IVF Store supports IVF and andrology laboratories with a broad range of laboratory supplies, cryopreservation products, embryo culture essentials, micromanipulation tools, and workflow solutions designed to help clinics operate with consistency, efficiency, and confidence. We are sharing this manual as part of our commitment to providing practical educational resources that support laboratory best practices, specimen safety, quality control, traceability, and day-to-day embryology operations.

On this page

• 1. Introduction
• 2. Philosophy and Mission
• 3. Recordkeeping
• 4. IVF Laboratory Preparation
• 5. Patient, Gamete, and Embryo Identification and Labeling
• 6. Sterile Technique
• 7. Sterile Filtration
• 8. Glassware Sterilization
• 9. Sterilization Guidelines
• 10. Semen Sample Tracking
• 11. Sperm Preparation for IVF – Ejaculated Samples
• 12. Sperm Preparation for IVF – Frozen Samples
• 13. Sperm Preparation for IVF – Testicular Sperm
• 14. Sperm Preparation for Intrauterine Insemination
• 15. Oocyte Retrieval
• 16. Conventional Insemination
• 17. Hyaluronidase and Oocyte Stripping
• 18. Intracytoplasmic Sperm Injection (ICSI)
• 19. Micromanipulation
• 20. Determination of Fertilization
• 21. Embryo Scoring
• 22. Assisted Hatching of Embryos
• 23. Embryo Biopsy
• 24. Embryo Cryopreservation and Vitrification
• 25. Embryo Transfer
• 26. Quality Control and Equipment Maintenance
• 27. Temperature Monitoring
• 28. Temperature and CO₂ Checks of Incubators
• 29. Digital Gas Analyzer
• 30. Calibration of Thermometers
• 31. pH Meter Calibration and Use
• 32. Inventory Control
• 33. Guidelines for Biohazardous Waste
• 34. Chemical Hygiene Program
• 35. Automatic CO₂ Incubators
• 36. Laminar Flow Hoods
• 37. Liquid Nitrogen Storage Tanks
• 39. Witness Workflow
• 40. Embryologist Training
• 41. Oocyte Donation Program
• 42. Forms
• 43. Quality Control Forms and Logs
• 44. Policies
• 45. Document Control

1. Introduction

1.1 Purpose

This manual establishes the policies, procedures and operational expectations for the IVF laboratory. It draws on current recommendations from the American Society for Reproductive Medicine (ASRM), the European Society of Human Reproduction and Embryology (ESHRE) and the Alpha Scientists in Reproductive Medicine to promote consistent, evidence‑based practice and optimize patient outcomes. The manual is intended to support safe and effective patient care by providing clear instructions for daily laboratory practice, specimen handling, culture, cryostorage management, quality systems and staff responsibilities.

1.2 Scope

The provisions of this manual apply to all embryology and andrology personnel, laboratory leadership, and any authorized staff member who handles patient specimens, performs laboratory procedures, enters records, performs quality checks or participates in laboratory support activities.

1.3 Objectives

The objectives of this manual are to:

• Standardize laboratory practice and provide a comprehensive reference for all staff.
• Support patient and specimen safety, including strict identity control and witness protocols.
• Maintain specimen integrity and traceability at all times by following best practices for labeling, documentation and chain‑of‑custody.
• Define consistent methods for preparation, culture, cryopreservation and transfer support.
• Support regulatory, accreditation and quality system compliance.
• Provide a training resource for new staff and support continuous professional development.

1.4 General Standard

All procedures described in this manual shall be performed only by personnel who are trained, authorized and currently competent to perform the assigned task. When the laboratory uses a site‑specific timing, concentration, setting, threshold, or grading scale, the currently controlled laboratory value shall be used and documented in the corresponding worksheet, log, validation record or appendix.

1.5 Safety Standard

All human specimens shall be treated as potentially infectious. Standard precautions, laboratory PPE requirements, and all applicable biosafety and occupational safety policies shall be followed during every procedure. When using cryogenic fluids, staff must wear appropriate personal protective equipment (PPE) including insulated gloves and face protection. Access to cryostorage areas should be restricted to authorized staff, and cryogenic tanks should be placed in well‑ventilated, visible areas with alarms and continuous monitoring to detect level loss.

2. Philosophy and Mission

2.1 Mission

The mission of the IVF laboratory is to provide accurate, safe and effective laboratory support for assisted reproductive treatment through consistent practice, meticulous specimen identification, validated laboratory methods and a strong commitment to patient care.

2.2 Core Commitments

The laboratory is committed to:

• Patient safety – protecting patients from harm through standardized procedures, adequate staffing, training and continuous quality improvement.
• Specimen integrity – preserving the viability and identity of gametes and embryos through meticulous labeling, chain of custody and witness procedures.
• Confidentiality – maintaining strict confidentiality of patient information and specimens.
• Scientific rigor – employing validated, evidence‑based methods and maintaining high technical standards.
• Accurate communication – ensuring clear and timely communication with clinicians, patients and each other.
• Continuous improvement – monitoring laboratory performance, participating in quality assurance/quality control (QA/QC) programs and adopting best practices from professional societies.
• Staff accountability – expecting every staff member to take responsibility for their actions, documentation and competency.

2.3 Scope of Services

The laboratory may provide services including:

• Semen handling and preparation.
• Oocyte identification and handling.
• Conventional insemination.
• Intracytoplasmic sperm injection (ICSI).
• Embryo culture and assessment.
• Assisted hatching.
• Embryo biopsy for genetic testing.
• Embryo transfer support.
• Embryo and oocyte cryopreservation (vitrification) and warming.
• Cryostorage inventory management.
• Specimen transport and offsite storage coordination.
• Quality control and equipment monitoring.

2.4 Leadership and Oversight

The laboratory director is responsible for oversight of laboratory operations, procedure approval, competency standards, validation of new methods, incident review and quality system performance. The medical director, where applicable, provides clinical oversight and supports alignment between laboratory and clinical care.

2.5 Quality Philosophy

The laboratory shall maintain a culture in which accuracy, preparedness, clear documentation and immediate escalation of concerns are expected. Deviation from written procedure is not permitted without authorization and documentation. Best‑practice guidelines from ASRM, ESHRE and Alpha shall inform all procedures, especially with respect to embryo culture conditions, grading criteria and cryostorage safety.

3. Recordkeeping

3.1 Purpose

To establish the requirements for creation, completion, review, storage, protection, retrieval, correction and retention of laboratory records.

3.2 General Policy

All laboratory activities must be documented in a complete, legible, accurate and timely manner. Records shall be sufficient to reconstruct what was done, by whom, when it was done, what specimen was involved and what the result or disposition was. Documentation is a professional responsibility and forms part of the legal medical record.

3.3 Types of Records

Records may include, but are not limited to:

• Patient laboratory worksheets.
• Semen receipt and preparation records.
• Retrieval and insemination records.
• Fertilization and embryo culture records.
• Cryopreservation, thawing and warming records.
• Embryo transfer records.
• Witness records.
• Equipment logs.
• Temperature logs.
• Gas check logs.
• Calibration logs.
• Maintenance records.
• Reagent and media traceability records.
• Inventory records.
• Training and competency records.
• Deviations, corrective actions and incident reports.

3.4 Required Elements

When applicable, laboratory records shall include:

• Patient identifiers (e.g., full name, date of birth, medical record number).
• Date and time of each activity or observation.
• Operator initials or signature.
• Witness initials or signature where required.
• Specimen type and stage.
• Stage or status of specimen (e.g., gamete, zygote, embryo, blastocyst).
• Media or reagent lot number when required for traceability.
• Storage location when relevant.
• Comments regarding deviations or unusual observations.

3.5 Completion of Records

Entries shall be made at the time the activity is performed or immediately thereafter. Blank spaces shall not be left in a controlled record when a field is required; if a field does not apply, it shall be marked according to laboratory policy. Electronic records shall maintain an audit trail of edits.

3.6 Corrections

Corrections must preserve the original entry. A single line shall be drawn through the incorrect entry when paper records are used, and the corrected information shall be entered with initials and date when required. Electronic corrections shall remain attributable to the individual making the change.

3.7 Storage and Retention

Records shall be stored securely to protect confidentiality, prevent unauthorized alteration, and allow timely retrieval. Retention periods shall follow laboratory policy and applicable legal and regulatory requirements; cryopreserved specimen records and associated consent forms are typically retained indefinitely or until disposition is completed.

3.8 What Trainees Should Understand

Good laboratory work is inseparable from good documentation. A procedure that is not documented clearly cannot be reconstructed or defended. Every critical movement of sperm, oocytes, embryos, cryodevices or biopsy samples must be traceable in the record.

4. IVF Laboratory Preparation

4.1 Purpose

To ensure that the laboratory, equipment, media, supplies and records are ready before any patient procedure or specimen handling begins.

4.2 Responsibilities

The embryologist assigned to opening duties is responsible for confirming laboratory readiness. All staff members are responsible for reporting any problem that may affect patient care, specimen safety or laboratory function.

4.3 Opening Procedure

At the start of each workday:

1. Perform hand hygiene and enter the laboratory according to laboratory dress and access requirements.
2. Confirm that the laboratory is clean, orderly and suitable for work.
3. Verify function of required equipment including microscopes, incubators, hot blocks, warming stages and any equipment needed for the scheduled cases.
4. Record temperatures of all monitored devices required by the daily QC schedule.
5. Verify incubator gas values using the approved digital gas analyzer. Incubators used for embryo culture should maintain a physiologic environment (approximately 37 °C, 5–6 % CO₂ and 5 % O₂ for tri‑gas incubators). ASRM guidelines recommend pH 7.25–7.35 for embryo culture media.
6. Confirm that gas supplies, alarms and back‑up systems are in normal status when applicable.
7. Confirm availability of pre‑equilibrated dishes, media, oil, sterile disposables, labels and forms. Culture dishes requiring gas equilibration should be prepared the day before intended use whenever possible to ensure stability.
8. Confirm that sharps and biohazard containers are present and not overfilled.
9. Review the daily clinical schedule and identify all procedures planned for the day.
10. Gather and verify case records for each scheduled patient. Assess whether all required consents, orders and instructions are present. Verify that cryopreservation consents clearly specify disposition options per ASRM guidelines.

4.4 Dish and Media Preparation

Media and dishes required for the day’s cases shall be prepared in advance according to the relevant procedure. Where equilibration is required, dishes shall be prepared early enough to ensure appropriate temperature and gas equilibration before use. Same‑day preparation should be limited to materials that do not require prolonged equilibration or to urgent clinical need. All prepared materials shall be labeled before use.

4.5 What to Check Before Procedures Begin

Before a case starts, the embryologist should be able to answer yes to the following:

• Are the right dishes prepared and equilibrated?
• Are all labels complete and correct and do they include two approved patient identifiers?
• Are the incubators functioning normally at 37 °C with correct gas conditions (5–6 % CO₂ and 5 % O₂)?
• Are the case worksheets and consents ready? Is the witness process available at critical steps?
• Are there sufficient supplies to complete the case without interruption?

4.6 Deviations

If any problem is identified that may affect specimen safety or laboratory readiness, patient specimens shall not be handled until the issue is assessed and resolved or formally escalated. Deviations shall be documented and investigated according to the quality management system.

5. Patient, Gamete, and Embryo Identification and Labeling

5.1 Purpose

To prevent specimen mix‑up by ensuring positive identification and traceability at every stage of laboratory care.

