Introduction
Despite the fact that intracytoplasmic sperm injection
(ICSI) allows successful fertilization in virtually
all cases of male subfertility it is evident that
ICSI is one of the most unphysiological methods
of assisted reproductive technologies (ART). It is
not only that major processes of in vivo fertilization
are circumvented, but ICSI also changes the
physiological conditions.
Firstly, incorporation of the unreacted acrosome
into the oocyte during ICSI could be potentially
hazardous to embryo development since there is an
increased risk to the formation of vacuoles in the
female gamete due to acrosomal enzymes (1, 2).
Additionally, in a mouse model Ca2+ responses significantly
differed in terms of duration, frequency
and amplitude between IVF and ICSI (3). Last but
not least, a dramatic change in gene expression has
been reported in ICSI as compared to the in vivo
situation in the mouse. Mostly, genes related to cell
function and development were found to be up- or
down regulated (4).
Apart from these physiological abnormalities
clinical embryologists applying routine sperm selection
criteria at ×400 magnification take a higher
risk of selecting male gametes defective incentrosomal
integrity (5, 6), genetical constitution (7),
phospholipase C zeta content (8), protamine ratio
(9) and/or DNA-methylation (10).
To avoid this scenario or at least to reduce the
potential effects of ICSI using suboptimal sperm,
every effort must be taken to deselect abnormal
and to accumulate good prognosis spermatozoa
(e.g., by applying particular sperm processing
methods). This quest for optimized male gamete
selection has been summarized as physiological
ICSI (11, 12).
In principle, four different parameters of sperm
morphology are conceivable that would allow to identification of physiological sperms: i. DNAintegrity
(13, 14), ii. Birefringence (15, 16), iii.
Maturity (17) and iv. High-resolution morphology
(18, 19).
The problem with DNA-strand break testing is
that it is almost impossible to do on living sperm
cells (20, 21). To put it differently, most current
tests irreversibly damage the sperm analyzed, thus,
it will not usable in ICSI. Polarization microscopy
for evaluating the highly ordered filaments of the
head and neck region requires special changes in
the setup of polarization microscopes which are
not manageable at all centers. Lastly, it seems that
checking hyaluronic receptor bonding capacity as
a major characteristic of sperm maturity is strongly
dependent on laboratory setup such as temperature
control.
Therefore, it is hardly surprising that ICSI at
very high magnification (at least ×6000) reflects
the majority of research on this matter in literature.
Intracytoplasmic morphologically selected sperm
injection (IMSI)
To overcome the limits of conventional microscopy,
Bartoovet et al. developed a method of unstained,
real-time, high magnification examination
of spermatozoa. This particular high magnification
scoring of spermatozoa is known under the term
motile sperm organelle morphology examination
(MSOME) and exclusively deals with the presence,
size, number, and location of vacuoles. Per
definition, MSOME criteria consider nuclear chromatin
content to be abnormal if the sperm head
contains one or more vacuoles (diameter of 0.78 ±
0.18 ìm) occupying more than 4% of the normal
nuclear area (22-24). It turned out that MSOME
represents reliable criteria as sequential analyses
of sperm samples from the same patients produced
similar results in terms of normal morphology and
presence of large vacuoles (25).
For the ease of adequate scoring, the MSOME
criteria have been used by several working groups
(26) to subgroup sperms into different classes
which helped to simplify statistics. In short, grade
I spermatozoa exclusively consisted of sperms
showing normal sperm head and absence of vacuoles,
thus, representing the optimal type. Grade II
gametes were made up of sperms showing a maximum
of two small vacuoles. Grade III spermatozoa
were characterized by the presence of more
than two small vacuoles or at least one large vacuole.
The worst grade IV showed large vacuoles in
consumption with head shape problems and other
abnormalities. Others used a similar model to classify
abnormal spermatozoa by their degree of vacuolization
(27). Cassuto et al. (28) introduced the
so-called HAVBIC criteria based on the detailed
analysis of head, acrosome, vacuoles, basis of the
sperm head, insertion which is the axial position of
the tail, and the presence of a cytoplasmic droplet.
