Chapter

Semen analysis and preparation

24 Aysha Itani

for example, illness with fever, medication, past med-

Introduction ical and surgical history and occupational and leisure
This chapter describes general semen analysis in detail activities. There are many everyday events that can
and discusses the various methods of semen prepara- affect the quality of sperm undergoing analysis, and
tion techniques that are currently being used in assis- some of these are discussed in Table 24.1.
ted reproductive laboratories (ART) today.
Male factor infertility contributes to approximately Sample production
one-third of all assisted reproduction referrals [1]. It is
Ideally, sample production rooms should be located as
therefore essential when investigating couples present-
close to the laboratory as possible so that samples can
ing for fertility treatment that an accurate analysis of
be analyzed as soon after liquefaction as possible. At
their semen is undertaken.
the Oxford Fertility Unit, hatches adjoin the labora-
The new edition of the WHO laboratory manual
tory so the sample can be handed directly to the labo-
for examination and processing human semen (WHO
ratory after collection. The semen sample should be
Manual) was released in 2010. This should be consid-
procured into a sterile single use container that has
ered the gold standard for all semen analysis and
been clearly labelled with the patient’s full name and
preparation methods and describes them all in detail.
original identifier. Washed containers should never be
Many ART laboratories still use the 1999 reference
used as they may contain soap or residue from its
manual, although changes arising from the 2010
previous contents. The sample should be produced
update are now beginning to phase in. Both sets of
by masturbation and the time taken noted. Whether
reference standards will be referred to in this chapter,
the whole sample was collected or not should also be
with the newer values clearly indicated in italics [2, 3].
noted on an accompanying slip. If the first part of the
Recent modifications to the WHO manual were essen-
ejaculate is missed it may influence analysis as the
tial, as earlier versions did not take into account the
latter part is less spermatozoa rich [3]. Some men
time to become pregnant (fecundity) when defining
often find it difficult to produce on their own, and on
the normal semen characteristics of a fertile man [4].
demand, and may need their partner to help them. If
Semen analysis condoms are to be used then silastic condoms without
spermicide must be utilized in order to avoid the toxic
There are four main stages of semen analysis:
effect of latex and spermicide which would obviously
1. Patient history be harmful to any sperm found within.
2. Sample production The average time for production is 10–15 minutes
3. Semen analysis [13]. If the patient takes significantly longer than this,
4. Interpretation of results then clinical decisions regarding the cryopreservation
of samples for use on the day of treatment should be
considered. This will both alleviate the stress upon the
Patient history patient of producing a sample on the day, and ensure
It is important to determine relevant patient history that a useable sample is available when required.
when analyzing semen, as sometimes very obvious In order to accurately assess and reproduce semen
factors can have significant implications on results, analysis results, samples should be produced only after

Textbook of Clinical Embryology, ed. Kevin Coward and Dagan Wells. Published by Cambridge University Press. 239
© Cambridge University Press 2013.
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 Section 4: ART: skills, techniques and present status

Table 24.1 Everyday events and their fertility implications

Event Fertility implication

Reproductive Previous pregnancies Male has been fertile in the past
history
Abstinence 2–7 days Longer periods of abstinence may reduce sperm motility, shorter peri-
ods may reduce total motile concentration and volume [5]
Diet Poor diet vs. well-balanced diet Good diet enhances sperm quality [6]
Caffeine Consuming increased amounts Increased levels of caffeine can negatively affect semen quality [7]
Alcohol Excessive alcohol consumption Decreases sperm motility [8]
Smoking Smoking cigarettes Decreases semen quality [8, 9]
Radiation/ Prior to chemotherapy or occupation Deleterious affects on semen quality [10]
chemical related
exposure
Driving Prolonged sitting Warms testicles and consequently decreases optimal sperm production
[11]
Medicine/past Vasectomy, congenital bilateral No sperm should be present in vasectomy and antisperm antibodies
surgery absence of vas deferens (CBAVD) may be present if reversed. CBAVD should result in azoospermia.
Steroids Use of steroids can show marked decrease in spermatogenesis [12]

