Human Reproduction, Vol.31, No.1 pp. 10– 23, 2016
Advanced Access publication on October 14, 2015 doi:10.1093/humrep/dev251
ORIGINAL ARTICLE Andrology
Another look at human sperm
morphology
J. Auger 1,2,*, P. Jouannet 3, and F. Eustache 2,4
1
Service d’Histologie-Embryologie, Biologie de la Reproduction/CECOS, Hôpitaux Universitaires Paris Centre, Site Port-Royal, 53,
Avenue de l’Observatoire, 75014 Paris, France 2INSERM U1016, Equipe "Génomique, Epigénétique et Physiologie de la Reproduction", Institut
Cochin, Université Paris Descartes, Paris, France 3Université Paris Descartes, Paris, France 4Service d’Histologie-Embryologie, Cytogénétique,
Biologie de la Reproduction/CECOS, Hôpitaux Universitaires Paris Seine-Saint-Denis, Site Jean Verdier, 93143 Bondy Cedex, France
*Correspondence address. Tel: +33-1-58-41-37-33; Fax: +33-1-58-41-15-65; E-mail: [email protected]
Submitted on June 17, 2015; resubmitted on August 29, 2015; accepted on September 15, 2015
study question: Can a standardized assessment of abnormal human sperm morphology provide additional useful information by
identifying men with more severe disturbances in different types of abnormalities?
summary answer: Definition-based categorization of sperm head, midpiece and tail defects has shown how differently these abnormalities are distributed in fertile men and other groups of men, thus providing high and low thresholds, a starting point for diagnosis or research
purposes.
what is known already: Several recent studies have reported indisputable genetic origins for various sperm defects. A few studies have
also identified associations between environmental factors and low percentages of morphologically normal spermatozoa. Nevertheless, with the exception of rare situations in which the vast majorityof spermatozoa have specific, easily characterized defects, such as ‘globozoospermia’, little attention
has been paid to the description and precise quantification of human sperm abnormalities. The lack of standardization in the phenotyping of sperm
morphological defects by conventional microscopy is a limiting factor for diagnosis and for intra- or inter-observer or centre consistency in studies
investigating the causal factors and possible functional consequences of the abnormalities detected. There are currently no baseline data for abnormalities of sperm morphology based on a standardized classification, in the general population, among fertile or other groups of men.
study design, size, duration: This study is based on detailed sperm abnormality datasets acquired by a standardized classification
method, from several groups of men, over the same 5-year period.
participants/materials, setting, methods: We studied cross-sectional data from fertile men (n ¼ 926), male partners from
infertile couples (n ¼ 1747) and testicular cancer patients (n ¼ 239). We used a standardized classification to analyse Shorr-stained slides, taking into
account all the abnormalities encountered.
main results and the role of chance: Most sperm defects were significantly more frequent in infertile than in fertile men, with
20–30% of infertile men having frequencies of abnormalities above the 95th percentile in fertile men for 9 out of the 15 categories of abnormalities.
Interestingly, several head abnormalities were significantly more frequent in patients with testicular cancer than in infertile men, highlighting the
particular impact of this condition on sperm morphogenesis. We used the 95th percentile in fertile men as the lower threshold and the 99th
percentile in infertile men as an extreme upper threshold, for the classification of morphological abnormality frequencies into three levels: low,
intermediate and high. The assessment of several semen samples, with or without a genetic background, for abnormal sperm morphology, based
on the percentage of normal spermatozoa, a teratozoospermia index, and the detailed profile of abnormalities categorized according to the three
levels proposed, has highlighted the value of detailed phenotyping for diagnosis and research purposes.
limitations, reasons for caution: The thresholds proposed for the various categories of sperm abnormality should be considered
relative rather than absolute, owing to the known sampling error related to the limited number of spermatozoa assessed per sample, or when studying
the general population or populations from regions other than Western Europe. The standardized assessment of abnormal sperm morphology
requires time and experience. We therefore suggest that this assessment is carried out during a first andrological check-up or for epidemiological
or research studies, rather than in the routine management of infertile couples for assisted reproductive technologies.
wider implications of the findings: The study design used for the fertile group of men was similar to that previously used for the
WHO reference values, providing a rationale for considering the 95th percentile in fertile men as the level below which abnormalities may be considered to occur at a frequency representing random background variations of a normal spermiogenesis process. The crude frequencies obtained, and the
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
All rights reserved. For Permissions, please email: [email protected]
11
Abnormal sperm morphology
three levels of abnormality frequency proposed for each standardized category of sperm defect, provide baseline data useful for diagnosis and a starting
point for future studies aiming to identify associations with genetic or environmental factors.
study funding/competing interest(s): Part of this study was supported by contract BMH4-CT96-0314 from the European
Union. The authors have no competing interests to declare.
Key words: fertile men / human semen / infertility diagnosis / multiple anomalies index / reference values / sperm defects / standardization
Introduction
Sperm morphology assessment, on a stained semen smear, reveals a plethora of morphological variations or abnormalities of the sperm head, midpiece and tail components. These abnormalities primarily reflect the
complexity of terminal sperm differentiation following several biochemical
and morphological changes (acrosome formation, flagellar development,
chromatin condensation and major reorganization of the nucleus and
cytoplasm) and the influence of multiple genetic or micro- or macroenvironmental factors modulating or disrupting this crucial stage in
morphogenesis.