5.2 Policy

No patient specimen shall be handled unless identity is confirmed and all receiving containers, devices and records are correctly labeled. The laboratory shall use a two‑person (or electronic witness) system to confirm identity at critical points including sperm preparation, insemination/ICSI, embryo movement, cryopreservation, thaw/warming and transfer.

5.3 Approved Identifiers

The laboratory shall use at least two approved patient identifiers on all patient‑specific materials and records. Identifiers may include full name, date of birth, medical record number, unique case number or barcoded ID. Identifiers must be legible, durable and consistent across all documents and containers.

5.4 General Labeling Rules

Before a specimen is introduced into any dish, tube, straw, cryodevice or other container, the item shall be labeled with approved identifiers. Labels shall be affixed before the specimen contacts the dish or container. Labeling must be done by the person handling the specimen and verified by the witness.

5.5 Items Requiring Labeling

Labeling is required for, as applicable:

• Collection containers (urine cups, semen containers, retrieval tubes).
• Sperm preparation tubes and devices.
• Oocyte dishes, insemination dishes, stripping dishes, injection dishes and culture dishes.
• Biopsy dishes and tubes.
• Cryopreservation devices (straws, cryoloops, carriers) and storage canes or goblets.
• Thawing or warming dishes and embryo transfer dishes.
• All related worksheets and records.

5.6 Verification Steps

Identity shall be checked:

• At specimen receipt.
• Before sperm preparation.
• Before insemination or ICSI.
• Before biopsy or assisted hatching.
• Before cryopreservation.
• Before thawing or warming.
• Before embryo transfer.
• Before specimen discard or release from storage.

5.7 Labeling Errors

Any discrepancy in labeling or identity must result in immediate suspension of specimen handling until the issue is resolved and documented according to the deviation process. Under no circumstances should a specimen with uncertain identity be used.

5.8 Teaching Point

Most serious laboratory errors begin as identity‑control errors, not technical errors. Labels must be correct before the specimen touches the dish. A witness is not a passive observer but an active participant verifying identity.

6. Sterile Technique

6.1 Purpose

To minimize microbial and particulate contamination during handling of gametes, embryos, media and sterile materials.

6.2 General Requirements

Open specimen handling shall occur only in approved clean work areas (e.g., laminar flow hoods, clean benches). Staff shall perform hand hygiene, wear required PPE (gowns, gloves, masks) and avoid unnecessary talking, coughing, rapid movement and traffic around open culture systems. Airflow and surface disinfection protocols shall be followed.

6.3 Preparation of Work Area

Before beginning work:

1. Remove unnecessary items from the work surface.
2. Disinfect the surface with approved laboratory disinfectant and allow to dry.
3. Arrange sterile and non‑sterile items so that sterile handling can proceed without cross‑contamination.
4. Ensure laminar flow hood or clean bench has run for the minimum pre‑use period and that airflow indicators are normal.

6.4 Handling of Sterile Items

Sterile packages shall be opened carefully to avoid touching the sterile portion. Any sterile item that is dropped, contaminated, expired, damaged or questioned shall be discarded. Pipettes must not contact non‑sterile surfaces. Culture dishes should remain covered except when pipettes are entering the dish.

6.5 During Procedures

Only materials required for the immediate procedure should be present. Open culture dishes shall remain uncovered only as long as necessary. Rapid movement, talking over open dishes and leaning into the hood should be minimized.

6.6 Teaching Point

Sterile technique in IVF is less about “theatrical” sterility and more about disciplined protection of highly sensitive specimens from preventable contamination. Awareness of one’s hands, arms, pipettes and airflow is critical.

7. Sterile Filtration

7.1 Purpose

To describe preparation of in‑house solutions requiring sterile filtration.

7.2 Procedure

1. Assemble all ingredients and verify identity and expiration.
2. Prepare the solution using the approved formula.
3. Mix until homogeneous.
4. Filter through an approved sterile filter of appropriate pore size into a sterile labeled container.
5. Label the container with solution name, date prepared, expiration date, preparer initials and any required storage condition.
6. Store according to the written storage instruction for that solution.

7.3 Rejection Criteria

Do not use the solution if there is precipitate, cloudiness, color change, damaged packaging or labeling error.

8. Glassware Sterilization

8.1 Purpose

To define the cleaning and sterilization process for reusable glassware that remains in laboratory use.

8.2 Procedure

1. Segregate used glassware from clean materials.
2. Wash with approved laboratory detergent.
3. Rinse thoroughly using the approved water source.
4. Inspect for residue or damage.
5. Dry completely.
6. Sterilize using the validated method for the item (e.g., autoclave, dry heat oven).
7. Store in a clean protected area until use.

8.3 Rejection Criteria

Do not reuse cracked, chipped, poorly cleaned or incompletely sterilized glassware.

9. Sterilization Guidelines

9.1 Purpose

To define general sterilization standards for reusable laboratory items.

9.2 Requirements

Sterilization method, exposure time, packaging, storage conditions and documentation shall be appropriate to the item. Sterilized items must remain protected until point of use. Package integrity must be checked before opening.

9.3 Teaching Point

A sterile item is not truly usable if sterility is lost during storage, transport or setup. Staff must protect sterilized items from contamination just as carefully as they sterilize them.

10. Semen Sample Tracking

10.1 Purpose

To maintain full traceability of semen specimens from receipt through final disposition.

10.2 Receipt Procedure

1. Confirm that the specimen container is labeled with approved identifiers and matches the requisition and patient record.
2. Record date and time of collection if available and date and time of receipt.
3. Record specimen source (e.g., fresh ejaculation, aspirate) and intended use.
4. Assign or confirm the laboratory case record.
5. Place the specimen in the designated location for processing.

10.3 Rejection Criteria

Specimens may be rejected or delayed if:

• Container labeling is incomplete or illegible.
• Specimen source is uncertain or consent is incomplete.
• Container integrity is compromised.
• Required collection criteria (e.g., abstinence period) are not met.

10.4 Chain of Custody

At all times, the specimen location and responsible staff member shall be known or readily traceable through records.

10.5 Teaching Point

Tracking starts before the microscope. It starts when the specimen is received and continues until final disposition.

11. Sperm Preparation for IVF – Ejaculated Samples

11.1 Purpose

To prepare ejaculated sperm for IVF by producing a clean, motile sperm fraction suitable for conventional insemination. The goal is not simply to spin or filter a specimen but to provide a sperm dose that matches the fertilization method safely and reproducibly.

11.2 Scope

This section applies to ejaculated semen samples processed for IVF. The laboratory may use either density gradient preparation or SwimCount® Harvester preparation. Density gradient preparation is generally preferred when the specimen contains substantial debris, round cells, variable liquefaction or mixed motility, and a concentrated motile pellet is needed. SwimCount® Harvester preparation is generally preferred when the specimen is adequately liquefied, has sufficient total motile sperm for device use and the goal is recovery of a motile sperm‑enriched fraction through migration into the collection chamber.

11.3 Initial Assessment

1. Verify patient identifiers on the specimen container, requisition and laboratory worksheet.
2. Confirm specimen type and intended use.
3. Assess the specimen for volume, liquefaction, viscosity and gross appearance. Allow the sample to liquefy fully before processing unless there is a clinical reason to proceed earlier.
4. Mix gently before aliquoting so the sample is representative.
5. Perform the required pre‑processing semen assessment, typically including concentration and motility and any additional parameters used by the laboratory.
6. Select the preparation method based on specimen quality, expected sperm recovery needs and the planned fertilization method.

11.4 Density Gradient Preparation

1. Label all tubes with approved patient identifiers.
2. Prepare the lower and upper gradient layers according to the gradient system in use.
3. Carefully overlay the liquefied semen sample onto the upper gradient layer.
4. Centrifuge using the laboratory’s standard gradient recovery setting, chosen to produce a clean motile pellet without excessive mechanical stress.
5. Remove the supernatant carefully without disturbing the pellet or recovered fraction.
6. Add sperm preparation medium to the recovered fraction.
7. Resuspend gently and centrifuge using the laboratory’s standard wash setting for sperm recovery.
8. Remove the supernatant and repeat the wash if needed to remove residual gradient material.
9. Resuspend the final pellet in the volume of medium required for insemination.
10. Assess the final preparation for concentration and motility and maintain under appropriate temperature and timing conditions until use.

11.5 SwimCount® Harvester Preparation

1. Verify patient identifiers on the specimen container, device and worksheet.
2. Confirm that the device is within expiration date, intact and suitable for clinical use.
3. Allow the semen specimen to liquefy fully unless otherwise directed.
4. Gently mix the liquefied specimen to ensure a representative sample without creating excessive bubbles.
5. Label all receiving tubes or dishes before beginning.
6. Prepare the device exactly as required by the manufacturer instructions and the laboratory workflow.
7. Load the semen specimen into the device using the volume designated by the current laboratory validation or manufacturer instructions.
8. Place the loaded device under the environmental conditions defined by the laboratory, usually controlled bench conditions unless incubated use has been validated.
9. Maintain the device undisturbed for the migration interval long enough to permit motile sperm migration into the collection chamber while avoiding unnecessary delay before insemination.
10. At the end of the migration interval, harvest the motile sperm‑enriched fraction from the designated collection chamber using a sterile pipette.
11. Examine the harvested fraction for concentration and motility.
12. If the harvested fraction is too dilute for conventional IVF, concentrate it by centrifugation using the laboratory’s standard wash setting, then resuspend the pellet in an insemination volume that permits the required sperm dose to be delivered to the insemination dish.
13. Hold the final preparation under controlled conditions until insemination.

11.6 Final Review

If the final preparation is inadequate for conventional IVF, notify laboratory leadership and the treating physician promptly to determine whether a change in plan is required (e.g., ICSI).

11.7 Teaching Point

The goal of sperm preparation is to provide a fraction that matches the fertilization method safely and reproducibly. Technique alone is not enough; one must understand the clinical context and adapt the protocol accordingly.

12. Sperm Preparation for IVF – Frozen Samples

12.1 Purpose

To prepare previously cryopreserved sperm for IVF after thawing.

12.2 Procedure

1. Verify the specimen designation against the patient record, thaw plan and storage inventory before removal from storage.
2. Confirm witness according to laboratory policy before thawing begins.
3. Retrieve the correct vial, straw or other storage unit from cryostorage and minimize warming during handling. Use proper PPE to protect against cryogenic exposure.
4. Thaw according to the standard laboratory temperature and timing sequence used for that specimen type. Avoid rapid warming or prolonged warming that may damage sperm.
5. Once thawed, transfer the sample to an appropriately labeled sterile tube.
6. Assess post‑thaw appearance, motility and concentration as applicable. Cryopreserved sperm often show reduced motility compared with fresh sperm.
7. If cryoprotectant removal is required, dilute or wash the sample gradually to reduce osmotic stress.
8. Centrifuge using the laboratory’s standard thaw‑preparation recovery setting when concentration or wash recovery is needed.
9. Remove the supernatant carefully and resuspend the recovered sperm fraction in approved insemination medium.
10. Reassess final concentration and motility. If the post‑thaw recovery is inadequate for standard IVF, consult with laboratory leadership and the treating physician about using ICSI or donor sperm.

12.3 Documentation

Post‑thaw concentration, motility, preparation method, final preparation findings and any significant limitation shall be documented.

