Human Reproduction Vol.19, No.10 pp. 2283–2288, 2004 DOI: 10.

1093/humrep/deh410
Advance Access publication July 8, 2004

Cytoplasmic droplets are normal structures of human

sperm but are not well preserved by routine procedures
for assessing sperm morphology

Trevor G.Cooper1,3, Ching-Hei Yeung1, Sabina Fetic1, Aligholi Sobhani2 and

Eberhard Nieschlag1
1
Institute of Reproductive Medicine of the University Clinic, Domagkstrasse 11, D-48129 Münster, Germany and 2Department

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of Anatomy, Tehran University of Medical Sciences, Tehran, Iran
3
To whom correspondence should be addressed. E-mail: [email protected]

BACKGROUND: There is a discrepancy between the use of terminology employed by clinicians and basic scien-
tists concerning the cytoplasmic droplets of sperm. Most clinicians consider their presence on sperm to be indica-
tive of abnormal sperm, whereas basic scientists consider them to be attributes of normal sperm. METHODS: The
presence of cytoplasmic droplets on human sperm was examined using conventional air-dried, fixed and stained
sperm smears and in living and fixed wet preparations. RESULTS: Cytoplasmic droplets were found on the
majority of motile sperm and in fixed preparations but only half of them were found in air-dried smears. There
was no relationship between the presence of abnormally large cytoplasmic droplets, indicative of abnormal sperm,
and the droplets found on living cells. CONCLUSION: The term ‘cytoplasmic droplet’ is confusingly used to
describe two different sperm structures: large amounts of retained, excessive cytoplasmic remnants, that survive
the air-drying procedure and are observed on abnormal sperm in conventionally stained sperm smears, and osmo-
tically sensitive vesicles that are present on normal living sperm. A plea is made to retain the term ‘cytoplasmic
droplet’ for the latter structure of normal sperm and to use the term ‘excess residual cytoplasm’ to describe the
abnormally retained cytoplasm observed on abnormal sperm in smears.

Key words: artefacts/cytoplasmic droplets/human sperm/morphology/nomenclature

Introduction Observations on human ejaculated sperm that have been

Cytoplasmic droplets are a normal component of mammalian swollen by the channel blocker quinine and that display poor
sperm, found at the neck of immature caput sperm and at the penetration of surrogate mucus suggest that volume regu-
end of the midpiece in mature cauda sperm. Their movement lation may play a role in human fertility (Yeung and Cooper,
along the midpiece during migration through the caput epidi- 2001; Yeung et al., 2003). The vast majority of literature on
dymidis is a characteristic feature of sperm maturation and ‘cytoplasmic droplets’ of human sperm considers them to be
they are retained on the majority of mammalian sperm stored indicative of abnormality with sperm being described as of
in the cauda epididymidis (Cooper and Yeung, 2003). There ‘diminished maturity’ (Gergely et al., 1999) or ‘immature
are fewer sperm with droplets in the bovine ampulla (Branton sperm’ (Ollero et al., 2000). The different terminology used
and Salisbury, 1947; Rao and Hart, 1948) and few on ejacu- to describe the cytoplasmic structure of human sperm (‘cyto-
lated sperm from bulls (O’Donnell, 1969), rams (White et al., plasmic droplets’, Rago et al., 2003), ‘cytoplasmic residues’
1959) and boars (Lasley and Bogart, 1944a,b). Recent studies (Keating et al., 1997), ‘residual sperm cytoplasm’ (Aitken
suggest that droplets may play an important role in fertility, et al., 1994), ‘abnormal retention of cytoplasmic droplets’
since sperm are subjected to a hypotonic challenge upon eja- (Zini et al., 2000), ‘retention of cytoplasm’ (Mak et al.,
culation into the female tract, and water would initially enter 2000) attests to the confusion surrounding this organelle.
the sperm’s cytoplasm, the bulk of which is in this organelle. Such disagreement in terminology is not helped by the
In transgenic mice, sperm that cannot maintain their volume different descriptions given by semen analysis manuals.
upon osmotic challenge exhibit flagellar angulation occurring According to the World Health Organization (1999), cyto-
at the site of the cytoplasmic droplet, and the mice are plasmic droplets observed in air-dried semen smears should
infertile (Cooper et al., 2004). only be considered morphological defects when large

Human Reproduction vol. 19 no. 10 q European Society of Human Reproduction and Embryology 2004; all rights reserved 2283
T.G.Cooper et al.

