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Reviews
Laboratory techniques for human embryos
Selmo Geber attended Medical School and residency in Obstetrics and Gynaecology in
Belo Horizonte, Brazil. After finishing, he continued his training at Hammersmith Hospital in
London, UK, where he performed research on preimplantation embryo development, PGD
and endometriosis, under the supervision of Professor Robert Winston and Dr Alan
Handyside. Returning to Brazil, Dr Geber completed his PhD degree and became adjunct
Professor at the Department of Obstetrics and Gynaecology at the Medical School of the
Federal University of Minas Gerais with undergraduate and postgraduate students. Since
1995, together with Dr Marcos Sampaio, he has been Director of ORIGEN at the Centre of
Reproductive Medicine, in Belo Horizonte and Rio de Janeiro, performing ~700 cycles of
IVF/ICSI per year. He has published two books on obstetrics and gynaecology, and written
many book chapters and articles nationally and internationally; he has nine scientific awards
Dr Selmo Geber and is a researcher of the national research council (CNPq). Research interests include
embryo development, PGD, endometriosis and cryopreservation of ovarian tissue and
transplantation.
Selmo Geber1,2,3, Liana Sales1, Marcos AC Sampaio1
1ORIGEN, Centro de Medicina Reprodutiva, Av Contorno 7747, Belo Horizonte, Minas Gerais, CEP 30 010 020, Brazil
2Departamento de Ginecologia e Obstetrícia da Faculdade de Medicina da Universidade Federal de Minas Gerais
3Correspondence: Fax: +55 31 32754204; e-mail: sjgeber@bhnet.com.br

Abstract
This review is concerned with laboratory techniques needed for assisted conception, particularly the handling of gametes
and embryos. Such methods are being increasingly refined. Successive stages of fertilization and embryogenesis require
especial care, and often involve the use of micromanipulative methods for intracytoplasmic sperm injection (ICSI) or
preimplantation genetic diagnosis. Embryologists must take responsibility for gamete collection and preparation, and for
deciding on the means of insemination or ICSI. Embryos must be assessed in culture, during the 1-cell, cleaving and
morula/blastocyst stages, and classified according to quality. Co-culture methods may be necessary. The best embryos for
transfer must be selected and loaded into the transfer catheter. Embryos not transferred must be cryopreserved, which
demands the correct application of current methods of media preparation, seeding and the correct speed for cooling and
warming. Before too long, methods of detecting abnormal embryos and avoiding their transfer may become widespread.

Keywords: embryo cryopreservation, embryo culture, fertilization in vitro, human embryos, preimplantation diagnosis of genetic
disease

Introduction have to be prepared for use in insemination or intracytoplasmic

The main objective of IVF laboratories is to mimic the conditions
in the oviduct and uterus. Inside the follicle, the oocyte matures
as a consequence of an LH surge. It is released from its follicle at
ovulation, and drawn into the oviduct by the activity of fimbrial
cilia. The oocyte is fertilized in the ampulla of the Fallopian tube,
while the initial cleavage divisions of the embryo occur within
the isthmus. At ~60 h, it enters the uterine cavity, when it has
approximately 8 cells. It grows to a blastocyst by day 5–6, and
implants in the uterus a few days later.
This paper is designed to set the scene for those unfamiliar with
IVF techniques. The IVF laboratory is responsible for the
collection of mature oocytes, fertilization, and cleavage in vitro,
and growth of embryos to blastocysts. While many of these
embryological changes occur normally in vitro, there are many
exceptions demanding novel forms of culture, or the use of
micromanipulation Accordingly, work begins immediately after
follicular aspiration with the identification of the oocytes.
Semen is prepared for insemination soon afterwards and may
This review will also be published in a Spanish textbook entitled Modern
Assisted Conception.
sperm injection (ICSI). Confirmation of fertilization is
performed ~19 h later by identifying the presence of two
pronuclei in the eggs. Carefully designed media are essential for
each of these stages, to support the continued growth of embryos
to blastocysts by day 5–6. Embryo transfer can be performed on
any day between day 1 and day 6 post-insemination, according
to the experience and choice of each centre. Detailed analyses
on the fundamentals of embryo growth and selection can be
found elsewhere (Boiso et al., 2002)
Gamete collection and preparation
Culture media
Adequate culture media are needed to sustain the gametes and
embryos in vitro. Extreme care is needed to ensure that highly
defined media are prepared with the greatest care, using highly
purified constituents. There is now considerable expertise in
the design and preparation of media, in many areas of
medicine. Earlier work on animals revealed the nature of 211
 Reviews - Laboratory techniques for human embryos - S Geber at al.