2–7 days of abstinence. Shorter periods of abstinence 1 hour after ejaculation, may indicate disorders of
may result in a reduction in both semen volume and accessory gland function, or infection.
sperm concentration. Conversely, increased periods of Semen analysis should take place as soon as lique-
abstinence may result in a higher percentage of non- faction has occurred, ideally 30 minutes after produc-
moving sperm. If additional samples are required, tion and certainly before 60 minutes have elapsed, in
then periods of abstinence should be consistent, in order to prevent the sample from evaporating and
order to permit comparable results [5]. deteriorating within the seminal plasma. The sample
should be removed from the incubator and inspected by
looking at the sample through the pot. This can estab-
Semen analysis lish sample presence and permit assessment of general
It is essential that at least two samples are analyzed in consistency. Due to the heterogeneous nature of semen,
order to gain a suitable assessment of the patient’s the sample should be mixed well by hand rotation prior
specific semen characteristics. Individual parameters to analysis. Samples should not be vortex-mixed as this
tend to be variable and in order to ascertain the will be detrimental to the sperm within.
characteristics of an average sample, it is best to collate Normal semen samples may contain gelatinous
the results of more than one sample. The normal bodies. While these can affect the production of wet
standards as defined in the WHO manual are listed preparation slides, they do not appear to have any
in Table 24.2. clinical significance. The presence of mucus streaks,
Basic semen analysis is made up of four compo- which may also be seen during initial analysis, can
nents: volume, sperm motility, count and morphol- interfere with motility parameters [3]. Normal semen
ogy. As soon as the sample is produced it should be is a homogenous grey-opalescent colour and varia-
placed in a warmed non-CO2 incubator to accelerate tions in this colour may be indicative of disease. If a
liquefaction. Liquefaction refers to the natural change sample is oligospermic then it is less opaque. A red/
in semen consistency over time from a gel-like vesic- brown colour indicates the presence of red blood cells.
ular secretion to a liquid [3, 5]. A non-liquefied sample This could be indicative of infection and should always
cannot be analyzed accurately as sperm are unable to be discussed with the patient and clinician to ascertain
move freely. Typical liquefaction time is 20 minutes that no serious conditions are evident. Semen can also
but liquefaction can occur almost immediately after have a yellow appearance due to jaundice or some
ejaculation. Delayed liquefaction of semen, more than vitamin supplements [3].
240

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 Chapter 24: Semen analysis and preparation

Table 24.2. WHO reference values

Parameter WHO 1999 [2] WHO 2010 [3]

Volume > 2ml 1.5ml
Appearance Grey and opaque Grey and opaque
pH > 7.2 > 7.2
Concentration > 20M/ml > 15M/ml
Motility > 50% progressively motile > 32% progressively motile
Morphology > 30% normal > 4% normal
Leucocytes < 1M/ml < 1M/ml

Volume and viscosity Abnormally increased viscosity is defined as when

the thread from the gravitating semen is more than
The first part of semen analysis is to determine volume
2 cm long [3]. Increased viscosity does not appear to
and viscosity. At the Oxford Fertility Unit, volume is
have any clinical significance; however, it does
calculated using a 5 ml or 10 ml graduated pipette. The
reduce initial movement parameters and conse-
normal volume for semen is between 2 and 5 mL (1.5
quently this reduces the capacity of sperm to pen-
ml). Low semen volume, together with an azoospermic
etrate the cervical mucus in vivo. After preparation,
sample, i.e. a sample containing no sperm, can be
and the removal of functional sperm from seminal
characteristic of ejaculatory duct obstruction or con-
plasma, normal movement parameters are returned
genital bilateral absence of the vas deferens. If the
[14].
volume is too high, this may indicate increased absti-
nence or inflammation of the accessory glands, espe-
cially if the pH is low [3].
The WHO manual recommends calculation of volume Sperm motility
by measuring the weight of the container containing the Two × 10 µl separate aliquots should be placed on a
sample. By using a container of known weight, the weight clean glass slide and covered with a 22 mm by 22 mm
of the sample can be extrapolated and the precise amount cover slip. This ‘wet prep’ can then be analyzed under a
of functional spermatozoa can be calculated as there will phase contrast microscope for presence of mucus
be no remnants in the pot or pipette. Volume loss can be in streaks, agglutination, presence of infection by
the order of 0.3 ml and 0.9 ml [3]. increased numbers of round cells and most impor-
While measuring volume, it is customary to meas- tantly the presence of spermatozoa. The required mag-
ure the pH of the sample using pH paper with a range nification is dependent on what is being observed, but
6.0–10.0. pH should also be analyzed within 1 hour of to detect the presence of bacteria, a minimum of 400×
production as pH is affected by the loss of CO2 that is recommended [3]. Sperm motility can be calculated
occurs following ejaculation and will therefore using this slide at a power of 200× magnification or
increase with time. 400× depending on concentration. Using a higher
Viscosity is then graded by allowing the sample to power will facilitate the analysis of concentrated
gravitate into its container and analyzing the stream. samples.
At the Oxford Fertility Unit viscosity is graded into The calculation of motility of ejaculated sperm is
four categories: an extremely important characteristic that has a high
1. Reduced, sample appears watery association with fertility [15]. It can be calculated
2. Normal, leaves the pipette in small, discrete drops using computer-aided sperm analysis (CASA), but in
3. Slightly increased – can be loaded into a pipette tip the clinical setting it is considered far more accurate to
easily determine motility manually using a cell counter and a
4. Greatly increased – does not load into a pipette tip phase contrast microscope.
easily (in this case cutting the end of the tip will In order to calculate motility, sperm are separated
into four categories: 241
facilitate the sample)
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 Section 4: ART: skills, techniques and present status

