COMPILED & EDITED BY: CIARA HUGHES ON BEHALF OF THE ASSOCIATION OF CLINICAL EMBRYOLOGISTS
Introduction175
A. Organisation and quality management175
1.Laboratory organisation175
2.Regulatory aspects175
3.Quality management system (QMS)176
4.Standard operating procedures176
5.Record keeping176
6.Evaluation and quality assurance (KPIs)176
B. Personnel and training177
1.Personnel177
2.Training in embryology techniques177
3.Training in ICSI technique177
C. Premises and environment178
1.The laboratory178
2.Safety179
D. Laboratory equipment, consumables, media and reagents179
1.Equipment179
2.Consumables, media and reagents180
E. Procedures181
1.Protective measures181
2.Infectious agents181
3.Identification of patients and their gametes and embryos182
4.Handling of oocytes and sperm183
5.Sperm preparation183
6.Oocyte retrieval184
7.Insemination of oocytes184
8.Scoring for fertilisation184
9.Embryo culture and grading185
10.Embryo transfer185
11.Cryopreservation185
12.Assisted hatching187
13.Embryo biopsy187
F. Research and the introduction of novel techniques187
1.Research187
2.Introduction of novel techniques187
Abbreviations188
References188
Appendix188
Introduction
The ACE Guidelines on Good Laboratory Practice were initially drawn up in 1996 and were considered as the minimal requirements for any laboratory offering assisted reproductive technology in the UK. However, since that time, the field of assisted reproduction has grown rapidly in the UK both in terms of training, regulations and technological advances and in March 2004, The European Parliament issued the directive 2004/23/EC entitled “On setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells”. This directive also covered human tissues and cells including fresh and frozen gametes, embryos and reproductive tissue. The UK, unlike many other countries within the EU was fortunate already to have in place a competent regulatory body, the Human Fertilisation and Embryology Authority (HFEA) which took over the role of overseeing the implementation of the directive. HFEA also published its 8th Code of Practice in October 2009 to reflect the changes in the HFEA Act 2008 and in HFEA policy.
In view of these developments, the ACE Guidelines on Laboratory Practice has been updated to reflect the changes in practice and to compliment the standards at both a national and European level. Although many clinics through their own willingness to improve, have implemented quality management systems voluntarily, the updated guidelines focus on the role and importance of quality management in the laboratory particularly in relation to key performance indicators (KPIs) and provide benchmarks which clinics should aim for.
The ACE certificate continues to be a comprehensive, established training programme that has delivered competent embryologists since 1996. The Association is also committed to the three year Scientist Training Programme as part of the Modernising Scientific Careers initiative. The training aspect of the guidelines addresses this recent, rapid evolution of training to become a Clinical Embryologist. Areas such as air quality, specific embryo grading schemes, CE marking of consumables and media, the introduction of electronic witnessing, research and novel techniques have all been updated.
These ACE guidelines endeavour to promote the highest possible level of standards to safeguard the couple undergoing assisted reproductive treatment, their gametes and embryos and the staff who are responsible for them.
A. Organisation and quality management
A1. Laboratory organisation
There should be a document which describes the organisation and function of the laboratory and its relationship to the clinical service(s) it supports including any additional services. The laboratory should have an internal organisational chart which clearly defines accountability and reporting relationships.
A2. Regulatory aspects
A2.1. All controlled activities relating to human gametes and embryos must be conducted according to the conditions of a licence granted by the Human Fertilisation and Embryology Authority. No such activities may be carried out except under the terms of such a licence.
A2.2. No person should carry out any clinical procedure, controlled or otherwise, involving gametes and embryos without having clear and direct evidence of relevant patient informed consent.
A2.3. All gamete and embryo procurement and handling must be carried out by persons with appropriate training and experience. This should only take place in conditions designated and licensed for that purpose by the HFEA.
A2.4. All procedures involving the initial procurement consent/collection, subsequent processing, mixing, movement of gametes or embryos from one culture vessel to another or in or out of cryostorage, must be witnessed by an appropriate person or validated witnessing system in accordance with current guidelines from the HFEA.
A2.5. Account should be taken of guidance and regulations issued by all relevant bodies including the Health and Safety Executive and the Medicines and Healthcare Products Regulatory Agency.
A2.6. Account should be taken of the EU parliament Directive 2004/23/EC on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells.
A2.7. Account should be taken of the EU Parliament Directive 2006/17/EC as regards technical requirements for the donation, procurement and testing of human tissues and cells.
A2.8. Account should be taken of the EU Parliament Directive 2006/86/EC as regards traceability requirements, notification of serious adverse reactions and events and technical requirements for the coding, processing, preservation, storage and distribution of human tissues and cells.
A2.9. All confidential information must be protected under the Data Protection Act 1998.
A3. Quality management system (QMS)
A3.1. The laboratory must participate in the centre’s quality management system and use this system to improve continually, the quality and effectiveness of the service provided in accordance with the conditions of this licence and the guidance on good practice as set out in the HFEA’s Code of Practice 8th Edition (Section 23.1).
A3.2. All laboratory activities should be carried out under a quality management system (QMS).
A3.3. Each clinic should have a designated quality manager (QM) who is responsible for the implementation and ongoing operation of the QMS. The head of the laboratory should work closely with the QM to ensure integration of the laboratory service into the QMS.
A3.4. Each laboratory should ensure that the quality system includes at least the following documentation:
A quality manual
Standard operating procedures for all activities
Guidelines
Training and reference manuals
Reporting forms
Donor records
Information on the final destination of gametes and/or embryos
A3.5.The laboratory should have quality objectives and strategic plans and ensure that there are resources available to implement and maintain them.
A3.6.The laboratory QMS should define clearly responsibilities, authorities and reporting relationships within the laboratory.
A3.7.There should be an internal and external audit plan which is implemented by the quality manager.
A3.8.The laboratory should have a validation master plan, and validation of equipment and processes should be complete.
A3.9.There should be a policy for handling and following up non-conformances.
A3.10.There should be a formalised document control and change request policy.
A3.11.A number of key performance indicators (KPIs) to allow evaluation and continuous quality improvement should be detailed and performed at specified time intervals. See section A6.
A3.12.The quality management system should ensure that there are third party agreements in place with all suppliers or service providers and that these are reviewed and renewed on a regular basis. There must also be third party agreements in place with external procurement sites that comply with the relevant legal requirements.
A4. Standard operating procedures
A4.1. All laboratory activities should be carried out according to written standard operating procedures in line with the centre’s quality system.