essential and non-essential constituents to sustain gametes and Water

embryos, and this has helped greatly in the preparation of
media for human embryos.
Chemical and organic constituents
The development of specific culture media began in the early
twentieth century, when various types of saline solutions
containing inorganic salts such as sodium, potassium, calcium,
magnesium and phosphate were developed. Different kinds of
culture media were tested using various cell types, embryos or
organ culture, many of them subsequently being modified.
Other culture media were developed to support human
embryos in vitro, or for other purposes such as for
crypreservation or for stem cells. Osmolarity of media was
adjusted to resemble animal, and later human serum (284 ± 2
mOsm/kg). Media were devised to maintain pH by means of
phosphate buffers for media used in air, or sodium bicarbonate
in media which were balanced with an atmosphere of 5% CO2
or 5% CO2, 5% O2 and 90% N2. In the early days of IVF, the
correct pH was achieved largely by varying the content of
sodium carbonate added to media. Energy was supplied by the
addition of glucose, lactate or pyruvate.
One of the most important constituents of culture media is
water. Since water is an excellent solvent and provides a
medium for most biological and chemical reactions, it is also
highly susceptible to contamination by more substances than
any other common solvent. Contaminants found in water can
be inorganic, organic particles and micro-organisms. These
contaminants may be naturally occurring in substances added
to make media, and must also be removed from water used for
preparing media to be used to sustain embryos.
Several systems for water purification have been developed.
Many culture media contain water that is deionized and/or
distilled at least five times. Others use highly purified water
produced by reverse osmolarity and special filtration systems.
It is important to bear in mind that water quality is an
important and constant limiting factor for the support of
embryo development. Very often, when results are lower than
expected in the IVF laboratory, there is a tendency to blame the
culture medium. Water plays a fundamental role in culture
media, so it is important for each laboratory to control its
purity.

Various supplements were added to produce balanced media. Proteins and amino acids
Albumin or serum was widely used to provide a nitrogen
reserve for protein synthesis, and to ensure correct buffering Various protein supplements can be added to culture media,
and stabilizing properties in media. Penicillin and/or including albumin, preferably human serum albumin, or
streptomycin were usually added, but may not be necessary in various sources of serum, including maternal serum or
ultra-clean laboratories. Consequently, many and varied umbilical cord serum. Synthetic preparations are also
culture media have been available within the last few years, as available. Initially most centres used human serum prepared
assisted reproduction expanded worldwide and methods of in-house. However, nowadays many centres have abandoned
embryo culture were refined. So far, there is no consensus on this approach due to the ready availability of synthetic
which medium should be chosen preferentially for IVF and preparations. Also, the presence of various
micromanipulation techniques. In this discussion, different inhibitor/detrimental factors in serum could increase the risk of
culture solutions will be classified into balanced salt solutions decreasing embryo quality and blastocyst formation. It is still
and complex media. controversial whether serum supplementation should be used.
For the in-vitro manipulation of human embryos, at least 7%
Among balanced salt solutions, Earle’s, T6, P1 and HTF serum is usually added to culture media.
(human tubal fluid) are recommended. These salt solutions
contain sodium bicarbonate, so it is important to maintain
them under 5% CO2 to avoid pH variation (Table 1). Complex Table 1. Commercially available balanced salt solutions.
culture media in their widest definition include the widely used
Ham’s F10 and Menezo’s medium. Recently, increased Reagents Earle’s HTF P1
attention has been given to more complex culture media, (mM) (mM) (mM)
prepared to prolong the period of culture time of human
embryos in vitro, and so sustain embryos for transfer to the NaCl 116.0 101.06 101.60
blastocyst stage. Currently, sequential cultures using two KCl 5.30 4.70 4.69
media are becoming popular. Recently, variations in various MgSO4 0.80 0.20 0.20
constituents have led to the introduction of media named G1
KH2PO4 – 0.37 –
and G2 (Gardner and Lane, 1998), although the physiological
basis of this approach and the withholding of information on CaCl2.2H2O 1.80 2.04 2.04
media contents have been challenged (Biggers, 2000, 2002). NaHCO3 – 25.00 25.00

Today, the addition of antibiotics, widely used in early phases NaH2PO4 0.90 – –
of IVF, is no longer a consensus requirement. Many Glucose 5.5 2.78 –
laboratories have improved the quality of their procedures and Pyruvate – 0.33 0.33
media, so the risk of contamination has decreased Lactate – 21.40 21.40
considerably. Moreover, the addition of antibiotics might
decrease the quality of embryos and impair blastocyst Taurine – – 0.05
formation (Magli et al., 1996). Many centres have therefore Sodium citrate – – 0.51
abandoned the use of antibiotics in culture media. Glutamine – – 1.00
212
 Reviews - Laboratory techniques for human embryos - S Geber at al.