Normal and abnormal sperm cells exhibit a number of features on
stained smears studied by conventional microscopy. Various sperm
morphology classifications have been proposed from the early 1950s
(notably with the remarkable contribution of MacLeod and Gold, 1951)
until the 1970s (reviewed in Mortimer and Menkveld, 2001) for categorizing spermatozoa according to these various aspects. Spermatozoa
retrieved from the uterine cavity or in the vicinity of oocytes recovered
from the female reproductive tract, and thus clearly with the potential
to migrate in the female genital tract and to bind to oocytes, were found
to be more homogeneous in appearance than those from a native
semen sample. This observation helped to define the appearance of
potentially fertilizing (morphologically normal) spermatozoa (Menkveld
et al., 1990; Liu and Baker, 1992). With the development of assisted reproductive technologies (ART) from the 1980s onwards, initially through IVF
and then with the development of ICSI in the 1990s, sperm morphology
analysis became important for prognostic reasons, but was mostly
restricted to assessments of the proportion of normal spermatozoa
(Ombelet et al., 1997). The sperm morphology assessment carried out
primarily in the context of ART is generally considered to constitute the
cornerstone of such analyses in humans. These methods have been fully
endorsed in the WHO laboratory manual for the examination and processing of human semen, as the reference method for assessing human
sperm morphology (WHO5: World Health Organization, 2010).
Several studies have identified genetic causes of various morphological
abnormalities of spermatozoa, such as globozoospermia and macrocephalic sperm, when these abnormalities concern the vast majority of the
sperm cells present (Guichaoua et al., 2009), and associations between
genetic, lifestyle or environmental factors and abnormal sperm morphology have also been reported (Wyrobek et al., 1983; Buck Louis et al.,
2014). However, the precise origins of many of the human sperm abnormalities observed are far from well understood, and the precise impact of
these abnormalities on sperm fertility potential also remains unclear.
As a consequence, it has recently been suggested that precise analyses
of the number of sperm abnormalities by conventional microscopy
would be a useful approach for diagnosis or research purposes
(Menkveld, 2013; Mitchell et al., 2015). The WHO5 report (World
Health Organization, 2010) has come to a similar conclusion, with its
statement that ‘categorizing all abnormal forms of spermatozoa may
be of diagnostic or research benefit’. WHO5 contains a rich iconography
on morphological abnormalities of spermatozoa, with legends providing
a simple conclusion: ‘normal’ or ‘abnormal’ spermatozoon, with comments in some cases, such as ‘pyriform’ and ‘amorphous’ head. This
document does not propose clear definitions of the various abnormalities of human sperm cells, and the corresponding ‘diagnostic or research
benefits’ are neither demonstrated nor discussed. This limits the
morphological evaluations of spermatozoa and comparisons of the
results obtained by different observers or in different studies (Wang
et al., 2014).
We have previously reported and used a method for categorizing
normal sperm morphology and the various sperm defects encountered
(Auger, 2010), in line with contemporary knowledge of sperm assembly
(Chemes and Rawe, 2003), the correspondence between the defects
observed on conventional microscopy and transmission electron microscopy (Bisson and David, 1975), and the functional consequences
of most of the defects categorized (Chemes and Alvarez Sedo, 2012).
One of the principal advantages of such a standardized categorization
is the provision of basic data about the distributions of various sperm
defects.
This study, based on our standardized method, was designed to establish reference ranges for morphological abnormalities of human spermatozoa, through the analysis of three large groups of men: fertile men, infertile
men and men with testicular germ cell tumours (TGCTs). Thresholds
for future clinical, epidemiological or basic studies are proposed and
discussed.
Materials and Methods
Subjects studied
Sperm morphology was assessed in three groups of men: a homogeneous
group of fertile men and two different groups of men of unknown fertility
status (male partners from infertile couples, referred to hereafter as ‘infertile
men’ for convenience, and men with testicular cancer).
The fertile men were the male partners of pregnant women participating
in a study investigating possible geographical differences in semen quality
(Jørgensen et al., 2001). Each of the men provided a single semen sample.
They were recruited from four different European cities in 1997– 1998.
These men had a mean age of 31 years (interquartile range (IQR): 24 – 38
years old) and their partners had become pregnant without the need for
fertility treatment (e.g. stimulation of ovulation, ART). In the protocol of
this international study, sperm morphology was analysed in a centralized
manner, in our laboratory, to minimize the well-known wide variability in
human morphology assessment by different observers and centres
(Ombelet et al., 1998; Eustache and Auger, 2003). A first analysis focusing
on geographical variation in sperm morphology and the influence of environmental and lifestyle factors has already been published (Auger et al., 2001),
but the distributions of the various sperm abnormalities were not reported.
In this study, only sperm morphology was assessed for couples for whom a
pregnancy was achieved within 1 year (n ¼ 926), in accordance with the
12
methodology used for the definition of the new WHO reference semen
values (Cooper et al., 2010).
The second population study consisted of male partners from infertile
couples referred to the reproductive biology laboratory of Cochin Hospital
Paris for routine semen analysis during the same time period. This group of
1747 men, for whom it was not possible to collect information about the duration of infertility of other clinical information in a systematic manner, was older
than the fertile men (mean: 36.5; IQR: 32–40). The dataset consisted of the
results of all consecutive routine semen analyses, with only the first semen analysis used in cases of repeated analyses for the same patient during the period.
The third group of men consisted of patients referred to the reproductive
biology laboratory of Cochin Hospital Paris by their urologist or oncologist,
during the same time period, for semen cryopreservation before antineoplastic treatment for a TGCT. Semen was collected before orchidectomy
(Petersen et al., 1999). These patients were younger than the men in the
other two groups (mean: 29.2; IQR: 25 – 33).
Pre-analytical steps in sperm morphology
assessment
All semen smears were prepared, fixed and stained according to the same
standardized procedure. Briefly, smears were prepared from a 10 ml wellmixed drop of semen, which was allowed to dry in air, and then fixed by
incubation for 1 h with a mixture of absolute ethanol (2/3) and acetic acid
(1/3), before Shorr staining (World Health Organization, 1992) by an
automated method (Sakura DRS601, Bayer Diagnostics, Puteaux, France)
ensuring the homogeneous staining of different preparations.
Methodology used for human sperm
morphology classification
The method used here for the assessment of human sperm morphology was
essentially the original method of classification described by David et al.
