Figure 1. Aniline blue-stained human sperm bound to a surface coated with hyaluronic acid. Modified form [1].

See Sperm selection for fertilization (I).


In assisted laboratories, semen samples are usually obtained after masturbation and the ejaculate is collected into a container. However, semen recovered from non-spermicidal condoms can be of higher quality because of the time producing the sample influences in the amount of seminal emission before ejaculation (2). Moreover, according to the World Health Organization criteria, laboratory manual for the examination and processing of human semen, the results of laboratory measurements of semen quality will depend on: completed collection of the sample, products of the accessory glands (that will dilute the concentration of epididymal spermatozoa), time past since last sexual activity, abstinence period and the size of the testis. All these factors, among others, will have an influence on the total sperm count per ejaculate (2).

Contrary to what is found in natural fertilization after ejaculation, there are no actual barriers to enhance semen capacitation in vitro. As a consequence, in assisted reproduction laboratories sperm must be separated from the seminal plasma within one hour after ejaculation. The purpose of semen processing is to increase the concentration of high-quality spermatozoa, and the method chosen will depend on the nature of the sperm, that will, in turn, determine which reproductive procedure will be performed (2).

Reports over the last two decades have made emphasis on the need for the optimal method for sperm selection to compile as many as possible of the following conditions: non-toxicity, ease to perform, inexpensiveness, suitability for high-throughput sample processing, accuracy in selecting the best subpopulation of sperm and ability to discard other cell types and seminal plasma as well as other substances that may harm the sperm (cryoprotectants, bioactive complements, motility enhancers) (3). Unfortunately, no known method to date successfully meet all the above, and so the selection of the most appropriate method normally depends on the specific procedure to follow.


“Conventional” techniques like the nowadays common washing swim-up (WSU) and differential density gradient centrifugation (DDGC) procedures are normally used for a variety of assisted reproductive procedures, although it is clear that not all of them will have comparable efficiency in selecting high-quality sperm. Some techniques are better indicated for certain conditions. For instance, high concentrations of highly motile sperm from normospermic men will show a good performance after WSU, and this will be suitable for regular IVF. On the contrary, severe oligospermic or asthenospermic individuals will require semen selection by using other techniques. The introduction of ICSI in the regular clinical practice enhanced the fertilization rates in the ’90s (4) but did not significantly increase live birth rates because of failures in early development, mostly due to the high incidence of structural chromosomal aberrations (5). This is a direct consequence of the inability of ICSI to specifically detect good quality spermatozoa.

In the following paragraphs, we discuss the appropriateness of different techniques for distinct assisted reproduction treatments, namely: in vitro fertilization (IVF), intrauterine insemination (IUI) and intracytoplasmic sperm injection (ICSI).


In the last few years, several studies have discussed about the best technique for sperm capacitation in vitro. Recently, Volpes and collaborators compared the effects of four methods used for sperm preparation, namely, direct swim-up, pellet swim-up, density gradient and density gradient followed by swim-up (6). The authors evaluated levels of DNA fragmentation using the sperm chromatin dispersion test for samples meeting the following criteria: minimum volume of 2 mL, minimum sperm concentration of 10 million/mL and minimum motility of 35%. The study showed lower DNA fragmentation after pellet swim-up and after density gradient followed by swim-up. However, the study highlighted the limitation that clinical outcomes from IVF/ICSI procedures were not correlated with DNA fragmentation in the sperm (6).

A different study compared sperm processing by WSU and DDGC in normospermic individuals, analyzing sperm motility, concentration, and morphology recovery rates (7). Conclusions were that, on the one hand, DGC was appropriate for males with low sperm concentration, since it yielded higher sperm concentration than the WSU technique. On the other hand, the latter facilitated a more efficient morphology-based sperm selection. Nevertheless, the main limitation of the study was the small number of patients and the fact that all of them were normospermic (7).

