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INTRACYTOPLASMIC SPERM INJECTION
History of ICSI
Extremely low sperm counts, impaired motility, and abnormal morphology represent
the main causes of failed fertilization in conventional IVF. Today, ICSI is the
ultimate option to treat these cases of severe male-factor infertility. One
single viable spermatozoon, preferably of good morphology, is
selected by the embryologist and injected in each oocyte available.ICSI is based on micromanipulation of oocytes and spermatozoa.
Initially, partial zona dissection (PZD) was established to facilitate sperm
penetration .
The barrier to fertilization represented by the zona pellucida
was disrupted mechanically so that the inseminated sperm cells
obtained direct access to the perivitelline space of the oocyte. Sub zonal
insemination (SUZI) represented the next step in micromanipulation techniques
. SUZI enabled the immediate delivery of several motile sperm
cells into the perivitelline space by eans of an injection pipette. ICSI is
even more invasive because a single spermatozoon is directly njected into
the ooplasma, thereby crossing not only the zona pellucida but also the
oolemma. ICSI had been first used successfully to obtain live offspring in
rabbits and cattle, and a reclinical evaluation was reported by the
Norfolk group . The first human pregnancies and births resulting from
this novel assisted-fertilization procedure were reported in 1992. Thereafter,
ICSI was revealed to be superior to SUZI in terms of oocyte fertilization
rate, number of embryos produced, and embryo implantation rate. As a result, ICSI has been used successfully worldwide to treat infertility
due to severe oligo-astheno-teratozoospermia, or azoospermia caused
by impaired testicular function or obstructed excretory ducts.
Since the first publication describing the ICSI procedure, minor
modifications contributed to reduced rates of oocyte degeneration, oocyte
activation (one-pronuclear), and abnormal fertilization (three-pronuclear).
Hyaluronidase may be responsible for oocyte activation; therefore, the
concentration
used during oocyte denudation and the exposure time of oocytes
to the enzyme have been reduced. The moment of denudation relative to
oocyte pick-up (immediately or four hours later) does not influence the ICSI
results. The orientation of the polar body during injection does, however,
influence embryo uality. Motile sperm cells are selected and immobilized prior
to injection. Cytoplasm aspiration to ensure oolemma rupture is critical to the
success of the ICSI procedure because the method of rupture has been correlated
with oocyte degeneration. Furthermore, the morphology of the injected
spermatozoon is related to the fertilization
outcome of the procedure as well as to the pregnancy outcome.
Indications for ICSI
Before the era of ICSI, attempts were made to modify and refine conventional
IVF to achieve increased rates of conception in cases of male-factor
infertility. Today, ICSI has clearly vershadowed the use of modified IVF
procedures (including high insemination concentration) for the treatment
of severe male-factor infertility. ICSI requires only one spermatozoon with
a functional genome and centrosome for the fertilization of each oocyte.
Indications for ICSI are not restricted to impaired morphology of the
spermatozoa,
but also include low sperm counts and impaired kinetic quality of
the sperm cells. ICSI can also be used with spermatozoa from the epididymis
or testis when there is an obstruction in the excretory ducts. Azoospermia
caused by testicular failure can be treated by ICSI if enough spermatozoa can be retrieved in testicular tissue samples. Table 1 gives an overview of
the current indications for ICSI.