12.4 Teaching Point

Thawed sperm may behave differently from fresh sperm. Recovery, motility and clinical usefulness can be meaningfully reduced after thawing. Clear communication with the clinical team is essential when the recovery is suboptimal.

13. Sperm Preparation for IVF – Testicular Sperm

13.1 Purpose

To process testicular or epididymal sperm specimens for IVF or ICSI.

13.2 Procedure

1. Verify specimen identity, specimen source and intended use before opening or processing the sample.
2. If the specimen is a fresh tissue sample, transfer a small portion to a sterile processing dish containing handling medium.
3. Using sterile needles or fine sterile forceps, gently tease or mince the tissue to release sperm while avoiding excessive shredding of the tissue.
4. If the specimen is an aspirate, mix gently and examine a small aliquot microscopically.
5. Examine the processed sample under the microscope for sperm presence, approximate abundance and motility when visible. Record whether sperm are present and motile.
6. If sperm are identified, isolate the most useful fraction by allowing larger tissue fragments to settle or by using a gentle wash and brief centrifugation step.
7. Remove excess debris when possible without risking loss of rare sperm.
8. Prepare the final suspension in a small volume of handling medium suitable for ICSI or other intended use.
9. If no motile sperm are seen initially, continue a careful microscopic search using the laboratory’s standard search method and consider reassessment after brief incubation. Communicate with the physician about potential use of immotile sperm or testicular sperm extraction.
10. Document the findings clearly, including whether sperm were present, whether motility was observed and how the sample was allocated for use or storage.

13.3 Special Considerations

These specimens may contain low sperm numbers and substantial cellular debris. Unnecessary handling shall be avoided and communication with the physician shall occur when sperm are extremely limited. Under no circumstances should testicular tissue from one patient be confused with that of another patient; strict labeling and witness procedures are required.

13.4 Teaching Point

With surgical sperm, the key skill is often careful searching and specimen preservation, not aggressive processing. Patience and communication with the clinical team are essential.

14. Sperm Preparation for Intrauterine Insemination

14.1 Purpose

To prepare sperm for intrauterine insemination (IUI) by producing a clean motile sperm fraction that is safe for uterine placement. The aims differ from IVF preparation; IUI preparation does not need embryo‑culture media or oil overlay but must remove seminal plasma and debris thoroughly.

14.2 Procedure

1. Verify specimen identity and intended use for IUI.
2. Allow the specimen to liquefy fully unless pre‑liquefied on receipt.
3. Perform the initial assessment required by laboratory policy, including volume and motility‑related observations.
4. Select the preparation method used by the laboratory for IUI, most commonly density gradient preparation or sperm wash.
5. If using density gradient preparation, layer the gradient, add the semen specimen, centrifuge, recover the motile fraction and perform one or more wash steps until the final preparation is clean and suitable for uterine insemination.
6. If using a wash‑only method, dilute the sample in wash medium, centrifuge, remove supernatant, repeat if required and resuspend the pellet in the final insemination volume.
7. Assess the final preparation for volume, concentration, motility and total motile sperm when applicable. A clinically acceptable dose is commonly around 1–10 × 10⁶ total motile sperm depending on patient factors and laboratory protocol.
8. Label the final preparation clearly and keep it under appropriate conditions until released to the clinical team.
9. Transfer the prepared specimen to the clinical team using the laboratory’s chain‑of‑custody process.

14.3 Teaching Point

IUI preparation is designed to produce a clean, clinically acceptable insemination sample for uterine placement, not an embryo‑culture sample. The final suspension should contain minimal debris and be free of seminal plasma to reduce cramping and prostaglandin effects.

15. Oocyte Retrieval

15.1 Purpose

To define the laboratory procedure for receiving follicular aspirates, identifying oocytes, washing cumulus‑oocyte complexes, placing oocytes into culture and documenting the retrieval outcome.

15.2 Scope

This procedure applies to all oocyte retrieval cases performed for IVF or ICSI.

15.3 Responsibility

Only trained personnel may receive follicular aspirates and identify oocytes. The embryologist assigned to retrieval is responsible for confirming patient identity, preparing the work area, receiving aspirates, identifying and washing oocytes, placing oocytes into the correct culture dish, documenting the count and communicating the final retrieval result to the clinical team.

15.4 Preparation Before Retrieval

Before the patient enters the procedure room or before aspirates are expected in the laboratory:

1. Review the patient schedule and confirm the planned case.
2. Verify that all retrieval dishes, holding dishes, culture dishes, collection tubes and worksheets are labeled with approved patient identifiers.
3. Confirm availability of warmed flushing or handling medium, pre‑equilibrated culture dishes, sterile handling pipettes and any other required supplies.
4. Confirm that the microscope, warmed stage, hot block or warming area are functioning and within acceptable temperature range.
5. Confirm that the laboratory or adjacent procedure support area is clean, organized and ready for immediate specimen handling.
6. Confirm that the witness process is available if required by laboratory policy at any retrieval‑related checkpoint.

15.5 Receipt of Follicular Aspirates

1. Confirm the patient identity with the clinical team before the first specimen is handled.
2. Receive each follicular aspirate promptly after collection.
3. Keep aspirate tubes in a warmed block or other approved temperature‑controlled area until examined.
4. Process each tube as soon as practicable; aspirates should be scanned continuously as they are received rather than allowed to accumulate.
5. Maintain the order of received tubes if required by laboratory workflow.

15.6 Identification of Oocytes

1. Gently pour or transfer the aspirate into the scanning dish or approved retrieval vessel.
2. Examine the aspirate systematically under the stereomicroscope at the magnification normally used by the laboratory for retrieval scanning.
3. Scan the entire dish in a consistent pattern so that no portion of the aspirate is missed.
4. Identify cumulus‑oocyte complexes (COCs) and distinguish them from blood clots, granulosa cell clumps, mucus or tissue fragments. According to the 2025 ESHRE/Alpha consensus, unusual COC characteristics such as very compact COCs, blood clots or hypergranular cumulus should be documented because they may affect fertilization outcomes. Morphological variations such as thick or dark zona pellucida, different perivitelline space sizes, irregular shapes, vacuoles, refractile bodies and cytoplasmic granularity generally do not preclude use but should be noted in the record. Giant oocytes (>180 µm) should be excluded from clinical use and very small or large eggs should be documented.
5. Carefully inspect dense cellular material or tissue fragments when an oocyte may be embedded.
6. Before discarding the original aspirate tube, inspect the tube wall and bottom for any COC that may remain adherent.
7. If flushing is performed or additional aspirate is received from the same follicle, continue scanning until all material designated for laboratory review has been examined.

15.7 Washing and Transfer to Culture

1. Pick up each identified COC using an approved handling pipette.
2. Transfer the complex through the laboratory’s designated wash droplets or dishes to remove excess blood and debris.
3. Place washed oocytes into the labeled pre‑equilibrated culture dish containing the approved culture medium.
4. Group oocytes in the dish according to laboratory workflow while preserving patient identity and traceability.
5. Return the culture dish to controlled incubation conditions promptly after transfer.

15.8 Retrieval Count and Communication

1. Maintain a running count of the number of oocytes identified.
2. Reconcile the total number of oocytes placed into culture before finalizing the retrieval record.
3. Communicate the final oocyte count to the clinical team using the approved communication method.
4. Document any unusual finding, including empty or poor‑yield aspirates, excessive blood, fragmented cumulus masses or concern regarding specimen condition.

15.9 Documentation

Document the following as applicable:

• Patient identifiers.
• Date and time of retrieval.
• Embryologist performing the procedure.
• Number of oocytes identified.
• Any flushing performed or notable handling issue.
• Final communication of the count to the clinical team.
• Any unusual observation (e.g., unusual COC morphology, presence of giant oocytes).

15.10 Teaching Point

A trainee should understand that retrieval scanning is a systematic search process. Missed oocytes usually result from inconsistent scanning technique, not from “invisible” specimens. Recording unusual COC characteristics supports clinical decision‑making and may inform fertilization strategy.

16. Conventional Insemination

16.1 Purpose

To define the procedure for inseminating oocytes with prepared sperm during conventional IVF and to provide clear guidance for choosing the appropriate oocytes, preparing the insemination dish, adding the sperm suspension and maintaining appropriate culture conditions until fertilization assessment.

16.2 Scope

This procedure applies to all oocytes designated for conventional insemination.

16.3 Responsibility

Only trained personnel may perform conventional insemination. The embryologist performing the procedure is responsible for confirming specimen identity, preparing insemination dishes, confirming sperm preparation suitability, inseminating the oocytes at the appropriate concentration, returning the dish to incubation, and documenting the procedure.

16.4 Timing

Conventional insemination shall be performed after oocytes have completed the laboratory’s standard post‑retrieval incubation interval. As an instructional standard, insemination is performed later on the day of retrieval after the oocytes have rested under stable culture conditions and the sperm preparation is ready. The timing of insemination should allow subsequent fertilization assessment at approximately 16–18 h after insemination (Day 1), consistent with ESHRE/Alpha guidelines.

16.5 Selection of Oocytes for Conventional Insemination

Oocytes designated for conventional insemination should be those assigned to standard IVF according to the treatment plan. In general, cumulus‑enclosed oocytes are inseminated without prior denudation unless the laboratory workflow or clinical plan requires otherwise. If there is concern regarding oocyte quality, excessive debris, unexpectedly poor sperm recovery, or the possibility that the insemination method should be changed, the embryologist shall review the case with laboratory leadership and the treating physician before proceeding. Giant oocytes or abnormally small oocytes should not be inseminated.

16.6 Preparation

Before insemination:

1. Confirm patient identity on the oocyte dish, sperm preparation tube and worksheet.
2. Confirm the required witness step according to laboratory policy.
3. Confirm the number of oocytes designated for conventional insemination.
4. Confirm that the insemination dish has been prepared with equilibrated medium under appropriate culture conditions.
5. Confirm that the sperm preparation has been assessed and is suitable for use. The sperm suspension should be free of debris and at a concentration sufficient to deliver the target insemination dose.
6. Confirm the target sperm concentration or insemination dose according to laboratory policy (e.g., 50 000–100 000 motile sperm per oocyte for conventional insemination). Adjust the dose when total motile sperm count is low.
7. Review whether the case includes any specific instruction regarding low sperm count, low motility, split insemination plan or fallback plan for ICSI.

16.7 Procedure

1. Remove the oocyte dish from the incubator only when ready to inseminate.
2. Review the oocyte identity again before sperm is added.
3. Place or confirm placement of the oocytes into the designated insemination droplet or well.
4. Inspect the dish to confirm that the number of oocytes present matches the expected count.
5. Mix the prepared sperm suspension gently so the suspension is uniform.
6. Add the appropriate volume of sperm suspension to the insemination droplet or well to achieve the target insemination concentration.
7. Observe briefly to ensure the sperm disperses appropriately into the insemination droplet rather than remaining as a concentrated bolus. If a bolus forms, gently mix with a pipette.
8. Confirm that the final insemination dish is properly labeled and contains the intended oocytes.
9. Return the dish promptly to the incubator. Minimize time outside the incubator to preserve pH and temperature.
10. Record the exact or approximate time of insemination according to laboratory practice.

16.8 What to Look For During the Procedure

During insemination, the embryologist should confirm that:

• The cumulus‑oocyte complexes appear intact and suitable for continued culture.
• The sperm suspension appears adequately motile for the intended IVF plan.
• There is no obvious dish contamination, evaporation, labeling discrepancy or handling error.
• The insemination droplet remains stable and properly overlaid if oil is used.

