Semen Semen preparation preparation

techniques for techniques for ARTARTGülnaz Şahin, MDGülnaz Şahin, MD

Ege University Family Planning Ege University Family Planning and Infertility Research and and Infertility Research and

Treatment CenterTreatment Center

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

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 19692. Edwards RG, Steptoe PC, Purdy JM: Establishing full term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol 19803. 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

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 by:Wood C, Trounson A. Springer-Verlag, Berlin; 1984

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)

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.

The quality of sperm samples is one of the factors determining successful assisted reproduction

(Ombelet et al. 2003)

Ejeculated semenEjeculated semen

Viscous liquid composed of mixture of Viscous liquid composed of mixture of testicular, epididymal secretions containing testicular, epididymal secretions containing spermatozoa and prostatic secretions produced spermatozoa and prostatic secretions produced at the time of ejeculation.at the time of ejeculation.

This seminal plasma contains substances which This seminal plasma contains substances which inhibit capasitation and prevent fertilizationinhibit 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).

The purpose The purpose

Concentrate the motile spermatozoa Concentrate the motile spermatozoa in a fraction which is free of seminal in a fraction which is free of seminal plasma and debrisplasma and debris

Maximalize the changes of Maximalize the changes of fertilization to provide as many fertilization to provide as many normally fertilized ooctes as possible normally fertilized ooctes as possible

Elemination of seminal PG, lymphokines, Elemination of seminal PG, lymphokines, cytokines and infectious agentscytokines and infectious agents

Reduce the number of free oxygen radicalsReduce the number of free oxygen radicals

Collection of spermCollection of sperm The male partner should collect semen into a The male partner should collect semen into a

sterile, clearly labelled disposible plastic jar with sterile, clearly labelled disposible plastic jar with in a room adjecent to the IVF laboratoryin a room adjecent to the IVF laboratory

The time of sample collection should be recorded The time of sample collection should be recorded on the labelon the label

Semen should be prepared soon after liquefactionSemen should be prepared soon after liquefaction If liquefaction delayed or specimen viscous; mixing If liquefaction delayed or specimen viscous; mixing

the specimen 1:1 with medium may help or the specimen 1:1 with medium may help or enzymatically liquefaction can be done (ie; enzymatically liquefaction can be done (ie; αα--amylase, hyolurinidase, tripsin based dissolving amylase, hyolurinidase, tripsin based dissolving sol.)sol.)

10 10 μμl of the sample is taken to check sperm l of the sample is taken to check sperm consentration and motilityconsentration and motility

Initial assesment of density and Initial assesment of density and motility allow to choose the most motility allow to choose the most appopriate method of sperm appopriate method of sperm preparationpreparation

The choice of sperm preparation The choice of sperm preparation method depens on,method depens on,

the motile count, ratio between the motile count, ratio between motile/immotile count, volume, motile/immotile count, volume, presence of antibodies, agglutination presence of antibodies, agglutination

ROS (reactive oxygen species) ROS (reactive oxygen species) induced damageinduced 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 impair—even totally destroy—their fertilizing cability.

Aitken RJ, Clarkson JS.; J Androl.1988

ROS induced damageROS 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)

Swim-upSwim-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

Standart swim-upStandart 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.

Direct swim-up from Direct swim-up from liquefied semenliquefied 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

Pellet and swim-up Pellet and swim-up

Alternatively, semen sample may be Alternatively, semen sample may be diluted and centrifuged and pellet diluted and centrifuged and pellet overlaid with mediumoverlaid with medium

Useful for viscous samplesUseful for viscous samples Not recommended when motility is very Not recommended when motility is very

poor or large degree of cellular poor or large degree of cellular contamination and debriscontamination and debris

Has disadvantage of peroxidative damage Has disadvantage of peroxidative damage during centrifugation with defective during centrifugation with defective sperm and white cells* sperm and white cells* *Aitken RJ, 1990

The sperm select systemThe 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 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. 1987;2:191–195.

Migration-sedimantationMigration-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, 74:834-835.