A4.2. All standard operating procedures must be fully version controlled and approved by the laboratory manager/lead embryologist and quality department.
A4.3. Standard operating procedures should include the scope and purpose, the equipment/reagents required, as well as the protocol to be followed.
A4.4. Standard operating procedures should be reviewed at least annually, and obsolete versions removed from circulation and archived.
A4.5. A regular systems audit should be carried out and documented to validate staff compliance with standard operating procedures.
A5. Record keeping
A5.1. All procedures should be recorded in standard format and include unique patient identifiers and the identity of the operator, as well as witnesses where relevant, and should be signed and dated.
A5.2. A system should be in place to identify and record batches of consumables. There should be a batch recall policy and system in place which should be tested periodically to ensure appropriate patient identification to recalled batches.
A5.3. The laboratory manual, containing all written procedures and all standard reporting forms should be available in the laboratory or be easily accessible on a computer in the laboratory under controlled software.
A5.4. A log of all procedures carried out should be maintained to permit continuous evaluation of results and generation of KPI’s.
A5.5. Patient donor records required for full traceability must be kept for a minimum of 30 years (or for a longer period as may be specified in Directions) after clinical use, or the expiry date, in an appropriate archive acceptable to HFEA. (HFEA COP 8th Ed. Section T48).
A5.6. Where computerised records are kept, an electronic back up should be carried out daily, with a copy of the data being kept in fireproof containers or in an approved remote location. If the clinic is operating a paperless system, then the data must be backed up daily using validated software and a remote server capable for storage for 30 years (or for a longer period as may be specified in Directions).
A6. Evaluation and quality assurance (KPIs)
A6.1. The laboratory should establish quality indicators for systematically monitoring and evaluating the laboratory’s performance. These key performance indicators should be evaluated on a regular basis and benchmarks established. It is recommended that established centres use the previous year’s results as their benchmarks. Suggested KPIs and reference ranges are outlined in Appendix 1.
A6.2. Intervals between analyses should be based on the laboratory caseload.
A6.3. Laboratories should calculate their own alert conditions for all parameters.
A6.4. Deviation from these benchmarks should warrant investigation in line with the clinic’s non-conformance policy.
A6.5. Embryologists should audit their personal performance on a regular basis, particularly in relation to all critical procedures.
A6.6. Competency assessment should be carried out on annual basis. This can be performed by direct observation by another laboratory staff member using the SOP to audit, with a subsequent analysis of the outcome.
A6.7. Analyses should take account of alterations in clinical procedures or personnel.
A6.8. Co-operation between disciplines should be facilitated in carrying out audits of unit and individual performance in order to maintain and improve outcomes.
B. Personnel and training
B1. Personnel
B1.1. The laboratory should establish documented procedures for staff management that ensure all staff have:
(a)an initial orientation and induction
(b)basic training and updated training as required
(c)on-going competence assessment with audits of this assessment
(d)an annual joint review (with their line manager)
(e)continuing education and professional development
(f)staff records
(g)appropriate access to meetings and communications
B1.2. All embryologists should have undergone a recognised training scheme such as the ACE Certificate in Clinical Embryology or the MSC training scheme for reproductive science. Embryologists who have trained overseas should be able to demonstrate their competence and apply for state registration via the overseas route.
B1.3. All clinical embryologists must be registered, or working towards, registration with the Health Professions Council within 1 year of becoming eligible.
B1.4. The individual responsible for the clinical embryology laboratory would be expected to:
possess an appropriate scientific or medical degree supplemented by training – preferably a higher degree and/or a recognised professional qualification
have a minimum of 4 years documented experience in carrying out assisted conception procedures
have state registration
have appropriate management, training, organisational and communication skills and experience
B1.5.A risk assessment should be carried out to ensure that there are adequate staffing levels with the appropriate amount of experience to undertake the laboratory workload according to the centre’s activity levels, including cover for transport/satellite centres, weekends and any out of hours service required.
B1.6.Staff numbers must be sufficient to provide cover for leave and sick leave without compromising performance or patient safety and each centre should have a contingency plan in the event of an emergency.
B1.7.All new staff must undergo a comprehensive orientation and induction programme and be appropriately trained and qualified for the tasks they are performing.
B1.8.No scientist shall undertake clinical work without supervision by a fully trained embryologist unless they are State Registered.
B1.9.All qualified scientific staff should be enrolled in a recognised programme for continuing professional education.
B1.10.There should be a method to ensure that individual responsibility of each member of staff and line of responsibilities are defined and documented in written procedures that are known and accepted by all members of staff.
B1.11.All staff should have an up-to-date job description and a contract of employment which clearly states the terms and conditions of employment including leave and sickness policies, out of hours cover and part-time regulations etc.
B1.12.In addition to clinical training for staff, other areas for training should be identified and organised depending on roles and responsibilities. Staff should have general training on manual handling, health and safety and where necessary, handling compressed gas, liquid nitrogen etc.
B1.13.There should be adequate personal security for staff.
B1.14.Staff should have sufficient locker space and toilet facilities available.
B1.15.In centres where staff may work alone, the centre should have a lone worker policy in place with the appropriate risk assessment carried out.
B1.16.Vaccination of personnel against Hepatitis B or other viral disease for which the vaccine is available is recommended.
B2. Training in embryology techniques
B2.1. Gametes and embryos may only be used, for the purpose of training persons in embryo biopsy, embryo storage or other embryological techniques and in those activities that are expressly authorised by the Authority. (HFEA COP 8th Ed. Section T93).
B2.2. The centre should have documented procedures for obtaining gametes or embryos to be used for research or training purposes and for obtaining written informed consent from donors for research and training purposes and ensuring that the gametes or embryos are only used in line with this consent. (HFEA COP 8th Ed. Section 22.2).
B2.3. Gametes and embryos may only be used, for the purpose of training persons in embryo biopsy, embryo storage or other embryological techniques, where both gamete providers have consented to the use of embryos, created using their gametes, for the purpose of training. (HFEA COP 8th Ed. Section T94).
B2.4. No gametes or embryos appropriated for the purpose of training staff in embryological techniques must be kept or used for the provision of treatment services. (HFEA COP 8th Ed. Section T94).
B2.5. The centre should have procedures in place which ensure that there is no conflict of interest between the use of gametes or embryos in training and the use of these gametes or embryos in the provision of treatment services.
B2.6. There should be a designated individual who is not directly involved in the patient’s treatment to discuss with the patient the training activity and the possibility of donating material for it. (HFEA COP 8th Ed. Section T95).
