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Assessment of male factor
Best Practice & Research Clinical Obstetrics & Gynaecology, 6, 26, pages 739 - 746
The assessment of male infertility is largely based around the examination of a freshly produced ejaculate by a trained technician according to laboratory methods agreed by the World Health Organization. Although many suggestions have been made to improve this approach, the basic techniques of semen analysis established in the 1950s are still being used. Although several putative tests of sperm function have been developed (e.g. the measurement of sperm hyperactivation, sperm acrosomal status, or sperm penetration through mucus or binding to zona pellucida), none have made it into routine clinical practice. Recently, several ‘new’ tests of sperm function and sperm selection have been developed. These include the use of microfluidic chambers, electrophoresis, the binding of sperm to hyaluronic acid, and high magnification sperm selection. Randomised-controlled trials are needed to evaluate these as a replacement or addition to routine semen analysis or current sperm preparation methods.
Keywords: male fertility, semen analysis, sperm DNA, sperm function, andrology.
Background to male infertility
The incidence of infertility in men is difficult to establish reliably, but current evidence suggests that up to 20–25% of young men have poor semen quality and, in 30–50% of couples undergoing in-vitro fertilisation (IVF), a male factor contributes to infertility. 1 Unlike the situation in some cases of female infertility (e.g. amenorrhea), possible male infertility is not outwardly obvious because, macroscopically, the ejaculates of fertile and infertile men appear the same. It is only when couples fail to achieve conception, that male infertility may be suspected and laboratory tests (e.g. semen analysis) are clearly required to establish this reliably. 2
In recent years, whether or not the incidence of male infertility has increased has been furiously debated. 1 This has largely been based on the hypothesis that, as yet, unknown factors in the environment are affecting the testicular development of an increasing number of male babies before birth. 3 It is proposed that after puberty such affected individuals are more likely to produce ejaculates with reduced sperm count and consequently are less fertile. Direct evidence to support this hypothesis, however, is lacking; moreover, more recent prospective data have suggested that semen quality in young men is not declining 4 in the way that was originally proposed from an analysis of retrospective studies. 5 Although increasing rates of urogenital defects and incidence of testicular cancer have been demonstrated, lending some support to the original theory, the cause of such urogenital conditions is clearly more complex than was first proposed. 6
As well as pre-natal exposures, the spermatogenesis of post-pubertal males can also be influenced by a number of medical and lifestyle factors. 7 For example, the ejaculates of men who have been treated with chemotherapy or radiotherapy typically have lower sperm concentrations than men not treated with these agents. 8 Similarly, men exposed to glycol ether in the workplace, 9 or men who have been infected with the sexually transmitted infectionChlamydia trachomatishave lower sperm concentrations.10 and 11Direct and independent effects on sperm motility, sperm morphology, or both, are less well described but equally important in their effect on male fertility.
Background to semen analysis
Antonie van Leeuwenhoek 12 first described human spermatozoa in 1678, although it wasn't until the 1950s when the first clinical descriptions of the relationship between semen quality and conception were made.13, 14, and 15In 1980, the World Health Organization (WHO) then published an internationally agreed ‘reference range’ designed to help clinicians make decisions using data on semen quality 16 ; over the next 30 years, four further updates17, 18, 19, and ∗20were produced as shown in Table 1 .
|Semen volume (ml)||–||≥2.0||≥2.0||≥2.0||≥1.5|
|Sperm concentration (×106/ml)||≥20||≥20||≥20||≥20||≥15|
|Total sperm number (×106)||–||≥40||≥40||≥40||≥39|
|Progressive Motility (%)||≥60||≥50||≥50||≥50||≥32|
|Normal morphology (%)||≥80||≥50||≥30||–||≥4|
|Vitality (% alive)||–||≥50||≥75||≥75||≥58|
|White blood cells||≤5.0||≤1.0||≤1.0||≤1.0||≤1.0|
|Antibody coated sperm (%)||–||≤10||≤20||≤50||≤50|
As these ‘ranges’ became widely used in clinical practice, two issues became apparent. The first was how the variation in the technique used in different laboratories could affect the semen analysis results being reported significantly. This was illustrated in a series of studies in the UK21 and 22and the USA, 23 leading to the establishment of training programmes24 and 25and external quality-assurance programmes26, 27, and ∗28in andrology. The second was that, even when semen analysis was carried out robustly, and with appropriate quality-control measures in place, a significant uncertainty could remain about the relationship between semen profile and the probability of conception.29, 30, and 31As a consequence, commentators argued the need to further revise the WHO ‘reference ranges’. To some extent, the publication of the 5th edition of the WHO manual in 2011 20 has addressed this problem in basing the reference ranges for the first time on ‘real world’ data. 32 This also introduced the importance of ‘confidence intervals’, allowing the user to understand the individual semen analysis values obtained in the context of measurement error ( Table 2 ).