In 2016, Yamanaka and coauthors attempted to determine the efficiency of combining both DGC and WSU techniques in reducing the number of sperm with abnormal nuclear morphology under the microscope (8). Results showed that the combination of the two approaches was better than one alone; both DNA fragmentation levels and sperm motility were improved compared to results after just DGC. Data also demonstrated that the combination of both techniques was efficient in enriching the sample with sperm with normal head and flagellum morphology (8).


A study by Karamahmutoglu on the most effective sperm preparation technique for IUI compared WSU vs. DGC (9). Even though data showed higher IUI success rates after having performed the DGC approach, no significant difference was found in the “mild male factor” subfertile group (with sperm count in the range of 5-15 million/mL). Moreover, other factors were affecting fecundity success rate, such as female age, number of cycles and type of infertility (9). These observations on the efficacy of DGC and WSU methods for IUI have recently been confirmed in a similar study by Butt and Chohan (10).


Sperm selection for ICSI is commonly carried out by the embryologist’s own judgement based on morphological criteria. This results in inconsistent decision-making and often selection of poor-quality sperm, since semen samples are considered morphologically normal with just 4% of normal-looking spermatozoa (2). Therefore functional, physiological and molecular traits of spermatozoa cannot be evaluated by ICSI, and so unnoticed DNA abnormalities (even specific causes of sterility) might be passed on to the offspring by the selected spermatozoon. Thus it is easy to understand why the embryology and reproduction community has been trying to develop new strategies to successfully select the best spermatozoa regarding phenotype, functional characteristics and genetic and molecular integrity.

Figure 2. Collection of studies that relate the effects of advanced methods of sperm selection and clinical outcomes (FR=fertilization rate; PR=pregnancy rate). Modified from [11]. *Please consult the original source for references therein.



There are currently three main groups of methods to facilitate selection of high-quality sperm, based on morphology, electrical charge and sperm surface maturity and organization (11, 12):

  • Difficulty to discern good quality spermatozoa by ICSI can be overcome by the motile sperm organelle morphology examination (MSOME) in real-time, which allows for observation of sperm ultramorphology at ~1,500x magnification. It evaluates morphological features like vacuoles or nuclear integrity. MSOME is similar in principles to the ICSI variation named intracytoplasmic morphologically selected sperm injection (IMSI). IMSI is used for evaluation of different sperm parameters such as acrosome, postacrosomal lamina, neck, flagellum, mitochondria and nucleus. The magnification reached is up to 6,000x, and it uses less expensive microscopy resources. Comparisons of clinical outcomes after ICSI vs. IMSI revealed that whereas fertilization rates are not significantly different, IMSI results in lower miscarriage occurrence and higher implantation rates (13), although no significant differences have been found between sperm prepared by WSU and by DGC for IMSI outcomes (14).
  • Discrimination of spermatozoa by an electrophoretical method is based on size and negative charge; it discards other cell types distinct from spermatozoa, and only those with a specific antigen on its membrane are retrieved (15). This antigen is CD52, a protein loaded onto the sperm head membrane during the maturation process within the epididymis (16), and its presence seems to be related to healthy and functional sperm (17). The selective nature of this method along with the inherent reduction of cytotoxic effects and DNA fragmentation levels could improve the quality of the sperm in the sample. The development of the technique raised certain concerns regarding adverse effects on sperm motility (15), although they might ultimately be overcome by the use of ICSI. In fact, positive results as a treatment for infertility were quickly observed using this method for semen preparation before ICSI (18).

A simpler and cheaper technique uses the electrokinetic potential of the sperm (19); the electric potential difference of the sperm membrane decreases with capacitation, which is used to pipette washed sperm into positively charged tubes, so that negatively charged (mature) sperm can be retrieved afterwards. However, the total sperm recovered by using this technique is low, which represents an important limitation (11).