B2.7. The person obtaining the consent for the use of the embryos in training should not be involved in the training project in order to reduce any conflict of interest. (HFEA COP 8th Ed. Section T95).
B2.8. The consent must be obtained by trained personnel in a manner and under terms that are easily understood by patients with each gamete provider being provided with the following information:
The nature of the training for which embryos will be used
That the decision whether to donate or not will not affect their treatment in any way
That they can vary or withdraw their terms of consent until the point the embryos are used in training
Whether any information will be fed back to them (HFEA COP 8th Ed. Section T97-T98)
B3. Training in ICSI technique
B3.1. Consent for the use of oocytes or sperm for ICSI training purpose, consent must be obtained according to the requirements outlined in B2.
B3.2. The injection of any sperm into oocytes for the purpose of training is not permitted even if they are partner donations unless this particular activity has been granted under a separate HFEA research licence.
B3.3. ACE recommends the use of beads or air bubbles for injection purposes.
B3.4. Oocytes
It is recommended that for the purpose of training, oocytes used must either be:
(a)oocytes that have been denuded in preparation for ICSI but found still to be immature (metaphase I or germinal vesicle stage) >48 h post hCG administration.
or
(b)mature oocytes (from either IVF or ICSI) that have been identified as unfertilised >48 h post insemination.
B3.5. No oocytes that have been used for the purpose of ICSI training can be used for human application.
C. Premises and environment
C1. The laboratory
C1.1. Embryology laboratories should operate as a clean room with an environment of a minimum of Grade D quality according to GMP. Optimally, the aim should be to have air quality at Grade C or above. If it is impractical to carry out a procedure involving the manipulation of gametes or embryos in a Grade C environment, it should be done in an environment of at least Grade D air quality. If the environmental air quality drops below Grade D during a procedure involving the manipulation of gametes or embryos, those gametes or embryos should be used in treatment only if the centre can assure itself that this poses no extra risk to the woman to be treated or to any resulting child.
C1.2. Air quality monitoring should be used as a routine measure of quality assurance (for example, through particle counts or the use of settle plates; recording any cultures observed).
The process of validating air quality should include:
(a)documenting culture conditions
(b)mapping temperature and using control charts to predict the effects of any change in procedures (HFEA COP 8th Ed. Section 15.18).
C1.3. The embryology laboratory should have adequate space to ensure safe and comfortable working conditions and be of a design that is appropriate for the volume and scope of the procedures performed.
C1.4. The laboratory must be secure and should be designed to minimise traffic in work areas.
C1.5.The location of storage areas and equipment should be planned for optimal efficiency in each working area.
C1.6.Laboratory design should be such as to facilitate cleaning to the required standards.
C1.7.Floors, walls and ceilings must have non-porous surfaces that can be cleaned easily.
C1.8.Separate office space should be available for carrying out administrative work.
C1.9.Access to the laboratory should take account of the need for environmental control and security.
C1.10.Where the laboratory is adjacent to the theatre area, traffic between the two should be limited to the minimum necessary.
C1.11.No unauthorised personnel should be able to enter the laboratory unaccompanied and no-one should enter the laboratory unless appropriately dressed.
C1.12.Only trained scientific, technical, medical or nursing staff, or staff in training who are under supervision, should be allowed to enter the laboratory while procedures are taking place.
C1.13.Visitors should never be left unsupervised in clinical laboratory areas.
C2. Safety
C2.1.It is the duty of all laboratory personnel to inform laboratory and/or centre management of any circumstances which, in their view, compromise the safety of laboratory personnel, and/or the safety and integrity of gametes and/or embryos in their care.
C2.2.The laboratory design should allow all procedures to be carried out without compromising the safety of staff, patients, or patients’ gametes or embryos.
C2.3.If it is the policy of the clinic to handle potentially infectious samples such as Hepatitis or HIV, the laboratory design should allow sample storage and processing to be isolated to prevent cross contamination. Separate storage vessels must be available for each infectious agent that is covered under the clinic’s acceptance policy. See Section E2.
C2.4.Equipment should be placed such that there is sufficient and safe operating space.
C2.5.Attention should be paid to the ergonomics of the operator, bench height, adjustable chairs, microscope eye height, efficient use of space and surfaces, sufficient air-conditioning with controlled humidity and temperature.
C2.6.Measures should be taken to minimise exposure of gametes and embryos to volatile organic compounds and other potentially toxic substances.
C2.7.Storage of frozen cells and tissues should be carried out in a separate, designated, secure and safe facility where possible.
C2.8.The location should allow the safe delivery of liquid nitrogen, with respect to staff and members of the public.
C2.9.There should be low-level extraction and airflow through the room, which is adequate for the volume of nitrogen stored.
C2.10.In the event of significant spillage, emergency procedures should be in place to allow the increased volume of gas generated to escape.
C2.11.The storage room should have an oxygen depletion monitor, linked to an external warning system. The oxygen detection cell must be regularly replaced in accordance with the manufacturer’s instructions.
C2.12.Staff who handle liquid nitrogen and/or compressed gases should have appropriate training. When either of these gases are handled, all appropriate safety precautions must be adhered to including the use of personal protective equipment.
C2.13.All cryostorage vessels should have an alarm system to alert staff to conditions outside safe limits, i.e. a rise in temperature or loss of nitrogen. This system should be operational full time, i.e. 24 h/365 days. The system should also be tested on a regular basis to ensure that it is functioning. Activation of these alarms should be tracked and trended to detect potential future vessel failure. This system must be capable of contacting laboratory staff remotely for any out-of-hours alarm activations.
C2.14.A procedure must be in place for dealing with cryovessel failure, to include precautions to be followed prior to entering a room where oxygen levels may have been compromised.
C2.15.All local health and safety rules should be followed and these should be available to all staff. Staff should be familiar with the Clinical Incident policy, e.g. needle stick injury.
C2.16.All staff should have access to relevant COSHH information.
C2.17.All staff should have appropriate manual handling training.
D. Laboratory equipment, consumables, media and reagents
D1. Equipment
D1.1.A user requirement specification (URS) should be completed prior to the purchase of any new equipment and the equipment should be CE marked.
D1.2.Any new piece of equipment installed should have an installation, operational and performance qualification (IQ, OQ, PQ) performed prior to usage in the clinical setting.
D1.3.There should be a yearly calibration, preventative maintenance and validation plan for all the equipment in the laboratory and these records should be stored by the laboratory or quality manager.
D1.4.All laboratory equipment should be fit for its purpose and easy to clean and disinfect. Records of cleaning and decontamination should be maintained and audited.