Table 1 Current Indications for Intracytoplasmic Sperm Injection
Ejaculated spermatozoa
Oligozoospermia
Asthenozoospermia (caveat for 100% immotile spermatozoa)
Teratozoospermia (_4% normal morphology using strict criteria-caveat for
globozoospermia)
High titers of antisperm antibodies
Repeated fertilization failure after conventional IVF
Autoconserved frozen sperm from cancer patients in remission
Ejaculatory disorders (e.g., electroejaculation, retrograde ejaculation)
Epididymal spermatozoa
Congenital bilateral absence of the vas deferens
Young syndrome
Failed vaso-epididymostomy
Failed vasovasostomy
Obstruction of both ejaculatory ducts
Testicular spermatozoa
All indications for epididymal sperm
Failure of epididymal sperm recovery because of fibrosis
Azoospermia caused by testicular failure (maturation arrest, germ-cell aplasia)
Necrozoospermia
ICSI with ejaculated spermatozoa can be used successfully in patients
with fertilization failures after conventional IVF and also in patients with
too few morphologically normal and progressive motile spermatozoa
present in the ejaculate (<500,000). High fertilization and pregnancy rates can be obtained when a motile spermatozoon is injected. Injection of only
immotile or probably non-vital spermatozoa results in lower fertilization
rates. In cases where only non-vital sperm cells are present in the ejaculate,
the use of testicular sperm is indicated. Other semen parameters, such as concentration, morphology (except for globozoospermia) , and
high titers of antisperm antibodies do not influence the success rates of
ICSI . Successful ICSI has also been described for patients with acrosomeless
spermatozoa . Any form of infertility due to obstruction of the excretory ducts
can be
treated by ICSI with spermatozoa microsurgically recovered from either the
epididymis or the testis. Obstructive azoospermia can result
from congenital bilateral absence of the vas deferens, failed vasectomy
reversal, or vaso-epididymostomy. When no motile spermatozoa can be
retrieved from the epididymis due to epididymal fibrosis, testicular spermatozoa
can be isolated from a testicular biopsy specimen.Testicular biopsy has also proven to be useful in some cases of
non-obstructive azoospermia . In patients with severely impaired
testicular function due to (incomplete) germ-cell aplasia (Sertoli-cell-only
syndrome), hypo-spermatogenesis, or incomplete maturation arrest, spermatozoa
may be recovered, sometimes only, after taking multiple biopsies.Testicular sperm recovery may not always be successful in all azoospermic patients.Cryopreservation of supernumerary spermatozoa recovered from
the epididymis or the testis is an important issue because microinjection
of cryo-thawed sperm cells can avoid repeated surgery in future
ICSI cycles.The ICSI procedure cannot be carried out in approximately 3% of
the scheduled cycles. The most common causes for cancellation are either
no cumulus-oocyte complexes or metaphase II oocytes are available, or no
spermatozoa are found in testicular biopsies of patients with nonobstructive
azoospermia.
Gamete Handling Prior to ICSI
A successful ICSI program depends on ovarian stimulation, which is
essentially similar to methods used for conventional IVF. Current ovarian
stimulation regimens use a combination of gonadotropin-releasing hormone
(GnRH) agonists or antagonists, human menopausal gonadotropin (hMG),
or recombinant follicle-stimulating hormone (recFSH), and human chorionic
gonadotropin (hCG), which allows the retrieval of a high number
of cumulus-oocyte complexes . Administration of GnRH agonists
allows for pituitary down-regulation to occur before the initiation of
exogenous FSH. Gonadotropin preparations or recFSH are administered to stimulate multiple follicle development. Ovulation is usually induced with
hCG (10,000 IU), which is administered when the serum estradiol level
exceeds 1000 pg/mL, and when at least three follicles of 18mm or more
in diameter are observed on ultrasound examination. The optimal time
for ultrasound-guided transvaginal oocyte aspiration is 36 hours after
hCG administration. On average, 11 cumulus-oocyte complexes per cycle
can be retrieved . After cumulus and corona cell removal, approximately
nine metaphase II oocytes per cycle are available for microinjection . Although hCG may be used for luteal-phase supplementation, exogenous
progesterone (administered intravaginally) is frequently applied in an
attempt to avoid the risk of hCG stimulation of remaining growing follicles.