(greater than one-third or one-half the sperm head size),

Table I. Characteristics of semen used in this study
implying that smaller droplets are not abnormal. The ESH-
RE/NAFA handbook (2002) confirms that sperm with Mean ^ SEM (range)
retained cytoplasm less than one-third the sperm head size Semen volume (ml) 4.3 ^ 0.3 (1.6– 7.6)
are normal but adds to the confusion by stating that residues Sperm concentration (106/ml) 50.1 ^ 7.6 (12– 201)
larger than this are abnormal ‘and classified as cytoplasmic Sperm count (106/ejaculate) 200.2 ^ 28.0 (24– 828)
Motility (%)
droplets’, associating that name with an abnormal structure. Grade a 30.8 ^ 2.0 (5–47)
Recent observations on osmotically sensitive ‘midpiece Grade b 19.3 ^ 1.4 (8–39)
vesicles’ have prompted the view that such vesicles may be a Grade c 8.0 ^ 0.6 (1–16)
Grade d 41.9 ^ 1.2 (25– 68)
normal component of human sperm (Abraham-Peskir et al., Normal morphology (%) 12.0 ^ 1.0 (2–27)
2002; Chantler and Abraham-Peskir, 2004), although these Abnormal residual cytoplasm 9.6 ^ 0.7 (3–20)
authors consider them distinct from ‘cytoplasmic droplets’ by Osmolality (mmol/kg) 336.1 ^ 3.2 (283– 395)
accepting that such ‘droplets’ survive air-drying and are
characteristic of immature sperm. provided in this study, as analysed according to the World Health