D1.5.Back up power supplies should be provided for all critical items of equipment, including incubators and monitoring equipment.
D1.6.Critical items of equipment such as incubators and cryostorage facilities, should be appropriately monitored, equipped with alarm systems, with effective procedures in place for dealing with alerts at all times. Any non-functioning equipment should be clearly identified as not in use and reported as a non-conformance to the quality team. Any such equipment should be re-validated before being bought back into service.
D1.7.Back-up facilities should be available in the event of critical equipment failure, e.g. no laboratory should have fewer than two incubators, and a spare cryostorage vessel should be available into which material from a failing vessel may be transferred.
D1.8.Gas cylinders should be placed outside the laboratory whenever possible, and secured in such a way as not to present a safety hazard.
D1.9.Automatic changeover units should be in place to ensure a continuous supply of gas to incubators.
D1.10.Standard operating procedures should be in place for cleaning, monitoring, recording equipment performance and the frequency of calibration/servicing.
D1.11.All incubators and heating appliances should have an alarm system to alert staff to conditions outside safe limits, i.e., a rise or drop in temperature, CO2 level or (if appropriate O2 levels). This system for the incubators should be operational during any period where gametes or embryos are being cultured. The system should also be tested on a regular basis to ensure that it is functioning. Activation of these alarms should be tracked and trended to detect potential future vessel failure. The information should be recorded, acceptance criteria defined and any deviations dealt with through the non-conformance system. This system must be capable of contacting laboratory staff remotely for any out-of-hours alarm activations.
D1.12.Incubators should be cleaned and tested for microbial contamination on a frequent basis. The frequency of cleaning should be determined by the manufacturer’s instructions, usage, and evaluation of previous microbial reports.
D1.13.Devices should be available to maintain the temperature of the media during each phase of the procedure when they are out the incubator, e.g. heated stages, warming blocks.
D1.14.A list should be kept under the document control system which has the emergency contact numbers for the relevant service engineers for critical equipment.
D1.15.An asset list should be kept for all equipment and a financial plan made for the replacement of old equipment.
D2. Consumables, media and reagents
D2.1.All consumables used should be of tissue culture grade and sterile. Optimally, consumables should be CE marked for the purpose of IVF use and be mouse embryo and endotoxin tested.
D2.2.Media to be used for the preparation, culture and storage of gametes and embryos should be of tissue culture grade and manufactured under conditions which follow good manufacturing practice (GMP).
D2.3.Commercial media should be CE marked for IVF use. If not, a risk assessment and justification for use should be carried out prior to usage.
D2.4.Documentation of quality control testing using an adequate bioassay system must be supplied by the manufacturer for each batch.
D2.5.Reagents and media should always be used prior to the manufacturer’s expiry date.
D2.6.Each laboratory should define its own acceptance and release criteria for critical media and have a change control policy in place for changing media to a different brand or supplier.
D2.7.Any additional reagents or media should be of a purity appropriate for the purpose.
D2.8.All media should be stored according to the manufacturer’s instructions or otherwise appropriately. Furthermore, media should be transported under manufacturer’s instructions and have cold chain storage traceability.
D2.9.All seals and packaging on commercial products should be checked on arrival. Any non-conformances should be documented, risk assessed and if necessary, the product should be returned to the supplier.
D2.10.Certificates of analysis and details of quality control measures should be supplied by manufacturers, and checked that they correspond with the batch delivered. Non-conformances should again be documented, risk assessed and if necessary the product returned to the supplier.
D2.11.A robust batch control system should be in place to ensure traceability of all media, media components, other reagents, plasticware or other consumables which will allow for a two-way audit and facilitate any batch analysis or recall.
D2.12.No products of human origin should be used in the manufacture of culture media unless it is from an appropriately screened, controlled source. Commercial suppliers of human serum albumin or media containing a serum derived protein source should supply evidence of screening according to the local rules for blood donors.
D2.13.Where it is necessary to add components to commercially supplied media in the laboratory, unless directed by the manufacturer it must be understood that this invalidates QC testing carried out by the manufacturer, and media will therefore need to be assessed by appropriate in house procedures.
D2.14.In order to maintain sterility, media should be processed in an appropriate laminar air-flow and handled using aseptic techniques. Training should be provided where necessary.
D2.15.Media should be used according to the manufacturer’s instructions. Any deviation should require a risk assessment and validation.
D2.16.All water used for cleaning equipment, humidifying incubators and filling water baths, should be of an appropriate purity.
E. Procedures
E1. Protective measures
E1.1.Protective measures should be in place to ensure aseptic conditions for gametes and embryos.
E1.2.All personnel entering the laboratory should wear a theatre hat, clean shoes or overshoes and a clean room overcoat or theatre scrubs as a minimum. In clean air laboratories appropriate clothes for the defined air grade should be worn. Personal Protective Equipment (PPE) should be available if handling compressed gas cylinders or liquid nitrogen. Ref also C1.1
E1.3.All personnel should follow a prescribed hand washing procedure when entering and leaving the laboratory.
E1.4.Personnel should only use cosmetics or toiletries that are un-perfumed.
E1.5.There should be no eating, drinking, chewing, smoking, applying of make up and no storage of food, drink, smoking materials or personal medication in the laboratory area.
E1.6.All biological material should be treated as a potential source of infection.
E1.7.Powder-free non-latex gloves should be worn for all aseptic procedures.
E1.8.All processing of gametes and embryos for clinical use should be carried out under conditions of suitable air quality, i.e. in Class II cabinets or equivalent as a minimum. The aim should be in line with Commission Directive 2006/86/EC which states that tissues and cells that are exposed to the environment during processing without a subsequent microbial inactivation process should be subjected to a Grade A environment as defined in the European Guide to Good Manufacturing Practice (GMP) with a background environment at least equivalent to Grade D in terms of particle and microbial counts. However, in certain situations, an air quality of Grade A standard is not indicated and in these circumstances it should be demonstrated and documented that the chosen environment achieves the quality and safety required for the type of gamete/embryo processing for human application.
E1.9.Particle counts and microbiological sampling should be carried out frequently to confirm the quality of air in the processing area and background air.
E1.10.All devices used for pipetting biological material, i.e. those devices that may be attached to disposable pipettes, should be cleaned and if necessary have filters replaced frequently.
E1.11.All centrifugation of biological material should be carried out in sealed vessels, within closed centrifuge buckets.
E1.12.All disposable material must be disposed of immediately after use in suitable containers.