If sperm recovery is poorer than expected or if the insemination dose appears inadequate, the embryologist should pause and review the plan before all oocytes are inseminated. Options include increasing the dose, switching to ICSI or splitting the cohort.

16.9 Post‑Insemination Handling

1. Maintain the dish under stable incubator conditions until the planned fertilization check (approximately 16–18 h after insemination). Avoid unnecessary disturbance of the insemination dish.
2. If any issue occurs during insemination, including dish contamination, labeling discrepancy or concern about sperm adequacy, notify laboratory leadership immediately and document the event.

16.10 Documentation

Document the following:

• Patient identifiers.
• Date and time of insemination.
• Number of oocytes inseminated.
• Sperm preparation used and target dose.
• Operator identity.
• Witness identity when required.
• Any deviation or unusual observation (e.g., low sperm count, changed plan, bolus not dispersing).

17. Hyaluronidase and Oocyte Stripping

17.1 Purpose

To define the procedure for removal of cumulus and corona cells from oocytes for maturity assessment and preparation for ICSI. Denudation should occur at the appropriate time after retrieval to allow the oocyte to complete its maturation.

17.2 Scope

This procedure applies to oocytes designated for denudation (typically those intended for ICSI or pre‑implantation genetic testing). Denudation is not performed on oocytes designated for conventional insemination.

17.3 Responsibility

Only trained personnel may perform oocyte stripping. The embryologist performing the procedure is responsible for preparing the stripping dish, controlling exposure to hyaluronidase, mechanically removing cumulus and corona cells, assessing oocyte maturity and documenting the result.

17.4 Preparation

Before stripping:

1. Prepare and label the stripping dish, wash droplets and post‑stripping culture dish.
2. Confirm that the hyaluronidase solution and associated media are within expiration and ready for use. Hyaluronidase should be used at the concentration validated by the laboratory (commonly 80–100 IU/mL) and exposure time should be minimized to reduce potential toxicity.
3. Confirm patient identity on all dishes and the worksheet.
4. Confirm the timing of the denudation procedure according to laboratory workflow; denudation is typically performed 3–5 h after retrieval to allow completion of meiosis I but before ICSI.
5. Arrange denuding pipettes from larger bore to smaller bore so that progressive removal of cells can be done efficiently.
6. Limit the number of oocytes handled at one time to the number that can be denuded promptly without prolonged exposure outside controlled conditions.

17.5 Procedure

1. Remove the oocytes designated for stripping from incubation when ready to perform the procedure. Keep them in a warmed environment until denuding begins.
2. Transfer the cumulus‑oocyte complexes into the hyaluronidase‑containing droplet or dish. Expose the oocytes only briefly (usually ≤30 s), just long enough to loosen the cumulus matrix so mechanical denudation can proceed.
3. Transfer the oocytes into wash droplets and begin mechanical denudation using denuding pipettes of appropriate bore size.
4. Move the oocytes through progressively smaller denuding pipettes as needed, using gentle aspiration and expulsion to remove residual cells.
5. Inspect each oocyte carefully once denudation is complete. Assess maturity based on the presence of the first polar body (metaphase II, mature), absence of polar body (germinal vesicle stage or metaphase I, immature) or degeneration.
6. Transfer the oocytes into the appropriately labeled post‑stripping culture dish and return promptly to the incubator.

17.6 Precautions

• Avoid prolonged exposure to hyaluronidase. Extended exposure can damage the zona pellucida and oocyte membranes.
• Avoid excessive mechanical stress when aspirating and expelling oocytes through pipettes.
• Do not strip more oocytes at one time than can be handled efficiently without prolonged exposure outside the incubator.
• If an oocyte appears fragile, abnormal or degenerating, document the finding and consult with laboratory leadership. Giant oocytes should not be injected.

17.7 Documentation

Document the following:

• Patient identifiers.
• Date and time of stripping.
• Number of oocytes denuded.
• Maturity findings (e.g., number of metaphase II, metaphase I or germinal vesicle oocytes).
• Operator identity.
• Any unusual observation or degeneration noted during stripping.

17.8 Teaching Point

The goal is complete but gentle denudation. Over‑handling can damage the same oocyte you are trying to prepare for ICSI. Proper timing and pipette size selection are critical.

18. Intracytoplasmic Sperm Injection (ICSI)

18.1 Purpose

To define the procedure for intracytoplasmic sperm injection of mature oocytes and to provide clear instruction for dish preparation, sperm selection, oocyte handling, injection technique and post‑injection review.

18.2 Scope

This procedure applies to all ICSI cases performed in the laboratory.

18.3 Responsibility

Only personnel specifically trained, authorized and currently competent in ICSI may perform this procedure. The embryologist performing ICSI is responsible for confirming identity, preparing the equipment and dishes, selecting sperm, injecting mature oocytes, returning injected oocytes to culture and documenting the procedure.

18.4 Preparation of Equipment

Before beginning ICSI:

1. Confirm function of the inverted microscope, heated stage, micromanipulators, holding pipette, injection pipette and any accessory equipment. Confirm that the heated stage is within the acceptable temperature range (usually 37 °C ± 0.5 °C).
2. Confirm that the injection dish, sperm handling dish and post‑injection culture dish are properly labeled.
3. Confirm that the required microtools are correctly mounted and functioning.
4. Prepare all media and oil droplets according to laboratory policy. Use fresh oil overlay and equilibrate injection and holding medium to the appropriate pH and temperature.
5. Confirm that the work area is organized so that identity control is maintained throughout the case. The dish containing oocytes should not be confused with any other case.

18.5 Preparation of Oocytes and Sperm

1. Confirm patient identity on all dishes, sperm materials and records.
2. Confirm maturity of the oocytes designated for injection (presence of first polar body indicates metaphase II). Do not inject immature (metaphase I or germinal vesicle) or degenerated oocytes.
3. Confirm the required witness step before injection according to laboratory policy.
4. Prepare the sperm sample in the sperm handling medium used by the laboratory. Select sperm according to laboratory training standards, favoring motile sperm with acceptable morphology when available. Immobilize sperm by tail tapping when necessary.

18.6 What to Look For Before Injection

Before injection begins, the embryologist should confirm that:

• The oocytes selected for injection are mature according to the laboratory’s maturity criteria (MII with first polar body). Giant oocytes should be excluded.
• The injection and holding pipettes are functioning smoothly without leaks or occlusion.
• The oocytes are not exposed to room conditions longer than necessary; incubation should be minimized before injection.
• The sperm population contains cells suitable for ICSI. When very few suitable sperm are available, the embryologist may inject non‑motile sperm after viability assessment or confirm an alternate plan (e.g., donor sperm).
• There is no dish labeling discrepancy or case mix‑up risk. Witness confirmation should occur.

If very few suitable sperm are present or if oocyte quality appears poor or fragile, the embryologist should proceed deliberately and document those concerns. Consultation with the physician may be necessary to adjust expectations.

18.7 Injection Procedure

1. Place a manageable number of mature oocytes into the injection dish. Keep remaining oocytes in the incubator until ready.
2. Position each oocyte with the holding pipette in the orientation preferred by the laboratory (e.g., polar body at 12 o’clock or 6 o’clock) to avoid injuring the spindle.
3. Immobilize the selected sperm if required by the ICSI method (e.g., by crushing the tail against the dish). Aspirate the sperm into the injection pipette.
4. Penetrate the zona pellucida and oolemma with the injection pipette using the laboratory’s standard micromanipulation technique. Depress the pipette plunger to deposit the sperm into the oocyte cytoplasm.
5. Withdraw the injection pipette carefully. Verify that the oolemma has resealed and that no cytoplasmic material is aspirated.
6. Inspect the oocyte briefly for survival and obvious lysis. If lysis occurs, document and continue with caution.
7. Transfer each injected oocyte to the labeled post‑injection culture dish. Keep the injection dish clean and ready for the next oocyte.
8. Continue until all designated mature oocytes have been injected or the case plan has been completed.
9. Return the dish promptly to the incubator.

18.8 What to Watch During Injection

The embryologist should monitor for:

• Oocytes that appear unusually dark, granular, vacuolated, fragile or difficult to penetrate. Over‑pressure or repeated attempts may cause lysis.
• Repeated membrane resistance or repeated collapse suggesting technical difficulty.
• Immediate lysis after injection, which should be recorded. Do not attempt to re‑inject the same oocyte.
• Sperm that are difficult to immobilize or poorly suited for injection. Consider alternative sources or consulting the physician.
• Excessive time outside the incubator for the case as a whole. Oocytes should remain outside the incubator only as long as necessary for injection.

When unexpected difficulty occurs, the operator should slow down, protect the remaining oocytes and seek assistance if needed according to laboratory policy.

18.9 Post‑Procedure Review

1. Confirm the number of oocytes injected.
2. Document any oocyte lysis, degeneration or technical difficulty.
3. Clean and secure equipment according to laboratory policy.
4. Ensure that the injected oocytes are returned to stable culture conditions without delay.

18.10 Documentation

Document the following:

• Patient identifiers.
• Date and time of injection.
• Number of mature oocytes available.
• Number of oocytes injected.
• Operator identity.
• Witness identity when required.
• Any abnormal observation, lysis, degeneration or notable technical difficulty.

19. Micromanipulation

19.1 Purpose

To define the general requirements for performance, quality oversight and monitoring of micromanipulation procedures including ICSI, assisted hatching, embryo biopsy and removal of lysed cell fragments from embryos.

19.2 General Requirements

Micromanipulation procedures shall be performed only by trained personnel with current competency. Before beginning any micromanipulation procedure, the operator shall confirm:

• Correct patient and specimen identity.
• Correct dish labeling and witness step completed.
• Acceptable condition of equipment and pipettes.
• Appropriate heated stage temperature (usually 37 °C).
• Appropriate media preparation (pH and temperature). Embryo culture media should maintain pH 7.25–7.35.
• Availability of witness support where required.

19.3 Quality Oversight

Performance shall be reviewed periodically using laboratory‑defined metrics, which may include:

• Oocyte survival after injection.
• Fertilization rate (normal 2PN zygotes) after ICSI or conventional insemination.
• Cleavage, morula or blastocyst development after ICSI.
• Embryo survival after assisted hatching or biopsy.
• Biopsy adequacy.
• Post‑manipulation embryo development and clinical outcomes.

19.4 Equipment Care

Micromanipulation equipment shall be cleaned, maintained and serviced according to laboratory policy. Damaged microtools or unstable manipulation systems shall not be used. Disposable pipettes should be changed frequently to prevent cross‑contamination.

19.5 Teaching Point

Micromanipulation requires calm workflow, meticulous identity control and deliberate movements. Speed without control is not a strength. Operators should develop dexterity through supervised training before practicing independently.

20. Determination of Fertilization

20.1 Purpose

To define the procedure for assessing fertilization status after conventional insemination or ICSI.

20.2 Timing

Fertilization assessment shall be performed on the morning after insemination or ICSI, typically at 16–18 h post‑insemination, consistent with the standardized post‑insemination interval used by ESHRE/Alpha (Day 1 at 16–17 h, Day 2 at 43–45 h, Day 3 at 63–65 h, etc.). Using standardized timing ensures that pronuclear status can be assessed consistently across all cases.

