infertility male factor best pract og 2012

7/30/2019 Infertility Male Factor Best Pract OG 2012

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Assessment of male factor

Allan A. Pacey, BSc, PhD *

Academic Unit of Reproductive and Developmental Medicine, Level 4 The Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK

Keywords:

male fertility

semen analysis

sperm DNA

sperm function

andrology

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, severalnew tests of sperm function and sperm selection have been

developed. These include the use of microfluidic chambers, elec-

trophoresis, 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.

2012 Elsevier Ltd. All rights reserved.

Background to male infertility

The incidence of infertility in men is difficult to establish reliably, but current evidence suggests that

up to 2025% of young men have poor semen quality and, in 3050% 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

* Tel.: 44 (0) 114 226 8290; Fax: 44 (0) 114 226 1074.

E-mail address: [email protected].

Contents lists available at SciVerse ScienceDirect

Best Practice & Research Clinical

Obstetrics and Gynaecologyj o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b p o b g y n

1521-6934/$ see front matter 2012 Elsevier Ltd. All rights reserved.

doi:10.1016/j.bpobgyn.2012.05.006

Best Practice & Research Clinical Obstetrics and Gynaecology 26 (2012) 739746
mailto:[email protected]:[email protected]:[email protected]://www.sciencedirect.com/science/journal/15216934http://www.elsevier.com/locate/bpobgynhttp://dx.doi.org/10.1016/j.bpobgyn.2012.05.006http://dx.doi.org/10.1016/j.bpobgyn.2012.05.006http://dx.doi.org/10.1016/j.bpobgyn.2012.05.006http://dx.doi.org/10.1016/j.bpobgyn.2012.05.006http://dx.doi.org/10.1016/j.bpobgyn.2012.05.006http://dx.doi.org/10.1016/j.bpobgyn.2012.05.006http://www.elsevier.com/locate/bpobgynhttp://www.sciencedirect.com/science/journal/15216934mailto:[email protected]


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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 envi-

ronment 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 inyoung men is not declining4 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 infection Chlamydia trachomatis have lower sperm concentra-

tions.10,11 Direct 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 Leeuwenhoek12 first described human spermatozoa in 1678, although it wasnt until

the 1950s when the first clinical descriptions of the relationship between semen quality and

conception were made.1315 In 1980, the World Health Organization (WHO) then published an inter-

nationally agreed reference range designed to help clinicians make decisions using data on semen

quality16; over the next 30 years, four further updates1720 were produced as shown in Table 1.

As these ranges became widely used in clinical practice, two issues became apparent. Thefirst 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 UK21,22 and the USA,23leading to the establishment of training programmes24,25 and external quality-assurance pro-

grammes2628 in 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.2931 As 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 201120 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).

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 theirattention to the potential value of assessing aspects of sperm DNA either as a routine part of semen

analysis or as a replacement to it.3335 This concept is underpinned by the logic that, although sperm

Table 1

World Health Organization reference ranges from 1980 to 2010 for the minimum semen quality thought to be compatible with

unassisted conception.1620

1980 1987 1992 1999 2010

Semen volume (ml) !2.0 !2.0 !2.0 !1.5

pH 7.27.8 7.27.8 7.2

Sperm concentration (106/ml) !20 !20 !20 !20 !15

Total sperm number (106) !40 !40 !40 !39

Progressive Motility (%) !60 !50 !50 !50 !32Normal 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

A.A. Pacey / Best Practice & Research Clinical Obstetrics and Gynaecology 26 (2012) 739746740


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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 rate38 and pregnancy loss in IVF39 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 tract43 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.

Microfluidic chambers

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,

Table 2

The current World Health Organization reference ranges for minimum semen quality (fifth centile of

a fertile population) showing the associated 95% confidence interval for each variable.20

Variable Fifth centile (95% confidence interval)

Semen volume (ml) 1.5 (1.41.7)

Sperm concentration (106/ml) 15 (1216)Total sperm number (106) 39 (3346)

Progressive motility (%) 32 (3134)

Total motility (%) 40 (3842)

Vitality (% alive) 58 (5563)

Normal morphology (%) 4 (34)

Table 3

Sperm function related to the mode of conception.43,45

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

Capacitation Y Y Y N

Responding to thermo and chemotactic cues Y Y Y NHyaluronic 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.

A.A. Pacey / Best Practice & Research Clinical Obstetrics and Gynaecology 26 (2012) 739746 741


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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 10 106 sperm 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 sperm19 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 ona chip48 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 byfish-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.

Electrophoresis

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 ml chambers separated by

a polycarbonate filter with a pore size chosen to allow the passage of sperm (about 5 mm) 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 1821 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, yetcould be achieved in a fraction of the time (5 mins v 20 mins) and provide comparable levels of fer-

tilisation (62.4% v 63.6%) and development of high-quality embryos (27.4% v 26.1%). Although indi-

vidual pregnancies from sperm isolated for ICSI using this technique have been reported,52

randomised-control data of live birth data are not yet available.

Table 4

Early sperm function tests with advantages and disadvantages of each. 46

Test Advantage Disadvantage

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.



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Hyaluronan binding

Hyaluronic acid or hyaluronan is the major glycosaminoglycan secretion of the cervix and the

cumulus-oopherus complex.53 During sperm transport in vivo, it is thought that sperm reaching the

egg bind to hyaluronic acid and, following their hyperactivation, this anchor facilitates their pene-

tration through the zona pellucida of the egg. In recent years, several investigators have shown thatimmature sperm with excessive cytoplasm and higher rates of aneuploidy have a dysfunctional ability

to bind to hyaluronic acid.54,55 Conversely, sperm bound to hyaluronic acid have been shown to more

likely have normal sperm morphology56 and also have more compacted chromatin and less residual

cytoplasm57 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% v 24%) and an improved live birth rate (23% v 18%) 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 corre-sponding drop in miscarriage rate (3.8% v 14.1%; 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 200400 magnification

normally used) might have significant benefits in selecting good from bad sperm. This was based on the

observationthat, at high magnification, thenucleus of some spermatozoa seemedto contain vacuoles that

could not be observed at lower magnification. So significant was theirfinding, that the paper was labelledby 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% v 26%) and the implantation rate (17.3% v 11.3%) for those couples whose

spermwere 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 analysis62 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 frag-

mentation 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% v 15.0%) and an

increase in the incidence of chaotic embryos (27.5% v 18.8%) in ICSI embryos compared with IMSI.65

This suggests indirectly that sperm with vacuoles may more likely be anupliod themselves or havedefects 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



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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 globo-

zoospermia, 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 defect69; however, in this instance, it

allowed fertilisation to take place without the need for any kind of artificial oocyte activation (byionophore), 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.

Conclusion

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 pregnancyloss, two professional organisations (ASRM and ESHRE) have not supported the routine measurement

of sperm DNA integrity as part of the infertility work-up.

Conflict of interest

None declared.

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Practice points

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 fromESHRE 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.

Research agenda

The relationship between sperm quality and spontaneous conception and intrauterineinsemination 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.



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