(1975) and then modified in the late 1990s to provide clear rules for the classification of normal and abnormal spermatozoa and for the categorization of
the various sperm defects observed in stained smears through conventional
microscopy (Auger et al., 2001). The various morphological categories are
shown in Fig. 1, with a summary of the definitions in the figure legend. According to this method, morphologically normal spermatozoa are categorized on
the basis of the same criteria proposed by WHO from the third edition of the
manual to the recent WHO5 (World Health Organization, 1992, 2010, respectively). However, unlike the WHO5 guidelines, our classification considered all borderline aspects to be normal, as in previous classification systems
(Freund, 1966; Eliasson, 1971) and as argued for in previous studies (Auger,
2010). Borderline aspects introduce marked uncertainty into classification
systems and are a major component of the overall intra- and inter-observer
variability in assessments without categorization rules. In the method used
here, a reticle eyepiece with a micrometre was used whenever the observer
had doubts about sperm size, in terms, for example, of the lengths of the
major axis or the minor axis of the head, and decision-support criteria
were defined for distinguishing borderline aspects of size and shape (see
Supplementary data, Fig. S1). This method was initially developed for diagnostic purposes. It therefore categorizes the head, midpiece and principal
piece defects most frequently observed in human sperm and known to correspond to particular aetiologies or ultrastructural alterations (Chemes and
Rawe, 2003). The use of a multi-entry grid, accounting for all abnormalities
and their combinations, without underestimating one abnormality with
respect to the others (e.g. a sperm cell can have residual cytoplasm in
excess as well as a bent tail), makes it possible to calculate an overall indicator
of the level of teratozoospermia, the Multiple Anomalies Index (MAI), equal
to the mean number of abnormalities per abnormal spermatozoon (Jouannet
et al., 1988; World Health Organization, 2010). Our classification considers
Auger et al.
true excess residual cytoplasm (ERC), but not physiological cytoplasmic
droplets (Cooper, 2005), as abnormal.
Analytical procedure
All stained smears were assigned to a pool of four experienced technicians
who assessed them unaware of sample origins, by using the combination of
a ×100 oil-immersion bright-field objective and ×10 eyepiece (×1000
final magnification). Each of these technicians routinely assesses two to five
stained smears daily, and their accuracy and reproducibility in assessments
of human sperm morphology, based on the classification described here,
are regularly proved to be consistent (an example for IQC is provided in
Supplementary data, Table S1).
Statistical methods
For the three groups of men studied, only data from stained smears for which at
least 100 spermatozoa could be classified were analysed. We also compared
each abnormality category within each group of men, between men with and
without normal sperm production and progressive motility, as defined in
WHO5 (normozoospermic men: at least 39 million spermatozoa per ejaculate
and at least 32% progressively motile sperm cells; World Health Organization,
2010). Age is not associated with sperm morphology, at least for the 25–41
years age group (Schwartz et al., 1984). We also found no association
between age and the extent of normal or abnormal morphology in the three
groups of men studied (data not shown). We therefore did not adjust comparisons for age. All statistical comparisons between fertile men, infertile men and
TGCT patients were performed with BMDP statistical software (Statistical Solutions, Cork, Ireland). The similarity of the mean values for the percentages of
normal spermatozoa, all morphological defects and MAI values between the
three groups was assessed by one-way analysis of variance (BMDP 7D subroutine), taking unequal variances into account (Brown–Forsythe test) when necessary. When the null hypothesis was rejected, post hoc Tukey tests were used for
pair-wise comparisons between groups of men. Crude data were used to determine the distribution profiles for each morphological abnormality category, MAI
and morphologically normal spermatozoa in the three groups of men studied.
The curves best fitting each distribution profile were then constructed with
the Fit-curve option of Sigmaplot software (Statistical Solutions).
By analogy with the WHO reference thresholds for semen characteristics
obtained for a similar population of fertile men (Cooper et al., 2010), we considered the 95th percentile for the various categories of abnormalities and
MAI, and the 5th percentile for the percentage of normal spermatozoa in
the fertile group as lower reference thresholds. Dichotomous categorization
has been shown to be of little overall clinical relevance for semen variables
(Björndahl, 2011). We therefore also used a second, experience-based,
higher threshold, set at the 99th percentile for the population of infertile
men, as an extreme level for the sperm abnormalities considered, potentially
with a specific origin and possible pathological implications. We then defined
three levels of sperm abnormalities on the basis of these two thresholds:
a low level of abnormality (LLA), below the 95th percentile in fertile men;
a high level of abnormality (HLA), above the 99th percentile in infertile men
and an intermediate level of abnormality (ILA), between the 95th percentile
in fertile men and the 99th percentile in infertile men. This ILA is probably nonrandom, particularly if it tends to increase towards extreme values. A preliminary validation of the utility of the three levels of abnormality proposed was
made on a sample of six men, one of whom was fertile, the other five having
specific teratozoospermia profiles with a known genetic background.
Ethical approval
The study was approved by the Cochin University Hospital for the three
groups of French men studied and by all collaborating institutions for the
fertile men from other European countries. Informed consent was obtained
from all participants.