20.3 Preparation

1. Prepare labeled assessment dishes if embryo movement is required by laboratory workflow.
2. Confirm patient identity on the dish and record. Witness if required.
3. Minimize the time the dish is outside the incubator. Set up the stereomicroscope or inverted microscope for pronuclear assessment (400–600×).

20.4 Procedure

1. Remove the dish from the incubator at the scheduled time.
2. Examine each oocyte or zygote using the microscope appropriate for pronuclear assessment.
3. Assess fertilization according to laboratory criteria. Normal fertilization is defined by the presence of two pronuclei (2PN) and two polar bodies. Abnormal patterns include 1PN, 3PN or higher, or absence of pronuclei (0PN). According to ESHRE/Alpha consensus, zygotes with a second polar body but no pronuclei should be recorded as “pronuclei not observed” rather than 0PN; some embryos with 1PN or partial pronuclei can develop normally and may be considered for transfer depending on clinic policy.
4. Record each oocyte as normally fertilized (2PN), abnormally fertilized (e.g., 3PN), unfertilized (0PN), pronuclei not observed or degenerated. Document the presence of second polar body, pronuclear size, alignment and nucleolar precursor body (NPB) patterns if part of the laboratory scoring system.
5. If required by laboratory workflow, move normally fertilized zygotes into the appropriate culture dish after assessment. Keep abnormal or unfertilized oocytes separate for subsequent disposition.
6. Return the culture dish promptly to incubation.

20.5 What to Look For

The embryologist should identify:

• Normal fertilization pattern according to the laboratory’s criteria (2PN with polar bodies, pronuclei of similar size and centrally aligned nucleoli).
• Abnormal pronuclear findings (e.g., 1PN, 3PN, pronuclear asynchrony, presence of vacuoles or refractile bodies). Note that some 1PN or pronuclei not observed zygotes may still be viable.
• Oocyte degeneration.
• Delayed or absent fertilization. If fertilization rates are low, review sperm preparation and oocyte quality and consult the physician.

20.6 Documentation

Document the fertilization findings for each case, including the total number of normally fertilized zygotes and any abnormal findings relevant to the clinical record. Fertilization assessment results should be available to inform embryo selection and patient counseling.

21. Embryo Scoring

21.1 Purpose

To define the procedure for assessment and documentation of embryo development and quality throughout culture and to provide trainees with guidance on what to examine, what to record and how those observations support clinical decision‑making. Scoring criteria are based on consensus guidelines from ESHRE/Alpha (2025) and ASRM.

21.2 Scope

This procedure applies to cleavage‑stage embryos, morulae, blastocysts and any cultured embryo assessed before transfer, biopsy, cryopreservation, extended culture or discard. Embryo assessment should not be performed more frequently than necessary; typical assessment time points are Day 2 (43–45 h), Day 3 (63–65 h), Day 4 (93–95 h) and Day 5 (111 h) post‑insemination.

21.3 Responsibility

Only trained personnel may assess and score embryos. The embryologist is responsible for using the laboratory’s grading system consistently, documenting the findings clearly and minimizing embryo exposure outside controlled culture conditions.

21.4 General Procedure

1. Remove the embryo dish from the incubator only when ready to assess.
2. Confirm patient identity on the dish and record. Witness if required.
3. Review the developmental day or time point before beginning assessment.
4. Examine embryos systematically so no embryo is missed. Use an inverted microscope at appropriate magnification.
5. Record stage and morphology immediately at the time of observation.
6. Assess only those features that are appropriate to the developmental stage. Avoid over‑interpretation of minor variation.
7. Return the embryo dish promptly to the incubator.

21.5 General Principles of Embryo Assessment

Embryo assessment should be consistent, deliberate and limited to observations that can be made reliably. Trainees should avoid over‑interpreting minor variation and should focus on reproducible features that affect stage assignment, quality grading and disposition decisions. A useful embryo assessment answers four questions:

1. What stage is the embryo at right now? (e.g., 4‑cell, 8‑cell, morula, blastocyst)
2. Does the morphology fit what is expected for that stage and day of development? (e.g., number of cells, compaction, blastocyst expansion)
3. Are there findings that suggest lower developmental potential or degeneration? (e.g., severe fragmentation, multinucleation, poor compaction)
4. Is the embryo currently best suited for continued culture, transfer, biopsy, cryopreservation or discard according to laboratory and clinical policy?

21.6 Cleavage‑Stage Assessment

At cleavage‑stage assessment (Days 2–3), document features such as:

• Cell number – Embryos with 4 cells on Day 2 and 8 cells on Day 3 are most likely to achieve blastocyst formation.
• Blastomere symmetry – Evenly sized blastomeres are associated with better prognosis; marked asymmetry suggests reduced potential.
• Degree of fragmentation – Fragmentation <10 % yields top ranking; 10–25 % is moderate; >25 % is considered poor.
• Evidence of multinucleation – Multinucleated blastomeres are associated with poor outcomes. Document presence or absence.
• Cytoplasmic abnormalities – Severe vacuolization, granularity or darkening may indicate poor quality. However, minor variations in perivitelline space, zona thickness or cytoplasm do not necessarily preclude use.

21.7 Morula and Blastocyst Assessment

At later‑stage assessment:

• Day 4 (morula) – Evaluate compaction status. Full compaction or early cavitation should be prioritized for transfer or freezing. Loose or incomplete compaction may indicate delayed development.
• Day 5/6 (blastocyst) – Assess blastocyst expansion stage and inner cell mass (ICM) and trophectoderm (TE) quality. According to the Gardner grading scale, expansion is graded from early to hatched blastocyst; ICM is graded A–C (1–3) based on size, compactness and morphology; TE is graded A–C based on number and cohesiveness of cells. Day of blastocyst formation is a key ranking factor: embryos that reach blastocyst stage on Day 5 generally have higher implantation potential than those on Day 6. TE grade and expansion stage are strongly associated with live birth.

21.8 What to Look For During Scoring

When scoring embryos, the embryologist should consider:

• Whether development is advancing appropriately for the culture day.
• Whether the embryo appears stable or degenerative (e.g., collapse, vacuoles, cytoplasmic granularity).
• Whether morphology supports continued culture or suggests a disposition decision.
• Whether multiple embryos in the cohort appear similar or whether one clearly stands out for transfer or biopsy planning.

Embryo scoring should remain descriptive and disciplined. It should support, not replace, clinical judgment and laboratory policy.

21.9 Use of Embryo Scores

Embryo scores shall support decisions regarding continued culture, transfer, biopsy, cryopreservation or discard according to laboratory and clinical policy. Embryo selection for transfer should consider not only morphological score but also day of development, patient history and clinic’s elective single embryo transfer policies.

21.10 Documentation

Document the embryo assessment clearly in the case record and in any embryo disposition or transfer planning worksheet used by the laboratory. Ensure that scores are recorded for each embryo at each assessment time point.

22. Assisted Hatching of Embryos

22.1 Purpose

To define the procedure for assisted hatching when clinically indicated and approved. Assisted hatching may be used in cases of advanced maternal age, previous implantation failure, thick zona pellucida or as part of trophectoderm biopsy preparation.

22.2 Scope

This procedure applies to embryos designated for assisted hatching according to the clinical plan and laboratory policy.

22.3 Responsibility

Only trained and authorized personnel may perform assisted hatching.

22.4 Preparation

1. Confirm patient and embryo identity.
2. Confirm witness according to laboratory policy if required.
3. Prepare the treatment dish and confirm function of the laser or other approved hatching system.
4. Confirm that the embryos designated for assisted hatching match the clinical plan.

22.5 Procedure

1. Move the embryo into the designated treatment dish.
2. Position the embryo so the zona can be treated at a site away from blastomeres or key cellular structures.
3. Focus the treatment area clearly.
4. Apply the hatching treatment used by the laboratory (e.g., laser ablation, partial thinning), creating either a zona thinning zone or a discrete opening of the size routinely used by the laboratory.
5. Reassess the embryo immediately after treatment for evidence of unintended damage.
6. Return the embryo promptly to the appropriate culture dish and incubator.

22.6 Documentation

Document the following:

• Indication for assisted hatching.
• Method used (e.g., laser, mechanical).
• Number of embryos treated.
• Operator identity.
• Any unusual observation during the procedure.

22.7 Teaching Point

The goal is controlled zona manipulation, not excessive treatment. Less is often better when the indication is valid. Over‑exposure to laser or acid Tyrode’s can damage embryos.

23. Embryo Biopsy

23.1 Purpose

To define the procedure for embryo biopsy for genetic testing while preserving accurate identity and embryo viability and to provide trainees with a clear understanding of both the technical workflow and the decision points that protect traceability.

23.2 Scope

This procedure applies to cleavage‑stage or trophectoderm biopsy procedures performed by the laboratory.

23.3 Responsibility

Only trained and authorized personnel may perform embryo biopsy. The embryologist performing the biopsy is responsible for confirming identity, preparing biopsy materials, obtaining the biopsy sample, maintaining correct linkage between the embryo and biopsy tube and documenting the procedure.

23.4 Preparation

Before biopsy:

1. Review the biopsy plan and confirm embryo identity.
2. Confirm that all biopsy tubes are labeled exactly according to the approved numbering or identification system. Use at least two identifiers and ensure the labels are durable at cryogenic temperatures.
3. Prepare the biopsy dish, post‑biopsy culture dish and all required records.
4. Confirm the required witness step before the biopsy begins and again at any required loading or tube confirmation step.
5. Confirm function of the microscope, micromanipulators, biopsy pipette, holding pipette and laser or other associated equipment.

23.5 Pre‑Biopsy Review

Before beginning, the embryologist should confirm:

• Which embryos are designated for biopsy and whether they meet the developmental criteria for the intended biopsy method (e.g., Day 5/6 blastocyst with expanded trophectoderm for TE biopsy).
• Whether the embryos are developmentally appropriate for the intended biopsy method (e.g., full compaction for cleavage stage biopsy; expanded blastocyst with visible TE cells for trophectoderm biopsy). Note that cleavage‑stage biopsy is less common but may be used in specific circumstances.
• That tube numbering, embryo numbering and worksheet numbering all align exactly. Use a witness to verify.
• That post‑biopsy embryo disposition is already planned (continued culture, cryopreservation or fresh transfer) and that cryopreservation inventory is ready.

A trainee should understand that the identity‑control portion of biopsy is as important as the technical biopsy itself.

23.6 Biopsy Procedure

1. Move the embryo designated for biopsy into the biopsy dish. Maintain temperature and pH.
2. Stabilize the embryo using the holding pipette or other support method.
3. If biopsy is performed at the blastocyst stage, orient the embryo so that the trophectoderm cells selected for biopsy are accessible and the inner cell mass is protected.
4. Create an opening in the zona or use a pre‑existing opening according to the laboratory method. Laser‑assisted hatching may be performed on Day 3 or Day 4 to facilitate TE biopsy.
5. Gently draw the selected cell or cells into the biopsy pipette.
6. Separate the selected cell or cells using the standard biopsy technique in use, typically with laser‑assisted or controlled mechanical separation.
7. Confirm that the embryo remains intact and transfer it promptly to the labeled post‑biopsy culture dish.
8. Proceed one embryo at a time unless another workflow has been specifically adopted and validated by the laboratory. Do not mix cells from different embryos.