E1.13.Needles and other sharp materials should be disposed of in appropriate containers with suitable precautions.
E1.14.Care should be taken when transferring gametes or embryos to cryo straws or other cryo-containers, to minimise the risk of contaminating the outside of the container.
E1.15.Consideration should be given to the use of high security devices which minimise contamination risk and the use of LN2 vapour storage where appropriate.
E1.16.Where material is received either from a satellite unit, or in cryopreserved form from another treatment centre, checks must be made to ensure that the material has been obtained and processed under appropriate conditions so as not to compromise the receiving laboratory. (HFEA COP 8th Ed. Section 16).
E1.17.The pre-acceptance checks should include as a minimum:
Viral screening information of clients at the time of procurement
Screening status of tank
The type of cryostorage (high security straws/vials)
Method of freezing (slow/vitrification)
Any batch recalls
Non-conformances during processing and storage
Cryovessel monitoring status
E1.18.It is recommended that a service level agreement should exist between importing/exporting clinics to ensure the definition of roles, responsibilities and future investigations and to comply with HFEA regulations.
E2. Infectious agents
E2.1.Screening patients and gamete donors for blood borne viruses and other sexually communicable diseases before processing or cryopreservation should be routinely adopted. For specific testing requirements refer to HFEA COP 8th Ed. (Section 11.15, T 52).
E2.2.Although all samples should be treated as potentially infectious, a mechanism should be in place to highlight to laboratory staff when a known positive sample is due to be received in the laboratory.
E2.3.The laboratory should have a clear policy on handling biologically hazardous samples.
E2.4.Any staff handling these samples must have appropriate training and personal protective equipment should be used.
E2.5.Material from patients known to carry infectious agents should only be handled under suitable conditions of containment as dictated by local and national guidance and regulation.
E2.6.Where patients who are known to carry HIV, hepatitis B or C are to be treated, attention must be paid to the guidance from the Advisory Committee on Dangerous Pathogens with regard to containment, and a local risk assessment must be carried out to establish safe and suitable procedures.
E2.7.Material from patients who present an infection risk should either be processed in a separate area, dedicated to the purpose, with separate, dedicated equipment, or, at a time when no other procedures are to be carried out. In this case, sufficient time must be allowed for thorough decontamination of all equipment and facilities before further procedures are allowed to continue.
E2.8.Where material from patients who present an infection risk is to be cryopreserved, attention should be paid to any possible risk of transmission, and existing regulations. Material from patients who have screened negative in testing for infectious agents should be stored separately from those who present a known risk of infection, or whose status is unknown. Samples that have tested positive for a blood borne virus but which have been processed and subsequently tested and found to be negative should be stored in a separate “washed” vessel and not with the positive or negative samples. Optimally separate “washed” cryostorage should be available for each blood borne virus. Collective storage of “washed” samples from multiple blood borne viruses should only be undertaken after a risk assessment has been carried out and under advice from a virologist.
E2.9.There should be a procedure for disposal of waste material from high-risk cases.
E2.10.There should be a validated de-contamination method for surfaces and equipment.
E3. Identification of patients and their gametes and embryos
E3.1.Valid informed patient consent must be confirmed prior to an embryologist performing a procedure.
E3.2.Centres should have a policy on when it is necessary to perform viral screening and the frequency for testing. For example, some clinics may not require screening for routine semen analysis or partner intrauterine insemination. However, each clinic must have risk assessed its own policy to ensure patient safety.
For couples undergoing IVF/ICSI or any cryostorage, the viral screen status should be confirmed prior to any procurement or processing of tissues in the laboratory and should be in date according to clinic policy.
E3.3.All material obtained from patients should be clearly labelled with at least the patient’s or donor’s first name and surname, date of birth and one unique identifier. This identifier could be the Hospital Number, NHS/CHI (Community Health index) number or a donor code (HFEA COP 8th Ed Section 18.20, 18.21).
E3.4.Part of the induction program for new staff should include training to ensure that all staff understand the principles of witnessing and follow the relevant protocol. Training of all staff in the identification and witnessing procedures is mandatory.
E3.5.Laboratories must have in place robust, effective processes to ensure that no mismatches of gametes or embryos or identification errors occur. Centres must double-check the identification of samples and the patients or donors to whom they relate at all critical points of the clinical and laboratory process. Witnessing protocols should be followed when any of the following clinical or laboratory procedures take place:
a.Collecting oocytes
b.Collecting sperm
c.Preparing sperm
d.Mixing sperm oocytes/injecting sperm into oocytes
e.Transferring gametes or embryos between tubes or dishes
f.Transferring embryos in a woman
g.Insemination of a woman with sperm prepared in the laboratory
h.Placing gametes or embryos into cryostorage
i.Removing gametes or embryos from cryostorage
j.Disposing of gametes or embryos
k.Transporting gametes or embryos
E3.6.These checks must be completed and recorded at the time the relevant clinical or laboratory process/procedure takes place. A record must be kept in each patient’s/donor’s medical record.
E3.7.Electronic systems such as bar-coding and radio frequency identification (RFID) are appropriate, subject to a risk assessment to ensure that any system introduced will not have a negative impact on gametes or embryos. The system must be deemed reliable and ensure that any electronic devices employed are safe. Furthermore, the system must be tested to ensure that the risk of false positive and negative matches and breakdowns is minimal. Integrated witness systems should be fully validated. Double witnessing is still required for entry, exit points and the mixing of sperm and oocytes.
E3.8.A hard copy of electronic witnessing should be retained.
E3.9.There should be a separate record of the name, job title and signature of everyone who carries out, or witnesses, laboratory and clinical procedures.
E3.10.Incubators should be organised to facilitate identification of patients’ gametes and embryos.
E3.11.Where donated gametes or embryos are to be used in treatment, only non-identifying information should be recorded in the records of the patient/couple receiving treatment.
E4. Handling of oocytes and sperm
E4.1.During procedures involving the handling or manipulation of oocytes or embryos, all suitable measures should be taken to control temperature and pH appropriately.
E4.2.Mouth pipetting should never be used for manipulating biological material. Only disposable pipetting devices should be used and disposed of immediately after single use.
E4.3.Material from one patient/couple only should be present in the working area while manipulations are being carried out.
E4.4.All dishes and tubes to be used in the preparation, manipulation and culture of patient material should be labelled with a minimum of the patient’s first name and surname, date of birth and 1 unique identifiers (Ref E3.3) which must be cross-referenced to the relevant documentation and to the patient/couple.