More recently, the clinical introduction of GnRH antagonists
allows a powerful and immediate suppression of pituitary gonadotropin
release and a rapid recovery of normal secretion of endogenous LH and
FSH . By making optimal use of ndogenous FSH, the amount of
exogenous FSH required for follicular growth could be substantially
reduced. A rapid recovery of pituitary LH and FSH release after cessation
of GnRH antagonist administration might permit the abandonment of
additional luteal-phase support. Fertilization by means of micromanipulation requires denudation of
oocytes (i.e., removal of the surrounding cumulus and corona cells). This
strategy allows not only precise injection of the oocytes, but also the
assessment
of their maturity, which is of critical importance for ICSI. Cumulus
and corona cells are removed using a combination of enzymatic and mechanical procedures. Both the enzyme concentration and the duration of
the exposure to the enzyme should be limited because they can result in
parthenogenetic
activation of the oocytes. Microscopic observations of the
denuded oocytes include assessment of the zona pellucida and the oocyte,
and the presence or absence of a germinal vesicle (GV) or a first polar body.
Ninety-five percent of the retrieved cumulus-oocyte complexes usually contain
an intact oocyte. The remaining 5% represent empty zonae, cracked
zonae, or morphologically abnormal oocytes.
Figure 1 shows different stages of oocyte nuclear maturity. On
average, 3.9% of the intact oocytes are at the metaphase I stage, having
undergone breakdown of the GV but not extrusion of the first polar body
. Approximately 10.3% of the intact oocytes are at the GV stage, and
about 85.8% of them are in the metaphase II stage, showing the presence
of the first polar body. ICSI is only carried out on metaphase II oocytes
because only such oocytes have reached the haploid state and, thus, can be
fertilized normally. Frequently, metaphase I oocytes achieve meiosis after a
few hours in vitro and are available for ICSI on the day of oocyte retrieval.
Despite lower fertilization rates (52.7 vs. 70.8%), injection of matured.
Figure 1 Oocyte maturity after cumulus and corona cell removal. (A) A germinal
vesicle (GV) stage oocyte is recognized by the presence of a typical GV. (B)
Oocytes
that have undergone GV breakdown but not yet extruded the first polar body are
called metaphase I oocytes. (C) A typical metaphase II oocyte displays the
presence
of a first polar body, which indicates that the oocyte is mature and has reached
the haploid state. Only metaphase II oocytes are submitted to intracytoplasmic
sperm injection.
metaphase I oocytes results in embryos of similar quality to metaphase II
oocytes at the moment of oocyte retrieval . Denuded and rinsed oocytes
are incubated until the time of microinjection.
For microinjection, spermatozoa from three different origins are
processed: ejaculated sperm and surgically retrieved sperm from the epididymis
or the testis. For all three categories, ICSI in combination with sperm
cryopreservation is currently used successfully . All patients selected
for ICSI with ejaculated semen undergo a preliminary semen assessment
prior to the treatment cycle to verify whether enough spermatozoa (preferably
motile) are present to perform ICSI.Routinely, sperm samples for ICSI are processed by density-gradient
centrifugation [using silane-coated silica particle colloid solutions ],
enriching the number of motile and morphologically normal sperm cells
needed for assisted reproduction. Only in cases of extreme oligozoospermia
(i.e., when gradient centrifugation results in an insufficient yield of sperm
cells for ICSI) is simple washing of the sperm sample performed to reduce
the loss of sperm cells for injection. Immediate injection of the oocytes is
then
indicated because sperm cells lose their initial motility and often die when the
sample is simply washed. This consequence can be ascribed to the presence of
reactive oxygen species and other damaging substances.
During microsurgical epididymal sperm aspiration, several
sperm fractions are collected into separate tubes. Sperm fractions with
similar concentration and motility are pooled, and a density-gradient
centrifugation
is performed. Micro-droplets of the re-suspended pellet are placed
in separate medium droplets adjacent to a central polyvinylpyrrolidone
(PVP) droplet in the injection dish. This facilitates the search for and the
selection of single motile spermatozoa. Spermatozoa are collected using a
testicular sperm extraction (TESE) pipette, which is larger in diameter than
an injection pipette (outer diameter 8-10 mm instead of 6-7 mm). The permatozoa
are then transferred to the PVP droplet and immobilized prior
to injection. Whenever possible, some of the freshly recovered sperm should
be frozen for later use in subsequent ICSI cycles, thereby avoiding repeated
surgical procedures.