Abnormal sperm morphology
13
Figure 1 Standardized classification of normal and abnormal human sperm morphology. Morphologically normal spermatozoa are defined as in the 5th
WHO manual (World Health Organization, 2010) except that all borderline aspects are considered normal (decision rules for borderline aspects are presented in Supplementary data, Figure S1); tapered head: increased major axis length and normal minor axis length; thin head: normal major axis length and
decreased minor axis length; microcephalic: decreased major and minor axes lengths; macrocephalic: increased major and minor axes lengths; multiple
heads: more than one head per sperm cell; abnormal post-acrosomal region: any type of outline and/or size and/or texture abnormality of the posterior
part of the head; abnormal acrosomal region: any type of outline and/or size and/or texture abnormality of the anterior part of the head; abnormal (or
excess) residual cytoplasm (or abnormal cytoplasmic remnant): residual cytoplasmic area . 30% of the head area, in any part of the sperm cell; thin midpiece: midpiece diameter, smaller than the principal piece diameter for . 40% of the whole midpiece length; bent/misaligned tail: may include neck anomalies, misalignment of the head major axis and midpiece axis or an acute angle (≤1208) between the head major axis and midpiece axis or an acute angle
(≤1208) between the midpiece and the principal piece axes ; no tail: isolated head, no tail observed; short tail: a tail length (midpiece + principal piece) no
more than five times the major axis length of the head; irregularly shaped tail: irregular or changing calibre along the tail; coiled tail: completely or partially
coiled tail, with the coil close to or around the head (flat coiling or coiling at the extremity of the tail reminiscent of hypo-osmotic coiling aspects should not be
included in this category); multiple tails: more than one tail per sperm cell. Many of the spermatozoa presented more than one type of abnormality. The
classification method encompasses all the abnormalities of each cell and the calculation of multiple anomalies index (MAI) (World Health Organization,
2010).
14
Auger et al.
three groups of men studied, the distributions of the incidence of abnormal post-acrosomal regions, abnormal acrosomal regions, bent or misaligned midpieces, coiled tails and of the MAI were also almost
Gaussian, but the curves were displaced to the right for infertile men
and TGCT patients in comparison with the curve for fertile men.
Other categories of sperm abnormalities followed an exponential
decay distribution profile that was generally less steep in infertile men
and TGCT patients than in fertile men. More than a quarter of TGCT
patients had values above the 95th percentile for fertile men, for the categories microcephalic sperm cells, abnormal post-acrosomal and acrosomal regions, ERC and short tail. Similarly, more than a quarter of the
male partners from infertile couples had a frequency of abnormalities
greater than that of fertile men for the categories abnormal postacrosomal and acrosomal regions, thin midpiece, bent or misaligned midpiece and short tail. In addition, some sperm defects, microcephalic
sperm cells, macrocephalic sperm cells, abnormal acrosomal regions
and ERC, were significantly more frequent in TGCT patients than in infertile men. The MAI value was higher than the 95th percentile for
fertile men in 47% of TGCT patients and in 28% of infertile men. Interestingly, in comparisons restricted to normozoospermic men, significant differences were still found between the three groups studied, for most of
abnormality categories (Table II).
Results
Fertile men
Distributions of morphologically normal spermatozoa and of the various
categories of sperm abnormality percentage and of MAI in the 926 fertile
men are presented in Table I. The 5th percentile for the percentage of
normal spermatozoa including all borderline aspects was 23% (95% CI,
20– 26). Head abnormalities were more frequent than midpiece and
principal piece defects. Texture or outline abnormalities of the acrosomal
or post-acrosomal regions were markedly more frequent among head abnormalities than head size abnormalities. Bent and coiled tails were the
most frequent flagellar abnormalities and were observed in most
samples. In most fertile men, abnormal sperm cells generally had fewer
than two associated abnormalities (95th percentile of MAI ¼ 1.92).
Comparison of fertile men, infertile men
and TGCT patients
The distribution profiles of the percentages of normal spermatozoa, all
morphological sperm abnormalities and MAI, in fertile men, infertile
men and TGCT patients, are presented in Fig. 2. The distribution of
the percentage of morphologically normal spermatozoa was essentially
Gaussian in the three populations, but with a sharp shift towards lower
values in infertile men and TGCT patients. More than 50% of TGCT
patients and almost 40% of infertile men had a percentage of morphologically normal spermatozoa below the 5th percentile for fertile men.
The frequency of each morphological abnormality was significantly
higher in infertile men and TGCT patients than in fertile men. In the
The usefulness of the proposed three levels
of abnormality
The broad overlap between the distribution profiles of the various
morphological abnormalities in fertile and infertile men precluded the
Table I Distributions of morphologically normal spermatozoa (including borderline cells) and categories of spermatozoa
with head, midpiece and principal piece abnormalities, in 926 fertile men.
Distribution range (percentiles)
...........................................................................................................................................
1st
5th [95% CI]
10th
25th
50th
75th
90th
95th
.............................................................................................................................................................................................
Normal spermatozoa
13
23 [20 –26]
29
40
52
Sperm defect categories*
99th
95th [95% CI]
90th
75th
50th
Tapered head
7
3 [2–4]
2
0
0
Thin head
21
14 [12 –16]
10
6
3
Microcephalic
11
7 [5–9]
5
3
Macrocephalic
3
1 [0–2]
0
0
Multiple heads
4
2 [1–3]
1
Abnormal post-acrosomal region
52
42 [39 –45]
37
61
68
71
25th
10th
5th
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
27
19
11
8
5
...........................................................................................................................................
Abnormal acrosomal region
74
60 [57 –63]
53
41
30
21
15
12
Excess residual cytoplasm
8
4 [3–5]
2
1
0
0
0
0
Thin midpiece
1
0
0
0
0
0
0
0
Bent or misaligned tail
16
13 [11 –15]
10
8
5
3
1
0
No tail
9
5 [4–6]
4
2
1
0
0
0
Short tail
4
1 [0–2]
0
0
0
0
0
0
Irregularly shaped tail
5
2 [1–3]
1
0
0
0
0
0
Coiled tail
26
17 [15 –19]
13
8
5
2
0
0
Multiple tails
3
1 [0–2]
0
0
0
0
0
0
Multiple Anomalies Index (MAI)
2.08
1.92
1.83
1.70
1.57
1.42
1.33
1.26
*
See Fig. 1 for definitions.
Abnormal sperm morphology
15
Figure 2 Curves best fitting the distributions of the various sperm morphology characteristics assessed in fertile male partners of pregnant women (black
line), male partners form infertile couples (blue line) and TGCT patients (red line). The vertical line represents the 95th percentile threshold in fertile men
(5th percentile for the percentage of morphologically normal spermatozoa), and the percentages in blue and red represent the percentages for infertile men
and TGCT patients, respectively, above the 95th percentile threshold (below the 5th percentile threshold for the percentage of normal spermatozoa).
