Clinical embryology has put into practice in the laboratories for assisted reproduction techniques
(ART) the recent discoveries regarding the physiology of early events in the development of
human life. The embryologist is responsible for the correct and justified application of ART in
The European Society of Human Reproduction and Embryology (ESHRE) wishes to give
guidelines which constitute the minimal requirements for any laboratory offering ART, with the
aim of implementing a quality control system for all the embryologists of ESHRE. The strict
application and further development of these guidelines will benefit all patients attending ART
A laboratory safety programme depends on every employee's participation and cooperation.
Non-compliance with safety precautions not only endangers the individual, but often
compromises the health and safety of fellow workers or patients and may result in damage or loss
of gametes and/or embryos. Relevant patient consent should be verified before commencing any
1. Staffing and direction
1.1. The laboratory is directed by an appropriately qualified and experienced scientist or medical
doctor according to national rules.
1.2. There are appropriate numbers of staff with the required experience to undertake the
workload of the laboratory.
1.3. All new staff are given a comprehensive orientation and introduction programme.
1.4. Continuing medical education (CME) is recommended for all personnel involved.
1.5. Individual responsibility of each member of staff and line of responsibilities are indicated in
a written procedure. www.shivaniscientific.com
2. Policies and procedures
2.1. All laboratory procedures must include provision for unique patient identification whilst
retaining patient confidentiality.
2.2. Laboratory results should be reported according to a written procedure. They should be
validated, dated and include unique patient identity. Any interpretation of results should be
accurate, comprehensive and clinically relevant.
2.3. There is a record of all reagents, calibration and quality control material.
2.4. There is a written, signed and dated protocol for every procedure, written transmission of
results and regular maintenance of equipment.
2.5. All the procedures should be gathered in a manual kept in the laboratory and available for
2.6. A log book should be maintained to permit a regular evaluation of the results.
3. Laboratory safety
3.1. Laboratory design
The embryology laboratory should have adequate space to follow good laboratory practice.
• The construction of the laboratory should ensure aseptic and optimal handling of gametes
and pre-embryos during all phases of the treatment.
• The location of storage areas and equipment such as incubators, centrifuges and cryo
equipment should be logically planned for efficiency and safety within each working area.
• Separate office space should be provided for administrative work, such as record keeping
and data entry.
• A general wet area in which washing of equipment, sterilization, etc., is performed,
should be separate from the embryo laboratory. Moreover, if fixatives are applied, these
analyses should be performed in a separate room in a fume-hood.
When commissioning the laboratory, thought should be given to the most recent developments in
equipment and facilities. Bench height, adjustable chairs, microscope eye height, efficient use of
space and surfaces, sufficient air-condition and the amount of daylight, all contribute to a
working environment that minimizes distraction and fatigue. Consideration should also be given
to local health and safety requirements.
3.2. Laboratory equipment
• The laboratory equipment used should be adequate for laboratory work and easy to clean
• Critical items of equipment, including incubators and frozen embryo storage facilities,
should be appropriately alarmed and monitored.
• All embryo laboratories should have an automatic emergency generator backup in the
event of power failure.
• A minimum number of two incubators is recommended. Gas cylinders should be placed
outside or in a separate room with an automatic backup system.
• Incubators should be frequently cleaned and sterilized. Nitrogen tanks should be cleaned
and sanitized at least every year.
3.3. Infectious agents
All assisted reproductive technologies involve handling biological material, and pose a potential
hazard of transmitting diseases to personnel. Sources of infection in this system are compounds
and follicle fluid of animal origin. Each unit should establish procedures and policies for the
safety of personnel, taking national and/or local safety regulations into consideration. Therefore:
• Vaccination of the personnel against hepatitis B or other available viral disease is
• Screening patients and gamete donors for HIV, hepatitis B/C and other sexually
transmitted diseases before treatment or gamete cryopreservation should be routinely
Although patients' admittance to IVF treatment cycles is regulated by physicians, the laboratory
staff must be informed about the risks of handling potentially infected biological material. The
following recommendations should be considered:
• If one or both partners are HIV-positive, IVF and embryo transfer should not be
attempted, unless local rules give authorization after extensive examination. Donor semen
is advised for a seronegative woman if her partner is seropositive, as HIV-1 viral particles
may be present in the cytoplasmic compartment of motile spermatozoa (Baccetti et al.,
1994 ). Therefore, the possibility exists that viral particles enter the oocyte during
fertilization either with conventional IVF or intracytoplasmic sperm injection (ICSI). The
fate of these particles in the resulting embryo is unknown.