  • Physiological traits of sperm are assessed by different methods, including hyaluronan binding assay (HBA) and phosphatidylserine (PS). Hyaluronic acid (HA) or hyaluronan is a negatively charged glycosaminoglycan present in the female reproductive tract and in the cumulus oophorus complex, and directs sperm binding to the zona pellucida (20, 21). The physiological intracytoplasmic sperm injection (PICSI) technique has shown spermatozoa binding to HA in vitro to be of better quality regarding nuclear maturation, membrane organization and remodelling and shaping than those who fail to bind to HA, resembling the sperm capacity of binding to the zona (22). Consequently, in recent years different studies have demonstrated that HA-binding spermatozoa display higher pregnancy rates, better embryo quality and an overall better clinical outcome (23, 24).

PS externalization to the outer sperm membrane is a typical apoptotic feature, which allows the cell to bind to magnetic beads conjugated with Annexin-V. This made it possible to develop a magnetic-activated cell sorting system (MACS) (25); the sperm suspension is incubated with the microbeads so that those apoptotic spermatozoa will bind to the beads, which will be subsequently retained within the MACS column in a magnet. Non-apoptotic sperm will then flow freely to be collected. Even though this technique enriches the sample in healthy sperm, it does not discard leukocytes or germ cells, and thus it must be combined with DDGC (26). A variation of the approach known as Annexin V glass wool (annexin V-GW) eliminates potential side effects of free magnetic beads (27), but still needs to be combined with repeated DGC cycles, which is actually not appropriate for oligospermic individuals (11).


The approach followed for semen preparation and selection for every patient/couple needs to be chosen upon a series of factors that mainly refer to the cause of infertility. The wide range of situations found regarding this topic makes it necessary to adopt a specific strategy every time. This will ultimately define the treatment to be applied and the techniques to be carried out subsequently.

Recent advances have developed new and better ways to detect good quality spermatozoa, minimizing DNA fragmentation and optimizing the rates of good morphology or motility, for instance. However, it is important to remember that there exists no particular strategy that always relates to the optimal clinical outcome. On the contrary, each situation must be considered in the light of the patient’s needs and characteristics, and so the technique for sperm preparation must be chosen accordingly.