(A) Head abnormalities, (B) midpiece and principal piece abnormalities, (C) Multiple Anomalies Index (MAI) and normal spermatozoa.
16
Figure 2 Continued.
Auger et al.
17
Abnormal sperm morphology
Figure 2 Continued.
definition of a single meaningful threshold for each abnormality category.
We found that the experience-based threshold chosen for the definition
of the HLA category (the 99th percentile in the population of infertile
men) differed considerably between abnormalities. For example, it
was 95% for abnormal acrosomal regions, but only 5% for macrocephalic
spermatozoa and 4% for a thin midpiece (Table III). Similarly, the threshold defining LLA (the 95th percentile for the population of fertile men)
was ≤3% for seven morphological abnormalities (tapered head, macrocephalic sperm, multiple heads, thin midpiece, short tail, irregularly
shaped tail and multiple tails), but as high as 60% for abnormal acrosomal
regions (Table III).
Figure 3 illustrates the possible relevance of the three levels of abnormality proposed for diagnosis or research purposes. It shows the sperm
morphology profiles for six men, one of whom was fertile, the other five
being infertile with several types of characterized teratozoospermia of
known genetic origin. The graphical representation of the abnormal
morphology profile shows both the exact level of each category of abnormality and the relative level according to the defined three-level/colour
ranges of abnormalities taken from the thresholds defined in the distributions in large groups of fertile and infertile men. This figure demonstrates
that, in such clinical situations, the simple recording of the percentage of
normal spermatozoa or the determination of MAI is not informative for
diagnosis (see Fig 3A, B and D). In contrast, the different categories of abnormality level—low (LLA), high (HLA) or intermediate (ILA)—based
on the frequencies determined according to our standardized definitions
for each category of abnormality, provided a clear picture of the morphological background noise (LLA for all abnormalities) characteristic of
fertile men, or of the combination of high levels of head or midpiece or
principal piece defects characterizing particular pathological morphologies. Examples C and D refer to well-known genetic ‘monomorphic syndromes’ (note the ‘normal’ MAI) that are easily discernible. Examples E
and F show more complex profiles (several yellow and red categories
and a high MAI) that deserve investigations. A recent study has shown
the genetic origin of the combination of the various tail defects; here
we show that noticeable levels of defects of the head or midpiece compartment may be associated with tail defects suggesting a possible different genetic origin. In addition, examples in Fig. 3 show that: (i) a relatively
high percentage of certain abnormalities may correspond to LLA and
therefore has little meaning as for the abnormality ‘Abnormal postacrosomal region’ (25– 30%, see A, D, B); (ii) a relatively low percentage of certain abnormalities may correspond to HLA and therefore
has an important meaning, for example ‘Thin midpiece’ or ‘Irregular
tail’ (B, for both) and (iii) some semen samples may have a rate of morphologically normal spermatozoa in the normal limits but may have a
HLA for some categories of anomalies (D and B). These examples
provide an indication of the potential usefulness of this approach for
future studies aiming to identify similar or new specific profiles related
to genetic or environmental factors.
Discussion
This study is the first to report the distribution of various head, midpiece
and principal piece defects encountered in human spermatozoa in large
groups of fertile men, infertile men and patients with a testicular cancer,
through the use of a standardized classification method. For all the characteristics studied, the distributions observed for infertile men and
TGCT patients deviated from that for fertile men, with several abnormality categories even displaying significantly higher frequencies in TGCT
patients than in infertile men.
Information obtained from a standardized
detailed sperm morphology assessment
The incidence of tapered heads was low in all three groups and lower
than published values (Andrade-Rocha, 2007), possibly due to the lack
of a precise definition of tapered heads in other studies (thin heads or
amorphous head shapes may have been included). Thin heads (normal
sperm head length but narrower width) have rarely been described,
probably owing to confusion with tapered heads. Thin heads were
more frequently observed in infertile patients and men with TGCT and
may reflect abnormal nuclear morphogenesis (Auger et al., 1990) and
fragmented DNA (Gandini et al., 2000). The relatively high incidence
of this abnormality in fertile men is probably dependent on complex, multiple, environmental factors rather than genetic determinants. A high incidence of thin heads has been reported in varicocele (Prasivoravong
et al., 2014) and experimental cryptorchidism (Mieusset et al., 1987).
18
Auger et al.
Table II Comparison of men with a normal total sperm count and motility according to World Health Organization (2010)
criteria, for the various standardized sperm morphological categories.
Fertile men
n 5 859
Infertile men
n 5 930
TGCT patients
n 5 127
F-Value
P-Value
.............................................................................................................................................................................................