The role of amino acids in culture media has received wide
attention following the introduction of closely defined
sequential media. Different combinations are recommended
for the various stages of culturing embryos to blastocysts by
days 5–6. The amino acid currently added to G1 and G2 media
are shown in Table 2. An energy substrate must be added to
culture media. Those chosen may differ according to the
growth stage of the embryos. It is commonly accepted that
pyruvate is the preferred source of energy for the early stages
of development. The question of glucose as a source of energy
has not yet been resolved. Glucose may be essential for semen
preparation and fertilization, since spermatozoa require
glucose to produce ATP for motility. Suitable media are also
essential to bind to the zona pellucida. Glucose may cause
embryo blockage (Conaghan et al., 1993), and lowering its
concentration in the media might enhance embryo
development. This possibility has been questioned recently
(Biggers, 2002), and more data are needed to clarify the role of
glucose for early embryos, since no advantages accrue for
embryo development and pregnancy with glucose-free culture
media. As embryos reach the blastocyst stage, however,
glucose is essential as the primary energy source.
Air contamination by chemicals
Chemical air contamination is quite common among
laboratories involved in assisted reproductive technology.
Cohen et al. (1997) have demonstrated that unfiltered outside
air may be cleaner than high efficiency particulate air filtration
(HEPA) filtered laboratory air or air obtained from incubators,
due to accumulation of volatile organic compounds derived
from adjacent spaces or specific laboratory products such as
compressed CO2, sterile Petri dishes and other materials or
Table 2. List of amino acids added to G1 and
G2 media for blastocyst culture.
G1 G2
Alanine Arginine
Asparagine Cystine
Aspartic acid Histidine
Glutamine acid Isoleucine
Glycine Leucine
Proline Lysine
Serine Methionine
Phenylalanine
Tryptophan
Tyrosine
Valine
Alanine
Asparagine
Aspartic acid
Glutamine acid
Glycine
Proline
Serine
devices known to release gaseous emissions. Isopropyl alcohol
was the most dominant product found, though it was not used
by the laboratory staff. Concentrations of this agent were low
in incubator air, indicating that it was probably absorbed by the
water in the pan or by culture medium. Measures to counter
chemical air concentration have been proposed. These
included the use of activated carbon, a potassium
permanganate filter and oxidizing material placed in the
central air handling systems, in separate free-standing units or
even inside the incubators.
Follicular aspiration and the pre-ovulatory
oocyte
For oocyte collection, many centres use HEPES-buffered
culture media in air in preference to carbonate buffers when
follicles are flushed to recover the oocyte when it is not found
in the first-drawn samples of follicular fluid. Media used for
aspiration do not require serum or energy supplements. It is,
however, fundamental to add a strong buffer preparation to
avoid pH variations. Heparin may be used to avoid the
formation of blood clots, as it has no apparent effect on
fertilization or cleavage in vitro (Boyers et al., 1987).
The identification of pre-ovulatory oocytes must be performed
simultaneously with follicular aspiration to decrease the time
of exposure of oocytes to variations in temperature and pH.
For the same reason, it is important that the laboratory be
placed as close as possible to the aspiration room. Oocyte
identification is performed initially by naked eye observation
of the cumulus–oocyte complex. Subsequently, confirmation
is made under a dissecting microscope. Oocytes are then
flushed to remove blood cells and debris, and placed in Petri
dishes or test tubes containing culture medium. As a source of
proteins for the oocytes and cleavage stage embryos is
contained within ooplasm, it is not necessary to use complex
culture media.
After identification, the oocytes can be classified according to
their degree of maturation. This classification can be
performed using morphological aspects, the appearance of the
cumulus–oocyte/corona radiata complex (Veeck et al., 1998)
or by directly identifying the polar body after removal of this
complex. This removal is performed routinely when there is an
indication for ICSI for cases of male factor infertility.
The cumulus–corona complex is removed enzymatically or
mechanically. Oocytes are placed in droplets of ~10 ml of
HEPES-buffered culture containing 80 IU/ml hyaluronidase
media and are subsequently flushed in the same medium
containing 10% serum through a narrow Pasteur pipette in
repetitive movements to remove the corona radiata.
