Applications
Note 190 | June 2008 - revised August 2012
Intracytoplasmic morphology selected sperm injection
(IMSI/BFS*) - an advanced technique for ICSI
Benjamin Bartoov, SPERMA LTD, Givat Shmuel, Israel
Ilka Schneider, Eppendorf AG, Hamburg, Germany
Abstract
In recent years, the techniques applied to help infertile couples having children of their own have improved considerably. Intracytoplasmic sperm injection (ICSI) has become a powerful means of overcoming male infertility in
patients. With the development of ICSI, the modern infertility lab typically includes micromani­pulation equipment
attached to inverted microscopes. A sophisticated advancement to ICSI is IMSI, intracytoplasmic morphologically
selected sperm injection, a method described by Bartoov et al. [1]. Prior to sperm injection the morphology of the
sperm cell is evaluated with high magnification DIC microscopy. Preceding studies have already demonstrated the
advantage of IMSI over the conventional IVF-ICSI procedure in terms of the pregnancy rate.
Introduction
Figure 1: Motile sperms with morphologically normal nuclear
shape but large nuclear vacuole.
* Bartoov Fertility system
It has been estimated that approximately 40 % of sterility
in couples can be attributed to male subfertility. ICSI
(Intracytoplasmic sperm injection) has raised hopes that
these couples can have children of their own. This method
of treating predominantly male-factor patients has achieved a breakthrough [2], and it has established itself as the
preferred method of treatment in the field of assisted reproduction. When ICSI is used in the routine way, one sperm
is taken from the sperm pool after routine selection under
a regular microscope that magnifies 200 to 400 times. The
birth rate resulting after ICSI treatment ranges between
10-30 % [3]. Sperm morphology has been recognized to be
a crucial factor for the efficiency rate of fertilization, pregnancy and birth rate [1;4]. Examination of spermatozoa with
the light microscopy can provide only limited information
on their internal structure. Using conventional magnification, only sperm morphology can be visualized, but not the
sperm nuclear abnormality. To overcome this drawback,
Bartoov et al. introduced a modified ICSI procedure: IMSI,
or ‘intracytoplasmic morphologically selected sperm injection’,
Application Note 190 | page 2
which is based on microinjection into retrieved oocytes of
spermatozoa with strictly defined morphologically normal
nuclei. These sperm are selected by MSOME, a method
of unstained, realtime, high magnification ‘motile sperm
organellar morphology examination’ [1], see Figure 1. The
optimized IMSI treatment does, in fact, result in significantly higher pregnancy rates of 66% versus 30%, compared
with conventional IVF-ICSI [5]. A great advantage of the
Eppendorf manipulators for this special procedure lies
within the small step resolution of 40 nm and the storage of
three working positions, thus enabling a sophisticated working under high magnification with sure instinct.
Materials and Methods
Devices (a typical workstation is shown in Figure 2)
• Inverted microscope Olympus IX71/81 equipped with
high power DIC (Nomarski/DIC) optics and Uplan Apo ×100
and × 40 oil/1.35 objective lens (Olympus, Germany)
• 2 Micromanipulators TransferMan NK 2 (Eppendorf AG,
Germany)
• Adapter for inverted microscope (Eppendorf AG,
Germany)
• 2 manual microinjectors CellTram Air and Oil (Eppendorf
AG, Germany)
• Video system, either analog or digital system
o 1. Analog system: ½ inch 3 × CCD color video camera
equipped with high resolution monitor, zoom ability of 45×.
o 2. Digital system: high quality digital camera + computer
with digital monitor as described above.
Consumables and media
• WillCo dish (Willco wells BV, Amsterdam, The Netherlands)
• IVF 4 Well Dish (Nunc, Hereford, UK)
• Sterile paraffin oil (OVOIL-100; Vitrolife, Kungsbacka, Sweden)
• Universal IVF or ISM1 medium (MediCult, Jyllinge, Denmark)
• Sperm preparation Medium (MediCult, Jyllinge, Denmark)
• Mineral oil (MediCult, Jyllinge, Denmark)
• PVP solution (ICSI-100, Vitrolife, Kungsbacka, Sweden)
• Sil-Select Plus (FertiPro N.V., Beernem, Belgium)
• EBSS (Ghent, Belgium)
Sperm preparation
The pre-selection procedure of motile high density spermatozoa for IMSI is performed on the basis of a two-layer
Sil Select density gradient system which consists of 1 ml
upper (low density) and 1 ml lower (high density) layers of
Figure 2: ICSI workstation
Eppendorf workstation on an Olympus IX81.
saline-coated colloidal silica particles suspended in HEPESbuffered Earle’s balanced salt solution (EBSS). The obtained
motile high density sperm fractions are used for further
MSOME preparation [5;6].
MSOME
The examination is performed in real time using high power
Nomarski optics enhanced by digital imaging to achieve
a magnification exceeding 6000 x. It has to be stressed
that preferably motile sperm are used for this examination, who under low light-microscopic magnification have a
high potential to be selected by routinely performed ICSI
(for the IMSI procedure sperm with poor motility can also
be selected, provided that they have a normal nucleus).