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The recommended clinical procedure for evaluating human Organization manual (1999), are presented in Table I.
seminal sperm (the production of air-dried smears before fix-
ation: World Health Organization, 1999) can cause morpho- Measurement of osmotic pressure
logical artefacts. For instance, it produces severely swollen The osmotic pressure of 10 ml semen was measured after lique-
sperm heads when applied to immature epididymal sperm faction with a vapour pressure osmometer (Wescor Vapro 5510;
(Yeung et al., 1997; Soler et al., 2000), yet no swollen sperm Kreienfeld Scientific Measuring Systems, Germany), calibrated
heads are observed if the cells are fixed before making the daily with a 290 mmol/kg standard solution. As the viscosity of
smears. Such a drastic procedure as air-drying has been semen retards saturation of the detection chamber, a timed delay of
shown to disrupt fragile, osmotically sensitive midpiece 2 min was employed before reading to ensure accuracy, as rec-
vesicles (Abraham-Peskir et al., 2002). Thus the apparent ommended by the manufacturer. BWW medium with osmotic press-
absence of droplets from a normal spermiogram may be an ure of 230 mmol/kg (BWW230) was made by reducing the amount
artefact of semen preparation for sperm morphological of NaCl.
analysis.
This report describes experiments performed in order to Microscopical observations
assess the presence of true (not abnormal) cytoplasmic dro- After liquefaction, semen samples were incubated for 15 min at
plets on human sperm in living, in fixed and wet, and in 378C and 3 ml were examined under a 22 £ 22 mm cover slip by
air-dried preparations. phase contrast microscopy (Olympus BH-20, Japan) with a £40
objective and £10 ocular on a heated stage (Mini-Tüb, Germany) at
378C. The percentage of motile sperm (WHO grades a þ b þ c)
Materials and methods was determined and immotile and motile sperm were separately
assessed for the presence of (I) cytoplasmic droplets (small, regular
Preliminary experiments distensions at the neck or midpiece: Figure 1a, c, e), (II) abnormal
Three observations on semen from one donor, a healthy father, residual cytoplasm (large, irregular material along the mid-piece:
revealed the presence of cytoplasmic droplets on human sperm. Figure 1d, h, i), (III) coiled or looped tails (Figure 1g, j, k) or (IV)
(i) An ejaculate was produced directly in a vessel containing 5% none of the above categories. To 10 – 20 ml of these samples was
(v/v) glutaraldehyde in phosphate-buffered saline (PBS) (Dulbec- added an equal volume of 7% (v/v) glutaraldehyde and after 60 min
co’s; Sigma, Germany) and aliquots of the material were com- at room temperature the fixed cells were washed by addition of 1 ml
pressed under a cover slip for microscopic evaluation. (ii) Within PBS, and centrifugation at 500 g for 5 min. The pellet was examined
30 s of production an ejaculate was incubated at 378C and within as a wet preparation at £400 magnification for the presence of cate-
1 min, and every 5 min for 30 min before and after liquefaction, gories I– IV above. Similar preparations were examined in which
50 ml was transferred to 500 ml 5% (v/v) glutaraldehyde fixative in semen was mixed with appropriate volumes of BWW230 to a final
Biggers– Whitten – Whittingham (BWW) medium (Biggers et al., osmolality of 290 mmol/kg, the osmotic pressure of cervical mucus
1971) and the percentage of sperm bearing droplets was ascertained (Rossato et al., 1996).
microscopically. (iii) An ejaculate was brought to the laboratory The routine air-dried, Papanicolaou-stained semen smears of the
within 75 s and incubated under 2 ml BWW medium [osmotic press- same ejaculates were examined at both £ 400 and £ 1000 magnifi-
ure (OP) 328 mmol/kg] containing 4 mg/ml bovine serum albumin. cation by the same observer of the wet, fixed preparations and
Sperm emerging from the liquefying ejaculate were evaluated in scored according to the same criteria for defining droplets. These
5 ml aliquots for motility and presence of droplets in unfixed, wet stained smears were also examined for the presence of normal cyto-
preparations. After liquefaction, routine semen smears for morpho- plasmic droplets, defined as being smaller than one-third to one-half
logy were made for evaluation of cytoplasmic droplets. the sperm head size, and also for the presence of abnormal cyto-
plasmic residues by experienced andrology technicians.
Ejaculates
Human ejaculates of widely differing quality were obtained with Statistics
informed consent from 37 patients attending the Institute of Repro- Differences between populations were assessed by paired or
ductive Medicine and from 12 student volunteers. Ten normozoos- unpaired t-tests, and relationships by linear regression. P , 0.05
permic samples were included and the characteristics of the semen was accepted as statistically significant.
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Figure 1. Examples of human ejaculated sperm observed in fixed wet preparations (phase contrast optics: a, b, h, j), live wet preparations
(differential interference contrast optics: d, e, f, i, k) and Papanicolaou-stained, air-dried smears (c, g). Examples of true cytoplasmic droplets
(a, b, c, e, f), abnormal cytoplasmic residues (d, h, i) and coiled tails (g, j, k). Bars ¼ 5 mm.

Results two liquefied samples (14.5, 10.0%) were far lower than
Preliminary observations those found in the glutaraldehyde-fixed samples examined at
1 min (65%) or 20 min (67%).
In order to determine if cytoplasmic droplets, as seen in other
mammalian species and considered as normal structures,
were present on human sperm, one ejaculate was fixed Sperm morphology and motility
immediately upon ejaculation and two were fixed within 75 s The majority of live sperm viewed in wet preparations in
of sample production. Observations on the sample delivered semen (mean 336 mmol/kg at the time of processing) and in
into fixative revealed the presence of droplets on the majority media of female tract tonicity (290 mmol/kg) were observed
of sperm within the unliquefied coagulum. When aliquots of to have cytoplasmic droplets at the neck regions, sometimes
an ejaculate were fixed at intervals before and after liquefac- extending along the length of the midpiece (Figure 1b, f).
tion, 47 – 65% of sperm had visible droplets over the 30 min The percentage of motile sperm with droplets significantly
examined. When sperm taken from the medium surrounding exceeded that of immotile cells in both semen and at the
a liquefying ejaculate were examined in wet preparations, osmolality of cervical mucus (290 mmol/kg) and there was
49 –57% of immotile sperm bore droplets, whereas 68 – 92% no difference in motility of sperm in these fluids (Table II).
of motile sperm did. The mean percentages of droplets The same sperm suspensions fixed in glutaraldehyde
observed in conventional Papanicolaou-stained smears of the also revealed cytoplasmic droplets on the majority of them.