Hyaluronidase is also present in the follicular fluid and
spermatozoa acrosome, to digest the cumulus–oocyte. For in-
vitro use, it is extracted from bovine testis.
After identification and classification, the oocytes can be
incubated in culture media at 37°C under an adequate gas
phase for between 1 and 8 h before insemination or ICSI
(Trounson et al., 1982).
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 Reviews - Laboratory techniques for human embryos - S Geber at al.
Semen preparation
In vivo, the fertilization process depends on the capacity of
spermatozoa to traverse cervical mucus and the uterine cavity,
and reach the ovulated oocyte in the Fallopian tubes. Only a
small proportion of the ejaculated spermatozoa reach the site
of fertilization in the ampulla. During passage through the
female reproductive tract, it is possible that spermatozoa are
capacitated, leading to the onset of hyperactivation. These
steps involve modifications in spermatozoa, including the
acrosome reaction and their traverse of the zona pellucida to
reach the oolemma. After sperm–oolemma binding, the entire
spermatozoon enters the egg and fertilization has begun.
The main objective when preparing semen for in-vitro
insemination is to remove seminal fluids that contain
substances possibly inhibiting the capacitation process. Also,
many dead cells, including leukocytes, epithelial cells, and
immotile and immature spermatozoa, are removed. High-
quality sperm samples will display progressive motility and
few anomalies.
The most widely used techniques for semen preparation are
swim-up and density gradient centrifugation through silicone-
coated silica particles. The swim-up technique is based on the
capacity of the spermatozoa with progressive motility to
migrate after centrifugation from the sediment to the overlying
culture medium. The density gradient centrifugation technique
is characterized by the utilization of columns with at least two
dilutions that, after centrifugation, separate seminal
constituents according to differential mass, so that
subsequently the most progressively motile spermatozoa will
be pelleted.
Culture media used for semen preparation by swim-up consist
of a physiological saline solution (listed above) supplemented
with serum. It must also contain an energy source that, in
combination with incubation at 37°C, might promote
hyperactivation within a period of 20–45 min. For density
gradient separation, the most widely used media are Percoll
and Pure-Sperm System.
Insemination in vitro and
intracytoplasmic sperm injection
into oocytes
Insemination
After identification, oocytes are placed in 20 ml droplets of
culture media under mineral oil in Petri dishes, at 37°C, under
a gas phase of 5% CO2. The previously prepared semen is
placed either in droplets of 20 ml in Petri dishes, or under
mineral oil, ready for insemination. Each droplet must contain
~50,000 spermatozoa for each oocyte. Spermatozoa can be
added to oocytes from 1 h to ~3–5 h after oocyte collection.
This period may be necessary to stabilize the chemical and
physical stress due to the novel osmolarity, temperature and
light exposure during insemination.
Usually, each oocyte is cultured singly in one droplet
containing the capacitated semen. Oocytes are exposed to
214 spermatozoa for 1–19 h at 37°C, under a gas phase of 5% CO2.
Some investigators propose that few spermatozoa, and a
briefer exposure to them, produces superior conditions for
fertilization. The oocytes are then moved to a new droplet of
culture media and ~19 h after insemination are checked for
fertilization, after careful mechanical removal of the corona
radiata.
Intracytoplasmic sperm injection (ICSI)
Since the first birth after ICSI published in 1992 (Palermo et
al., 1992), this method of assisted reproduction has been
applied worldwide in patients with severe male factor
infertility. ICSI can be used with spermatozoa isolated from
the ejaculate (either fresh or frozen–thawed), aspirated from
the epidydimis or collected during biopsies of the testis.
With washed, ejaculated spermatozoa, the best preparation is
achieved using swim-up or density gradient centrifugation as
described above. For spermatozoa obtained via epidydimal
aspiration, separation can be performed either by a micro-
swim-up technique or through sperm wash and
ultraconcentration, according to the initial concentration.
In the case of testicular biopsy, the fragment of testicle must be
washed and dissected using a pair of blades in a Petri dish. The
final material is incubated for ~2 h to allow the onset of
activation of the spermatozoa. After that period of time, the
material is ultraconcentrated and placed in microdroplets of
culture medium for identification and selection for injection
using ICSI.
Before sperm separation, oocytes are prepared for the
injection. They are placed in droplets of 80 IU/ml
hyaluronidase solution for 10–15 s, while aggressively
pipetting the cumulus–corona complex with a large flame-
polished Pasteur pipette. After 15 s, the oocyte is moved to a
fresh culture medium droplet where the remaining
cumulus–corona complex will be removed mechanically using
a thin flame polished Pasteur pipette. After complete removal
of the corona cells, oocytes are placed in a fresh droplet of
culture medium. Their nuclear maturity is then assessed by
identifying the first polar bodies in mature oocytes, which are
ready for ICSI at least 1 h later.