• If the male partner is infected by the virus of hepatitis B, the seronegative woman must be
vaccinated before IVF.
• If the woman is infected by the virus of hepatitis B, IVF is feasible but the couple must be
warned about the necessity of specific vaccination of the baby at birth. It has been
recently suggested that the use of ICSI in hepatitis B-positive patients could increase the
risk of bringing the virus into the oocyte from the surrounding fluid, possibly adhered to
the sperm membrane.
• If one partner is infected by the virus of hepatitis C, IVF may be performed after
• In case of seropositivity for syphilis, appropriate treatment must be given before IVF.
3.4. Protective measures
The purpose of the protective measures is also to ensure aseptic conditions for gamete and
embryos. The procedures should deal with, but not be limited to, the following:
• Use of laboratory clothing.
• Use of non-toxic (non-powdered) gloves and masks.
• Use of eye and face protection if cryogenic materials are handled.
• Use of vertical laminar-flow benches.
• Use of mechanical pipetting devices.
• Use of fume-hood in case of fixatives.
• Disinfection and sterilization of potentially infected equipment.
• Use of disposable material; after usage, it must be discarded immediately in the proper
waste containers. Potential infectious materials must be disposed of in a manner that
protects laboratory workers and maintenance, service, and housekeeping staff from
exposure to infectious materials in the course of their work.
• Needles and other sharps should be handled with extreme caution and discarded in special
containers. If possible, glassware should be omitted in the laboratory, otherwise the
Pasteur pipettes and broken glassware should be discarded in special containers.
Cross-contamination with infectious material from one patient to another could still occur during
the cryopreservation procedure when straws with semen or embryos are filled by dipping the
straw in patient medium with semen or embryos, sealing it and passing it into liquid nitrogen
without external disinfection (Tedder et al., 1995 ). Material stored in the cryopreservation tanks
should be kept in a way that avoids contact of the liquid nitrogen phase with the biological
substances. Safety cryo straws have been conceived in order to fulfil this requirement. Specimen
known to be contaminated should be stored in such high security straws.
4. Correct handling and identification of patients and their gametes and embryos
Written procedures should be present describing the various phases of IVF techniques. Rules
concerning the correct handling and identification of gametes and embryo samples should be
established by a system of checks and, where needed, double-checks. Proper training of all the
laboratory staff according to these procedures is mandatory.
• All material obtained from the patients, i.e. tubes with blood, follicular fluid and
spermatozoa, should bear unique identification of the treated couple.
• Incubators should be organized in order to facilitate identification of embryos, oocytes
• Verification of patients' identity should be performed at critical steps: before ovum
retrieval, at semen recovery and embryo transfer procedures.
• Double checks need to be considered at least at: insemination of oocytes, replacement of
embryos, embryo freezing and thawing.
Written validation of all critical steps in each patient's file is essential.
5. Culture media preparation and quality control testing
5.1. Disposables and reagents.
• Glassware and disposable items used for preparation of culture media should be of tissue
culture grade and dedicated for this purpose alone.
• Culture media, or reagents used in their preparation, should be of a purity appropriate for
5.2. Laboratory protocols should include in-house quality control procedures for culture media.
• All tissue culture media prepared in the laboratory should undergo quality control using
an appropriate bioassay system.
• When commercially produced media are used, integrity of the packages and appropriate
delivery conditions should be controlled.