Table II. Sperm motility in semen and Biggers–Whitten–Whittingham (BWW290) and the percentage of motile and immotile sperm
each displaying cytoplasmic droplets
Motility (%) Droplet-bearing sperm (%)

Motile sperm Immotile sperm

In semen 49.3 ^ 2.6 (10–87) 51.6 ^ 1.8 (21–72) 32.4 ^ 1.3 (15– 53)a
In BWW290 49.0 ^ 2.8 (11–82) 53.2 ^ 2.1 (10–87) 29.6 ^ 1.3 (15– 54)a

Values are mean ^ SEM (range) from 49 samples.

a
Significantly different between motile and immotile fractions, P , 0.05.

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T.G.Cooper et al.

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Figure 2. Relationship between the percentage of human ejaculated sperm bearing cytoplasmic droplets assessed wet after glutaraldehyde fix-
ation (abscissa) (£400 magnification, phase contrast optics; abscissa) and in air-dried, fixed, Papanicolaou-stained smears of the same ejacu-
lates (ordinate), observed at magnification £ 400 (X, phase contrast) and £ 1000 (X, oil immersion). The percentage of sperm with excess
residual cytoplasm (greater than one-third to one-half the size of the sperm head, X) showed no relationship with that of cytoplasmic droplets.
Regression analysis revealed correlation coefficients for ‘true’ droplets in stained smears against droplets in wet, fixed samples (n ¼ 48) of
0.500 (£ 400) and 0.432 (£ 1000).

In order to compare this value with that obtained from the Discussion
live wet preparations, a weighted mean was obtained by The results of this study indicate that ejaculated sperm from
multiplying the percentage of motile and immotile sperm by men possess true cytoplasmic droplets, which are normal
their respective percentages of droplets. This percentage structures of sperm in all other mammalian species, as judged
(40.5 ^ 1.5, n ¼ 50, mean ^ SEM) was not different (paired from wet preparations on living gametes and in rapidly fixed
t-test) from that of the glutaraldehyde-fixed sperm preparations. The percentage of motile sperm with droplets
(38.4 ^ 1.7). in both semen and medium of 290 mmol/kg being higher
than that of immotile cells suggests that they are not delete-
rious to vitality or motility and are clearly not a marker for
Sperm morphology in fixed and smeared preparations poor sperm quality. That the percentage of sperm bearing
The percentage of conventionally air-dried and Papanico- droplets in live preparations agreed with estimates obtained
laou-stained sperm revealed significantly fewer droplets than from glutaraldehyde-fixed preparations confirmed that such
observed in fixed wet preparations, whether observed at £ 400 droplets were not artefacts of cell fixation. Thus human
(magnification used for the fixed and wet preparations: 52%) sperm resemble other mammalian sperm in having a mid-
or £ 1000 (magnification used for routine semen analysis: piece cytoplasmic droplet; what differs is their position, at
51%), although there was a significant correlation between the neck rather than the end of the annulus, and that they
the percentages of droplets observed in the dried and wet remain attached to the spermatozoon in the ejaculate. Ultra-
preparations by the same observer [Figure 2: r ¼ 0.500 structural micrographs of well-fixed, human ejaculated sperm
(£ 40), r ¼ 0.432 (£100)]. When abnormal cytoplasmic resi- also demonstrate a cytoplasmic droplet at the neck (Holstein
dues (defined as greater than one-third to one-half the sperm and Roosen-Runge, 1981; Johnson, 1982; Neugebauer et al.,
head size: World Health Organization, 1999) were assessed 1990) as they do in the epididymis (Ånberg, 1957).
by experienced andrology technicians, the percentage was A related and important observation for the morphological
low (, 9.6 ^ 0.6) and bore no relation to the percentage of assessment of human ejaculates was the far lower percentage
true droplets found in wet and fixed preparations (Figure 2). of droplets on sperm observed in air-dried, Papanicolaou-
There was a statistically significant linear relationship stained smears. Indeed, the same observer counted far fewer
between the percentage of what the andrology technicians droplets in stained smears than in the live or fixed wet prep-
assessed as abnormal ‘cytoplasmic droplets’ and what were arations of the same ejaculates. This attests to the general
assessed as residual cytoplasm (category II) in 47 wet pre- inadequacy of the routine method for preserving structures
parations at £ 100 (r ¼ 0.584; data not shown). sensitive to the stresses accompanying air-drying before
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 Human sperm cytoplasmic droplets