E4.5.Labelling should be permanent and care should be taken to avoid devices capable of releasing volatile compounds which might erase the label.
E4.6.The embryologist responsible for each manipulation should be identified on all relevant records, and should sign/initial the laboratory records to acknowledge that they have carried out the procedure. They should also document the time and date of the procedure.
E4.7.When donated gametes and embryos are to be used, or where gametes or embryos are to be donated, material should initially be labelled with the donor’s identifiers and only labelled with the recipient’s details when the donation is confirmed with a double witness.
E5. Sperm preparation
E5.1.Semen samples should not be processed for any aspect of assisted reproductive treatment unless produced into an approved container legibly labelled with the patient’s identifying information.
E5.2.All samples should be clearly labelled with the male partner’s identifying information, the male partner should be asked to acknowledge that the sample is his, and sign the records.
E5.3.If a third party delivers the sample, they should be asked to sign that the sample has not been interfered with during transit.
E5.4.Samples should be produced on clinic premises. If, in exceptional circumstances, this is not possible, the patient must be supplied with an approved container and the circumstances recorded in the patient’s records (HFEA COP 8th Ed. Section 15.7). The patient must sign to confirm the sample’s ownership.
E5.5.The time of semen production should be recorded and the sample delivered to the laboratory within 1 h of collection. During transport the sample should be kept between 20 and 37°C. (WHO Manual, 2010).
E5.6.Patients should be given clear instructions regarding hygiene and that they should not use soap, creams, spermicidal condoms or lubricants.
E5.7.Where patients are unable to produce a sample by masturbation, non-spermicidal condoms may be supplied. However the sample must be removed from the condom as soon as possible. Lubricants that have been tested and confirmed non-toxic for sperm may be recommended in certain situations. The use of other devices is not recommended unless in exceptional circumstances.
E5.8.Standard operating procedures should be in place for the preparation of all sperm sample types – i.e. ejaculates, retrograde and surgically retrieved samples. Each SOP should be specific to the subsequent proposed treatment plan, i.e. IUI, IVF and ICSI.
E5.9.Semen assessment should be carried out according to a standard operating procedure using an inverted microscope. External and internal quality control schemes should be in place to limit inter-operator variation.
E5.10.The method of sperm preparation should be recorded, including any variations to the standard operating procedure. Efforts should be made to keep up to date with current sperm preparation methods and as a general rule, excessive centrifugation should be avoided.
E5.11.Records should be made of pre- and post-preparation sperm parameters, and of any dilution carried out.
E5.12.Where surgically retrieved sperm is used for treatment and the sample is of sufficiently good quality, the remaining sample should be cryopreserved to avoid further surgical procedures.
E5.13.Where a laboratory is equipped to handle samples positive for known infections these should always be prepared using an approved, validated washing procedure.
E5.14.Where donor sperm is used, the necessary identifying information must be recorded, including the donor and centre code.
E5.15.Sperm preparations should be protected from extreme temperatures and pH fluctuations (ABA Guidelines, 2012).
E5.16.If there is a history of difficulty in producing a sample, then arrangements should be put in place to obtain a frozen sample as a back-up.
E6. Oocyte retrieval
E6.1.Appropriate equipment should be available to maintain oocytes at 37°C during the oocyte retrieval and any subsequent transfer or manipulation in order to prevent damage to the oocyte.
E6.2.All media and consumables used throughout the procedure should be pre-warmed to 37°C.
E6.3.Efforts should be made to protect the pH of the oocytes during this procedure by the use of appropriately buffered media and/or the use of a dedicated IVF chamber.
E6.4.Aspirates from follicles should be assessed using microscopy with efforts taken to reduce exposure to light.
E6.5.Details of the procedure should be documented on the patient’s worksheet and any morphological observations noted.
E6.6.Detailed SOPs must be available and evidence of staff competence recorded
E7. Insemination of oocytes
E7.1.The laboratory should have strict criteria, which set out the circumstances under which IVF and ICSI are used. If a patient’s treatment is changed from the original plan, the patient should give consent, and the reasons why a change is recommended and documented placed in the patient notes. These consents should be obtained prior to anaesthetic administration for oocyte collection.
Standard IVF
E7.2.The origin of the sperm used should be recorded (ejaculated, frozen, donor etc).
E7.3.Records should be kept of the time of insemination and sperm concentration used.
E7.4.The number of motile spermatozoa used for insemination must be sufficient to optimise the likelihood of oocyte fertilisation without compromising embryo development. This is a critical step and must be double witnessed.
ICSI
E7.5.There should be a defined protocol for cumulus removal from oocytes prior to the ICSI procedure.
E7.6.A combination of the use of the enzyme hyaluronidase with physical denudation is recommended with care taken not to damage to oocyte. This can be achieved by limiting the concentration of the enzyme and the length of exposure time. Physical damage can be reduced by ensuring that the pipette selected is the appropriate bore size and that micromanipulation by the operator is not too vigorous.
E7.7.Records should be kept of the origin of the sperm used (ejaculated, frozen, surgically retrieved, donor etc.).
E7.8.The time commenced and time taken to complete the ICSI procedure should be recorded.
E7.9.Any observations made in relation to the morphology or maturity of the oocyte should be documented on the patient worksheet
E7.10.Viscous substances for the immobilisation of the sperm and control in the pipette can be used. These substances must be deemed suitable for this purpose by the manufacturer and the appropriate documentation provided to support this.
E7.11.Optimally, sperm selected for ICSI should have normal morphology and exhibit motility.
E7.12.Where there is no motility, a vitality test should be used for sperm selection.
E7.13.If no viable sperm are available from the ejaculate, the option of testicular sperm should be considered.
E7.14.Oocytes that have failed to fertilise by normal IVF or ICSI should never be re-inseminated (HFEA COP 8th Ed. Section 21.4 c).
E7.15.Modifications of ICSI, such as the use of IMSI or PICSI must only be implemented after adhering to section E14 “Introduction of Novel Techniques”which includes a risk assessment, adequate training and process validation and approval from HFEA if required.
E8. Scoring for fertilisation
E8.1.All oocytes that have been inseminated or injected with sperm should be examined for the presence of pronuclei and polar bodies between 16 and 20 h post insemination.
E8.2.The morphological status of each oocyte should be recorded.
E8.3.Oocytes with more than two pronuclei should be isolated from normally fertilised oocytes to avoid any risk of these being transferred to patients. This isolation should preferably be into a separate dish. Oocytes with one pronucleus should also be isolated and carefully checked for evidence of polar bodies to provide further information about fertilisation status. The transfer of embryos resulting from such oocytes should only take place in the absence of other suitable embryos (Feenan & Herbert, 2006).