Normal spermatozoa
51.3 + 14.4†
53 (41 –62)‡
36.4 + 16.4*
37 (24– 48)
28.6 + 13.2*,**
28 (20– 38)
Tapered head
0.7 + 1.5
0 (0–1)
1.2 + 2.6*
0 (0–1)
Thin head
4.5 + 5.1
3 (1–7)
Microcephalic
291
,0.0001
1.5 + 2.0*
1 (0–2)
18
,0.0001
7.4 + 8.0*
5 (2–10)
8.0 + 6.2*
7 (3–11)
55
,0.0001
2.4 + 2.6
2 (1–3)
4.5 + 5.7*
3 (1–6)
8.2 + 7.3*,**
6 (2–13)
89
,0.0001
Macrocephalic
0.3 + 0.8
0 (0–0)
0.4 + 0.9
0 (0–0)
0.7 + 1.1*,**
0 (0–1)
8
,0.0005
Multiple heads
0.4 + 1.0
0 (0–1)
0.7 + 1.8*
0 (0–1)
0.6 + 1.1
0 (0–1)
7
,0.001
Abnormal post-acrosomal region
20.5 + 11.2
18 (12 –28)
29.8 + 16.2*
28 (17– 40)
31.8 + 17.1*
29 (20– 41)
115
,0.0001
Abnormal acrosomal region
32.5 + 14.6
30 (22 –41)
42.4 + 18.1*
40 (29– 54)
55.6 + 16.0*,**
56 (43– 67)
165
,0.0001
Excess residual cytoplasm
1.0 + 1.7
0 (0–1)
2.5 + 3.2*
1 (0–3)
3.3 + 3.1*,**
3 (1–5)
105
,0.0001
Thin midpiece
0.1 + 0.3
0 (0–0)
0.3 + 0.6*
0 (0–0)
0.3 + 0.8*
0 (0–0)
31
,0.0001
Bent or misaligned tail
5.8 + 3.7
5 (3–8)
9.4 + 5.3*
8 (6–12)
7.4 + 4.9*,**
6 (4–11)
140
,0.0001
No tail
1.6 + 2.1
1 (0–2)
1.7 + 2.0
1(0– 2)
1.7 + 2.0
1 (0–2)
1
Short tail
0.3 + 0.8
0 (0–0)
1.0 + 1.7*
0 (0–1)
1.3 + 3.0*
0 (0–2)
68
,0.0001
Irregularly shaped tail
0.5 + 1.1
0 (0–1)
1.2 + 1.8*
0 (0–2)
0.9 + 1.7*
0 (0–1)
51
,0.0001
Coiled tail
6.3 + 5.6
5 (3–8)
8.1 + 5.9*
7 (4–11)
8.1 + 7.0*
6 (3–11)
21
,0.0001
Multiple tails
0.4 + 0.7
0 (0–1)
0.7 + 1.0*
0 (0–1)
0.7 + 0.8*
0 (0–1)
32
,0.0001
Multiple Anomalies Index (MAI)
1.58 + 0.20
1.57 (1.43 –1.71)
1.72 + 0.24*
1.71 (1.55– 1.86)
1.81 + 0.24*,**
1.78 (1.62– 2)
127
,0.0001
NS
†
Mean + SD.
Median (IQ range).
For each morphological category, differences between the three groups of men were investigated by one-way analysis of variance taking unequal variances into account (Brown–Forsythe
test). For pair-wise comparisons, post hoc Tukey tests were carried out, with: *P,0.01 in comparisons with fertile men, **P,0.01 in comparisons with infertile men
‡
For both situations, it was found to decrease a few months after ‘exposure’. Microcephalic spermatozoa which have an immature chromatin
(Karaca et al., 2014), a high level of DNA fragmentation (Tang et al.,
2010) and even abnormal nuclear protein profiles (Blanchard et al.,
1990) were two to three times more frequent in infertile than in fertile
men, as previously reported (Kalahanis et al., 2002). This frequency
was also higher in TGCT patients than in infertile men. The incidence
of macrocephalic sperm cells was low in all three groups as reported in
infertile men in a study based on the same classification (Guichaoua
et al., 2009). In this series, the maximal incidence was 12%. Higher frequencies are rarely observed and such cases result from specific gene deficiencies (Achard et al., 2007). Abnormalities of the acrosomal and
post-acrosomal regions were more frequent in infertile than in fertile
men. The frequency of abnormal acrosomal region was higher in
TGCT patients than in infertile men suggesting an additional role of
TGCT-related causal factors. The broad distributions of acrosomal
and post-acrosomal abnormalities whose definitions include size or
form or texture defects may reflect various aspects of sperm head morphogenesis possibly due to multiple micro-environmental and genetic
factors. The incidence of true ERC, not including physiological cytoplasmic droplets (Cooper, 2005), was very low in fertile men and between
the 75th and 95th percentile values of fertile men in unselected infertile
men. It was more homogeneously distributed in TGCT patients with a
higher mean value close to the 95th percentile for fertile men and
higher than in infertile men. TGCT may affect spermiogenesis, and this
high frequency may reflect Sertoli cell dysfunction in these patients
19
Abnormal sperm morphology
Table III Low, intermediate and high levels abnormalities (LLA, ILA and HLA, respectively) for each sperm abnormality
category based on the distributions in 926 male partners of pregnant women (F) and 1747 male partners from infertile
couples (I).
Morphology characteristic
Level of abnormality
........................................................................................................................................
LLA (≤95th
percentile F)
ILA (>95th percentile F; ≤99th
percentile I)
HLA (>99th percentile I)
(max value)*
.............................................................................................................................................................................................
Tapered head
≤3
.3
≤15
.15 (50)
Thin head
≤14
.14
≤43
.43 (68)
Microcephalic
≤7
.7
≤30
.30 (72)
Macrocephalic
≤1
.1
≤5
.5 (12)
Multiple heads
≤2
.2
≤9
.9 (33)
Abnormal post-acrosomal region
≤42
.42
≤83
.83 (95)
Abnormal acrosomal region
≤60
.60
≤95
.95 (99)
Excess residual cytoplasm
≤4
.4
≤18
.18 (44)
Thin midpiece
0
.0
≤4
.4 (17)
Bent or misaligned midpiece
≤13
.13
≤28
.28 (36)
No tail
≤5
.5
≤12
.12 (31)
Short tail
≤1
.1
≤14
.14 (57)
Irregularly shaped tail
≤2
.2
≤12
.12 (34)
Coiled tail
≤17
.17
≤37
.37 (71)
Multiple tails
≤1
.1
≤5
.5 (8)
*
Maximum values observed in this series.