• Documentation of quality control testing should be supplied by the manufacturer for any
commercially produced media employed. Correspondence with the delivered batch should
• Reagents and media should always be used prior to the manufacturer's expiry date.
5.3. Water used for the preparation of culture media should be of appropriate purity.
• If the water is purified on site, protocols must be in place for quality control of the water
system, including sanitization of the system, regular cartridge changes and replacement of
other components, endotoxin and bacterial screening.
• If culture grade water is purchased there must be strict control over storage and use in
relation to temperature and shelf-life.
5.4. There should be a systematic record of all batch numbers of reagents, media and disposables used in the
preparation of media with the date of their introduction to the system.
• Reagents should be designated exclusively for use in culture media.
5.5. Donor serum or follicular fluid are not recommended.
If they are used in culture media they should be screened according to the local rules for blood
• Human serum albumin (HSA) should come from a source screened as above. Commercial
suppliers of HSA or media containing a serum derived protein source should supply
evidence of such screening.
• Appropriate quarantining arrangements should be in place for donor use.
5.6. Oocytes may be cultured under equilibrated mineral oil.
The oil maintains temperature, osmotic pressure and pH during short-term manipulations of the
oocytes. Before use, it requires pre-equilibration in the incubator.
6. Handling of oocytes and spermatozoa
The laboratory procedures regarding the handling of gametes for assisted reproduction
techniques have been standardized to a large extent. The procedures should be easy, simple and
effective and must be performed in a laminar flow hood equipped with stages and heating blocks
pre-warmed at 37°C. Class II hoods should be preferred as they give protection to the operator.
Disposable items used in the laboratory preparation of culture media should be of tissue culture
6.1. Aseptic technique should be used at all times.
6.2. Appropriate measures should be taken to ensure that oocytes and embryos are maintained at
37°C during handling/observation using hot plates or other systems.
6.3. Tissue culture grade disposables should be used for handling body fluids/cells.
6.4. Pipetting devices (pasteurs, drawn pipettes, tips etc.) should be used for one procedure only,
should never be used for more than one patient, and should be disposed of immediately after use.
6.5. Simultaneous treatment of more than one patient should never be done in the same working
6.6. Identifying information marked on the culture dish/tube should be cross-referenced to the patient and
the patient's documentation.
• Procedures should be in place which ensure correct patient identification at all stages.
• Labelling of dishes/tubes containing oocytes, embryos, or spermatozoa should be
permanent. (Strong odour markers should be avoided.)
• Incubators should be organized in order to facilitate identification of embryos, oocytes
6.7. At each stage of the procedure the identity of the embryologist should be recorded.
7. Oocyte retrieval
7.1. Oocytes need to be kept at body temperature (36–37°C) as much as possible. There should
also be appropriate equipment in use to maintain this temperature when laboratory and egg
collection areas are on different sites. Scanning Petri dishes, collection tubes and heating blocks
are pre-warmed at 37°C. Follicular aspirates are checked for the presence of oocyte-cumulus
complexes under a stereo dissecting microscope with transmitted illumination base and heated
stage. Exposure of oocytes to light should be minimized where possible.
7.2. Where donor oocytes are to be used they should be labelled with the donor's name, or
reference code, at the time of oocyte retrieval, and only identified with the recipient's name at the
time of allocation.
8. Sperm preparation
Before starting a treatment cycle, semen analysis is performed according to the protocols
described in the World Health Organization manual (WHO, 1999).
The semen sample is collected in a sterile, plastic container without using condoms, creams or
lubricants. The container should be clearly labelled with the name of the couple.
8.1. Records should be kept of the period of abstinence, the type of container used (if this differs
from the norm), time and place of collection (with particular reference to samples produced
outside clinic facilities), and the time interval between collection and preparation.
8.2. Where donor spermatozoa is used, the necessary identifying information (donor code/clinic
code) must be recorded.
8.3. The method of sperm preparation should be recorded, including details of any variation on
the standard laboratory protocol.
8.4. A record should be kept of pre- and post-preparation sperm parameters and of any dilution
carried out prior to insemination.