fixing and staining. As even human sperm heads may expand Midpiece structures surviving the latter, drastic treatment
under these conditions (Yeung et al., 1997; Soler et al., would include abnormally large amounts of excess cytoplasm
2000), it should not be surprising that far less rigid not removed at spermiation, adhering to the midpiece and
and osmotically sensitive vesicles would collapse during staining green with Papanicolaou. As such excess residual
preparation. cytoplasm has been associated with sperm from smokers
These findings confirm and extend the observations of (Mak et al., 2000) and men with varicocele (Zini et al.,
osmotically sensitive ‘midpiece vesicles’ (MPV) extending 2000) and with deficiencies in sperm DNA (Fischer et al.,
along the length of the midpiece of human sperm in semen 2003) and phospholipid-bound docosahexanoic acid (Zini
and cervical mucus (Abraham-Peskir et al., 2002; Chantler et al., 2000), its presence is indeed indicative of abnormal
and Abraham-Peskir, 2004), which were also considered not spermiogenesis. Such sperm should not be described as of
detrimental to sperm and were not found in air-dried prep- ‘diminished maturity’ (Gergely et al., 1999) or as immature
arations. These authors considered the so-called MPV to be (Ollero et al., 2000), but merely abnormal, since they cannot
distinguishable from ‘cytoplasmic droplets’ by the absence of and do not undergo maturation in the epididymis.
visible content and their higher incidence than conventional A plea is made to adhere to a common nomenclature and

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‘cytoplasmic droplets’. MPV were found to have no content reserve the term ‘cytoplasmic droplet’ of human sperm to
as rendered visible by X-ray and differential contrast that defined for sperm from all other Eutherian species,
microscopy of wet preparations, whereas ‘cytoplasmic namely an anatomically normal, osmotically sensitive com-
droplets’ assessed in air-dried preparations were expected to ponent of a well-formed spermatozoon, produced by a func-
contain a proteinaceous content which is stained green with tional testicular tubule, and which does not survive well the
Papanicolaou dye. Others have considered swollen cyto- air-drying of semen smears. Conversely, the large, irregular
plasmic droplets to be artefacts of Percoll preparations ‘abnormal cytoplasmic droplet’ (World Health Organization,
(Arcidiacono et al., 1983), although they clearly can be 1999), that does survive air-drying and is stained in routine
observed in the absence of Percoll, and their detection by semen smears, should not be termed a cytoplasmic droplet;
confocal microscopy (Sofikitis et al., 1994) was also con- the term ‘excess residual cytoplasm’ (Aitken et al., 1994) is
sidered abnormal, presumably also because they resembled suggested to indicate the nature of this organelle, found on
large cytoplasmic remnants observed in air-dried pre- abnormally formed sperm liberated from a testis displaying
parations. damage to the seminiferous epithelium. As both true cyto-
We consider that MPV are ‘true’ cytoplasmic droplets as plasmic droplets and residual cytoplasm could be said to be
observed here (see Figure 1b, f), as they are present on living forms of midpiece vesicles, this term is rejected in favour of
gametes and neither survive air-drying well. Thus we dis- the long-existing term cytoplasmic droplet. Acceptance of
agree with Chantler and Abraham-Peskir (2004) that the two this terminology should obviate the confusion that has sur-
organelles are distinct and believe that this difference reflects rounded this topic in the past and reduce confusion arising
one of terminology: the abnormal (large, protein-rich, green- when the same term is used for two completely different
stained) ‘cytoplasmic droplets’ that survive the air-drying sperm structures.
procedure are considered by many to be the equivalent of
cytoplasmic droplets found on well-fixed sperm, whereas we
argue that they are excess residual cytoplasm retained by Acknowledgements
abnormal sperm produced by imperfect spermiogenesis. This We thank Ele Kürten, Sabine Rehr, Külli Nurmik and Daniele
view is supported by electron micrographs of well-fixed Hanke for performing the routine semen analysis.
semen that reveal the membranous components typical of a
true cytoplasmic droplet within a vesicle extending the entire
length of the human sperm midpiece (see Figure 3 in Smith References
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