The micromanipulation procedure is carried out on the heated
stage of an inverted microscope at ×400 magnification,
adapted with a pair of hydraulic micromanipulators and a
motor-driven coarse control. Initially, the micropipettes are
placed in each respective holder, i.e. the holding micropipette
is placed in the left holder and the injection micropipette in the
right holder.
The Petri dish used for micromanipulation is prepared using 5
ml droplets of culture media and 5 ml droplets of HEPES
buffered media containing 10% polyvinylpyrrolidone (PVP)
under mineral oil. Immediately before the injection procedure,
~1 ml of the sperm suspension is added to the PVP droplet.
This viscous solution is necessary to facilitate sperm handling
and oocyte activation, and to prevent spermatozoa from
sticking in the injection micropipette.
Oocytes are placed in the droplets of culture medium.
Spermatozoa that appear morphologically normal and have a
 Reviews - Laboratory techniques for human embryos - S Geber at al.
good progressive motility in the PVP are selected for injection.
The chosen spermatozoon is then gently compressed by the
micropipette tip in the sperm midpiece. This procedure
damages the sperm membrane and impairs motility of the
spermatozoon. The immobilized spermatozoon is then
aspirated, tail first, into the injection micropipette. Once in the
droplet containing the oocyte, the holding micropipette is
lowered and the oocyte is held in place. The injection pipette
is then pushed through the zona pellucida into the cytoplasm
and a single spermatozoon is injected.
Injected oocytes are moved to droplets of culture media under
mineral oil in Petri dishes, at 37°C, under a gas phase of 5%
CO2, on the following day. They are then assessed for
pronuclei formation and confirmation of fertilization.
Culture and manipulations of the
embryo
Embryo culture
Normal fertilization is confirmed by observing the presence of
two pronuclei and two polar bodies. The embryo is then kept
in culture until transfer. Culture medium can be selected from
one of those listed above, depending on laboratory experience.
Moreover, it is very important to maintain ideal conditions for
each embryo throughout the culture period including
temperature, pH, osmolarity, light exposure and respiration.
Implantation in vivo occurs when the embryo is at the
blastocyst stage, at approximately day 6 after fertilization.
Therefore many studies have been performed in order to
optimize the transfer of the embryos at this stage.
Theoretically, at this stage the embryos should be more able to
implant and tolerate conditions in the uterine lumen.
Historically, in assisted reproductive cycles, embryos have
been transferred to the uterus between day 1 and day 3.
Transfers on days 2–3 may be identical to the time that
embryos require to enter the uterus after passing through the
oviducts. Nevertheless, many human embryos can develop
from pronucleate stages to blastocysts in the uterine cavity.
Accordingly, many investigators believe that growth
conditions in vitro are sub-optimal for embryos when
compared with a uterine environment, and high pregnancy
rates are being achieved by transfers of single, selected
embryos (Gerris et al., 2001).
In the last decade, many groups have tried to achieve
pregnancies by delaying the transfer until day 5 or day 6 after
insemination (Boiso et al., 2002). Initial studies have
demonstrated the capacity of the embryos to reach blastocyst
stage in vitro, i.e. up to 50% of embryos in vitro (Geber et al.,
1995). However, only few pregnancies were initially reported
after the transfer of blastocysts cultured in the widely used
culture media. The use of co-culture techniques, using somatic
cells, was suggested as improving the development of embryos
to blastocysts and raising implantation rates after uterine
transfer. This method is now rarely used.
More recently, Jones et al. (1998) demonstrated that using a
sequential culture media makes it possible to achieve many
pregnancies by transferring embryos at the blastocyst stage at
day 5 or day 6. This idea is based on the premise that the
zygote needs more than one culture medium to take into
account its significant changes in its physiology and
metabolism during preimplantation development. Changing
embryo needs during growth in one culture medium might
impair the development of the cleavage stage embryo. In
support of this idea, the Fallopian tubes and uterus are known
to differ markedly in their physiology, so in vivo the embryo is
exposed to a changing environment. There are also many
advantages of culture in a cell-free medium. Under these
circumstances, there is no compromise between the needs of
the embryos and those of the somatic cells, and other
supplements can be added to the culture media. The need to
prepare somatic cells specially for co-culture is avoided and
there are no risks of contamination from an external cellular
source.