(a) Progressive motility > 20µm/s at 20°C The WHO 2010 has simplified the calculation of
(b) Slow or sluggish motility by grading sperm into three categories. (a)
(c) Non-progressive motility < 5µm/s Progressively motile, these sperm are actively moving
(d) Immotile and going either in a straight line or in a circle at any
speed, i.e. previous a and b. (b) Non-progressive motility,
To perform motility analysis, an area must be defined this includes all other types of motile sperm, i.e. shaking,
within the field of view (see Fig. 24.1). This area is twitching or moving in very small circles, and (c)
systematically scanned and all sperm of category ‘a’ immotile sperm, the lack of any movement at all.
and ‘b’ are scored and then the field is returned to It should be noted that the velocity of sperm
score and calculate the categories ‘c’ and ‘d’. To achieve is temperature dependent and can double when
an acceptably low counting error, at least 200 sperm assessed at body temperature as opposed to room
should be counted with a laboratory counter [3]. The temperature [16].
sperm counted should be spread over at least five fields
of view. These values are then expressed as a percentage.
This should be repeated on a second aliquot and the
Concentration
results compared. If there is less than 10% difference The determination of an accurate concentration is
between the highest categories on the two aliquots then important due to its association with fertilization
this is deemed acceptable; if not, then a third slide rates and time to conception [5]. A count is considered
should be prepared and analyzed and the results aver- normal if the concentration of sperm is greater than
aged. If it is not possible to count 200 sperm, this should 20M/ml (15M/ml). The concentration of sperm can be
be noted and the numbers not expressed as percentages. calculated in a variety of manners. The most accurate
and reproducible method of counting sperm is
through dilution to immobilize the sperm and to use
a haemocytometer. Due to the viscous nature of sperm
it is important to use a positive displacement pipette so
as to minimize errors in the dilution phase. A variety
of dilutions can be used, but if a 1 in 20 dilution is used
then the number of sperm in five large boxes of a
Neubauer Haemocytometer is the equivalent of the
number of sperm per ml (see Fig. 24.2). However, it
is critical not to overcount the number of sperm;
therefore only whole sperm should be counted (those
with heads and tails). In order not to count the sperm
twice, only sperm with the majority of its head in the
square should be counted and even then, only if it is on
one of the two boundary lines that have been allocated
to count. This will prevent overestimation.
Figure 24.1 Field of View. Lightened area is systematically scanned
for progressively motile and slow or sluggish sperm and then return
There are other methods for counting sperm, and
to field to calculate non-progressive and immotile sperm. these include the Makler chamber and Horwell

Figure 24.2 Haemocytometer and

representation of grid within. Indicating 25
large squares and 16 smaller ones, the cells
should only be counted when the majority
of sperm is within the square.