Density gradientsDensity gradients

Various gradient procedures Various gradient procedures It is rapid, relatively simple to performIt is rapid, relatively simple to perform Abnormal sperm, immotile sperm and Abnormal sperm, immotile sperm and

debris can largely eliminatedebris can largely eliminate Generally the recovery of motile sperm Generally the recovery of motile sperm

is greater with gradients but the is greater with gradients but the percentages of sperm with progressive percentages of sperm with progressive motility is usually lower than with motility is usually lower than with swim-upswim-up

Density gradientsDensity gradients Colloidal silica particles (coated with Colloidal silica particles (coated with

PVP:Percoll)PVP:Percoll) Silane coated silica particles (Isolate, Silane coated silica particles (Isolate,

PureSperm, SilSelect)PureSperm, SilSelect) Nycodenz (iodinated hydrocarbon), Ficoll, Nycodenz (iodinated hydrocarbon), Ficoll,

Highly purified arabinogalactanHighly purified arabinogalactan

The ejaculate is placed on top of the density media with higher density and is then centrifuged for 15–30 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

Density gradientsDensity 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.

Density gradientsDensity gradients Density gradients protect the sperm from Density gradients protect the sperm from

trauma of centrifugationtrauma of centrifugation High proportion of functional sperm can be High proportion of functional sperm can be

recovererecovere Two or three step gradients are simple and Two or three step gradients are simple and

highly effective in preparing motile sperm from highly effective in preparing motile sperm from suboptimal samplessuboptimal samples

In general longer centrifugation time increases In general longer centrifugation time increases the recovery of both motile and immotile spermthe recovery of both motile and immotile sperm

Debris, round cells, abnormal forms never Debris, round cells, abnormal forms never reach the bottom of the tube because of their reach the bottom of the tube because of their low density low density

Density gradientsDensity gradients

Gradients with larger volumes result in Gradients with larger volumes result in improved filtration, but decreased yieldimproved filtration, but decreased yield

Three layers of mini-gradient improve Three layers of mini-gradient improve filtration, recovery of sperm from severely filtration, recovery of sperm from severely oligospermic samplesoligospermic samples

Samples wiht large amont of debris should be Samples wiht large amont of debris should be distributed in smaller volumes over several distributed in smaller volumes over several gradientsgradients

Severely asthenoozospermic samples,with Severely asthenoozospermic samples,with normal density can also be distrubuted over a normal density can also be distrubuted over a series of mini-gradients series of mini-gradients

Density gradientsDensity gradients

Mini-gradient(95/70/50)Mini-gradient(95/70/50) Make layers with 0.3 ml f each sol:95,70,50Make layers with 0.3 ml f each sol:95,70,50 Dilute semen 1:1 with culture medium, Dilute semen 1:1 with culture medium,

centrifuge at 200 g for 10 minutes centrifuge at 200 g for 10 minutes Resuspend the pellet in 0.3 ml culture Resuspend the pellet in 0.3 ml culture

medium and layer over mini-gradientmedium and layer over mini-gradient Centrifuge at 600 g, for 20-30 minutesCentrifuge at 600 g, for 20-30 minutes Recover the pellet, resuspend in 0.5 ml Recover the pellet, resuspend in 0.5 ml

culture medium and assess count and culture medium and assess count and motility. Proceed exactly as for two step motility. Proceed exactly as for two step gradient prep; either centrifuge at 200 g gradient prep; either centrifuge at 200 g for 5 min and resuspended pellet, or layer for 5 min and resuspended pellet, or layer over the pellet for a swim upover the pellet for a swim up

Glass wool filtrationGlass 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 1994Berger T et al., Fertil Steril 1985

Glass wool filtrationGlass 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

Semen preparation techniques for intrauterine Semen preparation techniques for intrauterine inseminationinsemination

Boomsma CM, Heineman MJ, Cohlen BJ, Boomsma CM, Heineman MJ, Cohlen BJ, Farquhar CFarquhar C

SummarySummary The effectiveness of specific sperm preparation The effectiveness of specific sperm preparation

techniques for increasing pregnancy rates in techniques for increasing pregnancy rates in subfertile couples undergoing intrauterine subfertile couples undergoing intrauterine insemination (IUI) is unknowninsemination (IUI) is unknown

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

Cochrane Reviews March 2004

Which procedure for IUI?Which procedure for IUI?Swim-up/Gradient?Swim-up/Gradient?

Boomsma CM, Heineman MJ, Cohlen BJ, Farquhar C .Cochrane Reviews March 2004

Which procedure for IUI?Which procedure for IUI?Swim-up/Gradient?Swim-up/Gradient?