Testicular biopsy specimens, usually obtained by means of surgical
excisional biopsy, are shredded into small pieces with sterile microscope
slides on the heated stage of a stereomicroscope. The presence of spermatozoa
is assessed with an inverted microscope, which determines whether the surgical procedure can be stopped or whether extra biopsy pieces need to
be taken. The pieces of biopsy tissue are removed, and the medium is centrifuged
at 300g for 5 minutes. The pellet is then re-suspended for the ICSI
procedure. Single motile spermatozoa are collected in a manner similar to
the way epididymal sperm are, using separate medium droplets that contain fractions of the testicular sperm suspension. If no sperm cells can be found,
the tissue pieces can be treated with red blood cell lysis buffer or an
enzymatic collagen digestion medium . Lysis of excess red blood cells
may facilitate the search for sperm cells, and the use of enzymes may result in the recovery of otherwise inaccessible sperm cells that are initially
attached to the tissue. It is well known that it is not always possible to
retrieve testicular spermatozoa from biopsy specimens in patients with
nonobstructive
azoospermia due to germ-cell aplasia or maturation arrest.
In cases where testicular spermatozoa are retrieved under local anesthesia by
means of the fine-needle aspiration approach, the aspirated fractions
are immediately collected in the injection dish. No further sample processing,
except collecting the single motile spermatozoa with a microneedle
(TESE pipette), is needed prior to ICSI.
ICSI Procedure
For the ICSI procedure itself, an inverted microscope equipped with
micromanipulators
and microinjectors should be available . Magnification
capability of 200_ and 400_ is a prerequisite for precise procedures such
as ICSI. A heating stage on the inverted microscope maintains the temperature
at 37_C. Ambient temperature control is of vital importance for the
survival of oocytes, which are very sensitive to a decrease in temperature that
can cause irreversible damage to the meiotic spindle. The micromanipulators
allow three-dimensional manipulation (coarse and fine movements)
of the holding and injection pipette on the left- and right-hand side,
respectively.
The microinjectors are used to either fix or release the oocyte with the
holding pipette, or to aspirate and inject a spermatozoon with the injection
pipette. The injectors can be air-filled or filled with mineral oil. A
micrometer
controls the plunger. The whole setup is placed on a vibration-proof table to
avoid possible interfering motion. Several companies supply micro-tools for
holding and injection; however, some centers still prepare their own microtools,
which demands extra effort, time, and specialized equipment.
The ICSI procedure involves the injection of a single motile spermatozoon
into the oocyte. The procedure is carried out in a plastic microinjection
dish containing micro-droplets covered with mineral oil. A fraction (_1 mL)
of the sperm suspension is added to the periphery of the central PVP droplet.
Separate medium droplets are used in cases of epididymal or testicular
sperm. The oocytes, denuded from their surrounding cumulus and corona
cells, are placed in the eight surrounding medium droplets. The viscous
character of the PVP solution slows down the motility of sperm cells, thereby
facilitating the manipulation. It also allows better control of the fluid in the
injection needle and prevents sperm cells from sticking to the pipette.
During ICSI, the following steps can be distinguished: selection and
immobilization of a viable sperm cell, correct positioning of the oocyte prior
to injection, and rupture of the oolemma prior to the release of the sperm
cell into the oocyte.
Figure 2 illustrates the whole injection procedure. In the injection
pipette, which is filled with PVP, a single living, morphologically normal
spermatozoon is aspirated. Viability is evidenced by the motility of the
sperm cell, even if it is only a slight twitching of the tail. The sperm cell is
then released in a perpendicular position to the injection pipette, which
facilitates immobilization. Immobilization of a sperm cell involves rubbing
the tail with the pipette against the bottom of the dish, which results in a
breakage at one point, preferably below the midpiece. Immobilization of
spermatozoa has been proven to be important for oocyte activation, which
is achieved by release of sperm cytosolic factors via the ruptured membrane.