242

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 Chapter 24: Semen analysis and preparation

Fertility Counting chamber, both of which have a midpiece should be 0.6 microns in width, regular and
ten by ten counting grid and use undiluted semen. its length should be in the order of approximately
The advantage of this method, i.e. the ease and speed 4 microns.
of use, is counteracted by a substantial error rate as The tail, or principal piece, should be uniform
care must be taken not to overfill these chambers. along its length and thinner than the midpiece. It
When overfilled, the chambers do not allow for a should be unbroken and have no kinks or coils and
single layer of sperm to be created and thus introduce be approximately ten times the size of the head, i.e.
a source of error. While these techniques may be quite 45 microns long. Various abnormalities of head, neck,
usual for a busy, non-specialized laboratory, they midpiece and tails exist. Full and concise descriptions
should not take the place of the method recommended are available in the WHO manual [3].
by the WHO manual for accurate analysis [17]. Analyses involve both stained and unstained meth-
ods. There are many commercially available kits avail-
able to stain sperm. It is possible to perform simple
Sperm morphology morphological assessment upon raw semen. However,
Sperm morphology is important in the evaluation of a while such practice will not be accurate for diagnostic
semen sample; however, there is no clear boundary laboratories, it may be sufficient in a clinical setting. If
between fertility and infertility with normal sperm. If there is an increase of grossly abnormal sperm, then
there are grossly abnormal morphological defects, ICSI (intracytoplasmic sperm injection) may be recom-
then the fertilizing capacity is greatly diminished. mended. This is especially true for globozoospermic
The variable morphology of human sperm makes the samples where the acrosome is missing and conven-
analysis quite difficult and there are various common tional fertilization would not normally be possible.
classification systems with the more recent editions of Commercially available kits assist with morpho-
the WHO manual quoting the Kruger method and the logical assessment by differentiating between various
more strict Tygerberg Criteria. components of the individual sperm (head, acrosome,
A normal, human sperm has a smooth, regular oval midpiece and tail) by staining, e.g. Diff-Quik,
shape, with a well-defined acrosome area that takes up TestSimplets (MidAtlantic Diagnostics, Inc, Mount
approximately 40–70% of the head area. The head Laurel, NJ) or SpermMac (Autosperm, Fertipro,
should be whole and contain no vacuoles. Normal Lotenhulle, Belgium). Slides are prepared in accord-
sperm are approximately 4.1 microns in length with a ance with the manufacturer’s instructions. These are
median width of 2.8 microns [3] (see Fig. 24.3). The then scored according to normality or not. Staining
methods are particularly useful as the sperm are
immobilized and clearly identifiable.

Other aspects of analysis

In addition to concentration, count and morphology,
there are further investigations that can be performed
in order to make a complete analysis of semen quality.

Anti-sperm Antibodies (ASABs)

Mature sperm are produced after puberty and because
of this they are sometimes recognized as foreign pro-
teins by the immune system. While the sperm are
within the testicle they are protected by tight junctions
of the Sertoli cells. When there is a breach of this
‘blood-testis’ barrier, an immune response may
occur. The most common reasons for this are vasec-
tomy, testicular biopsy and testicular injury, e.g. tor-
sion and infection [18]. Antibodies are then produced 243
Figure 24.3 Normal sperm and median lengths and secreted into the prostatic and seminal fluids.
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 Section 4: ART: skills, techniques and present status

