The Mens Fertility Forum - Mensfe
Welcome, Guest. Please login or register.

Login with username, password and session length
News: This forum is now live for members so please register and make a difference!
   Home   Help Search Login Register  
Pages: [1]
Author Topic: Novel technologies for selecting the best sperm for in vitro fertilization and i  (Read 14192 times)
Sr. Member
Posts: 314

« : »

The increasing focus on developing new tools to more accurately diagnose and select individual sperm before intracytoplasmic sperm injection will allow us to counsel and treat couples with greater confidence and efficiency. Current sperm selection techniques are based on the premise that if an ejaculated spermatozoon has cleared spermatogenesis with the correct morphology and/or membrane properties then it is most likely normal. Techniques that are designed to prepare a clean “normal” sperm population or that assist in selecting an individual “normal” spermatozoon are currently being investigated. The use of techniques, including density-gradient preparation, electrophoretic separation, microfluidics, high-magnification sperm morphology selection, and hyaluronic acid binding, is discussed. The research evidence that supports the interrelated developmental and genetic integrity of the selected sperm, particularly sperm DNA damage and clinical outcome evidence are presented.

Key Words: Sperm selection, ICSI, male infertility, sperm morphology

 Discuss: You can discuss this article with its authors and with other ASRM members at

One of the most dramatic technological breakthroughs in assisted reproduction technologies (ART) was the introduction of intracytoplasmic sperm injection (ICSI) in the early 1990s (1). After numerous attempts to successfully treat male factor infertility through epididymal aspirations (2), zona dissection, and injection of multiple sperm into the perivitelline space (3), only the use of ICSI provided pregnancy rates that were equivalent to those in routine in vitro fertilization (IVF).

The use of ICSI is sometimes thought to defy the basic evolutionary paradigm of “survival of the fittest,” which was coined in the late 1800s. Although its meaning has been challenged and changed it is evident that many of the techniques we currently use in ART do not adhere to this phrase. In particular, it could be argued that many of the techniques that we use introduce gametes into the genetic pool that would have a lower chance to participate in natural fertility. Numerous follow-up studies have failed to show any major concerns regarding ICSI offspring. In some of the major follow-up reports conducted by Belgian authors, they found that in 8-year-old ICSI children pubertal staging was similar between ICSI and spontaneously conceived (SC) children. Neurologic examination also failed to show important differences between ICSI and SC children. ICSI children did not require more remedial therapy or surgery or hospitalization than SC children. However, a major congenital malformation was experienced in 15/150 compared with 5/147 SC children (P<.05) (4). Further follow-up showed that ICSI and SC children show similar cognitive and motor development up to the age of 10 years (5).

The success of ICSI has surprised many; however, concerns are still prevalent. A recent study examining more than 300,000 births from a South Australian registry concluded that the increased risk of birth defects associated with IVF was no longer significant after adjustment for parental factors. The risk of birth defects associated with ICSI remained increased, after multivariate adjustment, although the possibility of residual confounding could not be excluded (6).

Some concern over the use of “poorer-quality” sperm are warranted, but we are yet to define adequately what is present in a spermatozoon that may adversely impact fetal outcome. Conversely, numerous groups are now working on methodologies to try and select “normal” spermatozoa so as to limit the possibility of an adverse outcome after ICSI.

Back to Article Outline

“Poorer-quality” sperm

What is a poor-quality spermatozoon? In a normal human ejaculate there are well over 40 million spermatozoa, but when spermatogenesis is compromised a greater percentage of sperm in the ejaculate begin to show a range of abnormalities. These include membrane, mitochondrial, centriolar, nuclear, and chromosomal anomalies. How can one select spermatozoa to eliminate these anomalies? Given that the object is to isolate live spermatozoa, the approaches used have been based on looking at morphology or a membrane property. A major approach in the discovery of sperm biochemical markers, independently from sperm concentration and motility in the semen, is based on the recognition that there are objective markers of sperm function that focus on elements of spermatogenesis and spermiogenesis that are abnormal in the process of sperm development from spermatogonium to spermatozoa. The basis of this approach is that if an ejaculated spermatozoon has cleared spermatogenesis with the correct morphology and/or membrane properties, then it is most likely normal. Huszar et al. (7) have worked on this basis in first identifying cytoplasmic retention as an abnormal morphologic feature and finally progressing through many years' work to develop the hyaluronic acid (HA)–binding assay, which will be discussed subsequently.

Back to Article Outline

Different types of ejaculated spermatozoa

When spermatozoa arrive in the ejaculate, they have progressed through spermatogenesis and traversed through the male reproductive tract fluids. The result can be a population of spermatozoa that is heterogeneous regarding quality. Major mechanisms of inducing abnormalities in spermatozoa during either the production and/or the transport of sperm cells can include: 1) apoptosis or anomalies during the process of spermatogenesis; 2) DNA strand breaks produced during the remodeling of sperm chromatin during the process of spermiogenesis; 3) post-testicular DNA fragmentation induced mainly by oxygen radicals during sperm transport through the seminiferous tubules and the epididymis; 4) DNA fragmentation induced by endogenous caspases and endonucleases; 5) DNA damage induced by radio and chemotherapy; and 6) DNA damage induced by environmental toxins (Cool.

