Practical Guide to Sperm Analysis

Basic Andrology in Reproductive Medicine

Nicolás Garrido, Rocío Rivera, Nicolás Garrido, Rocío Rivera

• Publisher: CRC Press
• Year: 2017
• ISBN:
  9781351647670
• Edition: 1
• Topic: Medicine
• Subtopic: Gynecology, Obstetrics & Midwifery

This is a reference manual for daily use in the Reproductive Medicine or Andrology laboratory, which goes beyond the literature available in the scientific journals by compiling insights into a detailed and applied clinical approach. All established practitioners in Reproductive Medicine will find much of practical relevance about the latest insights into sperm selection and analysis.

1

The Usefulness of Sperm Viability Testing in Reproductive Technology: The Hypo-Osmotic Swelling Test, Laser and Motility Stimulants

Denny Sakkas

The Scientific and Biological Bases of Testing Sperm Viability

It is commonly accepted that the standard World Health Organization (WHO) criteria for sperm number, motility, and morphology are a good, although not always perfect, indication of a male’s fertility status. The current criteria¹ have recently been changed from the 1999 to 2010 WHO recommendations, whereby semen volume (2–1.5 mL), sperm concentration (20–15 million per mL), progressive motility (505%–32%), and normal forms (14%–4%) have all been decreased, respectively. Although the current criteria suggest a certain volume, number, motility, and morphology, it must be emphasized that this is an indication that the male may have difficulties to father a child rather than predicting fertility.

Fortunately, the majority of these males that fall below these semen parameters will conceive even though it may take longer or they will need assistance through one of the assisted reproductive technology methods. Some, however, will be diagnosed with extremely low or absolute asthenozoospermia, and even with the assistance of intracytoplasmic sperm injection (ICSI) it becomes difficult to treat this condition.

These extreme cases of ICSI where there are no motile spermatozoa, extremely few motile spermatozoa, only twitching spermatozoa, or when sperm numbers are extremely low now represent the most challenging treatment situations. The advent of no motile spermatozoa may arise due to a number of scenarios not excluding immotile cilia syndrome, which is thought to be present in approximately 1 in 5000–6000 men.^2,3 More frequently, the presence of no motile spermatozoa will arise after sperm are extracted from the testes or epididymis or after a low-quality sample is frozen and thawed.

Along with the presence of no motile spermatozoa is also the inability to distinguish if those spermatozoa are in fact viable or dead. It is well accepted that the arbitrary selection of an immotile spermatozoon and attempt at fertilization by ICSI will provide significantly lower chances of success.^4–8 The challenge, however, is how to treat these men, indeed the question is, “When an ICSI technician is deprived of the chance to choose a motile spermatozoa how does he/she find a live sperm?” This chapter discusses the various options that allow in vitro fertilization (IVF) clinics to improve the chances of success for couples in which the male has extremely poor sperm parameters that limit the chance of selection of a viable sperm.

Analytical Techniques to Test Sperm Viability

The standard and recommended viability test for sperm is that recommended by the WHO manual. The test in effect identifies which sperm have an intact membrane by either excluding a particular dye or hypotonic swelling. Briefly, dye exclusion means that any damaged plasma membrane will allow entry of membrane impermeant stains; it entails a vitality test using eosin–nigrosin where live spermatozoa will have white heads and dead spermatozoa will be red. Eosin alone¹ is also an option for testing viability. Some commercially available options are provided, for example, Sperm VitalStain from Nidacon, Sweden.