Increased fertilization rates with ICSI have been reported for the following
aggressive damage to the sperm tail plasma membrane.
After immobilization, the sperm cell is again aspirated (now tail-first)
to allow the injection of a minimal volume of medium together with the
sperm cell. The oocyte is held in position by means of minimal suction by
the holding pipette. The polar body is located at the six o0clock position,
which avoids damage to the spindle. Experiments in our laboratory
using Hoechst dye-stained oocytes for microinjection clearly showed no
interference with the spindle if oocytes are injected with the polar body at
the six o0clock position. If both the holding pipette and the oocyte are in
perfect
focus, the injection needle containing the immobilized sperm cell near
the tip can be introduced in the equatorial plane of the oocyte at the three
o0clock position. Permanent focus of the injection pipette tip ensures that
the needle remains in the equatorial plane of the oocyte.
Figure 2 Intracytoplasmic sperm injection procedure. (A) A single motile
spermatozoon
is selected and immobilized by pressing its tail between the microneedle and
the bottom of the dish. The sperm cell is then aspirated tail-first into the
injection pipette.
(B) Using the holding pipette, the mature oocyte is fixed with the polar body at
the 6 o0clock position. The sperm cell is brought to the tip of the injection
pipette.
(C) The injection pipette is introduced at the 3 o0clock position and rupture of
the
oolemma is ascertained by slight suction. Then the sperm cell is delivered into
the oocyte with a minimal volume of medium; afterwards, the pipette can be
carefully
withdrawn. (D) A single sperm cell can be appreciated in the center of the
ooplasma. injection pipette. In contrast, the oolemma is not always immediately pierced
by simple injection of the needle and often minimal suction needs to be
applied. The ooplasm then enters into the injection pipette, and sudden
acceleration
of the flow indicates membrane rupture. The aspiration is immediately
stopped, and the sperm cell is then slowly released into the oocyte with a
minimal
volume of medium and the pipette can be withdrawn carefully.
Different patterns of oolemma breakage have been described, depending
on whether the ooplasm breaks during insertion of the pipette, whether
slight or stronger aspiration of the ooplasrm is needed, or whether breakage
of the oolemma in another place has to be attempted. Immediate
rupture of the oolemma without any aspiration has been associated with
lower oocyte survival rates.
Fertilization and Embryo Cleavage After ICSI
After the injection procedure, oocytes are rinsed and cultured in micro-droplets
covered with lightweight paraffin oil. The conditions are similar to
those employed for IVF inseminated oocytes: the oocytes are kept at
37_C in an atmosphere of 5% O2, 5% CO2, and 90% N2. Injected oocytes are examined for integrity and fertilization about 16-18 hours after
ICSI. An average damage rate of approximately 9% of the injected
oocytes can be expected, irrespective of the origin of the sperm used.
Oocytes are considered normally fertilized when two individualized or fragmented
polar bodies are present together with two clearly visible pronuclei
(2-PN) that contain nucleoli.
The fertilization rate after ICSI is usually expressed per number of
injected oocytes and ranges from 57% to 67% according to the sperm origin. As shown in Figure 3, abnormal fertilization may occur, reflected
by one-pronuclear (1-PN) oocytes (about 3% of the injected oocytes).
These oocytes are likely to be parthenogenetically activated as a result of
mechanical or hemical factors. The occasional finding of threepronuclear
(3-PN) oocytes (about 4%) after injection of a single
spermatozoon into the ooplasm is probably caused by failure of extrusion
of the second polar body at the time of fertilization. Neither type of
embryo resulting from 1-PN to 3-PN oocytes is transferred to patients.