E8.4.Oocytes showing no fertilisation within the expected time frame should be observed for delayed fertilisation, 2nd polar body extrusion and/or cleavage. This timing must be clearly documented and only in the absence of other suitable embryos, would these by considered for transfer (Feenan & Herbert, 2006).
E9. Embryo culture and grading
E9.1.Records should be kept of the development of each fertilised oocyte/embryo and the stage at which any embryo is transferred or cryopreserved. The location of each embryo in the culture dish should also be noted depending on the lab culture policy.
E9.2.The information recorded should include at a minimum:
Cell number
Degree of fragmentation
Evidence of multi-nucleation
Blastocyst morphology (if applicable)
Removed cytoplasm description
E9.3.ACE recommends that clinics standardise their grading scheme according to the joint system developed in 2011 by ACE/NEQAS.
E9.4.Embryo grading should be carried out according to a standard operating procedure using an inverted microscope and external and internal quality control schemes should be in place to limit inter-operator variation.
E9.5.Co-culture of embryos with cells other than those originating directly from the couples being treated should not be carried out.
For embryo grading:
For blastocyst grading:
E10. Embryo transfer
E10.1.In an effort to reduce multiple pregnancies, clinics should have a strict policy in relation to the number of embryos to be transferred with the option of elective single embryo transfer being highly recommended.
E 10.2.Embryo transfer should be carried out using a sterile, single use catheter designed for the purpose, that has been tested using an appropriate bioassay and CE marked for clinical use.
E10.3.The embryo transfer room should be adjacent to the laboratory. Where the transfer room is not immediately adjacent to the laboratory, measures must be in place for maintaining temperature and pH during transport.
E10.4.Embryo transfer is a critical witnessing step and identification of the patient and embryo(s) to transfer should be performed by two competent staff members. Electronic witnessing on its own at this point is not acceptable.
E10.5.Selection of embryos for transfer must be carried out in accordance with a written standard operating procedure.
E10.6.Consent to embryo transfer and the number of embryos to be transferred must be confirmed prior to the transfer commencing by the embryologist and the clinician or nurse carrying out the procedure.
E10.7.Details of the number of embryos transferred, the time of transfer and the medical staff involved should be documented.
E10.8.Notes regarding the clinical procedure should be documented for future transfers.
E11. Cryopreservation
E11.1.The cryopreservation program is a crucial element of any successful clinic and permits the storage and subsequent use of supernumerary embryos thereby facilitating the strategy to minimise multiple births.
Cryopreservation can also be used to:
establish a fertility reserve for those undergoing chemo or radiotherapy
delay transfer for medical reasons such as a risk of OHSS
quarantine potential material for use in donation or surrogacy
E11.2.The laboratory should have a clear policy on the tissue and cells it is licensed to cryopreserve. Ovarian and Testicular tissue as cells of the germ line, fall within the definition of gamete in the Human Fertilisation and Embryology Act 1990 (as amended) and so are subject to the same storage requirements as sperm and oocytes. HFEA COP 8th Ed. (Section 17.10). Furthermore, the storage and subsequent use for human application of ovarian and testicular tissue is an activity which is licensed by the Human Tissue Authority (HTA). The relevant licence must but be applied for and granted before any procurement takes place.
Any subsequent thawing or transplantation of this tissue must be in line with Section E14.
E11.3.The lab should have a clear policy on the type and quality of embryos suitable for freezing in line with the latest research and information to ensure good survival rates and implantation potential after thawing.
E11.4.The lab should have a clear protocol on their chosen method for freezing, i.e. vitrification or slow-cooling. This process should be validated within each clinic setting prior to implementation.
E11.5.All staff should be fully trained in the safe use of cryogenic equipment, including pressurised nitrogen delivery vessels, nitrogen dewars and if necessary nitrogen vapour storage systems. They should be aware of the limitations of the equipment, potential pitfalls and procedures to cope with emergency situations or catastrophic failure and have SOPs in place.
E11.6.All centres storing gametes and embryos should have effective alarms and monitoring systems to ensure the safety of cryopreserved gametes and embryos. These systems should have:
(a)local alarms (i.e. on individual dewars for temperature or liquid nitrogen level)
(b)an auto-dial facility or similar (e.g. link to alarm board) to contact staff outside normal working hours
(c)adequate staffing and funding to implement formal emergency procedures, including the provision of on-call arrangements, and
(d)adequate spare storage space or vessels to enable transfer of samples if a vessel fails
E11.7.Containment or plastics for the cryopreservation of oocytes or embryos should be used according to manufacturers’ instructions. The integrity of plastic-ware should withstand long-term storage at ultralow (liquid or vapour nitrogen) temperatures and should have effective sealing methods to reduce the risk of leakage and cross contamination.
E11.8.Embryos should be maintained in storage at temperatures below −130°C.
E11.9.Vacuum lined storage vessels (dewars) should be used for storing embryos in liquid nitrogen or the vapour phase of nitrogen. These vessels should be purchased from reputable manufacturers and be fit for the purpose of gamete and embryo cryostorage. These cryovessels should be regularly inspected and linked into a suitable equipment replacement cycle. Any catastrophic vessel failure should be reported to the Medicines and Healthcare products Regulatory Agency (MHRA).
E11.10.Receptacles should be clearly and permanently labelled with the date of storage, first name and surname, date of birth and a unique identifier (Ref E3.3). Care should be taken to keep-up to date with any developments with a unique European Coding system that could be implemented for non-partner donation.
E11.11.Documentation should include the method of freezing, type and batch number of cryoprotectant used and the stage of embryo development.
E11.12.The number of embryos in each straw or vial (which should not exceed two), and the number of straws or vials stored per patient should be recorded.
E11.13.Documentation should also include the date and time of freezing, the storage period for which the patient/couple has consented, and the location of the embryos stored.
E11.14.Documentation of thawing procedures should include morphological changes seen during and after thawing, percentage cell survival, and the duration of culture prior to transfer.
E11.15.Documentation of thawing procedures should also record the number of embryos thawed, survival rate, and the number transferred.
E11.16.Where embryos have been created using donated gametes the relevant donor codes should also be included in the documentation.
E11.17.Documentation of thawing procedures should also incorporate an amendment of the storage inventory.
E11.18.All the above should be recorded in the patient’s records and in independent laboratory or cryobank records.
E11.19.An audit of all stored material should be carried out every two years at a minimum, cross-referencing stored material with storage records, and storage consents.