23.7 What to Watch During Biopsy

The embryologist should pay attention to:

• Whether the embryo tolerates manipulation well or collapses.
• Whether the inner cell mass remains protected during trophectoderm biopsy.
• Whether the number of cells removed is appropriate for the testing method (commonly 5–8 TE cells for blastocyst biopsy).
• Whether there is any risk of mixing samples or misaligning embryo and tube numbering.
• Whether the biopsied embryo remains viable in appearance after the procedure.

If identity becomes uncertain or if the embryo is too fragile, stop and consult laboratory leadership.

23.8 Loading the Biopsy Sample

1. Prepare the corresponding labeled biopsy tube with proper buffer.
2. Move the biopsy sample into the tube using the loading technique in current use (e.g., microcapillary pipette or washing into PCR tube). Avoid contamination with maternal DNA or other embryos.
3. Confirm that the embryo number, tube number and worksheet match exactly.
4. Confirm witness at the required checkpoint.
5. Secure the tube according to the testing laboratory and laboratory‑specific requirements (e.g., freeze or ship as per genetic testing lab instructions).

23.9 Post‑Biopsy Handling

1. Return biopsied embryos promptly to the incubator or proceed to cryopreservation according to the clinical plan.
2. Package and store or ship the biopsy samples according to the approved workflow. Maintain chain of custody and appropriate temperature (e.g., refrigerated or frozen transport).
3. Reconcile the number of embryos biopsied with the number of tubes prepared and loaded.

23.10 Documentation

Document the following:

• Patient identifiers.
• Embryos biopsied, including embryo numbering or labeling system used.
• Biopsy tube identifiers and genetic test requisition.
• Number of cells removed when applicable.
• Operator identity.
• Witness identity.
• Destination laboratory if applicable.
• Final status of each biopsied embryo (e.g., culture, frozen).

24. Embryo Cryopreservation and Vitrification

24.1 Purpose

To define the procedure for cryopreservation of embryos using the laboratory’s vitrification or other approved cryopreservation method, with enough detail that trainees understand both the sequence of steps and the reasons why careful timing, identity control and storage accuracy are critical. Cryopreservation should follow ASRM and ESHRE guidelines, including obtaining written consent that specifies disposition options in the event of death, separation or abandonment.

24.2 Scope

This procedure applies to all embryos designated for cryopreservation.

24.3 Responsibility

Only trained and authorized personnel may cryopreserve embryos. The embryologist performing the procedure is responsible for confirming identity, preparing solutions and cryodevices, processing embryos through the cryopreservation sequence, assigning the storage location and documenting the procedure.

24.4 Preparation

Before cryopreservation:

1. Confirm patient identity, embryo identity and the clinical plan for cryopreservation. Verify that written cryopreservation consent forms specify disposition options (e.g., future use, donation, research or discard) according to ASRM guidelines.
2. Confirm witness according to laboratory policy.
3. Prepare and label all cryodishes, cryodevices (e.g., cryoloops, straws), storage records and associated worksheets before embryos are moved. Labels must be legible, water‑resistant and durable at cryogenic temperatures.
4. Confirm that all cryopreservation solutions are correctly prepared, within expiration and at the required handling condition (e.g., room temperature or warmed). Confirm pH and osmolarity.
5. Confirm that the storage tank, cane, goblet or other storage hardware is available and that the target storage location has been assigned. Ensure cryotank level probes and alarms are functional and that the tank is monitored continuously.
6. Arrange the workspace so embryos can be processed one at a time or in small controlled groups without confusion. Ensure proper PPE (cryogloves, face shield) and that the workstation is free of clutter.

24.5 Embryo Selection

1. Confirm which embryos are designated for cryopreservation based on the laboratory and clinical plan.
2. Verify that embryo identity and numbering in the dish match the worksheet or disposition record. Use witness verification.
3. Record the number, stage (e.g., blastocyst grade) and quality of embryos selected for cryopreservation. Consult the Gardner grading system for blastocyst quality.
4. Confirm that the embryo stage and quality are appropriate for the cryopreservation approach in use (e.g., expanded blastocysts are preferred for vitrification; cleavage‑stage cryopreservation may be used selectively).

24.6 General Principles of Vitrification

Vitrification is a rapid cryopreservation process designed to prevent ice crystal formation by exposing embryos to cryoprotectant solutions and then cooling them extremely quickly. The key technical priorities are:

• Maintaining correct embryo identity at all times.
• Moving through the solution sequence in the correct order and for the correct duration.
• Avoiding excessive carryover volume when transferring embryos between solutions and onto the cryodevice.
• Avoiding prolonged exposure to concentrated cryoprotectants, which can be toxic.
• Loading the cryodevice efficiently with minimal fluid volume.
• Recording the exact storage location immediately.

24.7 Cryopreservation Procedure

1. Move the embryo or embryos into the first equilibration solution using a handling pipette that permits gentle transfer with minimal carryover volume. Allow the embryo to equilibrate for the standardized interval (commonly 2–3 min) to allow the initial osmotic response.
2. Transfer the embryo or embryos to the second equilibration solution or the vitrification solution for the brief exposure interval used by the laboratory. This step should be performed efficiently; prolonged exposure to concentrated cryoprotectant can reduce survival.
3. During final exposure, prepare the cryodevice and ensure that the label matches the embryo identity and the worksheet. Load the embryo onto the cryodevice in the minimum fluid volume required by the device workflow. Remove excess medium to achieve rapid cooling and avoid ice formation.
4. Confirm the cryodevice identity and embryo assignment immediately before final cooling (plunge into liquid nitrogen or closed‑system cooling). Use witness verification.
5. Complete the vitrification step by placing the loaded cryodevice into liquid nitrogen or into the closed‑system cooling step used by the laboratory, depending on the device in use.
6. Secure the cryodevice in the assigned protective hardware if applicable (e.g., straw sleeve, goblet holder).
7. Transfer the cryodevice to the assigned storage cane, goblet or rack. Place the cryodevice into the assigned tank position without delay. Record the exact storage location immediately in the cryostorage log and electronic inventory.
8. Repeat for each embryo or group according to laboratory policy. Do not allow multiple embryos to wait in cryoprotectant beyond the validated exposure time.

24.8 What to Watch During Cryopreservation

During cryopreservation, the embryologist should pay close attention to:

• Whether embryos are moving through the solutions in the correct sequence and for the correct time.
• Whether carryover volume is minimized during transfers. Use pipettes appropriate for the embryo stage.
• Whether the final loading volume on the cryodevice is appropriately small (ideally <0.1 µL) to allow rapid vitrification.
• Whether the embryo assignment matches the cryodevice label exactly. Witness verification is mandatory.
• Whether the cryodevice reaches the assigned storage location without delay. Do not leave loaded devices on the bench.
• Whether any embryo appears fragile, degenerative or difficult to handle. Consider discarding poor‑quality embryos rather than exposing them to cryoprotectant.

A trainee should understand that many cryopreservation errors are not dramatic technical failures but small lapses in timing, labeling or location recording. Continuous level monitoring and remote alarms should be in place to detect tank problems.

24.9 Post‑Procedure Reconciliation

1. Confirm that the number of embryos cryopreserved matches the number documented on the worksheet and in the cryostorage inventory.
2. Confirm that all cryodevice identifiers match the worksheet and storage record. Reconcile any discrepancy immediately.
3. Confirm that the storage location entry is complete and accurate. Include tank number, cane/goblet position and slot number.
4. Reconcile the post‑cryopreservation embryo disposition for the case (e.g., embryos cryopreserved, embryos transferred, embryos discarded).

24.10 Documentation

Document the following:

• Patient identifiers.
• Date of cryopreservation.
• Operator identity.
• Witness identity.
• Number of embryos cryopreserved.
• Developmental stage and quality of each embryo or group as required.
• Cryodevice identifier.
• Storage tank and position location.
• Any deviation or unusual observation during cryopreservation (e.g., embryo collapse, cryodevice damage).

25. Embryo Transfer

25.1 Purpose

To define laboratory support for embryo transfer, including verification, embryo selection, catheter loading and post‑transfer confirmation, with enough detail that trainees understand not only the sequence of actions but also the points where accuracy and timing matter most.

25.2 Scope

This procedure applies to all fresh and frozen embryo transfer cases supported by the laboratory.

25.3 Responsibility

Only trained personnel may perform embryo transfer support. The embryologist is responsible for confirming identity, preparing embryos for transfer, loading the catheter, supporting the physician during transfer, confirming whether embryos remain in the catheter after transfer and documenting the procedure.

25.4 Preparation

Before the transfer:

1. Review the transfer paperwork and embryo inventory.
2. Confirm the transfer plan, including the number and stage of embryos to be transferred and whether genetic results or other selection criteria apply.
3. Verify the patient name and date of birth with the patient and clinical team.
4. Retrieve the correct embryo transfer dish from the incubator. Confirm dish identity against all transfer documents with a witness according to policy.
5. Prepare the catheter, syringe and required sterile materials. Prime the catheter with culture medium and remove air bubbles according to the laboratory method. Some laboratories use small air separators to visualize the embryo column; others use medium only.
6. Confirm the physician is ready before loading the catheter.
7. Keep embryo exposure outside the incubator as short as practical during the loading process.

25.5 Embryo Selection

1. Identify the embryo or embryos designated for transfer.
2. Confirm that the selected embryos match the transfer plan and the record. Use witness verification.
3. Review embryo stage, quality and day of development before loading. High‑quality embryos (e.g., Day 5 Grade 4AA blastocysts) are preferred for single embryo transfer.
4. Move the selected embryo or embryos into the transfer droplet using approved handling technique.

25.6 Catheter Loading

1. Prepare the catheter using transfer medium and remove air bubbles according to the laboratory method.
2. Draw the transfer medium into the catheter in the sequence used by the laboratory, typically including medium segments and, if part of the method, small air separators to help visualize the transfer column.
3. Load the embryo or embryos into the catheter using the loading volume in current use (commonly 10–30 µL). Ensure the embryo is positioned in the correct part of the column.
4. Inspect the loaded catheter as appropriate before handing it to the physician. Confirm identity and that the catheter is not kinked or leaking.
5. Hand the catheter to the physician using sterile technique and without unnecessary delay.

25.7 What to Watch During Transfer Support

The embryologist should pay attention to:

• Whether the correct embryo or embryos were selected and loaded.
• Whether the catheter column appears correctly loaded and free of bubbles or debris.
• Whether there are excess bubbles or loading irregularities that may interfere with the transfer.
• Whether the timing from dish removal to catheter handoff is kept as short as practical.
• Whether the catheter returns promptly for embryo retention check.

25.8 Post‑Transfer Check

1. Receive the catheter immediately after transfer.
2. Examine the catheter under the microscope according to laboratory policy.
3. Confirm whether any embryo remains in the catheter or associated flush medium if used. If an embryo is retained, notify the physician immediately and follow the repeat transfer procedure. Document the retention event.

25.9 Documentation

Document the following:

• Patient verification (name, date of birth).
• Witness identity.
• Date and time of transfer.
• Number and stage of embryos transferred.
• Embryo quality (e.g., blastocyst grade). Document whether embryos were thawed/warmed successfully.
• Catheter check result (e.g., empty, embryo retained).
• Operator identity.
• Any unusual event or repeat loading.

26. Quality Control and Equipment Maintenance

26.1 Purpose

To define the quality control, preventive maintenance, calibration, troubleshooting and corrective action requirements for equipment and monitored systems used in the IVF laboratory.