8.5. In case of surgically retrieved spermatozoa, exceeding spermatozoa after insemination
should be cryopreserved for further assisted reproductive cycles; this will avoid repeated surgery.
9. Insemination of oocytes
9.1. Preparation of spermatozoa for insemination.
The method of preparation is designed according to individual samples. A trial preparation prior
to the treatment cycle may be advisable in order to choose the most adequate technique. Sperm
preparation aims to:
• concentrate and select the active and motile spermatozoa;
• get rid of seminal plasma, debris and contaminants;
• select against abnormal forms.
A frozen backup sample may be requested for those patients who anticipate the possibility of
Different methods have been proposed for sperm preparation. Among them, the swim-up
technique and the discontinuous density–gradient centrifugation are the most used (Burkman et
al., 1984 ; McClure et al., 1989 ; Van der Zwalmen et al., 1991).
As a general rule excessive centrifugation should be avoided especially in oligospermic samples
in order to avoid increasing the concentration of reactive oxygen species.
Since January 1997 the Percoll that was used in the discontinuous density–gradient
centrifugation procedure has been prohibited for clinical purposes.
The preparation of spermatozoa retrieved by microsurgical operations requires specific protocols
(Devroey et al., 1994 ; Silber et al., 1994 , 1995 ; Oates et al., 1997 ; Kupker et al., 1998 ).
9.2. A record should be kept of the time of insemination and the sperm concentration used. For
conventional insemination, the number of spermatozoa must be high enough to yield the best
fertilization rate without compromising embryo development.
9.3. For the intracytoplasmic sperm injection (ICSI) procedure, it is important to select vital
spermatozoa, as evaluated by their motility. Where only non-vital sperm cells are present in the
ejaculate, the use of testicular spermatozoa may be proposed.
9.4. Preparation of oocytes for ICSI. Removal of cumulus–corona (CC) cells.
Oocytes are denuded from the surrounding cumulus and corona cells using an enzymatic
procedure with hyaluronidase, followed by mechanical denudation using a pipette with an inner
diameter of ±150 µm. Both the enzyme concentration and the duration of exposure to the enzyme
should be limited (Van de Velde et al., 1998 ). Care needs to be taken in order to avoid damage
to the oocytes, which can result from too vigorous pipetting or from a pipette diameter which is
too small. After rinsing several times in washing medium drops, the denuded oocytes are
observed under the inverted microscope at x200 magnification and cultured until the time of
9.5. The injection procedure.
During ICSI, the following points are important:
• the selection and immobilization of a viable sperm cell;
• the correct positioning of the oocyte prior to injection;
• the rupture of the oolemma prior to the release of the sperm cell into the oocyte.
Polyvinylpyrrolidone (PVP) can be used to facilitate the manipulation of spermatozoa and to
control the fluid in the injection pipette, limiting the volume injected into the oocyte.
10. Scoring for fertilization
10.1. All oocytes that have been inseminated or micro-injected should be examined for the
presence of pronuclei at 16–20 h post-insemination.
10.2. The status of each oocyte should be recorded.
10.3. Oocytes with one or more than two pronuclei should be isolated from normally fertilized
oocytes (Plachot et al., 1992).
10.4. Oocytes showing no signs of fertilization should be maintained in culture and observed for
late appearance of pronuclei.
10.5. The normally fertilized oocytes are removed from the insemination medium and transferred
into new dishes with pre-equilibrated fresh growth medium.
11. Embryo culture and embryo transfer
The scoring of embryos at regular intervals provides the possibility to the embryologist of
tracking embryo development.
11.1. The stage of embryo development at the time of transfer should be documented. Embryos
can be grown to day 5 or 6 for a transfer at the blastocyst stage. Sequential media (one medium
from days 1–3 and a second one from days 3–5 or 6) allow the growth of blastocysts with a high
implantation rate replacing the need for co-culture systems (Guerin and Nicollet, 1997 ; Gardner
et al., 1998 ).