These antibodies may cause sperm to agglutinate, pre- infiltration of leucocytes that are present in semen.
venting forward progression and thus diminishing ROS have been associated with roles in capacitation
their fertilizing capacity, or become cytotoxic and kill and cell signalling pathways when present at low levels
sperm. Some antibodies prevent sperm-cervical [22]. However, ROS can also be generated in excess
mucus interaction or prevent zonal binding. If there when certain samples are prepared on density gra-
is an increase in ASABs, then it is of clinical advantage dients. Spermatozoa within these samples have the
to use ICSI in order to reduce the risk of failed fertil- capacity to create additional ROS. This is a sign of
ization that could be associated with poor sperm impaired sperm maturation or abnormal morphology
motility or binding [19]. and has been associated with male factor infertility.
The Mixed Agglutination Reaction (MAR) test is These sperm retain excessive levels of residual sperma-
one of the many ways to test for antisperm antibodies. tid cytoplasm in the midpiece and are also associated
This test is performed by mixing a small aliquot of with reduced fertilizing ability [23].
semen with latex beads that are coated with immuno- ROS are metabolites of oxygen and include hydro-
globins. If antibodies are present then the motile gen peroxide, nitric oxide, superoxide anion, along
sperm form clumps with the beads; if not, then the with hydroxyl and hydroperoxyl radicals. This is crit-
sperm will continue to swim freely about the slide [19]. ical for the andrology laboratory because when ROS
However, most laboratories now use the Immunobead are present at high levels they can cause pathological
test as this can determine which antibodies (IgA, IgG cellular damage. Oxidative damage to cellular lipids,
and IgM) are present. In this test, semen is combined proteins and DNA can occur. Although most cells
with latex beads coated with IgA or IgG and incubated. have anti-oxidant defence mechanisms, when these
If antibodies are present then the beads attach directly fail, sperm function can be impaired.
to the sperm. This test provides more information
than other tests as the beads can attach to the head,
neck, midpiece or tail and thus determine the specific DNA fragmentation
location of the ASABs [20]. Human sperm chromatin is highly condensed, but single
and double helix breaks can occur over time. Normally
these are repaired by integral mechanisms; however, at
Sperm vitality some stage, this damage becomes non-repairable and
The vitality of sperm is normally tested by assessing can be transmitted to any resulting embryos [24].
the ability of the plasma membrane to prevent the A high level of DNA fragmentation within human
introduction of certain dyes or stains. Sperm vitality spermatozoa may represent a cause of male factor infer-
is not routinely analyzed in andrology laboratories. tility that is overlooked by normal semen analysis. The
However, such methods can be useful in identifying fertilizing capacity of sperm with increased DNA frag-
rare cases of necrospermia, as opposed to total sperm mentation is not always affected; however, increased
motility, e.g. Kartagener Syndrome [4]. genetic abnormalities will have significant consequences
The Hypo-Osmotic Swelling Test (HOST) is on embryo development to blastocyst stage. There is
another means of identifying sperm vitality. This test also a poor prognosis for a successful pregnancy due
is based on the ability of live sperm to withstand to an associated increase in miscarriage rates [24].
moderate hypotonic conditions by measuring the abil- In order to measure the levels of DNA fragmenta-
ity of the sperm plasma membrane to transport water. tion in a sample there are many tests available, e.g.
Dead sperm, whose membranes are no longer intact, TUNEL, COMET or commercially available kits such
do not exhibit swelling and thus can not function as Halosperm [Halotech Dna, Sl, Madrid] that uses the
during the fertilization process. Live sperm will show Sperm Chromatin Displacement test. In these tests,
controlled swelling and their tails will curl. When the semen is tested for the levels of DNA fragmentation
sperm are placed back in normal media, the tails by immersing sperm into an inert gel. The cells are
should recover and they can be used for ICSI [21]. treated in order to denature the DNA in those exhibit-
ing high levels of fragmentation. The nuclear proteins
are washed away and the slide stained. Sperm with
Reactive oxygen species minimal or no DNA fragmentation will show large
244 The generation of Reactive Oxygen Species (ROS), or halos, whereas those with increased fragmentation
free radicals, is normally associated with the may not show a dispersion halo at all.
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 Chapter 24: Semen analysis and preparation

Table 24.3 Summary of results and treatment options

Condition Description Treatment option

Normozoospermia Production of sperm in normal numbers and motility IUI or conventional IVF
Oligozoospermia Low sperm count. Concentration < 20 Mill/ml ICSI Backup
(Either conventional IVF or ICSI depending on
severity on day of clinical treatment)
Asthenozoospermia Reduced sperm motility. Progressive motility < 50% ICSI if severe
Teratozoospermia Reduced numbers of sperm with a normal appearance ICSI if severe
(morphology). Normal morphology < 30%
Cryptozoospermia Apparent azoospermic sample but where sperm are ICSI – In these cases it may be best practice to
found during analysis only after centrifugation freeze a sample prior to treatment to ensure
availability of sperm on day of treatment
Necrozoospermia Samples with only dead sperm (not necessarily just Treatment with their own sperm is not presently
immotile sperm) an option
Globozoospermia Often referred to as ‘round-head’ defect. Sperm ICSI
morphological defect where the acrosome is absent
and sperm usually have small round heads
Aspermia No ejaculate, i.e. complete lack of semen, e.g. ICSI if sperm is found
retrograde ejaculation
Azoospermia No spermatozoa in the ejaculate obstructive: where If sperm found then ICSI
sperm are created, but cannot mix with the rest of the
ejaculatory fluid due to a physical obstruction (e.g.
CBAVD) non-obstructive: where there is a problem
with spermatogenesis