|Variable||Fifth centile (95% confidence interval)|
|Semen volume (ml)||1.5 (1.4–1.7)|
|Sperm concentration (×106/ml)||15 (12–16)|
|Total sperm number (×106)||39 (33–46)|
|Progressive motility (%)||32 (31–34)|
|Total motility (%)||40 (38–42)|
|Vitality (% alive)||58 (55–63)|
|Normal morphology (%)||4 (3–4)|
It has long been recognised that semen analysis only goes so far in providing a physical description of the ejaculate. Therefore, in an attempt to improve on this, many investigators have now turned their attention to the potential value of assessing aspects of sperm DNA either as a routine part of semen analysis or as a replacement to it.33, 34, and ∗35This concept is underpinned by the logic that, although sperm may appear motile and have normal morphology, if their DNA is damaged or inappropriately packaged, fertilisation, embryo development, or both, may fail because the oocyte only has a limited capacity to repair the damage. 36 Sperm DNA may be damaged by a variety of environmental and lifestyle factors, 37 and substantial data have reported the relationship between sperm DNA packaging and fragmentation and live birth rate 38 and pregnancy loss in IVF 39 and intracytoplasmic sperm injection (ICSI). 40 The problem is that significant debate about which tests may be clinically useful remains. Interestingly, both the American Society for Reproductive Medicine (ASRM) 41 and the European Society for Human Reproduction and Embryology (ESHRE) 42 have both published recent position statements indicating that they do not yet support the use of sperm DNA testing on a routine basis.
Background to sperm function tests
Sperm function tests differ from the measurements undertaken at semen analysis, as they set out to try and examine aspects of sperm biology that have physiological relevance as sperm ascend through the female reproductive tract 43 or are used in the various techniques of assisted conception. 44 Clearly, during natural (unassisted) conception, sperm need to undertake a greater range of functional steps (e.g. passage through cervical mucus or the ability to migrate through the utero-tubal junction) than sperm that are used in assisted conception (e.g. ICSI) 45 as shown in Table 3 . Some of the main sperm function tests developed over the past few years are presented in Table 4 , 46 and the advantages and disadvantages of each are highlighted. Although it can be seen that many putative tests of sperm function have been developed, however, none have yet been universally accepted and incorporated into routine clinical practice. Therefore, it is clear that a need remains for new sperm function tests to be developed. The following sections outline some of the promising avenues.
|Coitus||Intrauterine insemination||In-vitro fertilisation||Intracytoplasmic sperm injection|
|Initiation of sperm motility at ejaculation||Y||Y||Y||N|
|Passage through cervical mucus||Y||N||N||N|
|Transport through the uterus||Y||N||N||N|
|Passage through the utero-tubal junction||Y||Y||N||N|
|Sperm epithelial contact in the fallopian tube||Y||Y||N||N|
|Responding to thermo and chemotactic cues||Y||Y||Y||N|
|Hyaluronic acid binding in the cumulus-oopherus complex||Y||Y||Y||N|
|Hyperactivation and penetration of the zona pellucid||Y||Y||Y||N|
|Decondensation of sperm DNA||Y||Y||Y||Y|
‘Y’, indicates that a particular sperm function is needed for the given mode of conception; ‘N’ indicates that it is not.
|Cervical mucus penetration and motility||Penetration of mucus by sperm correlates well with IVF outcome.||Supply of suitable mucus difficult to co-ordinate. Although other substitutes exist, they have not been adequately trialled.|
|Hyperactivation||Ability of sperm to hyperactivate correlates with IVF outcome.||Requires sophisticated and expensive computer-assisted sperm analysis machines.|
|Sperm zona interaction||Defective zona binding can predict fertilisation failure in IVF.||Obtaining a reliable supply of zona for use is logistically and ethically challenging.|
|The acrosome reaction||Shows some prediction with IVF outcome.||Time consuming and a range of putative agonists exist. Lacks dynamic range.|
|Zona free hamster penetration assay||Correlates well with outcome of spontaneous pregnancy and IVF.||Requires use of laboratory animals and in some countries (e.g. UK) requires specific licencing and monitoring by regulatory authorities.|
|Reactive Oxygen Species||Can provide accurate data on oxidative stress and correlates with DNA damage.||Requires specific laboratory equipment (e.g. luminometer) and is time consuming.|
IVF, in-vitro fertilisation.
