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.
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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

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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

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