However, MSOME examination usually is performed
utilizing an inverted light microscope
equipped with high-power Nomarski optic enhanced
by digital imaging to achieve a magnification
of up to ×6300.
Injection of spermatozoa selected by the above
mentioned MSOME criteria culminated in a modified
ICSI technique called IMSI (29). Its introduction
in the field of ART definitely facilitated the
observation of live human spermatozoa, particularly
by showing sperm vacuoles not necessarily
seen at lower magnification, prior to injection in
the oocyte. It soon turned out that precise morphological
integrity of the human sperm nucleus is an
important parameter associated with pregnancy
rate (29, 30). In detail, the inventors of the IMSI
technique showed a significantly increased pregnancy
rate in IMSI (66%) as compared to routine
ICSI (30%). The associated implantation rate was
even the 3-fold (9.5 vs. 27.9%). In case that no optimal
sperm was available for IMSI an increase in
abortion rate from 10 to 57% was described (30).
Of course it has to be kept in mind that early
research in this field has more or less been performed
by one working group, thus, a potential
bias in their interpretation of the data cannot be
excluded (e.g., an abortion rate as high as 57% is
incredibly high).
Where do vacuoles come from?
IMSI is a rather time-consuming procedure since
selecting enough morphologically normal spermatozoa
for injection according to the above mentioned
MSOME criteria may take up to 2 hours
(31). It has been suspected that this prolonged process
of searching for spermatozoa at high magnification
might damage the male gametes since it
had been observed that after 2 hours on the micro scope’s heated stage sperm nucleus vacuolization
showed a significant increase (32).
Recently, Neyer et al. (33) nicely highlighted
that sperm head vacuoles are rather not affected
by in vitro conditions as analyzed by a system of
microchannels. This sperm-microcapture area allowed
observation of the same living human spermatozoon
over a period of 24 hours. In a series of
experiments it was demonstrated that neither temperature
nor oxidative stress led to the formation
of de novo vacuoles. On the other hand, induction
of the acrosome reaction using the ionophore
A23587 did not lead to disappearance of vacuoles
(33). Recently, it was shown that freezing-thawing
procedures also did not influence the relative vacuole
area in the anterior, medial or basal part of the
sperm head (34).
Based on these data it appears evident that vacuoles
are structures being pre-existing and their
number cannot be altered by in vivo conditions.
This is in line with data from a Japanese group
(35) who found that the distribution of vacuoles in
ejaculated (98.3%), epididymal (87.5%), and testicular
sperm (87.5%) and in spermatids (33.7%)
was directly correlated to maturational stage of
the germ cell. In other words, the closer a sperm
gets to ejaculation, the higher is its risk to bear one
or more vacuoles. It seems that particularly the
time of acrosome reaction is critical. Kacem et al.
(36) reported that of all acrosome-reacted sperm
analyzed 70.9% were free of vacuoles, whereas in
those sperms showing incomplete acrosome reaction
or an intact acrosome at all the corresponding
percentage was only 39.3%. This strongly suggests
that IMSI selects acrosome-reacted spermatozoa.
And it is in line with Montjean et al. (37)
who found that sperm vacuoles are associated with
acrosomal and capacitation status, that is to say,
they appear to be a reflection of normal sperm
physiology. However, at least in globozoospermia
a non-acrosomal origin of vacuoles is discussed
(38).
Where are vacuoles located?