Several research groups have attempted to develop chambers through which sperm might be allowed to swim as a way mimicking their passage through the female reproductive tract. For example, a novel device designed as a ‘home test kit’ was developed in 2006, 47 in which ejaculated sperm were exposed to hyaluronic acid inside a capillary channel at the end of which any motile sperm were allowed to react with a monoclonal antibody to CD59 tagged with a colloidal gold label. If sufficient numbers of antibody coated sperm (calibrated at 10 × 106sperm per ml or greater) became trapped in a nitrocellulose lateral flow strip at the end of the capillary, then a red line appeared in a window alerting the user that at least the then WHO minimum number of motile sperm 19 were present. Although impressive, devices such as this were semi-quantitative, and it was only a matter of time before developments of electronic technology allowed for more quantitative solutions to be proposed.
One example in its early stages is the development of a semen analysis ‘lab on a chip’ 48 where silicon and glass chips containing micro-channels of various complexities (straight or branched) were made, and which the sperm could be allowed to swim down. When combined with small electrodes alongside the channels, the change in impedance observed as individual sperm passes can give information about cell size, membrane and cytoplasm. Furthermore, it has been shown that this technique can be successfully used to discriminate between sperm, leucocytes and polystyrene beads. This is similar in approach to the methods used by fish-counting machines in rivers, 49 although clearly working at the microscopic scale. Although much development work remains to be carried out, early results are impressive, showing good correlation between traditional measures obtained at semen analysis and those recorded on the new chip. If perfected, such technology may radically alter the methods of semen analysis currently being used today.
In a development related to the use of microfluidic devices described above, a novel system to separate functional sperm on the basis of their size and electronegative charge using electrophoresis was proposed in 2005. 50 A device was developed comprising two 400 μl chambers separated by a polycarbonate filter with a pore size chosen to allow the passage of sperm (about 5 μm) but exclude leukocytes, precursor germ cells (e.g. spermatogonia), or both. When a sample was introduced into the ‘inoculation chamber’ and an electric field applied (75 mA at a variable voltage from 18–21 volts for up to 900 s at 23 °C), it was shown that a purified suspension of sperm could be obtained in the collection chamber. Moreover, such sperm had lower levels of DNA damage. When compared with traditional techniques such as density gradient centrifugation, 51 the results obtained were highly comparable, yet could be achieved in a fraction of the time (5 minsv20 mins) and provide comparable levels of fertilisation (62.4%v63.6%) and development of high-quality embryos (27.4%v26.1%). Although individual pregnancies from sperm isolated for ICSI using this technique have been reported, 52 randomised-control data of live birth data are not yet available.
Hyaluronic acid or hyaluronan is the major glycosaminoglycan secretion of the cervix and the cumulus-oopherus complex. 53 During sperm transportin vivo, it is thought that sperm reaching the egg bind to hyaluronic acid and, following their hyperactivation, this anchor facilitates their penetration through the zona pellucida of the egg. In recent years, several investigators have shown that immature sperm with excessive cytoplasm and higher rates of aneuploidy have a dysfunctional ability to bind to hyaluronic acid.54 and 55Conversely, sperm bound to hyaluronic acid have been shown to more likely have ‘normal’ sperm morphology 56 and also have more compacted chromatin and less residual cytoplasm 57 than unbound sperm.
On the basis of these observations, the binding of sperm to hyaluronic acid has been proposed as a new sperm function and selection test for use in ICSI, and dishes in which micro-drops of hyaluronic acid have been placed are now commercially available and being used. Early studies to examine the efficacy of this approach have shown that selecting sperm with this method results in a higher number of grade 1 embryos for transfer (36%v24%) and an improved live birth rate (23%v18%) compared with selecting sperm using traditional methods. 58 In a larger randomised-controlled trial currently under way, early data from 802 ICSI cycles suggest a 13% increase in clinical pregnancy rate and a corresponding drop in miscarriage rate (3.8%v14.1%;n = 168) when sperm for injection were selected on the basis of their hyaluronic-acid-binding characteristics. 59 Further trials are currently ongoing to evaluate this approach further.