Cochrane Database of Systematic Reviews, 2004

SpermSperm processing processing

Samples with an acceptable number Samples with an acceptable number of motile sperm ( > 20 millions / ml ) of motile sperm ( > 20 millions / ml ) can be processed efficiently by sperm can be processed efficiently by sperm wash twice and swim-up. wash twice and swim-up.

Poor quality semen samples should Poor quality semen samples should be processed using density gradient be processed using density gradient centrifugation DGC.centrifugation DGC. Morshedi M et al, 2003 Morshedi M et al, 2003

Sperm preparation for Sperm preparation for ICSI ICSI

Combination of methods can be useCombination of methods can be use Extremely Extremely

oligospermic/asthenozoospermic oligospermic/asthenozoospermic samples cannot be prepared by samples cannot be prepared by density centrifugation or swim-updensity centrifugation or swim-up

High-speed centrifugation High-speed centrifugation and washingand washing

Cryptozoospermic samples which must Cryptozoospermic samples which must be prepared for ICSI can be centrifuged be prepared for ICSI can be centrifuged directly at 1800 g for 5 minutes or directly at 1800 g for 5 minutes or diluted with medium and then diluted with medium and then centrifuged at 1800 g for 5 min.centrifuged at 1800 g for 5 min.

Wash the pellet with small volume of Wash the pellet with small volume of medium(0.5 ml) and centrifuge at 200 g medium(0.5 ml) and centrifuge at 200 g for 5 min. for 5 min.

Recover this pellet in a minimal volume Recover this pellet in a minimal volume of medium(20-50 of medium(20-50 μμl), and overlay with l), and overlay with mineral oil mineral oil

No motile spermNo motile sperm

It may possible to see slight tail It may possible to see slight tail movement in a medium drop without movement in a medium drop without PVP. PVP.

If absolutely no motile sperm are found, If absolutely no motile sperm are found, immotile sperm may be used. The immotile sperm may be used. The fertilization rate with immotile sperm is fertilization rate with immotile sperm is generally lower. Previous assesment generally lower. Previous assesment with a vital stain may be helpful before with a vital stain may be helpful before deciding ICSI treatment deciding ICSI treatment

No motile spermNo 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).

PDE inhibitorPDE inhibitor

As pentoxifylline stimulates motility without altering the sperm membrane, it appeared as an ideal substance to initiate motility in immotile spermatozoaThis 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, 17:194-199.Nodar F et al, Fertil Steril 1999, 71:1149-1152.

HOS (hypo-osmotic swelling HOS (hypo-osmotic swelling test)test)

Assesses the osmoregulatory ability of Assesses the osmoregulatory ability of the sperm, and the functional integrity the sperm, and the functional integrity of its membranesof its membranes

Can be used to discriminate viable Can be used to discriminate viable sperm from non-viables in a sample sperm from non-viables in a sample which has zero motility which has zero motility

In hyposmotic environment sperms In hyposmotic environment sperms react by swelling of the tail. Dead react by swelling of the tail. Dead spermatozoa whose plasma membranes spermatozoa whose plasma membranes are no longer intact do not show tail are no longer intact do not show tail swelling swelling Jeyendrn RS, Van der Ven HH, et al. (1984):J Reprod Fertil 70:219–

228.

No motile spermNo motile sperm Shown significantly elevated fertilization and Shown significantly elevated fertilization and

pregnancy rates when oocytes were injected with pregnancy rates when oocytes were injected with HOS-positive spermHOS-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, 7:474-484.

**Asch R et al, Hum Reprod 1995**Jurisicova A et al, Mol Hum Reprod 1996**Simerly C et al., In:Genetics of Human Male Fertility Edited by: Barratt C et al, Paris; 1997:258-286.**Aitken RJ et al, Biol Reprod 1998, 59:1037-1046.