  1. Torabi F, Binduraihem A, Miller D. Sedimentation properties in density gradients correspond with levels of sperm DNA fragmentation, chromatin compaction and binding affinity to hyaluronic acid. Reprod Biomed Online.2017;34(3)298-311.
  2. World Health Organization DoRHaR. WHO laboratory manual for the examination and processing of human semen (fifth edition)2010. 287 p.
  3. Ortega N, Bosch P. Methods of Sperm Slection for In Vitro Fertilization. In: Friedler S, editor. In Vitro Fertilization – Innovative Clinical and Laboratory Aspects: InTech; 2012. p. 168.
  4. Palermo G, Joris H, Devroey P, Van Steirtteghem A. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992;340:17-8.
  5. Bonduelle M, Van Assche E, Joris H, Keymolen K, Devroey P, Van Steirtteghem A, et al. Prenatal testing in ICSI pregnancies: incidence of chromosomal anomalies in 1586 karyotypes and relation to sperm parameters. Hum Reprod. 2002;17(10):2600-14.
  6. Volpes A, Sammartano F, Rizzari S, Gullo S, Marino A, Allegra A. The pellet swim-up is the best technique for sperm preparation during in vitro fertilization procedures. J Assist Reprod Genet. 2016;33(6):765-70.
  7. Fácio C, Previato L, Machado-Paula L, Matheus P, Araújo Filho E. Comparison of two sperm processing techniques for low complexity assisted fertilization: sperm washing followed by swim-up and discontinuous density gradient centrifugation. JBRA Assist Reprod. 2016;20(4):206-11.
  8. Yamanaka M, Tomita K, Hashimoto S, Matsumoto H, Satoh M, Kato H, et al. Combination of density gradient centrifugation and swim-up methods effectively decreases morphologically abnormal sperms. J Reprod Dev. 2016;62(6):599-606.
  9. Karamahmutoglu H, Erdem A, Erdem M, Mutlu MF, Bozkurt N, Oktem M, et al. The gradient technique improves success rates in intrauterine inseminatino cycles of unexplained subfertile couples when compared to swim up technique; a prospective randomized study. J Assist Reprod Genet. 2014;31(9):1139-45.
  10. Butt F, Chohan MA. Comparative efficacy of density gradient and swim-up methods of semen preparation in intrauterine inseminatino cycles. J Pak Med Assoc. 2016;66(932).
  11. Said TM, Land JA. Effects of advanced selection methods on sperm quality and ART outcome: a systematic review. Hum Reprod Update. 2011;17(6):719-33.
  12. Henkel R. Sperm preparation: state-of-the-art, physiological aspects and application of advanced sperm preparation methods. Asian J Androl. 2012;14(2):260-9.
  13. Souza Setti A, Ferreira R, Paes de Almeida Ferreira Braga D, de Cassia Savio Figueira R, Iaconelli AJ, Borges EJ. Intracytoplasmic sperm injection outcome versus intracytoplasmic morphologically selected sperm injection outcome: a meta-analysis. Reprod Biomed Online. 2010;21:450-5.
  14. Borges EJ, Setti AS, Vingris L, Figueira RC, Braga DP, Iaconelli AJ. Intracytoplasmic morphologically selected sperm injection outcomes: the role of sperm preparation techniques. J Assist Reprod Genet. 2013;30(6):849-54.
  15. Ainsworth C, Nixon B, Aitken R. Development of a novel electrophoretic system for the isolation of human spermatozoa. Hum Reprod. 2005;20:2261-70.
  16. Schroter S, Derr P, Conradt H, Nimtz M, Hale G, Kirchhoff C. Male-specific madification of human CD52. J Biol Chem. 1999;274:29862-73.
  17. Giuliani V, Pandolfi C, Santucci R, Pelliccione F, Macerola B, Focarelli R, et al. Expression of gp20, a human sperm antigen of epididymal origin, is reduced in spermatozoa from subfetile men. Mol Reprod Dev. 2004;69:235-40.
  18. Ainsworth C, Nixon B, Jansen R, Aitken RJ. First recorded pregnancy and normal birth after ICSI using electrophoretically isolated spermatozoa. Hum Reprod. 2007;22:197-200.
  19. Chan PJ, Jacobson JD, Corselli JU, Patton WC. A simple zeta method for sperm selection based on membrane charge. Fertil Steril. 2006;85:481-6.
  20. McLeskey S, Dowds C, Carballada R, White R, Saling P. Molecules involved in mammalian sperm-egg interaction. Int Rev Cytol. 1998;177:57-113.
  21. Toole B. Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer. 2004;4:528-39.
  22. Parmegiani L, Cognigni G, Bernardi S, Troilo E, Ciampaglia W, Filicori M. ‘Physiologic ICSI’: hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality. Fertil Steril. 2010;93:598-604.
  23. Mokanszki A, Tothne E, Bodnar B, Tandor Z, Molnar Z, Jakab A, et al. Is sperm hyaluronic acid binding ability predictive for clinical success of intracytoplasmic sperm injection: PICSI vs. ICSI? Syst Biol Reprod Med. 2014;60:348-54.
  24. Beck-Fruchter R, Shalev E, Weiss A. Clinical benefit using sperm hyaluronic acid binding technique in ICSI cycles: a systematic review and meta-analysis. Reprod Biomed Online. 2016;32:286-98.
  25. Grunewald S, Paasch U, Glander HJ. Enrichment of non-apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting. Cell Tissue Bank. 2001;2:127-33.
  26. Said T, Grunewald S, Paasch U, Glander H, Baumann T, Kriegel C, et al. Advantages of combining magnetic cell separation with sperm preparation techniques. Reprod Biomed Online. 2005;10:740-6.
  27. Grunewald S, Miska W, Miska G, Rasch M, Reinhardt M, Glander H, et al. Molecular glass wool filtration as a new tool for sperm preparation. Hum Reprod. 2007;22(5):1405-12.