Improved Isolation of Sperms from Crude Testicular Sperm Extracts (TESE)
Markus Montag, Department of Gynecological Endocrinology & Reproductive Medicine, University Clinics of
Bonn, Germany
Abstract
A major reason for infertility is reduced sperm quality. In the majority of patients with impaired sperm parameters,
intracytoplasmic sperm injection can overcome the underlying problem, provided that motile spermatozoa are
present in the ejaculate. In cases of azoospermia, sperm may be retrieved from testicular biopsy material. This
application note focuses on the requirements and procedures related to testicular sperm extraction and injection.
Introduction
The introduction of intracytoplasmic sperm injection (ICSI)
in 1992 was a breakthrough in the treatment of male infertility
[1]. Men with only few spermatozoa in the ejaculate
could father a child following the injection of a single viable
sperm into an oocyte and subsequent embryo transfer. In
1995, ICSI was also applied to men presenting with azoospermia
[2]. These men have no spermatozoa in the ejaculate
at all, either due to an obstruction of the vas deferens
(obstructive azoospermia) or due to impaired testicular
sperm production where only some areas within the testis
exhibit a focal spermiogenesis (non-obstructive azoospermia).
In these patients testicular tissue can be surgically
retrieved and spermatozoa present in the tissue can be prepared
by different methods for subsequent sperm injection.
Usually the testicular tissue itself or the sperm suspension
derived from a testicular tissue preparation can be divided
into several aliquots and cryopreserved for later use. Thus
the stimulation of the woman may proceed once the presence
of sperm in the testis is confirmed and thus helps
to avoid an unnecessary hormonal stimulation in case of
unsuccessful sperm retrieval.
Approximately 10 % of all cycles where ICSI is applied
are performed with spermatozoa retrieved from the testis.
Although the injection procedure itself is identical to the
one in routine ICSI with ejaculated sperm, the isolation
of spermatozoa from the testicular tissue preparation is
dependent on the number and quality of sperm present in
the preparation. As testicular sperm are either immotile or
present only with local motility, one must actively search
for the sperm in a specially prepared dish. Compared to
standard ICSI, TESE requires additional manipulation steps
like the search for spermatozoa, the identification of sperm
viability and the successful retrieval of sperm from the tissue
preparation. Thus it greatly benefits from the proper
instrumentation which makes the procedure as economic
as possible.
Eppendorf offers a number of solutions which assist in the
successful and easy isolation of testicular spermatozoa
– from the micromanipulator with custom defined capillary
positions up to special capillaries designed for efficient
retrieval of sperm from even crude tissue preparations for
subsequent injection.
Materials and Methods
Compared to standard ICSI, TESE requires additional manipulation
steps like the search for spermatozoa, the identification
of sperm viability and the successful retrieval of
sperm from the tissue preparation. Thus it greatly benefits
from the proper instrumentation which makes the procedure
as economic as possible.
Devices
- Inverted microscope with a heated plate and Hoffmann
contrast objectives
- Laser system (OCTAX Laser Shot™ System, MTG, Germany)
- 2 TransferMan® NK 2 micromanipulators (Eppendorf,
Germany)
- CellTram® Air microinjector for holding the embryo,(Eppendorf, Germany)
- CellTram® vario microinjector for selection and transfer of
sperms
Consumables and media
- Light mineral oil (e.g., M-8410; Sigma-Aldrich, Munich,
Germany)
- Shallow Petri dishes, tissue-culture-grade
- IMSI/TESE Tip (transfer capillaries; Eppendorf, Germany)
- VacuTips (holding capillaries; Eppendorf, Germany)
- TransferTips® (ICSI) (injection capillaries; Eppendorf,
Germany)
- Culture media (HEPES-buffered, supplemented with
antibiotics, protein and pyruvate, e.g. Gamete buffer;
COOK IRELAND LTD., Ireland)
- ICSI media (culture medium supplemented with PVP, e.g.
Sperm medium; COOK IRELAND LTD., Ireland)
Microinjection dish preparation
It is essential to heat all media and oil to 37 °C prior to use.
Place one droplet of PVP (5-10 μ L) in the center of a Petri
dish and several droplets of buffered medium (5–10 μ L) for the
oocytes. Three to four larger droplets (50 μ L) will be needed
for the testicular preparation from which the spermatozoa are
retrieved. Then the droplets are completely covered with light
mineral oil to maintain the stability of droplets as well as the
temperature and osmolarity.
Once prepared, and dependent on the buffered medium used,
the microinjection dish may be placed into the incubator or
onto a warming plate until required.
Immediately after thawing of the testicular tissue preparation,
aliquots should be pipetted at different dilutions into the large
(50 μ L) droplets of the microinjection dish and the spermatozoa
should be allowed to settle for another 5-10 minutes. In
cases with immotile spermatozoa in the preparation, a preincubation
of the loaded dish for another 1-2 hours could be
helpful to support that some sperm become motile.