(Russell et al., 1989). However, it remains unclear whether and how this
disease interferes with sperm morphogenesis. Most infertile men had an
incidence of ERC below 20%. Only a few cases had a markedly higher frequency of ERC, from 30 to 44%, probably corresponding to a highly specific context or causal factor. In these extreme cases, the high incidence
of ERC may jeopardise fertilizing potential due to the ability of sperm with
ERC to generate high levels of ROS (Rengan et al., 2012). A bent midpiece showed a higher incidence than other midpiece and principal
piece abnormalities (95th percentile value of 13% for fertile men). This
may partly reflect the inclusion in the same category of midpieces bent
at the neck or principal pieces and misalignments, and it may hide
more subtle differences between the groups of men studied. In a study
based on the same approach, the incidence of bent midpiece was
increased during and decreased after experimental cryptorchidism
(Mieusset et al., 1987). With the exception of an absent tail, the incidence
of principal piece abnormalities was higher in infertile men and TGCT
patients than in fertile men. Detailed high-magnification examination,
by light microscopy, of flagellar morphology on semen smears obtained
and stained according to a rigorous procedure (World Health Organization, 2010) can be used to detect pathological or genetic conditions
when a certain category of tail abnormality is present at high frequency
by the approach described here (Mitchell et al., 2015), or by the
detection of associations of various tail abnormalities at high levels. For
example, an abnormal frequency of thick or irregular tails of normal
length, as observed by light microscopy, has led to the description of a
new type of sliding spermatozoa, characterized by periaxonemal
defects at the TEM level (Feneux et al., 1985). Men with sperm cells presenting a combination of five morphological defects of the sperm tail, as
categorized in our study, were found to have mutations of DNAH1
encoding an inner arm heavy chain dynein (Ben khelifa et al., 2014).
According to our definition, coiled tails category includes coiling with
the head inside or outside the coils, two forms of coiling with different
origins (Yeung et al., 2009). The incidence of all coiled tails in the infertile
population, with a mean value of 10%, was similar to that reported by
Yeung et al. (3% head-in-coil and 8% other coiled sperm) for a comparable population and higher than in the fertile men studied (mean ¼
6.5%). The percentage of men with more than 20% of spermatozoa
with coiled tails was 10% for infertile men and 11% for TGCT patients,
an incidence similar to that reported by Yeung and co-workers. This abnormality is positively associated with heavy smoking, and may fluctuate
with levels of neutral a-glucosidase, suggesting the negative influence of a
deleterious epididymal environment (Yeung et al., 2009). The incidence
of multiple tails was very low in all three populations, typically below 1%,
the maximum incidence in this series being 8%. A higher incidence, which
may affect the vast majority of the spermatozoa, is rare and associated
with macrocephalic heads in the macrocephalic sperm syndrome
caused by homozygous mutations of the aurora kinase C gene (Dieterich
et al., 2007).
Clinical relevance of a standardized and
detailed assessment of normal and abnormal
sperm morphology
The 5th percentile value of normal spermatozoa in our population of
fertile men, taken as the reference threshold, as in WHO5, was 23%.
The similarity in the groups of fertile men studied here and in the study
by Cooper et al. (2010) made it possible to compare our 23% threshold
with the 4% threshold obtained using the WHO5 criteria that consider all
20
Auger et al.
Figure 3 Examples of six profiles of morphological sperm abnormalities with the corresponding percentage of normal spermatozoa and MAI, based on
the standardized classification, with a representation of low (green), intermediate (yellow) and high (red) levels of abnormality, based on the distributions in
fertile and infertile men (see Statistical methods). (A) A fertile man with low levels of abnormality; (B) an infertile patient with a normal frequency of normal
spermatozoa and MAI despite an abnormally high level of double-headed spermatozoa; (C) an infertile patient with a globozoospermia-like profile (Guichaoua et al., 2009) and no normal sperm cell, and a relatively low MAI due principally to very high levels of microcephalic sperm cells, essentially combined
with an abnormal acrosomal region; (D) an infertile patient with fibrous sheath dysplasia (Chemes et al., 1987), despite normal levels for the percentage of
normal spermatozoa and MAI; (E) an infertile patient with high levels of multiple morphological abnormalities of the flagella (Ben Khelifa et al., 2014) and an
exceptionally high level of thin midpieces, resulting in low numbers of normal spermatozoa and a high MAI; (F) an infertile patient with MMAF together with
high levels of thin and microcephalic heads resulting in a very high MAI. ERC, excess residual cytoplasm; DFS, dysplasia of the fibrous sheath; MMAF, multiple
morphological abnormalities of the flagella.
21
Abnormal sperm morphology
borderline aspects to be abnormal. The marked difference between
these two values could correspond primarily to the inclusion or exclusion
of the borderline aspects in the normal sperm categorization (Eliasson,
2010; Handelsman and Cooper, 2010). The use of a set of criteria for
borderline aspects (Rothmann et al., 2013) could be useful in answering
the question of whether the borderline aspects per se matter or not.
A recent epidemiological study (Buck Louis et al., 2014) showed that
normal sperm morphology, whether according to the traditional
WHO approach (including borderline forms) or to the ‘strict criteria’
(excluding borderline forms, as in WHO5), displays a similar level of association with time to pregnancy. However, a possible bias in this study
cannot be ruled out, because a given laboratory does not generally use
both approaches during routine semen analysis. Using the 5th percentile
value of normal spermatozoa in fertile men as a threshold, more than
one-third of unselected infertile men and half of TGCT patients were
below this threshold. No such discrimination was reported when using
the ‘strict criteria’ classification, for comparisons of fertile men and
selected infertile men (Guzick et al., 2001) or with use of the reported
4% threshold for morphologically normal sperm cells according to the
pregnancy rate in IUI (Deveneau et al., 2014).