Interpretation of results plasma should be the lowest possible in order to max-

imize sperm yield and minimize the production of ROS.
Once semen analysis has been completed, a recom-
mendation for treatment protocol can then be made
and options considered (see Table 24.3). Preparation methods
There are four main preparation methods:

Sperm preparation 1. Dilution and washing

2. Sperm migration
Seminal fluid contains a wide range of factors other
than spermatozoa that must be removed in order to 3. Density gradient
optimize the number of sperm available for fertiliza- 4. Commercially available products
tion. Vitality and motility diminish in time. This is due Whichever method is utilized, all plasticware, glass
to the presence of decapacitation factors within the and media should be checked for toxicity to sperm
seminal plasma. It is therefore essential that samples and embryos, with a sperm survival test, prior to
are prepared as soon as possible after production in first use.
order to remove spermatozoa from the seminal plasma The choice of preparation method will generally
and its harmful effects. The presence of even a small vary between laboratories but essentially it will depend
amount of seminal plasma within the preparation can on the sample itself. At the Oxford Fertility Unit, most
prevent capacitation so it is important that a clean samples are prepared on a dual density gradient.
preparation is produced in order to concentrate the However, a severe oligoasthenospermic sample is
optimal sperm for fertilization [25]. best prepared on a 40% gradient as this will condense
While preparing semen, damage to sperm must be the sample and the viable sperm will usually swim out
minimized by avoiding large fluctuations in temperature in long drops. A long drop is an elongated drop of
and unnecessary centrifugation. The centrifugal forces media under oil where the preparation is placed. The
used while separating motile sperm from the seminal 245
drops are allowed to settle and are then examined.
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 Section 4: ART: skills, techniques and present status

Figure 24.4 Sperm migration. Semen is placed under media

and the motile sperm allowed to swim up with time.

Sperm, if present, can then be individually picked up motile portion of sperm move up into media and then
using the injection pipette if required for ART. after 30–45 minutes, depending on the quality of the
sample, the supernatant is retrieved and centrifuged at
Dilution and washing 500g (0.5 rcf) for 5 minutes in order to concentrate the
sample. A motility and concentration analysis should
Raw semen is diluted with a large volume of culture
then be performed in order to calculate the number of
media and separated. The advantage of this technique
motile sperm present. The whole process should be
is that it is a simple procedure that washes away the
performed at 37°C [17].
seminal plasma. The disadvantage is that all sperm,
There are many advantages of a swim up, the
including dead sperm, are pelleted, thus decreasing the
biggest being that it creates a highly motile prepara-
fertilizing capability by impairing the functional
tion. This method is beneficial for use on samples that
motility of the good sperm. It also does not reduce
have an increased amount of cellular debris or round
ROS and should only be used with ICSI [4].
cells as these do not clog up the gradients. It is also a
suitable technique to use for samples that do not
Sperm migration (swim up) respond very well to the centrifugation method and
Here, functional sperm are separated by migration. remains cost effective as it does not require expensive
Small aliquots of liquefied semen are placed under a gradients or chemicals other than the culture media.
layer of culture media and left to swim out. This is However, the disadvantages of this method are that
usually done by carefully using a syringe and a needle it is not suitable for every sample. Very viscous sam-
filled with semen. It is important to ensure that there ples respond poorly to this technique as the semen
are no air bubbles in the syringe and that the semen is tends to pull up into the media and thus ‘contaminate’
expelled slowly. Once the allocated amount has been the spermatozoa/media solution. Prolonged exposure
placed under media, the needle is sharply pulled of sperm to the seminal plasma means that samples
upwards, avoiding drawing any semen up. It is possi- with significant levels of ASABs are not suitable to be
ble to create the swim up by layering culture media used with this technique, for, ideally, the sperm should
over the semen; however, care must be taken not to be removed as quickly as possible from the semen [25].
dislodge the lower layer. Multiple tubes can be used in Swim ups are also not overly suitable for oligospermic
order to maximize sample yield. The tubes should be samples as they have notoriously low yields and thus
246 left at an angle in order to increase semen/media inter- insufficient sperm may be retrieved. The removal of
face (see Fig. 24.4). The samples are left so that the the supernatant must be done very carefully in order to
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 Chapter 24: Semen analysis and preparation