26.2 Scope

This section applies to all equipment used for specimen handling, culture, storage, environmental monitoring, cryostorage and laboratory support, including incubators, warming devices, microscopes, laminar flow hoods, refrigerators, freezers, pH systems, gas analyzers, thermometers and liquid nitrogen storage tanks.

26.3 Responsibility

The laboratory director or designee is responsible for oversight of the quality control and maintenance program. Staff members assigned to daily, weekly, monthly or periodic equipment checks are responsible for completing those tasks on schedule, documenting results and immediately reporting any out‑of‑range or abnormal finding.

26.4 General Requirements

All equipment used in the laboratory shall:

• Have a unique equipment identifier when practical.
• Have an installation or validation record where required.
• Have a defined monitoring and maintenance schedule.
• Have written instructions for routine use and basic troubleshooting.
• Be labeled or otherwise identified when out of service.
• Be removed from clinical use if function is uncertain or out of acceptable range.

26.5 Daily Quality Control Principles

Before patient‑use procedures begin each day:

1. Confirm that each required piece of equipment is powered on or in normal operating state.
2. Check displays, alarm indicators and environmental readings where applicable.
3. Record required temperature, gas or status values in the QC log.
4. Review results for acceptability before using the equipment for patient specimens. For example, incubators should be at 37 °C ± 0.5 °C with 5–6 % CO₂ and 5 % O₂.
5. If a result is abnormal, investigate before proceeding. Do not place specimens into equipment with unknown or out‑of‑range conditions.

26.6 Preventive Maintenance

Preventive maintenance shall be performed according to the laboratory schedule and manufacturer recommendations. Maintenance may include cleaning, filter replacement, calibration, lubrication where appropriate, inspection of moving parts, alarm testing, replacement of worn accessories and professional service. Cryostorage tank alarms should be tested quarterly and tanks should be checked three times per week or continuously monitored with level probes.

26.7 Out‑of‑Range Results or Equipment Failure

If a monitored value is outside the acceptable range or an equipment malfunction is suspected:

1. Recheck the result to exclude user or recording error.
2. Determine whether any patient specimen or active culture may have been affected. Move affected specimens to alternate validated equipment if necessary.
3. Remove the equipment from service when appropriate.
4. Notify laboratory leadership immediately.
5. Document the event, corrective action and whether patient specimens were affected.
6. Do not return the equipment to service until acceptable function has been confirmed.

26.8 Documentation

Quality control and maintenance documentation shall include:

• Equipment identification.
• Date and time of the check or service.
• Measured values or status findings.
• Initials or identity of the person performing the task.
• Corrective action when required.
• Service provider information when outside service is used.

26.9 Teaching Point

Good QC is proactive. The aim is to identify drift early, before patient specimens are affected. Staff should not ignore alarms or out‑of‑range readings.

27. Temperature Monitoring

27.1 Purpose

To ensure that all monitored equipment and controlled areas used for specimen handling, culture, storage and laboratory support remain within acceptable temperature limits.

27.2 Scope

This section applies to incubators, refrigerators, freezers, warming stages, hot blocks, ovens, room temperature areas, transport devices and any other equipment or area monitored for temperature.

27.3 General Principles

Temperature‑sensitive equipment shall be checked at the frequency defined by the laboratory schedule. At a minimum, all equipment essential to patient specimen handling and culture shall be checked before clinical use for the day.

27.4 Procedure

1. Use the designated thermometer, display, monitoring system or validated measurement device for each unit.
2. Read and record the temperature carefully.
3. Compare the observed value with the acceptable range assigned to that equipment. For incubators, acceptable temperature is typically 37 °C ± 0.5 °C.
4. Initial or sign the temperature log.
5. If the temperature is outside range, repeat the check and confirm whether the result is real.
6. If the result remains abnormal, follow the out‑of‑range procedure (see 27.5).

27.5 Out‑of‑Range Response

If temperature is outside acceptable range:

1. Confirm that the reading was taken correctly.
2. Check whether doors or lids were recently opened.
3. Verify whether the equipment display matches the independent reading when applicable.
4. Move patient materials if continued safe use is uncertain.
5. Notify laboratory leadership.
6. Document the event and corrective action.

28. Temperature and CO₂ Checks of Incubators

28.1 Purpose

To verify that incubators maintain the environmental conditions required for embryo culture.

28.2 General Standard

Incubators used for embryo culture must be checked routinely for temperature and gas conditions before use and at the frequency defined by laboratory policy. As a general practice, culture incubators should be reviewed at the start of the day and again whenever there is concern about performance, alarm condition or recovery after prolonged opening.

28.3 Daily Procedure

1. Review the incubator display for temperature and gas values.
2. Record the displayed values on the incubator QC log.
3. Verify gas concentration using the approved digital gas analyzer according to the established schedule. For tri‑gas incubators, target settings are 5–6 % CO₂ and 5 % O₂; pH of the media should be maintained at 7.25–7.35.
4. When an independent temperature verification device is used, record that value as well.
5. Compare all readings with the acceptable range assigned to that incubator.
6. Confirm that alarms are active and not in fault status when applicable.

28.4 If the Incubator Is Out of Range

1. Recheck the display and independent verification values.
2. Confirm that the door is closed and the incubator has had adequate recovery time if recently opened.
3. If the incubator remains out of range, do not place new patient specimens into the unit.
4. Transfer existing culture dishes to a validated alternate incubator if specimen safety may be affected.
5. Notify laboratory leadership immediately.
6. Document the deviation and corrective action.

29. Digital Gas Analyzer

29.1 Purpose

To define the use of a digital gas analyzer to independently verify incubator gas levels.

29.2 Scope

This section applies whenever incubator gas conditions are verified independently of the incubator’s built‑in display.

29.3 Preparation

Before use:

1. Confirm that the analyzer is within its required calibration or verification interval.
2. Confirm adequate battery charge or power supply.
3. Inspect tubing, ports and any filters for visible damage or blockage.
4. Zero or initialize the analyzer as required by the manufacturer instructions.

29.4 Procedure

1. Connect the analyzer according to the manufacturer instructions and the laboratory setup.
2. Sample gas from the designated incubator port or location.
3. Allow the reading to stabilize fully before recording the value.
4. Record the result in the QC log for the corresponding incubator.
5. Repeat for each incubator requiring gas verification.
6. After use, clean, store and recharge the analyzer as required.

29.5 Abnormal Results

If the gas reading is outside acceptable range:

1. Repeat the reading to exclude operator error.
2. Confirm whether the incubator display shows a similar value.
3. Notify laboratory leadership if the result remains abnormal.
4. Remove the incubator from clinical use if safe culture conditions are not confirmed.

30. Calibration of Thermometers

30.1 Purpose

To ensure that thermometers and temperature measurement devices used in the laboratory provide accurate readings.

30.2 Scope

This applies to all independent thermometers, probes and reference temperature devices used for verification of laboratory equipment.

30.3 Procedure

1. Identify the thermometer or probe to be verified.
2. Compare it against the laboratory’s traceable reference standard using the approved calibration setup.
3. Record the observed value of the device and the reference value.
4. Determine the difference between the device reading and the reference. If within tolerance, document the result and return it to service. If outside tolerance, label it out of service or apply the approved correction factor if permitted by laboratory policy.
5. Document the date, result and initials of the person performing the verification.

30.4 Frequency

Thermometers shall be verified at installation, at defined intervals thereafter and whenever the accuracy of the device is in question.

31. pH Meter Calibration and Use

31.1 Purpose

To define the calibration and use of the pH meter in the IVF laboratory.

31.2 Preparation

Before use:

1. Confirm that the meter is clean and functioning.
2. Confirm that the probe is properly stored and not damaged.
3. Assemble the required calibration buffers (typically pH 7.00, 7.35 and 9.00 or as recommended).
4. Allow buffers and samples to reach the appropriate temperature if required by the laboratory method.

31.3 Calibration Procedure

1. Rinse the probe with purified water and blot gently.
2. Place the probe in the first calibration buffer.
3. Allow the reading to stabilize and complete the calibration step according to manufacturer instructions.
4. Rinse and repeat with the second buffer and, if required, a third buffer.
5. Confirm that the meter accepts the calibration and shows acceptable electrode response.
6. Document the calibration.

31.4 Sample Measurement

1. Rinse the probe after calibration.
2. Place the probe into the sample to be measured.
3. Allow the reading to stabilize.
4. Record the pH value. For embryo culture, pH 7.25–7.35 is typically targeted.
5. Rinse the probe after the measurement and return it to storage solution as required.

31.5 If Calibration Fails

If the pH meter does not calibrate properly:

1. Recheck the buffers and probe condition. Replace buffers if expired or contaminated.
2. Clean the probe if appropriate.
3. Replace the probe or remove the meter from service if acceptable calibration cannot be achieved.

32. Inventory Control

32.1 Purpose

To maintain adequate stock of approved laboratory materials while ensuring traceability, expiration control and proper storage.

32.2 Receipt of Materials

When supplies are received:

1. Inspect the shipment for damage or temperature excursion when applicable.
2. Verify the item identity against the order.
3. Check lot number and expiration date.
4. Confirm that the correct item was received in acceptable condition.
5. Record receipt according to the laboratory inventory process.
6. Store the item under the correct conditions immediately (e.g., refrigerated, frozen, room temperature).

32.3 Storage and Rotation

1. Store materials according to manufacturer instructions (e.g., protect from light, maintain at 2–8 °C).
2. Segregate patient‑use media and reagents from non‑clinical materials when appropriate.
3. Rotate stock so earliest expiration is used first unless other control measures are in place.
4. Date items when opened if required by policy.
5. Remove expired, damaged or recalled materials from service immediately and document their disposition.

32.4 Shortage Prevention

The laboratory shall monitor stock levels sufficiently to prevent interruption of patient care. Low‑stock items shall be reordered before critical shortage occurs.

33. Guidelines for Biohazardous Waste

33.1 Purpose

To define the segregation, packaging, storage and disposal of biohazardous waste generated in the laboratory.

33.2 General Procedure

1. Place contaminated disposable materials into designated biohazard waste containers.
2. Place sharps only into approved sharps containers. Do not recap needles.
3. Do not overfill waste or sharps containers. Close and replace containers when they reach the defined fill level.
4. Close and replace containers when they reach the defined fill level.
5. Package and store waste for removal according to institutional and regulatory requirements. Maintain chain of custody and documentation for all hazardous waste.

33.3 Spill Response

Biohazard spills shall be contained, disinfected and documented according to the laboratory spill response procedure. Staff should be trained in using spill kits and disinfectants.

34. Chemical Hygiene Program

34.1 Purpose

To define the core chemical safety requirements for the laboratory.

34.2 General Requirements

1. All chemical containers shall be labeled with the identity of contents, hazard warnings and date opened.
2. Safety data sheets (SDSs) shall be accessible to staff.
3. Chemicals shall be stored according to hazard class and compatibility (e.g., acids separate from bases, oxidizers away from organics).
4. Staff shall use required PPE when handling chemicals (e.g., gloves, eye protection, lab coats).
5. Waste chemicals shall be disposed of according to institutional and regulatory requirements. Do not pour chemicals down drains unless permitted.

34.3 Training

Staff shall receive chemical hygiene training before working independently with laboratory chemicals. Training should include hazard identification, proper handling, emergency procedures and waste disposal.