Abnormal headsAbnormal heads In cases with 100% abnormal head In cases with 100% abnormal head

anomalies, the fertililization and anomalies, the fertililization and implantation rates lower, individual implantation rates lower, individual judgment should be applied to each judgment should be applied to each case, with several assessment of several case, with several assessment of several different semen samplesdifferent semen samples

Globoozospermia,100% head anomaly, Globoozospermia,100% head anomaly, where all sperm lack an acrosomal cap; where all sperm lack an acrosomal cap; debate continues as to whether it is debate continues as to whether it is ethically advisable to offer treatment to ethically advisable to offer treatment to these manthese man

Retrograde ejeculationRetrograde ejeculation

Bladder shoul emptied via catheter, Bladder shoul emptied via catheter, approximately 20 ml of culture approximately 20 ml of culture medium then installedmedium then installed

After ejeculation, bladder is again After ejeculation, bladder is again emptied, entire sample centrifugedemptied, entire sample centrifuged

Resulting pellet can then be Resulting pellet can then be resuspended in medium and resuspended in medium and processed on appopriate density processed on appopriate density gradients gradients

Retrograde ejeculationRetrograde ejeculation

1 gm bicarbonate PO night before 1 gm bicarbonate PO night before and morningand morning

Split the urine 10 ml. fractions and Split the urine 10 ml. fractions and santrifugate at 300 g, 5 minsantrifugate at 300 g, 5 min

Resulting pellet can then be Resulting pellet can then be resuspended in medium and resuspended in medium and processed on appopriate density processed on appopriate density gradients gradients

Azoospermia: epididymal Azoospermia: epididymal sperm sperm

Can be obtained by open microscopic Can be obtained by open microscopic surgery or by percutaneus puncture surgery or by percutaneus puncture using by needle to aspirate fluidusing by needle to aspirate fluid

If large numbers of sperm are found, If large numbers of sperm are found, they can processed by density gradient they can processed by density gradient or swim-upor swim-up

Samples with fewer sperm can be Samples with fewer sperm can be washed and centrifuged with IVF washed and centrifuged with IVF medium, and concentrated sample is medium, and concentrated sample is than added to microdroplets in the than added to microdroplets in the injection dish. injection dish.

Azoospermia: Testicular Azoospermia: Testicular spermsperm

Testicular tissue is obtained either by open biopsy or Testicular tissue is obtained either by open biopsy or percutaneus fine-needle aspirationpercutaneus fine-needle aspiration

Place tissue into a small petri dish of warm culture Place tissue into a small petri dish of warm culture media with hepes, albuminmedia with hepes, albumin

Dissect and squeeze tubules using fine gauge needlesDissect and squeeze tubules using fine gauge needles Transfer raw suspension to a test tube and vortex for 30 Transfer raw suspension to a test tube and vortex for 30

ss Depending on concentration, motility, amount of debris, Depending on concentration, motility, amount of debris,

either use directly after wash and resuspension or either use directly after wash and resuspension or seperate on a density gredientseperate on a density gredient

Leave sperm to incubate to allow sperm to gain motility Leave sperm to incubate to allow sperm to gain motility up to 24 hours in culture media with albumin at 37up to 24 hours in culture media with albumin at 37°°C C under 5% CO2under 5% CO2

for same day use, prepare plate for ICSI and leave at for same day use, prepare plate for ICSI and leave at 3737°°C in Hepes buffered culture media with albumin C in Hepes buffered culture media with albumin

enzimatic digestion or enzimatic digestion or mechanical preparation of mechanical preparation of

testicular tissue testicular tissue

The optimal method of obtaining suitable The optimal method of obtaining suitable sperm from testicular tissue is still under sperm from testicular tissue is still under debatedebate

Mechanical preparation by mincing andMechanical preparation by mincing and

shredding *shredding * Enzimatic digestion by using collagenase **Enzimatic digestion by using collagenase **

It has been shown no advantages one over It has been shown no advantages one over the other preparation method***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. Biology;Hum Reprod,2002

sperm morphology and sperm morphology and genotypegenotype

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

Sperm DNA damageSperm 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)

Sperm DNA aneuploidySperm DNA aneuploidy

Sperm cromatin Sperm cromatin condensationcondensation

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 <30%, while levels above these values would be indicative of a semen sample containing spermatozoa with abnormal chromatin

Hammadeh ME. Int Hammadeh ME. Int JAndrol.2001;24:360-368JAndrol.2001;24:360-368

Hammadeh ME. Int Hammadeh ME. Int JAndrol.2001;24:360-368JAndrol.2001;24:360-368

Hammadeh ME. Int Hammadeh ME. Int JAndrol.2001;24:360-368JAndrol.2001;24:360-368

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

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

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.

(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)

MACSMACS 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)

MACSMACS 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.

PICSIPICSI 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 UniversityK. C.Worrilow, H. T. Huynh, et al.; ASRM 2007 October OP

PICSIPICSI

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

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

THANK YOUTHANK YOU