Preparation of the microinjection capillaries and storing of
positions
The IMSI/TESE Tip microcapillary has to be fitted and equilibrated
before starting the retrieval of spermatozoa from testicular
biopsy preparations.
Since the holding
pipette, which is needed later on for the ICSI
procedure, is much bigger than the injection pipette, it can be
used as a guide for positioning and equilibrating.
First, the microcapillaries have to be fitted into the universal
capillary holder, which is connected to the microinjector via
a tube. When working with oil-filled systems (e.g., CellTram
vario), it must be ensured that no air bubbles are in the
system. The capillaries are then gently pushed past the sealing
rings inside the tool holder.
After attaching the universal capillary holder to the X head of
the TransferMan NK 2, the alignment needs to be controlled.
The injection angle can be adjusted independently via the
knurled screws and the angle mark on the X head (Fig. 2).
It is also necessary to prime micropipettes with medium
before use so that the gametes which shall be manipulated
never come into contact with air or oil. Usually, equilibration
is achieved
using ICSI media.
The TransferMan® NK 2 can store up to 3 positions. The
capillary can be moved easily in any direction (x/y/z) by
means of a joystick. By pressing the joystick button twice
(double-click) the capillary can be returned to a preset
position. Usually one position is set as a “parked” position,
Position 1, which is stored slightly above the droplet so that
the pipettes do not interfere with the droplets as the dish is
moved around the stage, while another one serves as the
“working” position.
The working position (Position 2) is chosen
in the focal plane.
Sperm retrieval
Due to the use of large volume droplets for searching spermatozoa,
the density can be adjusted so that the amount of
cellular debris is in a range which enables the identification
of spermatozoa at the bottom of the dish. As testicular
sperm are not necessarily motile, special attention has to be
paid during the searching process. Once a sperm has been
allocated, the IMSI/TESE capillary can be lowered into working
position 2. It is recommended to suck the sperm with
the head first into the IMSI/TESE capillary, as this is more
efficient and reduces the amount of debris which will be
drawn into the capillary (compare Fig. 3).
However, due to the large diameter of the IMSI/TESE capillary,
sperm retrieval is very easy and clotting of the capillary
is not an issue. In some rare cases highly motile sperm
can also be found. As these are moving very fast in the
PVP-free culture medium, it can be very difficult to catch
the sperm among the other cells. In this case one may
choose to temporarily immobilize the sperm with a single
laser shot which allows an easy retrieval into the IMSI/
TESE capillary (for details see [3]). In case of highly immotile
spermatozoa, the laser system can also be applied to
investigate sperm viability. If a single laser shot is applied
directly to the sperm tail, a change in the tail geometry
(e.g. curling) indicates that the sperm membrane is still
intact and the sperm cell is still viable and can be used
for later injection (for details see [4]). Spermatozoa may be
sampled in the PVP droplet or, if more time is required, in
a medium droplet. Once enough sperm are isolated, the
IMSI/TESE capillary must be replaced with a standard ICSI
capillary and the oocytes can be loaded into the microinjection
dish.
Even if immotile sperm are injected, it is recommended to
treat the sperm like in a standard ICSI procedure, meaning
that the sperm tail is compressed towards the bottom of
the dish to cause a membrane break which assists in the
fertilization process. Microinjection of testicular spermatozoa
therefore is essentially done in the same way as
with ejaculated spermatozoa. Please refer to procedure as
described in [5].
Corresponding author
Markus Montag • Department of Gynecological Endocrinology & Reproductive Medicine
University Clinics of Bonn, Germany, Sigmund-Freud-Str. 25, D-53105 Bonn
Phone: +49 228 287 15449; Fax: +49 228 287 14651 • e-mail: markus.montag@ukb.uni-bonn.de
Literature
[1] Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon
into an oocyte.
Lancet 1992 Jul 4;340(8810):17-8
[2] Tournaye H, Camus M, Goossens A, Liu J, Nagy P, Silber S, Van Steirteghem AC, Devroey P. Recent concepts in the
management of infertility because of non-obstructive azoospermia.
Hum Reprod. 1995 Oct;10 Suppl 1:115-9
[3] Montag M, Rink K, Delacrétaz G, van der Ven H. Laser-induced immobilization and plasma membrane permeabilization
in human spermatozoa.
Hum Reprod. 2000 Apr;15(4):846-52
[4] Aktan TM, Montag M, Duman S, Gorkemli H, Rink K, Yurdakul T. Use of a laser to detect viable but immotile spermatozoa.
Andrologia. 2004 Dec;36(6):366-9
[5] Andrulat H, Voss S. Intracytoplasmic Sperm Injection (ICSI) - Procedure and equipment.
Eppendorf Application Note
09, June 2006
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