E11.20.Systems should be in place to ensure that no embryos or gametes are stored beyond the storage period consented to by the patients or donors.
E11.21.Laboratories storing gametes and/or embryos for patients whose future fertility may be impaired by a medical condition or procedure should divide individual patients’ samples into separate storage vessels, in case of dewar failure.
E12. Assisted hatching
E12.1.There are conflicting reports about the clinical efficacy of this procedure. ACE recommends that if clinics choose to employ this technique they have defined selection criteria and validate the method used in terms of safety and outcome.
E12.2.The patient/couple’s records should include clear documentation that assisted hatching has been carried out, including the stage of development, method used and the operator.
E13. Embryo biopsy
E13.1.The clinic should adhere to the most recent HFEA Code of Conduct guidelines with respect to any embryo testing programme.
E13.2.Temperature and pH should be maintained during all manipulations.
E13.3.Microtools should be sterile and single use only.
E13.4.Breaching the zona may be carried out by mechanical, chemical or laser methods.
E13.5.Exposure to chemicals used to breach the zona should be limited to the minimum which is effective.
E13.6.Cells removed for this procedure must be collected using appropriate devices with labelling to ensure traceability; every step where cells or embryos are moved from one container to another must be witnessed; culture conditions must be designed to ensure traceability.
E13.7.The biopsy procedure may be carried out by:
Polar body removal
Single/double blastomere biopsy at the day 3 stage
Trophectoderm biopsy at the blastocyst stage
E13.8.Embryo biopsy for PGD or PGS should be carried out in association with an accredited genetics laboratory.
E13.9.Where embryos are found to be abnormal by PGS or PGD, confirmation of diagnosis should be sought where possible.
E13.10.Embryo biopsy should only be carried out for genetic tests specifically approved for that unit by the HFEA.
E13.11.Genetic Counselling should be available to any couple who have been identified as having an inheritable disease.
F. Research and the introduction of novel techniques
F1. Research
F1.1.For any research project which involves the use of gametes or embryos, it is necessary to apply to the HFEA for a research licence which is valid for up to three years.
F1.2.All research activity must adhere to the mandatory requirements of the HFEA Code of Practice 8th Ed Section 22.
F1.3.The legislation currently prohibits:
(a)embryos being placed in any animal
(b)embryos that are not human being placed in a woman
(c)gametes that are not human being placed in a woman
(d)mixing human gametes with animal gametes, except when carrying out the “hamster test” in line with a licence
(e)embryos being kept or used after 14 days from when the process of creating the embryo began, or after the primitive streak has appeared (if earlier than 14 days)
(f)embryos intended for a research project being used for any purposes other than those of that research project an embryo created or obtained for research being placed in a woman
(h)keeping the results of the “hamster test” after any research is complete or, in any event, after the two cell stage
(i)a research licence being used for any project other than the one specified in that licence
(j)research activities being carried out on premises other than those specified in the licence
(k)research activities being carried out under the supervision of anyone other than the specific person designated in the licence
(l)a treatment licence authorising the activities of a research project, and
(m)a research licence applying to more than one research project.
F1.4.ACE are supportive of well designed research studies which have the appropriate ethical approval.
F2. Introduction of novel techniques
F2.1.There should be an implementation plan (IQ, OQ, and PQ) before any new procedure is employed by the lab.
F2.2.This should include the change control request, changes in SOPs, the likely benefits and a training program for staff.
F2.3.Before implementing any new procedure, the process modified must be validated and documented.
F2.4.No new techniques that are licensable under the terms of the HFE Act should be introduced unless they fall within the terms of an existing licence.
Abbreviations
ACE Association of Clinical Embryologists
COP Code of Practice
COSHH Control of substances hazardous to health
EU European Union
GMP Good manufacturing practice
HFEA Human Fertilisation and Embryology Authority
HTA Human Tissue Authority
ICSI Intracytoplasmic sperm injection
IMSI Intracytoplasmic morphologically-selected sperm injection
IQ Installation qualification
IVF In vitro fertilisation
KPI Key performance indicator
MSC Modernising scientific careers
OQ Operating qualification
PGD Preimplantation genetic diagnosis
PGS Preimplantation genetic screening
PQ Performance qualification
QM Quality manager
QMS Quality management system
RFID Radio frequency identification
SOP Standard operating procedure
URS User requirement specification
Appendix 1
ACE laboratory key performance indicators*.
OVERALL
Based on patients under the age of 40 who had at least three oocytes collected or more.
ACE laboratory key performance indicators*.
FRESH CYCLES
These should be calculated for both D2/3 and D5/6.
FROZEN CYCLES
These should be calculated for both D2/3 and D5/6.
References
- ABA Guidelines. (2012). Laboratory Andrology Guidelines for Good Practice Version 3.
- Feenan, K. & Herbert, M. (2006). Can ‘abnormally’ fertilized zygotes give rise to viable embryos? Human fertility (Cambridge, England), 9, 157–169.
- Health and Safety Commission: Safety in Health Services Laboratories. “Safe working and prevention of infection in clinical laboratories”. 1991 HMSO.
- HFEA Code of Practice (8th Edition). Available at: http://www.hfea.gov.uk/docs/8th_Code_of_Practice.pdfCode of Practice
- Human Fertilisation and Embryology Act 2008.
- Human Tissue Authority. Available at:http://www.hta.gov.uk
- Magli, M. C., Van den Abbeel, E., Lundin, K., Royere, D., Van der Elst, J., & Gianaroli, L.; Committee of the Special Interest Group on Embryology. (2008). Revised guidelines for good practice in IVF laboratories. Human reproduction (Oxford, England), 23, 1253–1262.
- Magli, M. C., Ferraretti, A. P., Crippa, A., Lappi, M., Feliciani, E., & Gianaroli, L. (2006). First meiosis errors in immature oocytes generated by stimulated cycles. Fertility and sterility, 86, 629–635.
- Tedder, R. S., Zuckerman, M. A., Goldstone, A. H., Hawkins, A. E., Fielding, A., Briggs, E. M. et al. (1995). Hepatitis B transmission from contaminated cryopreservation tank. Lancet, 346, 137–140.
- Van de Velde, H., De Vos, A., Joris, H., Nagy, Z. P., Van Steirteghem, A. C. (1998). Effect of timing of oocyte denudation and micro-injection on survival, fertilisation and embryo quality after intracytoplasmic sperm injection. Human reproduction, 13, 3160–3164.
- WHO Manual 5th Edition, 2010.