T. Huynh, et al. ASRM 2007
October OP
65
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66
  • The highes quality spermatozoa in the
    ejaculate are the most electronegative (Kirchhoff
    and Schroter, 2001 Giuliani et al., 2004
    Ainsworth et al., 2005) and spermatozoa can be
    separated from other contaminating
    electronegative cells (such as leukocytes)by
    their small cross-sectional size
  • rapid (5 min), efficient and selective, with
    no contaminating cells from TESE biopsy material

67
  • THANK YOU
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