Current ideas thus propose that embryos are kept in culture
media from day 1 after insemination until day 3, as classically
performed. They are then transferred to the subsequent culture
media until day 5 or 6. This second phase culture media
contains amino acids and vitamins to allow protein synthesis.
It is also important to change the embryos every two days in
order to avoid ammonia formation as a consequence of amino
acid degradation (Gardner and Lane, 1998).
Transferring embryos to the mother at the blastocyst stage
could increase implantation rates, since they will be
synchronized with the endometrium. Moreover, the most
viable embryos are transferred, having been selected according
to their capacity to develop in vitro and therefore more likely
to implant. Improved implantation rates and using better
criteria for embryo selection might reduce the number of
embryos to be transferred, even to a single blastocyst, and so
reduce the incidence of multiple pregnancies. Other
advantages of the blastocyst transfer include a longer period to
assess the growth of embryos in vitro for cryopreservation and
possible improvements in preimplantation diagnosis
technique. Remaining embryos can be cryopreserved,
although it is getting late in development for this procedure
unless it is by day 5.
Co-culture of embryos with somatic cells
Most stages of early human embryonic development occur
within the oviduct, including fertilization, syngamy, cleavage
and compaction. The fertilized egg is in contact with tubal
fluid containing many micro- and macromolecules, many of
which are still unknown. However some of these products
could have a role in embryo development in vivo. As cited
above, the main objective in an IVF laboratory is to mimic
conditions within the Fallopian tubes. It was therefore
suggested that contact between the embryo and different tissue
cells either human or animal (co-culture), might recreate the
in-vivo environment (Boiso et al., 2002). Improved embryo
development and implantation rates could thereby be possible.
The methodologies involved in such forms of culture evoke
much discussion and disagreement.
Co-culture experiments can be conducted using cumulus cells,
granulosa cells, fetal bovine uterine fibroblasts, oviduct
epithelial cells, human endometrial cells, Vero cells, ovarian
cancer cells and combinations of these cells. It is important to
place embryos in close contact with the co-cultured cells for 215
 Reviews - Laboratory techniques for human embryos - S Geber at al.
one or more days until they reach the morula or even
blastocyst stage, and are ready to transfer or freezing. The
main problem related to the co-culture methodology is the
elevated risk of contamination after culturing animal or human
tissue cells, and its subsequent consequences. Therefore, co-
culture should be considered as an alternative only in special
situations such as implantation failure and blastocyst transfer.
Moreover, several studies have demonstrated that there is no
difference in blastocyst formation and pregnancy rates when
comparing co-culture and sequential media methods.
Zona drilling
Some IVF clinics still use micromanipulation to drill the zona
pellucida to induce assisted hatching, or to remove one or
more embryo cells for preimplantation diagnosis. It is
performed either during cleavage or at the blastocyst stage,
when embryos are selected for transfer (Geber and Sampaio,
1999). Embryos are initially transferred into drops of HEPES
buffered medium (Earle’s or a similar medium) in a Petri dish
under mineral oil, in order to minimize pH variation. The
micromanipulation procedure is carried out on an inverted
microscope at ×400 magnification, adapted for a pair of
micromanipulators. Micropipettes are prepared before
transferring the embryos to the biopsy dish.
Once prepared, embryos are initially immobilized by suction
on a flame-polished holding pipette held in the left
micromanipulator. The second micromanipulator with a
double holder controls a drilling pipette (internal diameter 10
µm) containing acid Tyrode’s solution (pH 2.2) and a sampling
pipette (internal diameter 30 µm) containing buffered medium.
The acid Tyrode’s solution with its chemical action can be
replaced by a 0.5% solution of the enzyme preparation
pronase, in Earle’s medium. An alternative to drilling is the use
of lasers.
Drilling must be controlled very gently in order to avoid
making a large hole in the zona or even the lysing blastomeres.
The drilling pipette is then placed in close contact with the
zona pellucida and a hole made with a controlled stream of
acid Tyrode. In the case of assisted hatching, the embryos are
now returned to culture medium for a later transfer. In the case
of preimplantation diagnosis, after penetrating the zona the
pipette is removed, and the sampling pipette is pushed through
the hole. One or two cells judged to be at the equivalent of the
8-cell stage are removed by gentle suction and sent for genetic
diagnosis. The biopsied embryos are then returned to culture
medium. After obtaining the genetic results, unaffected
embryos are selected for transfer, which can be performed
either on same day (day 3) or at the blastocyst stage.
Removal of fragments
Another alternative to improve implantation rates especially
after freezing–thawing, is the removal of small cytoplasmic
fragments, after assisted hatching. This is not a well-defined
method and represents some risks for damageing normal
blastomeres and thus impairing embryo development.