It seems that the actual location of all types of
vacuoles is random. Information on the actually
preferred site of vacuoles is scarce. Tanaka and
co-workers (35) calculated that >60% of all vacuoles
are found in the acrosomal region which approximately
represents the anterior two thirds of
the sperm head. Dividing the sperm head area into
three sections, 40.9% of normal-shaped sperm had
vacuoles in the tip, 74.9% in the middle region
and only 4.3% in the posterior area. Irrespective
of their location, a total of 92.8% of vacuoles were
small and only 4.6% were large (39). In a paper
of Perdrix et al. (40) 38.0 ± 5.10% of motile spermatozoa
obtained after gradient density centrifugation
showed a large vacuole occupying >13%
of the total sperm head area. Again these vacuoles
could mainly be detected in the anterior (45.7 ±
2.9%) and median sperm head (46.1 ± 3.0%). This
statement is somewhat in contrast to the transmission
electron microscopy (TEM) data from the
same study which indicated that the large vacuoles
were exclusively present in the nucleus. This
could be due to an incomplete reconstruction of all
TEM sections since a maximum of 1.5μm (70 nm
sections ×20 cuts) of the sperm diameter was depicted.
The total dimension in the posterior region
would be around 3μm (41). Alternatively, this is a
problem with all the vacuolization data presented
so far, interpretation of the images was based on
the assumption that vacuoles in sperm have the
same background as vacuoles found in oocytes,
e.g., being cavities within the cytoplasm (42).
More recent work applying sophisticated technologies
such as three-dimensional deconvolution
and atomic force microscopy clearly showed that
in "all vacuolated spermatozoa the acrosome was
intact, the plasma membrane was sunken but intact
and the large vacuole was identified as an abnormal,
thumbprint-like nuclear concavity covered by
acrosomal and plasmic membranes" (41, 43).
Clinical consequences of vacuoles
There is a relative heterogeneity between semen
samples, so that the frequency by which good
spermatozoa can be selected varies greatly among
patients. De Vos et al. (44) demonstrated that only
5/350 (1.4%) male factor patients have to face
the problem that no sperms better than grade III
(26) are available for injection. This automatically
means that at least in some poor prognosis patients
only suboptimal sperm can be injected.
Things get clearer if the attention is drawn to
a model in which actively damaged spermatozoa
were used (45-48). This approach exclusively ended
up with normal fertilization and cleavage, while
blastomere number on day 3 (48) and blastocyst development (45, 46) were very much related to
the degree of damage. Although the above cited
colleagues actively damaged the DNA of the spermatozoa,
rather than injecting vacuolized sperms,
there is evidence that a possible negative paternal
effect on in vitro development will not develop
before embryonic genome expression. This would
explain why no studies are available indicating a
correlation between sperm vacuolization and fertilization
rate (26, 44, 49, 50). In addition, no advantage
of IMSI over ICSI was seen in terms of day 2
morphology (51).
Vanderzwalmen et al. (26) reported identical
numbers of zygotes and developmental rates up to
day 3 between IMSI and ICSI irrespective of the
fact whether grade I or IV sperms were injected.
However, blastocyst formation was found to be superior
in IMSI. The worse the grade of the injected
spermatozoon was, the lower was blastulation
(56.3, 61.4, 5.1, 0%). Blastulation was also significantly
increased in IMSI as compared to ICSI in
the paper of Knez et al. (49). Interestingly, time
lapse analysis revealed that blastocysts from grade
I spermatozoa required the shortest mean time for
all developmental events as compared with blastocysts
from spermatozoa of other classes showing
vacuoles (52). In some developmental phases
there was a 10h-lag between embryos from grade I
and IV which led to the observation that only early
blastocyst could be seen on day 5 if grade IV spermatozoa
were used for ICSI.
The higher availability of blastocysts for transfer
could be one reason for the observed increase in
pregnancy rate. Apart from the manuscripts discussed
above, numerous authors have suggested
an advantage of IMSI over ICSI in different patient
cohorts (53). In a group of 125 couples (54),
IMSI improved clinical outcomes (implantation,
pregnancy) without affecting biological outcomes
(fertilization and cleavage rates, embryo morphology).