Intracytoplasmic morphologically selected sperm injection
In 2002, Bartoov et al. 60 suggested that the selection of sperm for injection into oocytes during ICSI by observing them at a magnification of over 6000 times (compared with the ×200–400 magnification normally used) might have significant benefits in selecting good from bad sperm. This was based on the observation that, at high magnification, the nucleus of some spermatozoa seemed to contain ‘vacuoles’ that could not be observed at lower magnification. So significant was their finding, that the paper was labelled by the journal in the section ‘Breakthroughs in andrology’, and the publication of the first randomised-controlled trial in 2008 seemed to support that claim, showing a statistically significant improvement in the clinical pregnancy rate (39.2%v26%) and the implantation rate (17.3%v11.3%) for those couples whose sperm were selected by intracytoplasmic morphologically-selected sperm injection (IMSI) compared with traditional ICSI. 61 Subsequently, these observations have been supported by a number of studies, such that a recent meta analysis 62 has concluded that significant improvement has been achieved in implantation (OR 2.72; 95% CI 1.50 to 4.95) and pregnancy rates (OR 3.12; 95% CI 1.55 to 6.26) when carrying out IMSI. In addition, the miscarriage rate seems significantly decreased (OR 0.42; 95% CI 0.23 to 0.78) in IMSI cycles, although the investigators concluded that further randomised-controlled trials were needed.
Given these promising results, it is interesting to speculate how the observed ‘vacuoles’ relate to sperm function. It has been proposed that they might be visible markers of either DNA fragmentation, aneuploidy, or errors of chromatin packaging.
To test the first of these suggestions, studies have correlated the results of DNA fragmentation tests, such as the terminal transferase dUTP nick end labelling (TUNEL) assay, and found that only the sperm containing the largest vacuoles (> 50% of the nuclear volume) correlate with a high rate of DNA fragmentation. 63 This is supported by a study in which individual sperm were stained for DNA fragmentation and then examined for vacuoles. Of 227 sperm showing large vacuoles, only seven (3.1%) were TUNEL-positive, suggesting that DNA fragmentation is not linked to the presence of vacuoles. 64
The relationship between the ploidy of embryos derived from IMSI and conventional ICSI has been examined, and shows a significant increase in sex chromosome aneuploidy (23.5%v15.0%) and an increase in the incidence of ‘chaotic embryos’ (27.5%v18.8%) in ICSI embryos compared with IMSI. 65 This suggests indirectly that sperm with vacuoles may more likely be anupliod themselves or have defects of the sperm centriole that affect the first mitotic division of the embryo.
Finally, to examine the relationship between vacuoles and chromatin packaging, the pattern of chromomycin A3 in relation to the presence or absence of vacuoles was examined, 66 and a statistical correlation was found between the two, supporting the hypothesis that vacuoles may represent abnormal chromatin packaging. This suggests that vacuoles may be a nuclear thumbprint linked to the failure of chromatin condensation. 67
In a final variation on the IMSI technique, a recent case report showed how, in a case of globozoospermia, IMSI was used to select sperm with a small bud of acrosome that was present in about 1% of ejaculated sperm. 68 Normally, globozoospermia is a sterilising defect 69 ; however, in this instance, it allowed fertilisation to take place without the need for any kind of artificial oocyte activation (by ionophore), and a successful pregnancy and birth followed. Clearly, in this instance, the use of IMSI was not to detect nuclear defects, but observe the fine structure of sperm organelles.
Semen analysis remains the main technique of assessing male fertility, and revisions to the WHO manual and the publication of revised reference ranges has been a useful step forward and are, for the first time, evidence-based. Although many putative tests of sperm function and selection have been proposed, relatively few have made it into routine clinical practice; randomised-controlled trials of IMSI, however, and the binding of sperm to hyaluronan, are ongoing. Perhaps, surprisingly, although many studies that damage to sperm DNA correlates well with clinical pregnancies and early pregnancy loss, two professional organisations (ASRM and ESHRE) have not supported the routine measurement of sperm DNA integrity as part of the infertility work-up.
- Semen analysis remains the main diagnostic test for establishing male fertility and infertility.
- Semen analysis should be carried out according to WHO (2010) methods, in a laboratory with suitably skilled staff that takes part in external quality assurance programmes for semen analysis.
- Although many tests of sperm DNA fragmentation are available, best-practice guidelines from ESHRE and the ARSM do not support their routine use.
- Evidence from meta-analysis suggests that IMSI may increase pregnancy rates and decrease miscarriage rates.
- The clinical value of other putative tests of sperm function remains to be established through appropriate randomised-controlled trials.
- The relationship between sperm quality and spontaneous conception and intrauterine insemination and IVF.
- Randomised-controlled trials of sperm DNA integrity and assisted conception.
- Effectiveness of IMSI and binding on ICSI outcomes.
- Basic biology of sperm interaction with the epithelium of the female reproductive tract.
Conflict of interest
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