35. Automatic CO₂ Incubators

35.1 Purpose

To define operation, monitoring, cleaning and maintenance requirements for automatic CO₂ incubators.

35.2 Start‑of‑Day Check

1. Confirm that the incubator is powered and functioning normally.
2. Review the displayed temperature and gas values.
3. Confirm alarm status and that CO₂ and O₂ supplies are adequate.
4. Record required QC values (temperature, CO₂ and O₂ concentration).

35.3 Use During the Day

1. Open doors only as long as necessary. Minimize disturbance to maintain stable environment.
2. Return culture dishes promptly after assessment or handling.
3. Avoid unnecessary traffic between incubators. Do not cross‑contaminate surfaces.
4. Do not overload the incubator beyond its validated capacity. Maintain spacing for airflow.

35.4 Cleaning and Maintenance

1. Clean and disinfect interior surfaces according to the maintenance schedule. Use non‑toxic disinfectants validated for incubators.
2. Remove shelves or trays for cleaning as required.
3. Replace filters, water pans or related components as required. Use sterile water for humidification.
4. Document all scheduled maintenance and service. Do not return incubators to clinical use until cleaning is complete and quality control checks are satisfied.

36. Laminar Flow Hoods

36.1 Purpose

To define use and maintenance of laminar flow hoods or equivalent clean workstations used for open specimen handling.

36.2 Start‑Up Procedure

1. Turn on the hood and allow it to run for the minimum period required by laboratory policy (commonly 15–30 min) before use.
2. Confirm airflow status if the hood provides airflow indicators. Do not use if airflow is inadequate.
3. Disinfect the work surface with appropriate disinfectant and allow to dry.
4. Arrange only the items required for the procedure. Do not clutter the work area.

36.3 Use During Procedures

1. Keep materials arranged to avoid blocking airflow. Do not place large items at the front or back of the hood.
2. Perform manipulations well within the clean zone (usually the center of the work surface). Avoid movements that disrupt laminar flow.
3. Minimize rapid movements that may disturb airflow (e.g., quick arm motions). Avoid talking, coughing or unnecessary conversation over open specimens.
4. Avoid placing unnecessary papers or bulky items in the hood. Remove waste promptly.

36.4 Shutdown and Maintenance

1. Remove waste and used materials at the end of work.
2. Disinfect the surface after use.
3. Maintain certification and service records. Hoods should be certified at least annually or according to regulation.
4. Remove the hood from patient use if airflow or certification status is uncertain. Notify laboratory leadership.

37. Liquid Nitrogen Storage Tanks

37.1 Purpose

To define monitoring, filling, maintenance and recordkeeping for liquid nitrogen (LN₂) storage tanks used for reproductive specimens.

37.2 Routine Monitoring

1. Check tank level or the approved monitoring parameter at the schedule defined by the laboratory (e.g., three times per week or continuous monitoring). Use calibrated level probes or dipsticks.
2. Record the value or status in the tank log.
3. Confirm alarm status if the tank is alarmed. Alarm systems should be tested quarterly.
4. Inspect the tank exterior for frost pattern changes, physical damage or other abnormal conditions.
5. Confirm that the tank remains above the laboratory refill threshold and within normal operating condition. Fill the tank before it reaches the minimum safe level.

37.3 Filling Procedure

1. Confirm the tank identity before filling.
2. Use appropriate PPE (cryogloves, face shield, protective apron).
3. Fill the tank using the laboratory’s safe liquid nitrogen filling process (e.g., slow fill to avoid thermal shock, avoid overfilling or splashing).
4. Avoid overfilling or splashing. Maintain ventilation in the filling area.
5. Recheck the level after filling if required by workflow. Record the fill event.

37.4 Abnormal Tank Conditions

If unexpected level loss, unusual frost, suspected leak or alarm occurs:

1. Notify laboratory leadership immediately.
2. Assess whether specimens need transfer to an alternate tank. Prepare an emergency transfer plan.
3. Document the event and action taken. Consider contacting the tank manufacturer or facility maintenance for inspection.

38. Liquid Nitrogen Storage, Use, Transportation, and Tank Monitoring

38.1 Purpose

To define safe handling, movement, transport and monitoring of liquid nitrogen and cryostorage systems. Cryostorage safety is critical; tank failures can result in catastrophic loss of embryos and gametes.

38.2 Personal Safety

Personnel handling liquid nitrogen shall use appropriate PPE including insulated gloves, eye protection and protective clothing as required. Work shall occur in well‑ventilated areas. Oxygen monitors should be installed in enclosed rooms containing multiple tanks to detect dangerous O₂ depletion.

38.3 Internal Movement of Specimens

1. Confirm specimen identity and intended destination before movement. Use two identifiers.
2. Prepare the receiving tank or location before removing the specimen from storage.
3. Move specimens quickly and carefully to minimize warming. Keep cryodevices below the vapor line of LN₂ whenever possible.
4. Confirm placement in the new location immediately. Verify that the new tank is at an appropriate level and temperature.
5. Update the inventory record at once. Document the transfer, time and personnel involved.

38.4 Transport to or from Offsite Storage

1. Confirm patient authorization and documentation where required. Consent forms should specify whether offsite storage is allowed.
2. Reconcile the inventory before transport. Verify each specimen being transferred.
3. Prepare the transport vessel and confirm it is suitable for cryogenic transport (dry shipper or validated container). Pre‑cool the vessel.
4. Verify each specimen being transferred. Label the transport vessel with patient identifiers and destination facility.
5. Maintain chain of custody throughout transport. Document personnel handling and times of departure/arrival.
6. Reconcile inventory again at receipt into the destination storage system. Verify that specimens were not compromised during transport.
7. Document the date, personnel involved, specimens transferred and final location.

39. Witness Workflow

39.1 Purpose

To define the witness process for critical specimen handling steps. The witness process ensures that specimen identity is verified by two qualified individuals or by an electronic witness system at defined checkpoints.

39.2 General Standard

A second qualified staff member, or an approved electronic witness system when in use, shall confirm specimen identity at critical steps. The witness must independently verify the match between the patient identifiers on the specimen, receiving container and record.

39.3 Critical Witness Points

Witnessing shall occur, as applicable, before or during:

• Sperm preparation release for use.
• Conventional insemination.
• ICSI.
• Embryo movement for assisted hatching or biopsy.
• Biopsy sample loading.
• Cryopreservation and thawing/warming.
• Embryo transfer preparation.
• Specimen discard or disposition.
• Release from storage or transfer to offsite storage.

39.4 Witness Procedure

1. The operator presents the labeled materials and record.
2. The witness independently reads the identifiers on all relevant items and compares with the patient record. Do not simply ask, “Does this look right?”; the witness must actively verify.
3. The witness confirms the match verbally or through the approved system.
4. The witness documents the check according to laboratory policy (initials, date and time or electronic signature).
5. If there is any discrepancy, the procedure stops until the issue is resolved.
6. The operator does not proceed until the witness step is complete.

39.5 Teaching Point

A witness is not a passive observer. The witness performs an independent identity check. Complacency undermines the purpose of witnessing.

40. Embryologist Training

40.1 Purpose

To define orientation, training, competency assessment and continuing education requirements for embryology personnel.

40.2 Initial Training

Before independent work, personnel shall complete:

1. Laboratory safety orientation.
2. Biohazard training.
3. Chemical hygiene training.
4. Occupational safety training required by the institution.
5. Review of the procedure manual.
6. Supervised observation of assigned procedures.
7. Supervised hands‑on practice for assigned procedures.
8. Documented competency assessment (e.g., demonstration of proficiency in sperm preparation, oocyte handling, embryo culture, ICSI under supervision).

During supervised training, the trainee should first observe the procedure, then perform portions of the procedure under direct supervision, then perform the full procedure while being evaluated for technique, identity control, documentation and response to unexpected findings. Training should include instruction on the latest consensus guidelines for embryo grading and cryostorage management.

40.3 Ongoing Competency

Competency shall be reassessed at defined intervals and whenever a new method, device or major workflow change is introduced. Continuing education should include review of professional society guidelines and participation in proficiency testing.

40.4 Training Records

Training records shall identify the procedure taught, trainer, dates of observation and performance, competency outcome and authorization for independent performance. Records must be retained according to laboratory policy.

40.5 Teaching Point

Training is complete only when the trainee can perform the procedure accurately, document it correctly and recognize when to stop and ask for help. Competency includes adherence to identity control and witness protocols.

41. Oocyte Donation Program

41.1 Purpose

To define laboratory controls applicable to donor oocyte cases.

41.2 General Requirements

Donor oocyte cases shall follow the same standards for specimen identification, traceability, witnessing, storage and documentation used for all other laboratory cases, with any additional donor‑specific consent, infectious disease screening and regulatory documentation requirements incorporated into the case workflow.

41.3 Laboratory Workflow

1. Confirm donor and recipient documentation according to the case plan. Donor anonymity should be maintained if applicable.
2. Label all materials clearly to preserve donor‑recipient traceability. Use separate color codes or numbering schemes if needed to differentiate donors.
3. Maintain careful separation of donor case materials from other active cases. Do not co‑incubate donor and autologous oocytes in the same incubator shelf unless identity control is robust.
4. Apply required witness steps at all critical points (retrieval, fertilization, cryopreservation, transfer).
5. Document donor oocyte allocation, insemination, cryopreservation, transfer or discard according to the case plan. Retain donor consents and infectious disease screening results as required by regulation.

42. Forms

The laboratory shall maintain controlled forms for patient information, embryo culture, cryopreservation, thawing or warming, transfer, specimen tracking, HCT/P labeling when required and donor eligibility or ineligibility documentation when applicable. Forms shall be current, legible and completed in full. Electronic forms should maintain audit trails and secure electronic signatures.

43. Quality Control Forms and Logs

Controlled QC forms and logs shall be maintained for room temperature, water quality, drying oven temperature, deep freezer temperature, hot block temperature, incubator temperature, daily temperature records, gas checks, maintenance records, calibration records, tank monitoring and other required monitored parameters. Entries shall be made at the time the activity is performed or immediately thereafter. Logs should be reviewed regularly to identify trends or deviations.

44. Policies

44.1 Exposure Control

The laboratory shall maintain an exposure control policy addressing bloodborne pathogens, PPE, exposure response, spill response and waste handling. Staff should know how to access and use spill kits and eye wash stations.

44.2 OSHA and Safety Compliance

The laboratory shall comply with applicable occupational safety requirements including staff training, hazard communication, accident prevention and incident reporting.

44.3 Latex Allergy

The laboratory shall maintain practices to reduce latex exposure for patients and staff with known or suspected sensitivity. Use latex‑free gloves and equipment when necessary.

44.4 Manufacturer Recalls and Product Alerts

The laboratory shall maintain a process for identifying, reviewing, documenting and acting upon manufacturer recalls and product alerts affecting laboratory materials or equipment. Action may include removal from service, quarantining and contacting the manufacturer.

44.5 Adverse Events

The laboratory shall maintain procedures for documenting, investigating and reporting adverse laboratory incidents and adverse patient reactions in accordance with applicable policy and regulation. Lessons learned should feed into quality improvement.

45. Document Control

This manual is a controlled document. Revisions shall be reviewed and approved before implementation. Superseded versions shall be removed from active use and archived according to document control policy. The current approved version shall be available to authorized staff. Staff should be notified of revisions and trained on new procedures as necessary.