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Semen preparation techniques for ART Gülnaz Şahin, MD Ege University Family Planning and Infertility Research and Treatment Center.

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Presentation on theme: "Semen preparation techniques for ART Gülnaz Şahin, MD Ege University Family Planning and Infertility Research and Treatment Center."— Presentation transcript:

1 Semen preparation techniques for ART Gülnaz Şahin, 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 Edwards RG, Steptoe PC, Purdy JM: Establishing full term human pregnancies using cleaving embryos grown in vitro. Br J Obstet Gynaecol 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 by:Wood 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. 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 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 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 Maximalize the changes of fertilization to provide as many normally fertilized ooctes as possible Elemination of seminal PG, lymphokines, cytokines and infectious agents Elemination of seminal PG, lymphokines, cytokines and infectious agents Reduce the number of free oxygen radicals 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 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 The time of sample collection should be recorded on the label Semen should be prepared soon after liquefaction Semen should be prepared soon after liquefaction If liquefaction delayed or specimen viscous; mixing the specimen 1:1 with medium may help or enzymatically liquefaction can be done (ie; α-amylase, hyolurinidase, tripsin based dissolving sol.) If liquefaction delayed or specimen viscous; mixing the specimen 1:1 with medium may help or enzymatically liquefaction can be done (ie; α-amylase, hyolurinidase, tripsin based dissolving sol.) 10 μl of the sample is taken to check sperm consentration and motility 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 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 choice of sperm preparation method depens on, the motile count, ratio between motile/immotile count, volume, presence of antibodies, agglutination 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 destroy their 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 Alternatively, semen sample may be diluted and centrifuged and pellet overlaid with medium Useful for viscous samples Useful for viscous samples Not recommended when motility is very poor or large degree of cellular contamination and debris 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* 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 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.

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, 74:

20 Density gradients Various gradient procedures Various gradient procedures It is rapid, relatively simple to perform It is rapid, relatively simple to perform Abnormal sperm, immotile sperm and debris can largely eliminate 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 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 PVP:Percoll) Colloidal silica particles (coated with PVP:Percoll) Silane coated silica particles (Isolate, PureSperm, SilSelect) Silane coated silica particles (Isolate, PureSperm, SilSelect) Nycodenz (iodinated hydrocarbon), Ficoll, Highly purified arabinogalactan 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 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

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 Density gradients protect the sperm from trauma of centrifugation High proportion of functional sperm can be recovere 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 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 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 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 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 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 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 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 sol:95,70,50 Make layers with 0.3 ml f each sol:95,70,50 Dilute semen 1:1 with culture medium, centrifuge at 200 g for 10 minutes Dilute semen 1:1 with culture medium, centrifuge at 200 g for 10 minutes Resuspend the pellet in 0.3 ml culture medium and layer over mini-gradient Resuspend the pellet in 0.3 ml culture medium and layer over mini-gradient Centrifuge at 600 g, for minutes Centrifuge at 600 g, for 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 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 Useful for samples with very low counts and poor motility Very effective in removing debris, requires several hours of preparation Very effective in removing debris, requires several hours of preparation Wash the sample by dilution and cent.twice Wash the sample by dilution and cent.twice Resuspend the pellet in a reduce volume of medium 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 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. Leave at room temp for 3-24 hours. Carefully aspirate motile sperm by using a fine drawn pipette 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 insemination Boomsma CM, Heineman MJ, Cohlen BJ, Farquhar C Summary Summary The effectiveness of specific sperm preparation techniques for increasing pregnancy rates in subfertile couples undergoing intrauterine insemination (IUI) is unknown 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. 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 ( > 20 millions / ml ) can be processed efficiently by sperm wash twice and swim-up. Samples with an acceptable number of motile sperm ( > 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 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 Combination of methods can be use Extremely oligospermic/asthenozoospermic samples cannot be prepared by density centrifugation or swim-up 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. 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. 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 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. 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 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, 17: Nodar F et al, Fertil Steril 1999, 71:

41 HOS (hypo-osmotic swelling test) Assesses the osmoregulatory ability of the sperm, and the functional integrity of its membranes 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 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 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 70:219–228.

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43 No motile sperm Shown significantly elevated fertilization and pregnancy rates when oocytes were injected with HOS-positive 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, 7: **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: **Aitken RJ et al, Biol Reprod 1998, 59:

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 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 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 Bladder shoul emptied via catheter, approximately 20 ml of culture medium then installed After ejeculation, bladder is again emptied, entire sample centrifuged After ejeculation, bladder is again emptied, entire sample centrifuged Resulting pellet can then be resuspended in medium and processed on appopriate density gradients 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 1 gm bicarbonate PO night before and morning Split the urine 10 ml. fractions and santrifugate at 300 g, 5 min 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 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 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 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. 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 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 Place tissue into a small petri dish of warm culture media with hepes, albumin Dissect and squeeze tubules using fine gauge needles Dissect and squeeze tubules using fine gauge needles Transfer raw suspension to a test tube and vortex for 30 s 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 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 37°C under 5% CO2 Leave sperm to incubate to allow sperm to gain motility up to 24 hours in culture media with albumin at 37°C under 5% CO2 for same day use, prepare plate for ICSI and leave at 37°C in Hepes buffered culture media with albumin for same day use, prepare plate for ICSI and leave at 37°C in Hepes buffered culture media with albumin

49 enzimatic digestion or mechanical preparation of testicular tissue enzimatic digestion or mechanical preparation of testicular tissue The optimal method of obtaining suitable sperm from testicular tissue is still under debate The optimal method of obtaining suitable sperm from testicular tissue is still under debate Mechanical preparation by mincing and Mechanical preparation by mincing and shredding * shredding * Enzimatic digestion by using collagenase ** Enzimatic digestion by using collagenase ** It has been shown no advantages one over the other preparation method*** 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. Biology;Hum 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 <30%, 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.2001;24:

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57 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) annexin V 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

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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 THANK YOU


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