Therefore, more studies are needed to confirm the real place of
this technique in assisted reproduction.
216
Artificial hatching and zona removal
It is essential for implantation that the blastocyst hatches from
the zona pellucida. The mechanisms of hatching are still not
fully understood, and it is possible that the endometrium might
play a role in the in-vivo hatching process. Thus we might
assume that repetitive implantation failures can be related to
the difficulty of the blastocysts to hatch in vivo. Hence, the
hatching process can be either assisted as described above or
performed by the total removal of the zona pellucida. This can
be achieved enzymatically by placing selected fully expanded
blastocysts in 0.5% pronase in Earle’s medium for 1–2 min at
37°C under 5% CO2. Zona free blastocysts are then washed
and incubated in blastocyst medium until transfer. This
methodology might be used in special situations such as zona
hardening and implantation failure. No improvement in
pregnancy rates during routine IVF has yet been demonstrated
(Sampaio and Geber, 2001).
Embryo selection and transfer
Embryo transfer needs care and attention. It is the last step on
a long journey, and mistakes can jeopardize all the effort of
patients, clinicians and scientists in achieving good quality
embryos (Boiso et al., 2002).
First, deciding how many embryos to transfer, and when to
carry out transfer, are fundamental decisions in the IVF
laboratory. Embryo transfer might be performed at any time
from day 1 to day 6 post-insemination. Embryos must be
evaluated twice or more each day. This process begins at the
pronuclear stage by observing the disposition of the pronuclei
and the polarization of the nucleoli (Scott and Smith, 1998;
Lundqvist et al., 2001). Some embryos may divide to 2-cell on
day 1, although an average time is ~26 h after insemination.
At day 2, the first cleavage division occurs. After this, embryos
double their number of blastomeres each day. Thus embryos
have an expected 2–4 cells at day 2, 6–10 cells on day 3,
become morula at day 4 and blastocysts at day 5 and 6. With
embryo transfer at day 2 or day 3 after insemination, a higher
number of normal blastomeres is often a good prognosis. Since
cell divisions follow a synchronized and symmetrical form, it
is essential to note how embryos with 8 cells at day 2 are
cleaving faster then expected. They should not be considered
for transfer. Embryos with odd numbers of blastomeres
probably have cells with slower division than others.
Embryo morphology is another important criterion during
transfer, based on the blastomere regularity, cell size and the
presence of cytoplasmic fragments. According to this
classification, the best embryos are those having blastomeres
of the same size and without fragments. Greater degrees of
fragmentation and blastomere irregularity give a poor
prognosis of pregnancy. The presence of multinucleated
blastomeres should also be recorded. When possible, these
embryos should not be considered for transfer.
During embryo development, the thickness of the zona
pellucida is reduced naturally, perhaps in order to facilitate the
hatching process before implantation. Embryos with thicker
zonae might thus have a poor prognosis for implantation, since
their hatching might be impaired. The combination of all these
 Reviews - Laboratory techniques for human embryos - S Geber at al.
criteria in order to select the most likely embryos to implant
might improve pregnancy rates.
When transferring blastocysts, morphology may be the most
important criterion to be considered. An expanded blastocele is
essential, the inner cell mass (ICM) should be well defined,
and trophectoderm (TE) cells must be regular and smooth
(Table 3). Hatching blastocysts may be preferable for transfer
with an increased probability of implantation, but this
approach has not been studied in detail. Uterine embryo
transfer via the cervical canal is the most widely used
technique for replacement. It must be performed using a no
touch method after embryos are drawn gently into a transfer
catheter, filled with culture media supplemented with ~50%
serum.
Cryopreservation of embryos
The cryopreservation of any cell involves crucial factors such
as the formation of intracellular ice and cellular damage due to
an increased osmolarity of the freezing medium associated
with slow cooling. These problems stress the need for starting
with a safe cryopreservation procedure. Special media are
prepared that are capable of cryoprotection. Many other
variables are important during the freezing process, such as the
time of seeding, dehydration speed and cooling speed.
The use of cryoprotectants for freezing embryos is essential for
good results. These substances protect the cells from adverse
consequences of the freezing process and generally have three
properties. They pass through membranes passively, have a
water displacement action and are cytotoxic at elevated
concentration. The most commonly used cryoprotectant for
assisted reproduction procedures are dimethyl sulphoxide
(DMSO), glycerol and propanediol.