Unfortunately, the use of any stain precludes an individual sperm from being used clinically. Hence, the act of staining, although providing information about viability, will not be useful. Another test that is commonly used to assess viability is the hypo-osmotic swelling test (HOST), which acts because sperm with intact membranes are not leaky and will swell as they are able to retain fluid leading to coiling of the tail. The test was first described by Jeyendran et al.⁹ in 1984 and is a good indicator of the functional integrity of the sperm membrane.⁶ Its use in being able to select viable nonmotile sperm is popular because of the simplicity of the test. One problem with the test is that it may be less accurate when frozen–thawed spermatozoa are assessed as they experience a higher rate of spontaneously developed tail swellings and that this can exaggerate the HOST score.¹⁰

Clinical Options with Nonmotile Spermatozoa

As seen in the earlier section, nonmotile spermatozoa do not necessarily mean dead spermatozoa, hence when applying ICSI to these cases it is virtually imperative that this distinction be made. The term extreme ICSI was coined recently in an article by Palermo et al.¹¹ The extreme in this article refers more to treatment of men with severely compromised spermatogenesis, including those with virtual azoospermia or nonobstructive azoospermia requiring an extreme search for spermatozoa. In their study they came to the conclusion that in testicular sperm extraction (TESE) patients there was a decrease in pregnancy rate (44%–23%) with increasing time of search for sperm prior to ICSI. More importantly they concluded that, even with extreme ICSI, there is a preference to use spermatozoa that display motility characteristics as even if motility is poor—or there is twitching—it still displays proof of cell viability. Hence, there is a preference in the clinical setting to always use motile sperm, regardless of the quality of motility.

There are, therefore, a number of choices when faced with the prospect of no motile sperm in a sample to be used for ICSI.

1. Select sperm randomly from the nonmotile population.
2. Select sperm using a test that can indicate viability without damaging the sperm, for example, HOST or laser-assisted selection.
3. Stimulate the nonmotile sperm so that it achieves some motility.

Clinical Implications of Using Immotile Sperm for ICSI

No large specific data sets exist on the use of totally immotile spermatozoa for ICSI as in many cases an attempt is always made to select some motile spermatozoa; some cases proceed with a mix of embryos fertilized by both motile (twitching) and immotile spermatozoa. The data, however, are conclusive that the inability to identify motility in a sperm prior to ICSI is detrimental. In an initial study by Nijs et al.,¹² they found that both initially immotile and totally immotile spermatozoa had the capacity to fertilize an oocyte after ICSI, whatever their origin, testicular or epididymal. Totally immotile ejaculated spermatozoa fertilized significantly fewer oocytes after ICSI when compared with initially immotile ejaculated spermatozoa. Embryos of lower quality tended to be produced when totally immotile spermatozoa of any origin were used compared with embryos resulting from initially immotile spermatozoa. Pregnancy rates were also severely reduced when totally immotile sperm were used from the epididymis and ejaculate. Another early study also showed similar tendencies. The microinjection of completely immotile spermatozoa in 11 couples who underwent an initial ICSI cycle with 100% immotile freshly ejaculated spermatozoa resulted in pronuclear fertilization in only a total of 18/145 (12.4%) injected oocytes.¹³ None of these cycles resulted in a pregnancy. Although the earlier studies showed that fertilization was not an ultimate impediment when using immotile spermatozoa, a study by Liu et al.⁵ has shown that one of the major factors influencing fertilization failure after ICSI was the presence of only immotile sperm.

Clinical Implications of Selecting Viable Immotile Sperm for Intracytoplasmic Sperm Injection

A number of methods have been adopted to select immotile sperm using a test that can indicate viability without damaging the sperm. The most widely and traditionally used test is HOST, which acts as a surrogate measure of sperm membrane integrity or viability. The HOST has been used historically in cases of sperm samples with 100% immotile cells, including those from patients with Kartagener’s syndrome.^9,14,15

Recently, it was also reported that the HOST can identify individual spermatozoa with minimal DNA fragmentation,¹⁶ and with traits of apoptosis, abnormal head morphology, nuclear immaturity, or membrane damage.^17,18 When used for immotile sperm the results indicate that HOST is beneficial for testicular sperm; however, when applied to ejaculated sperm the results are less convincing. Sallam et al.¹⁹ performed a randomized controlled trial in a total of 79 couples with immotile testicular spermatozoa treated with ICSI and examined HOS. In the first group, spermatozoa used for injection were selected using the modified HOST, whereas in the second group spermatozoa were selected based on their morphology. The fertilization rate was significantly higher in the HOST group (43.6%) compared with the no-HOST group (28.2%), wherea...