Since live-birth rate represents the ultimate success
of ART, IMSI studies with this parameter as
primary outcome are of particular interest. Recently,
Greco et al. (50) compared IMSI with good
prognosis (class I and II) and bad prognosis sperm
(class II and IV). In this publication the "late" outcomes
like implantation (23.1 vs. 7.0%), clinical
pregnancy (41.7 vs. 17.1%), and live-birth rate
(36.7 vs. 14.3%) were statistically significantly
higher in the patients with better MSOME quality
as compared to the bad prognosis counterpart.
Others (55-57) confirmed a significant trend towards
a higher live-birth rate with IMSI (21-38 vs.
12-20%).
There is evidence that IMSI favors injection of
genetically normal spermatozoa (58). In this article
major malformations were significantly reduced in
IMSI newborns versus ICSI ones (1.3 vs. 3.8%).
This could be associated with an increased incidence
for sex chromosome aneuploidy and chaotic
embryos in ICSI but not in IMSI cycles (59). A
possible impact of IMSI on gender of the offspring
is still under discussion (60, 61).
How do vacuoles affect performance of the
sperm?
With the above mentioned data in mind, it seems
to be very likely that IMSI based on MSOME (29),
HAVBIC (28) or Vanderzwalmen criteria (26) deselects
spermatozoa of reduced potential.
Indeed, Garolla et al. (61) analyzed mitochondrial
function and aneuploidy rate in preselected
sperms and claimed better results in these MSOME
sperms as compared to an unselected counterpart.
The aneuploidy part of the study was later confirmed
by another working group (40). However,
it is evident that IMSI does not decrease the aneuploidy
rate in patients who are heterozygous for
reciprocal translocations (62). In addition, sperm
maturity as assessed as hyaluronic acid binding
and sperm nucleus normalcy rate seem to go hand
in hand (63).
Beyond that it was emphasized that strand breaks
occur more frequently in spermatozoa with large
nuclear vacuoles (64). Numerous papers jumped
on the bandwagon and stated that DNA integrity
and sperm head vacuolization are negatively correlated
(65-68).
More recently, a growing body of colleagues did
not support the said correlation between the presence
of sperm head vacuoles and other sperm parameters
(69-71). One explanation for this divergence
could be the fact that the vast majority of
studies analyzed DNA fragmentation and sperm
vacuolization in two different populations of the
same ejaculate not allowing for proper prediction.
As far as known, there are only few studies fulfilling
the requirement of performing different methods on the same spermatozoon. This is the only approach
to ensure insights on whether a vacuolized
spermatozoon reflects other abnormalities (DNA
integrity, maturity, chromatin status, aneuploidy)
as well.
Those that did fulfill this prerequisite did not find
any correlation between vacuolization and strand
breaks or aneuploidy (39, 41). The comparable incidence
of structural DNA breaks in sperms with
or without a large vacuole was 9.1 and 4.4%, respectively
(41). These results strongly indicate that
DNA damage is not responsible for or associated
with sperm head vacuoles.
Since it is clear that small as well as large
vacuoles are in fact indentations of the sperm
head (41, 43) chromatin displacement and failure
in chromatin condensation is a threatening
consequence which in turn could cause epigenetic
effects. This unique finding places special
emphasis on those publications that noted an association
between vacuoles and chromatin status
(40, 41, 61).
Prospective comparison between ICSI and IMSI
Certainly there was a hype about IMSI in the
past. This method was chosen to overcome problems
related to oocyte quality (72), maternal age
(73), and complete developmental arrest (49).
In particular at the beginning of the IMSI era, it
seemed that IMSI was the solution for all problems;
however, the more information was gathered
on the nature of vacuoles and their possible impact
on sperm physiology the less publications could
be placed.
This declining interest in IMSI is reflected in two
meta-analyses. The first one of Souza Setti et al.