About 4000 motile high density sperm cells, obtained from
the above fraction, are transferred to a 4 µl observation
microdroplet of sperm preparation medium containing PVP
solution. In order to estimate the morphological state of the
sperm nucleus, one has to follow the motile sperm cell by
moving the microscopic stage in the x, y and z directions
for about 20 seconds. To reduce PVP toxicity, which has to
be used to slow down highly motile sperm from disappearing from the monitor screen, the concentration of the PVP
is adjusted to a minimum (range 0 – 8 %). The observation
microdroplet is placed in a sterile, glass-bottomed dish
under sterile paraffin oil. The sperm cells, suspended in the
observation microdroplet, were used for individual retrieval
by MSOME.
Application Note 190 | page 3
The criteria for a normally shaped nucleus by MSOME are
smooth, symmetric and oval configurations (previously defined by scanning electron microscopy [1;5;7]). For MSOME,
the average length and width of this configuration were
estimated in 100 spermatozoa with an obviously normal
nuclear shape [8]. During sperm selection, a fixed, celluloid
form of a sperm nucleus representing the normal criteria is
superimposed on the examined cell; any sperm cell with a
nuclear shape has to be excluded from selection if it varies
in length or width by 2 SDs from the normal mean axes
values. Also, all sperm with more than one vacuole or with
vacuoles occupying more than 4 % of nuclear area have to
be discarded [7].
Sperm retrieval after MSOME
MSOME selected sperm cells are retrieved from the observation droplet and placed into a recipient selection droplet, containing 4 μl of sperm preparation medium in the same petri
dish. This procedure is performed using the Eppendorf TransferMan NK 2 Micromanipulation System, which is equipped
with a sterilized, non-angulated, glass microcapillary with a
12 µm inner diameter and 45° bevelled, non-spiked tip.
Prior to the experiment the microcapillary has to be fitted,
aligned and equili­brated before starting the MSOME procedure. It is also necessary to prime micropipettes with medium
before use so that the selected sperm and later on the
retrieved oocytes never come into contact with air or oil.
The glass microcapillary was inserted from above in a z-axis
movement into the observation droplet. Since the TransferMan NK 2 can store up to 3 positions, one position is set as
a parking position above the droplet and another one serves
as the actual working posi­tion inside the droplet in the focal
plane of the sperms next to the bottom of the petri dish. By
pressing the joystick button twice (double-click) the capillary
can be returned to a preset position. The capillary can be
moved easily in 40 nm resolution steps in any direction (x/y/z)
by means of a single joystick. This feature enables the fast
and precise retrieval of the highly morphologically qualified
sperm cells visualized on the monitor.
Microinjection
The retrieved, cumulus-free ova were placed into drops of
sperm preparation medium prepared in the same glass dish
with the recipient droplet. The latter contained the sperm cells
morphologically selected for ICSI. A schematic overview of
the prepared petri dish is shown in Figure 3.
Figure 3: Petri dish prepared for IMSI procedure.
Red circle: droplet with unselected sperm, retrieved via pre-selection procedure of motile high density spermatozoa on the basis of
a two-layer Sil Select density gradient system; pink circle: recipient
droplet for selected spermatozoa; blue circles: droplets with retrieved oocytes for sperm injection; purple droplet: additional PVP.
The microinjection procedure is performed according to
standard procedures at the usual magnification of 200
– 400x [9]. A typical injection is demonstrated in Figure 4.
Each microinjected oocyte is immediately transferred to a
4-well dish, incubated in 0.5 ml of universal IVF or ISM1
medium and covered with 0.5 ml of mineral oil at 37 °C with
an atmosphere of 5 % CO2.
Figure 4: The spermatozoa is deposited towards the center of
the aspirated oocyte.
Application Note 190 | page 4
Results and Discussion
In our studies [1;8;10], we came to the conclusion that the
MSOME criteria for normally shaped nuclei are smooth,
symmetric and oval configuration, with average length and
width limits and a homogeneity of the nuclear chromatin
mass, with no regional nuclear disorders, and containing no
more than one vacuole, which occupies no more than 4% of
nuclear area. During selection, a fixed, transparent, celluloid
form of a sperm nucleus fitting the normal nuclear shape is
superimposed on each examined cell. Spermatozoa which
vary in length or width by two standard deviations from the
normal mean axes values have to be considered as abnormally shaped cells. In order to investigate the homogeneity
of the chromatin mass, we are using the front and side
Figure 5: The spermatozoa are captured in small bays extruding
from the rim of the observation droplets.
views of the differential interference contrast (DIC) optics,
thus being able to distinguish between the topography of
the chromatin vacuoles, which are always surrounded by a
smooth chromatin mass, and the topography of the regional
disorders of the nucleus, which look like craters or extrusions.
No other observation methods, including electron microscopy, can do so, since only MSOME is performed in real time
on motile spermatozoa, which, during observation, change
position (see Figure 5).
Thus, sperm motility is a must at least for the precise detection of nuclear content. In order to immobilize and transfer
the selected motile sperm, fast, but ultra-fine resolution
movement of the transfer capillary and the possibility to
store working and storage position is a prerequisite, especially when working under thus high magnification condition.