Before starting embryo freezing, it is crucial to select the best
embryos to be frozen. The quality of the embryos plays a great
part in the success of the procedure and will avoid pointless
freezing and thawing. Only grade I or II embryos should be
considered for freezing. Once embryos are selected, it is
important to choose an adequate cryoprotectant according to
the development stage of the embryos. Propanediol is used for
embryos in the initial cleavage stages, i.e. from the pronuclear
Table 3. Possible markers for blastocyst
viability (Bongso, 1999).
Morphological characteristics
ICM size
Thin zona
Single large blastocoele
‘Sickle-shape’ in TE cells
Cleavage speed
Cavitated, expanding – day 5 (a.m.)
Fully expanded on day 5 (p.m.)
Hatching on day 5/6 (p.m./a.m.)
Smooth hatching: no collapse of blastocoele
stage to the 8-cell stage. In the case of blastocysts, glycerol
gives the best results. DMSO can be used for all stages and
even for oocyte cryopreservation.
Real damage to the embryo can occur during thawing besides
during freezing. As with freezing, it is important to adjust
thawing speed to the freezing speed. The most important part
of the freezing process is the gradual dehydration of the cells
if the temperature transition is reached by slow cooling.
Thawing must then also be carried out slowly, in order to allow
rehydration to occur gradually. After thawing, embryos are
placed in culture media for 24 h before transfer. This permits
the best cleaving embryos to be chosen, and might enable good
rates of implantation to be achieved.
Methods of vitrification are still being developed, but it cannot
be long before they are introduced into the routine laboratory
care of cleaving embryos and blastocysts. Various methods
involving specially constructed cryo-loops, and optimal stages
for the procedure are being developed (Cho et al. 1999; Lane
et al. 1999).
Conclusions
The main objective of an IVF laboratory is to mimic the in-
vivo environment of the female reproductive tract. Culture
media for gamete collection, preparation and for embryo
development can be classified into complex media and
balanced salt solutions. One of the most important constituents
of culture media is water. Since water is an excellent solvent
and provides a medium for most biological and chemical
reactions, it is also highly susceptible to contamination by
more substances than any other common solvent. It is also
very important to supply culture media with glucose, lactate or
pyruvate for energy uptake. It is still controversial whether
serum supplementation should be used. Using sequential
culture media, it is possible to achieve many pregnancies by
transferring embryos as blastocysts on day 5 or day 6.
For the ICSI procedure, oocytes are prepared for the injection
in droplets of hyaluronidase, while aggressively pipetting the
cumulus–corona complex with a large flame-polished Pasteur
pipette. HEPES-buffered media containing 10% PVP is
necessary to facilitate sperm handling and oocyte activation,
and to prevent spermatozoa from sticking in the injection
micropipette.
Drilling the zona pellucida by micromanipulation is performed
nowadays for assisted hatching and for embryo biopsy during
preimplantation diagnosis. Embryos are initially immobilized
by attachment to a suction pipette, and drilling is performed
using acid Tyrode’s solution, pronase or lasers. In the case of
assisted hatching, embryos are returned to culture medium for
a later transfer. In the case of preimplantation diagnosis, the
pipette used to deliver acidified medium to penetrate the zona
pellucida is removed, and the sampling pipette is pushed
through the hole. One or two cells judged to be at the
equivalent of the 8-cell stage are removed by gentle suction
and sent for genetic diagnosis After obtaining the genetic
results, unaffected embryos are selected for transfer, which can
be performed either on same day (day 3) or at blastocyst stage.
Chromosome counts on excised blastomeres are now helping
to raise implantation rates in older mothers by rejecting those 217
 Reviews - Laboratory techniques for human embryos - S Geber at al.
embryos found to have cytogenetic anomalies (Obasaju et al.,
2001). An alternative for assisted hatching is to remove the
whole zona at the blastocyst stage and to transfer the zona-free
blastocysts.
Embryo selection for transfer can be performed on day 1 by
observing the disposition of the pronuclei and the polarization
of the nucleoli; at day 2 and day 3 normal cleavage can be
observed, as well as morphological characteristics such as
blastomere size and fragmentation. At day 5 or 6, when
transferring blastocysts, morphology may also be an important
criterion to be considered. An expanded blastocele is essential,
the inner cell mass should be well defined, and trophectoderm
cells must be regular and smooth.
The cryopreservation of embryos involves crucial factors such
as the formation of intracellular ice and cellular damage due to
an increased osmolarity of the freezing medium associated
with slow cooling. These problems stress the need for starting
with a safe cryopreservation procedure. Special media are
prepared that are capable of cryoprotection. Many other
variables are important during the freezing process such as the
time of seeding, dehydration speed and cooling speed.
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