(74) postulated higher rates of implantation [odds
ratio (OR: 2.72)] and pregnancy rates (OR: 3.12),
but a lower miscarriage rate (OR: 0.42) in IMSI
cases as compared with ICSI cases. The problem
with this meta-analysis, however, is that it was
only based on a limited number of three prospective
trials (29-31).
A more recent meta-analysis, a Cochrane Review
(75), based on nine prospective randomized
controlled trials did not support previous results.
In detail, as many as 1002 IMSI and 1012 ICSI
cycles were compared. Neither live birth [risk ratio
(RR: 1.14) ] nor abortion rate (RR: 0.82) could be
altered by the IMSI technique. None of the studies
included reported congenital abnormalities. Incredibly,
the fact that clinical pregnancy rate was
significantly increased (RR: 1.29) did not keep the
authors from downgrading the quality of this evidence
because of imprecision, inconsistency, and
strong indication of publication bias.
A recent sibling-oocyte study on more than
3,000 oocytes compared conventional ICSI with
a sperm selection method using higher magnification.
No differences in oocyte fertilization rate or
in embryo quality were observed. Also, the clinical
pregnancy rate and the implantation rate per embryo
transferred were similar for IMSI-only and
ICSI-only transfers; thus, these data do not support
any benefit of IMSI in a non-selected population
with fresh ejaculated sperm containing ≥1 million/
ml (44). IMSI in another prospective randomized
trial (76) did not show a significant improvement
in the clinical outcome compared with ICSI although
the authors found trends for higher implantation
(28.9 vs. 19.5%), clinical pregnancy (54.0
vs. 44.4%) and live birth rates (43.7 vs. 38.3%) in
the IMSI group.
Conclusion
With no clear consensus regarding the effect
of IMSI on implantation or pregnancy rates,
IMSI is most likely a procedure in ART to be
reserved for specific cases (77). Even if IMSI
is chosen as the method of choice, in the vast
majority of cases several sperms of good morphology
(e.g., grade I and II) will be collected
and used for injection. Thus, the question arises
when to apply IMSI at all.
There is evidence that inflationary routine application
of MSOME criteria in order to select
the best spermatozoon is not required (78). In
255 couples attempting their first ART attempt
for male infertility a prospective randomized
trial was performed to compare the clinical
outcomes of IMSI and ICSI and to evaluate
the influence of sperm characteristics on these
outcomes. It turned out that the results of IMSI
were similar to the ICSI ones whatever the degree
of sperm DNA fragmentation, nuclear immaturity
and sperm morphology. These results
show that IMSI instead of ICSI has no advantage
in the first ART attempts (79), thus supporting the results of Balaban et al. (76) and De Vos et
al. (44).
However, in a subsequent cycle to a failed ICSI,
pregnancy and delivery rates were significantly
higher for patients deciding to switch to the IMSI
technique as compared with patients staying with
ICSI (80). In an additional prospective study in
which couples acted as their own controls, 75 infertile
couples were offered IMSI after at least two
previous treatment failures (81). IMSI seemed to
give better embryo quality and more blastocysts,
which allow more embryo transfers at the blastocyst
stage and consequently an increased pregnancy
rate.
In contrast to a recent review of Boitrelle et al.
(82), El Khattabi et al. (57) reported identical live
birth rates in IMSI (21%) and ICSI (22%) in cases
with repeated implantation failure. Yet, another
subgroup with male factor infertility benefitted
from the IMSI technique since there was a significant
improvement in live birth rate (38 vs. 20%).
These findings are in line with those reported by
others (52, 76).
In cases of severe male factor infertility, such
as patients with high sperm DNA fragmentation
rates, selection of normal spermatozoa with a
vacuole-free head using IMSI yields the greatest
likelihood of obtaining pregnancies. Successful
pregnancy and healthy childbirth were
also obtained in a case of total globozoospermia
after MSOME/IMSI without assisted oocyte activation
(83). Despite conflicting published results
teratozoospermia is the preferential indication
for MSOME and IMSI, but this has to be
confirmed in future studies.