The incidence of various sperm abnormalities or the resulting MAI
may be associated with the probability of pregnancy (Jouannet et al.,
1988; Slama et al., 2002; Buck-Louis et al., 2014). However, this does
not imply that a single category of abnormalities considered independently of other co-variables could serve as a powerful fertility prognostic
tool. Thus, none of the abnormality categories studied can be used in isolation, to discriminate, with high specificity and sensitivity, between the
male partners of pregnant women, and infertile men or testicular
cancer patients, as previously reported for other semen characteristics
(Ombelet et al., 1997; Björndahl, 2011). However, high frequencies of
some crucial categories of abnormalities may reflect overall changes to
the spermiogenic process, with functional consequences resulting in a
lower probability of natural conception within a short period of time
(Jouannet et al., 1988), or a lower rate of fertilization (Jeulin et al.,
1986). This should be borne in mind in the management of infertile
couples. Is it reasonable to determine the profile of all morphological abnormalities systematically in ART? Indeed, the standardized assessment
of sperm abnormalities requires attention to detail, rather than a simple
dichotomous classification of normal and abnormal spermatozoa. Training and quality control are required (Eustache and Auger, 2003). In our
experience, acceptable levels of variability can be achieved after an
initial period of theoretical and practical training no longer than that for
the evaluation of sperm motility. Quality controls (e.g. in Supplementary
data, Figure S1) indicate non-critical variations provided that regular
quality controls ensure the maintenance of competence (the episodic
discrepancies observed are no greater for the assessment of sperm
defects than for normal sperm assessments). A detailed standardized assessment of morphology abnormalities could be recommended for the
first andrology check-up for infertile couples, regardless of whether
ART is to be carried out. It may be useful if carried out in addition to
an andrological check-up in which medical history, lifestyle factors and
work environment are considered. In contrast, owing to the known stability of sperm morphological features, a detailed morphological assessment is not required in the subsequent follow-up or during ART.
However, if a factor (fever, medication, temporary exposure to toxicants
etc.) is thought to have influenced the sperm morphological profile, a
repeat assessment should be considered.
Towards a new quantification of sperm
abnormalities for clinical and research
purposes
We propose three levels of defects based on the percentiles for fertile
and infertile men: a low level ≤95th percentile for fertile men; an intermediate level .95th percentile for fertile men and ,99th percentile for
infertile men and a high level ≥99th percentile for infertile men. This
three-level stratification of morphological abnormalities is potentially
useful for studies considering genetic determinants or the role of environmental factors capable of disrupting sperm morphogenesis, as illustrated in Fig. 3. The low level of abnormality essentially corresponds
to non-specific background noise. In contrast, the HLA, which excludes
most fertile men and corresponds to extreme values in the infertile population, may be considered a biomarker of a causal genetic or environmental factor requiring investigation. Despite, the large overlap between
fertile and infertile men with intermediate levels of abnormality, the intermediate category should not be considered as a random signal in any of
the male populations studied (see Fig. 3E and F).
The potential of a standardized assessment
of sperm defects for epidemiological, genetic
and basic studies
A few studies have reported associations between the extent of various
sperm defects and pathological conditions, such as varicocele or cryptorchidism (Auger et al., 2001; Andrade-Rocha, 2007; Cakan et al.,
2008), environmental or occupational exposures (Bigelow et al., 1998;
Gebreegziabher et al., 2004; Hansen et al., 2010). However, the imprecise definition of sperm defects in most of these studies constitutes a
confounding factor. An interesting alternative approach for basic or
epidemiological studies is the combination of light microscopy with computer vision, which is highly accurate and precise, provided that preanalytical and analytical set-ups are optimized (Bellastella et al., 2010).
Several reports have demonstrated the relevance of this approach for
such studies (Schrader et al., 1990; Fenster et al., 1997). The measurement of various morphometric features of all sperm cells and the visual
estimation of well-characterized abnormalities may be complementary
approaches as recently reported (Buck Louis et al., 2014). However, it
should be pointed out that computer vision is mainly applied for the
measurement of a set of features mainly of the sperm head compartment, and not the midpiece and principal piece. In contrast, the visual approach, although not allowing precise and reproducible measurements
of the various sperm compartments, is more appropriate than computer
vision for pattern recognition of an entire sperm cell. Overall, the choice
of the method used depends on the laboratory equipment (only a minority of reproductive laboratories have analytical cytometry facilities) and of
the aims of the study (Auger, 2010, for a detailed comparison of the
advantages and limits of visual and computer-aided approaches). Only
rare morphological syndromes such as globozoospermia, macrocephalia, decapitated sperm syndrome and fibrous sheath dysplasia have
been described in men consulting for infertility, and several causal mutations have been identified (Coutton et al., 2015). However, these mutations do not account for all cases and the variability of clinical forms
observed suggests that other mutations or other genes may be involved.
Partial forms of these syndromes in which apparently normal spermatozoa coexist with those of the morphological type of interest are less rarely
22
observed. More generally, particular combinations of abnormalities with
high frequencies may reflect complex and variable genetic disorders. For
this reason, a detailed, standardized analysis of the sperm defects,
accounting for the respective percentages of each defect, may help to distinguish between phenotypes.
In conclusion, the sole assessment of the percentage of morphologically normal spermatozoa, as usually performed in infertile couples before
ART, and also in epidemiological studies, conceals the differential effects
of exposures or genetic determinants on head or tail compartments and
substructures, and on sperm fertilizing ability. Based on our results
showing noticeably different distributions of the sperm abnormalities
in fertile men, infertile men and testicular cancer patients, a standardized
assessment of morphological abnormalities of spermatozoa including the
use of a trichotomous categorization of abnormalities based on their
level of occurrence is proposed. It would be useful at a first andrological
check-up rather than during the routine management of infertile couples
by ART. This approach cannot provide information about possible functional consequences and it is clearly not dedicated to deciphering the
underlying modes of action of various pathological or environmental conditions on the sperm morphogenesis process. However, by improving
sperm phenotyping, it constitutes a useful step forward for clinical,
genetic and epidemiological studies.
Supplementary data
Supplementary data are available at http://humrep.oxfordjournals.org/.
Authors’ roles
J.A. and F.E. designed the study, verified all data collected and carried out
the data analysis. J.A., F.E. and P.J. interpreted the data, wrote the article
and prepared the tables and figures.
Funding
Part of this study was supported by contract BMH4-CT96-0314 from the
European Union.
Conflict of interest
None declared.
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