avoid dislodging the seminal layer. It is best prac- for example, silicon particles are costly and not all
tice to remove little and often in order to max- samples respond well to the centrifugation technique.
imize the success of the technique. It is also time In these cases it is possible to perform a gradient
consuming because of the incubation time. followed by swim up in order to maximize the num-
ber of motile sperm available for use.
The density gradient is prepared in the follow-
Density gradient ing way. The lower, or more dense, layer is usually
placed into the bottom of a sterile conical tube.
The most common method of sperm preparation
This is usually 1–3ml 80% silica particles but can
in the ART laboratory is selective washing. This
be 90% or 95%. The tube is then swirled to allow
utilizes density gradient centrifugation to fraction-
friction between the tube and the top meniscus to
ate subpopulations of sperm. During centrifugation
reduce. Carefully, the upper layer is added by
the sperm reach a point in the density gradient
swirling the solution slightly above the interface
that matches their own density. Motile sperm are
layer and allowing it to slide down the walls of
separated from immotile sperm and seminal
the tube creating a clean interface. It is possible to
plasma by high-speed centrifugation through dif-
underlay the heavier layer, but care must be taken
ferent gradients of commercially bought silane
not to blow bubbles which will compromise the
coated silica particles, e.g. Puresperm (Nidacon
interface. The semen is added on top, the tube
International AB, Gothenburg, Sweden). Normal
closed and then centrifuged at 300g (0.3 rcf) for
sperm with more condensed DNA travels further
20 minutes. Following centrifugation, the upper
to the bottom of tube during centrifugation. Round
layer and interface should be carefully removed
cells and abnormal forms with cytoplasmic drop-
and then discarded. The pellet should be removed
lets never make it to the pellet. However, overcen-
with a soft tip and then washed twice in order to
trifugation can cause ROS buildup if sufficient
remove any remaining silica particles (see Fig.
care is not taken. Care should also be taken to
24.5). At the Oxford Fertility Unit, we use 2 × 5
avoid overloading the gradient as this can ‘block’
min washes at 500g. Following the final wash, the
the interface layer and prevent the passage of func-
sample is resuspended in an appropriate volume
tional sperm [5, 17].
depending on the technique to be used. For IVF a
The advantages of density gradients are numer-
concentration of 5–10M/ml and for ICSI, 1–2M/ml
ous; they are suitable for all samples including vis-
is suitable.
cous samples and are a lot quicker than a swim up to
Forty per cent centrifugation: this is a variation of
perform. There is usually an increase in functional
the dual density technique described above and is used
sperm that can be retrieved and hence a good overall
for removing seminal plasma from severe oligoastheno-
yield and the gradients generally prevent the produc-
teratozoospermic (OATS) or blood/tissue from
tion of ROS [26]. However, there are disadvantages,

Figure 24.5 Density gradient. Following

centrifugation, normal sperm are collected
at the base of the tube in a pellet which
should be removed and then washed to
remove any remaining silica particles.

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 Section 4: ART: skills, techniques and present status

surgically retrieved sperm. This method concentrates now be achieved electronically using a commercial
samples to facilitate the recovery of sperm and should product.
only be used for ICSI. It is vital that any sperm preparation tubes are
labelled clearly and it is good practice to only prepare
Commercially available products one sample in a hood at any one time. The Oxford
Fertility Unit uses electronic witnessing for its sperm
There are many commercially available products to
preparation. Preparation tubes are electronically
select sperm, most of which are based on the filtration
tagged, as is the sample production pot. These are
column method. Motile sperm are separated from the
both verified by a second physical witness at the start
non-motile fraction by a series of densely packed glass
of the proceedings to ensure accuracy.
wool fibres. The principle behind this is based on the
motility of sperm themselves and the ability of the
glass wool to act as a filter as the sperm swim through Summary
the narrow fibres. The advantages are that these meth- Semen analysis and preparation are important aspects
ods are simple to perform, there is usually a good yield of any assisted reproduction laboratory. Their results
and ROS are significantly reduced. However, these have significance whether in the clinical or diagnostic
methods are expensive and the final preparation may settings. It is therefore vital that these techniques are
not be as clean as the more conventional methods carried out accurately and consistently, following
mentioned previously [17, 27]. a training programme, with suitable quality control
procedures in place to maintain standards.
Quality control and assurance
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