Post-fertilization, about 90% of 2-PN oocytes obtained by ICSI enter
cleavage, resulting in multicellular embryos. Cleavage characteristics of the
fertilized oocytes are evaluated daily. Normally developing, good-quality
embryos reach the four-cell and eight-cell stage, respectively, on day 2
and in the morning of day 3 postmicroinjection. Numbers and sizes
of blastomeres and the presence of anucleate cytoplasmic fragments are
recorded. The cleaving embryos are scored according to equality of size of
the blastomeres and proportion of anucleate fragments. Type A (excellent
quality) embryos do not contain anuclear fragments. Type B (good
quality) embryos have a maximum of 20% of the volume of the embryo
filled with anucleate fragments. In type C (fair quality) embryos, anucleate
fragments represent 21% to 50% of the volume of the embryo. Type D (poor
quality) embryos have anucleate fragments present in more than 50% of the volume of the embryos. These embryos cannot be used for transfer to the
patients. Embryos in the former three categories (type A, B, and C) are eligible
for transfer.
Figure 3 Fertilization outcome after intracytoplasmic sperm injection. (A)
Oocytes
are considered normally fertilized when two individualized or fragmented polar
bodies are present together with two clearly visible pronuclei (2-PN) that
contain
nucleoli. (B) Abnormal fertilization may occur as one pronuclear (1-PN) oocyte,
probably due to parthenogenic activation. (C) The occasional finding of three
pronuclear
(3-PN) oocytes after injection of a single spermatozoon into the ooplasm is
probably caused by non-extrusion of the second polar body at the time of
fertilization.
Figure 4 Embryo cleavage after intracytoplasmic sperm injection. Only embryos
resulting from normally fertilized oocytes (A) will be transferred to patients.
Embryo
cleavage is evaluated daily. Two-cell embryos (B), four-cell embryos (C), and
eightcell
embryos (D) are usually obtained on day 1 (late afternoon), on day 2, and in the
morning of day 3, respectively. The blastomere number is recorded and the
embryos
are scored accordingly to equality of size of the blastomeres and the presence
of
anucleate cytoplasmic fragments. On day 4 (sometimes already on day 3), a
certain
degree of compaction can be observed (E). For blastocyst (F) scoring, the
classification
system introduced by Gardner and Schoolcraft is used. Embryo transfer is
usually done on day 3 (eight-cell stage) or day 5 (blastocyst stage).
Nowadays, most centers perform embryo transfers on day 3 or day 5
after oocyte retrieval. At that time, the embryos are expected to be at the
eight-cell stage. Because the embryonic genome is fully activated after
the eight-cell stage , it may indeed be beneficial to evaluate embryos
at least until after the transition from maternal to embryonic genome,
making it possible to identify those embryos with a better developmental
potential. The number of embryos transferred depends on the age of the
woman and on the rank of trial. In women under 37 years of age who are
undergoing a first or second ICSI attempt, preference is given to transfer
of only two excellent or good-quality embryos. In other cases, three or more
embryos may be placed into the uterus. Higher pregnancy rates can be
obtained when elective transfer of two or three embryos is possible.
However, the number of embryos transferred should be limited in order to
avoid multiple pregnancies.
Today, commercially available sequential culture media allows the culture
of human embryos up to the blastocyst stage (day 5 or 6). On day 4
(sometimes already on day 3), a certain degree of compaction can be
observed. Compaction in the mammalian pre-embryo is a fundamental event that leads to the formation of the trophectoderm, the inner cell mass
and the blastocoele. Full compaction (16-32-cell stage) is followed by
immediate cavitation and blastocoele expansion . For blastocyst scoring,
the classification system introduced by Gardner and Schoolcraft can
be used. A distinction between early and expanded blastocysts is made, and the latter category is further scored, according to the quality of the inner
cell mass and the trophectoderm. The possibility of prolonged human
embryo culture allows for day 5 or blastocyst transfers. Preferably, expanded
blastocysts with a cohesive trophectoderm and a clear inner cell mass
are transferred. Possible advantages of blastocyst transfer are better embryo
selection and better synchronization between embryo and endometrium,
which may result in higher mplantation rates per blastocyst transferred
. This in turn would allow transfer of fewer embryos, thereby decreasing
the number of multiple pregnancies.
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