We try to collect two highly morphologically selected sperm
cells for each ova expected to be retrieved. It could be
that in the male partner’s semen we will not be able to find
morphologically qualified sperm cells, and then we select
‘second best’. We could show [8;10] that in comparison to
ICSI performed with spermatozoa selected by conventional
techniques (microscope magnification of 200–400 times),
our new method exhibits a higher pregnancy rate per cycle
(66 % versus 30 %) and a significantly lower abortion rate
per pregnancy (9 % abortion rate versus 33 %).
In conclusion, microinjection of sperm with abnormal shape
or nuclear vacuoles appears to reduce pregnancy outcome.
This drawback can be prevented by morphological sperm
selection based on MSOME. To this date, some 500 babies
have been born in Israel after IMSI treatment, some 200
more in Europe.
Application Note 190 | page 5
Literature
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Bartoov B, Berkovitz A, Eltes F, Kogosowski A, Menezo Y, Barak Y. Real-time fine
morphology of motile human sperm cells is associated with IVF-ICSI outcome. J Androl 2002; 23(1):1-8.
Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single
spermatozoon into an oocyte. Lancet 1992 ; 340(8810):17-8.
Nyboe Andersen A, Goossens V, Gianaroli L, Felberbaum R, de Mouzon J, Nygren KG.
Assisted reproductive technology in Europe, 2003. Results generated from European
registers by ESHRE. Human Reproduction 2007; 22(6): 1513–1525.
Bartoov B, Eltes F, Pansky M, Langzam J, Reichart M, Soffer Y. Improved diagnosis of male fertility potential via
a combination of quantitative ultramorphology and routine semen analyses. Hum Reprod 1994; 9(11):2069-75.
Bartoov B, Berkovitz A, Eltes F, Kogosovsky A, Yagoda A, Lederman H, et al. Pregnancy rates are higher with
intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection.
Fertil Steril 2003; 80(6):1413-9.
Berkovitz A, Eltes F, Yaari S, Katz N, Barr I, Fishman A, et al. The morphological normalcy of the sperm nucleus
and pregnancy rate of intracytoplasmic injection with morphologically selected sperm.
Hum Reprod 2005;20(1):185-90.
Bartoov B, Fisher J, Eltes F, Langzam J, Lunenfeld B. A comparative morphological analysis of abnormal
human spermatozoa. In: Insler V, Bettendorf G, eds. Advances in Diagnosis and Treatment of Infertility.
Amsterdam: Elsevier; 1981:355–373.
Berkovitz A, Eltes F, Yaari S, Katz N, Barr I, Fishman A, et al. The morphological normalcy of the sperm nucleus
and pregnancy rate of intracytoplasmic injection with morphologically selected sperm.
Hum Reprod 2005;20(1):185-90.
Andrulat H, Voss S. Intracytoplasmic Sperm Injection (ICSI) - Procedure and equipment.
Eppendorf Application Note 09, June 2006.
Berkovitz A, Eltes F, Ellenbogen A, Peer S, Feldberg D, Bartoov B. Does the presence of nuclear vacuoles
in human sperm selected for ICSI affect pregnancy outcome? Hum Reprod 2006;21(7):1787-90.
More information (including how to become a BFS* certified specialist) can also be found on
http://www.bartoov-sperma.com/eng/eng_home.html and www.eppendorf.com/imsi
* Bartoov Fertility system
Application Note 190
Ordering Information
Description
TransferMan® NK 2 *
Proportional micromanipulator for
suspension cells and microdissection
Manual pressure device for the reliable
holding of suspended cells (e.g. oocytes)
Manual pressure device for the reliable
holding of suspended cells (e.g. oocytes)
25 glass capillaries for injecting sperm
into an oocyte (ICSI) rigid parallel flange,
sterilized, tip angle 35°
25 glass capillaries for injecting sperm into
an oocyte (ICSI) flexible flange, sterilized, tip
angle 35°
25 glass capillaries for injecting sperm into
an oocyte (ICSI) rigid flange, sterilized, tip
angle 35°
25 glass capillaries for holding large cells
(e.g. oocytes) sterili­zed, tip angle 35°
Adapter for any inverse microscopes
CellTram® Air *
CellTram® Oil *
TransferTip®-RP * and **
TransferTip®-F * and **
TransferTip®-R * and **
VacuTip™ * and **
Microscope adapter
Order no.
international
5188 000.012
Order no.
North America
920000011
5176 000.017
920002021
5176 000.025
920002030
5175 114.000
930001074
5175 106.008
930001031
5175 113.004
930001066
5175 108.000
930001015
available on request
WillCo dish is a registered trademark of Willco wells BV, Amsterdam, The Netherlands.
ISM1 medium is a trademark of MediCult, Jyllinge, Denmark.
ICSI-100 is a trademark of Vitrolife, Kungsbacka, Sweden.
* This product is registered in Europe as a medical device (according to Medical Device Directive MDD/93/42/EDD). This
product is not registered in the U.S. as a medical device and does not have a 510(k) registration. For resarch use only. Not
for use in human medical applications.
** Proven non-cytotoxicity by the mouse embryo development test
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