11.2. In some countries the maximum number of embryos transferred is established by the local
legislation. As a general recommendation, it is advisable not to exceed two. In cases where more
than two embryos are replaced, the couple has to be extensively informed on the risks of multiple
gestations. When blastocysts are available, the number of blastocysts transferred should be
limited to two. The supernumerary embryos may be cryopreserved, donated to research or
discarded, according to the couple's wishes and to the national legislation.
11.3. The patient records for embryo transfer should include details of:
• batch number and type of media used for transfer;
• time from oocyte retrieval to transfer;
• time from oocyte insemination to transfer;
• the number and developmental stage of embryos at transfer;
• fate of excess embryos;
• type of catheter used for transfer.
11.4. If the laboratory is some distance from the embryo transfer room, arrangements should be
made to maintain temperature and pH by the use of mobile equipment whilst transporting
11.5. Sterile disposable catheters should be used for transfer.
12. Cryopreservation of embryos
Embryo freezing can be performed at different stages: zygotes, embryos in early cleavage (day 2
or day 3), blastocysts. Embryos displaying a high degree of fragmentation or very slow or
blocked cleavage rate should be discarded from storage procedures due to the low survival rate
reported after thawing.
In some countries, the application of cryopreservation procedures is regulated by law.
12.1. Techniques and facilities for cryopreservation of embryos should be available in each IVF centre with
the aim of:
• cryopreserving spare embryos after transfer;
• delaying embryo transfer in a subsequent cycle if the patient becomes ill or develops
ovarian hyperstimulation syndrome (OHSS);
• storing the embryos generated from donated oocytes in order to allow a 6 month
quarantine so that the potential donors may be controlled for infectious diseases prior to
12.2. Several protocols have been formulated depending on the embryo cellular stage, type of
cryoprotectant, and speed of cooling (Testart et al., 1986 ; Ménézo et al., 1992 ; Veeck et al.,
12.3. In order to minimize any risk of transmission of infection via liquid nitrogen, embryos should be stored
in specific receptacles (i.e. straws, vials etc.) that can be sealed effectively.
• Transfer of embryos to receptacles should be by a method which avoids contamination of
the external surface.
• Sealing should be checked carefully before freezing.
12.4. Patients having transfer of thawed embryos ideally should be screened for hepatitis B and C, and HIV.
• When a patient is known to be a source of infection risk, a system of separate storage
should be considered.
12.5. Documentation on stored embryos should include:
• the type and batch number of cryoprotectant used;
• the stage of embryo development;
• the number of embryos in each straw/vial, which should not exceed two;
• the number of straws/vials stored per patient.
12.6. Straws/vials containing embryos must be clearly and permanently labelled with reference
to patient details and their unique identification code.
12.7. Storage records should be kept in both the patient's individual records and the storage
records for individual nitrogen banks.
12.8. An annual audit of stored gametes and embryos must be carried out, cross referencing
contents with storage records.
12.9. Storage records should include precise details of the location of the vials/straws.
12.10. Documentation of thawing procedures should include morphological changes seen during
thawing and the time period of culture prior to transfer.
13. Assisted hatching
This technique has been designed with the aim of improving embryo hatching and possibly
implantation. However there are conflicting reports about its clinical efficacy.
13.1. Three methods are being used; the mechanical technique, that is partial zona dissection with
glass microneedles (Cohen et al., 1990 ), the chemical assisted hatching using acidic Tyrode's
(Cohen et al., 1992 ), and the laser assisted hatching (Strohmer and Feichtinger, 1992 ).
13.2. Special care should be taken to avoid damage to the embryo during the procedure.
Novel techniques have been proposed during the last few years aimed at expanding ART indications:
freezing of oocytes and ovaries, in-vitro maturation of oocytes, intracytoplasmic transfer, spermatid
injection, embryo implantation. Reports continue to accumulate from animal studies and preliminary
data. However, additional studies are necessary before admitting these procedures to the standard
technique protocols which are routinely performed in IVF laboratories.