Many of the diagnostic techniques available can assess whether some of these abnormalities have occurred, but they fail to proactively allow for the selection of spermatozoa before treatment. For example, one area of sperm structure that has generated increasing interest in sperm assessment is that of the sperm nuclear DNA/chromatin structure. In 1980, Evenson et al. (9) published a manuscript in Science titled “Relation of mammalian sperm chromatin heterogeneity to fertility.” Their study used flow cytometry measurements of heated sperm nuclei to reveal a significant decrease in resistance to in situ denaturation of spermatozoal DNA in samples from bulls, mice, and humans of low or questionable fertility compared with others of high fertility. They showed that there were changes in sperm chromatin conformation that were related to the diminished fertility. They then went on to suggest that flow cytometry of heated sperm nuclei could provide a new and independent determinant of male fertility.

In addition to that original methodology, numerous tests have been developed for the analysis of sperm nuclear DNA fragmentation (Cool. These tests include TdT-mediated dUTP nick-end labeling (10), Comet (11), chromomycin A3 (CMA3) (12), in situ nick translation 13, 14, DNA breakage detection–fluorescence in situ hybridization (15), sperm chromatin dispersion test (SCD) (16), and sperm chromatin structure assay (SCSA) 17, 18. A recent meta-analysis (19) concluded that sperm DNA damage is associated with a significantly increased risk of pregnancy loss after IVF and ICSI. The protocol of analysis may, however, prove to be more important, because it was shown that the sperm DNA fragmentation values of the actual aliquots used for IVF insemination were significantly correlated with pregnancy outcome (20). This is in sharp contrast to the results reported by Bungum et al. (21), where no correlation was found between sperm DNA fragmentation values in the samples used for IVF, as measured by the SCSA test, and pregnancy outcome. Overall, some controversy still remains about the utility of these tests (22).

It is important to note that in general, patients requiring ICSI will present with higher levels of sperm DNA damage (Fig. 1). In techniques such as IVF or ICSI, where a lot of the natural selection and sperm competition is bypassed, it becomes more important to remove or isolate DNA damaged sperm for treatment. To date, no sperm test can directly gauge the degree of sperm DNA damage in a single sperm and isolate it, so we are forced to examine surrogate markers that may reflect sperm DNA damage or other detrimental sperm qualities.
View full-size image. View Large Image
 Download to PowerPoint

Figure 1

The proportion of 95 men with >20% (dark gray) and <20% (light gray) of their spermatozoa being positive for sperm DNA damage as assessed by the TdT-mediated dUTP nick-end labeling assay in relation to a sperm count of (A) <20 million/mL and (B) >20 million/mL..

Back to Article Outline

Selecting the best sperm

The strategy to select the best sperm or to eliminate those that are abnormal from the population can be performed with the use of various approaches. The population of sperm as a whole can be prepared or individual sperm can be isolated. The approaches published to date reflect the following strategies to select or deselect sperm largely on the basis of morphology or membrane characteristics.
Future approaches to sperm selection

A number of new technologies are being developed that may also serve the purpose of allowing better sperm selection before classic IVF or ICSI. Other molecular techniques may allow the analysis of sperm populations in the future. For example, DNA methylation patterns of key developmental genes have been shown to differ in spermatozoa, which may affect embryo development (60). Interesting results are also being generated from proteomic (61) and RNA (62) analysis of sperm, which will create a database for identifying key factors implicated in defective sperm function and aid in both diagnosis and treatment. This approach may already be bearing fruit: Enciso et al. (63) have recently presented evidence that a novel peptide-based stain could be designed that is capable of detecting DNA damage in individual sperm cells. Evaluation of sperm DNA fragmentation with the use of this peptide could eventually allow this approach to be applied to the selection or deselection of viable spermatozoa with intact DNA for use in ICSI and/or IMSI.

Back to Article Outline


The effect of the paternal genome on reproductive outcome is definitely coming under more scrutiny. Although the evidence is overwhelming in animal models, the effect of the paternal genome is less evident and possibly more subtle in the human. ICSI and the use of compromised sperm populations is definitely creating some concern regarding its effect on offspring. The overall results point to incremental improvements in pregnancy rates when particular patients are selected for treatment with the use of these technologies. As these novel sperm selection methods are developed, the challenge will be in choosing which patients to apply different strategies for (Table 1). Some of these technologies will be more applicable broadly, whereas others may suit only a specific subset of patients. The increasing focus on developing new tools to more accurately diagnose and select individual sperm before ICSI will allow us to counsel and treat couples with greater confidence and efficiency and to allay the fears that we are passing on a compromised paternal genome when treating men with the use of ART.

« Last Edit: by mensfe_admin » Logged
Pages: [1]
Jump to:  

Powered by MySQL Powered by PHP Powered by SMF 1.1.21 | SMF © 2015, Simple Machines Valid XHTML 1.0! Valid CSS!